(ACS Symposium Volume 207) Harvey W. Blanch, E. Terry Papoutsakis, and Gregory Stephanopoulos (Eds.)-Foundations of Biochemical Engineering. Kinetics and Thermodynamics in Biological Systems-American .pdf

of
Biochemical Engineering
In Foundations of Biochemical Engineering; Blanch, H., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
Foundations
of Biochemical Engineering
Kinetics and Thermodynamics
in Biological Systems
Harve W Blanch EDITOR

University of California, Berkeley
E. Terry Papoutsakis, EDITOR
Rice University
Gregory Stephanopoulos, EDITOR
California Institute of Technology
Based on the 1982 Winter Symposium
of the ACS Division of Industrial
and Engineering Chemistry jointly
sponsored by the Division of Microbial
and Biochemical Technology,
Boulder, Colorado,
January 17-20, 1982
ACS SYMPOSIUM SERIES 207
AMERICAN CHEMICAL SOCIETY
W A S H I N G T O N , D.C. 1983

Library of Congress Cataloging in Publication Data
American Chemical Society. Division of Industrial
and Engineering Chemistry. Winter Symposium
(1982: Boulder, Colo.)
Foundations of biochemical engineering.
(ACS symposium series, ISSN 0097-6156; 207)
Includes bibliographies and index.
1. Biochemical engineering—Congresses
I. Blanch, Harvey W., 1947
E. Terry, 1951- . III.
American Chemical Society. Division of Industrial
and Engineering Chemistry. V. American Chemical
Society. Division of Microbial and Biochemical Tech-
nology. VI. Title. VII. Series.
TP248.3.A48 1982 660'.63 82-20694
ISBN 0-8412-0752-6
Copyright © 1983
American Chemical Society
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FOREWORD
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PREFACE
THIS V O L U M E IS BASED on the 1982 Winter Symposium. Following recent

tradition, this Winter Symposium was organized to provide a forum for
expression of new ideas and new research approaches.
This volume discusses the fundamentals of biotechnology; that is,
kinetics and thermodynamics in biological systems. State-of-the-art keynote
chapters by established researchers are employed to provide the proper
perspective and direction
approaches.
In the last few years biotechnology has grown tremendously in scope
and significance, both as an active research area and as an industrial
activity. There has also been a clear trend toward the exploration of the
most sophisticated aspects of biological growth: tissue cultures, active
biomolecules, antitumor compounds, chemical feedstocks from biomass,
and gene cloning, to mention only a few. This trend has been accompanied
by the increased use of computers in the design and control of the bio-
logical reactor.
The explosive growth of the field and the use of computers have
magnified, however, a fact that had been realized for quite some time by
a few. That is, that the kinetics and thermodynamics of biochemical
reactions and biological growth are understood only primitively. We should
remind ourselves that biological systems are immensely more complicated
than the nonbiological systems upon which most of chemical technology is
based. We should not forget that until quite recently there were relatively
few active investigators in biotechnology throughout the world, and that
most of these were microbiologists by training. This has changed dramat-
ically, however, as biotechnology has become a common word among
biochemists, cell geneticists, molecular biologists, gene doners, engineers,
protein chemists, and others.
In preparing this book, we strived to stress two things:
• the interdisciplinary nature of biotechnology. We thus made every

effort to bring together prominent researchers from both the life
sciences and biochemical engineering.
• the need to examine the fundamentals of biotechnology more sys-
tematically and vigorously. We believe that no structure can grow
well and last long without good foundations.
ix

The topics in this volume represent both keynote chapters and chapters on
new research. They have been arranged in six sections, such that each
section builds on the preceding one. The volume begins with an examina
tion of fundamental biochemical events in the cells. It proceeds with the
examination of the interaction of the biochemical events that constitute the
growth of an individual cell; then it examines homogeneous and hetero
geneous cell populations. The book continues with thermodynamic aspects
of biological growth, and ends with the application of all of the above to
bioreactor design.
Obviously, completeness has been sacrificed to diversity. We felt that
the examination of the interactions among these fundamental aspects was
more important than looking in depth at only a few of these aspects. Most
of you would agree that ongoing research is a vital and exciting process,
consisting of active and ofte heated interaction f data ideas d minds
We hope that the reader
in this process.
In putting together the symposium on which this book is based, we
relied on the judgement, help, and advice of many colleagues, to whom we
express our sincere thanks. We also thank the organizing committee of
the Winter Symposia of the Division of Industrial and Engineering
Chemistry and, in particular, the former chairman of the committee,
Nicholas A . Peppas. Thanks are also due to R. W. Eltz, chairman of
the Division of Microbial and Biochemical Technology of the American
Chemical Society, joint sponsor of the symposium. We are sincerely
indebted to Steve Drew of Merck, Sharp & Dohme Research Labora
tories for bringing us the private sector's views on the symposium's theme.
HARVEY W. BLANCH
University of California
Berkeley, CA
E. TERRY PAPOUTSAKIS
Rice University
Houston, TX
GREGORY STEPHANOPOULOS
California Institute of Technology
Pasadena, CA
June 1, 1982

ACKNOWLEDGMENTS
Financial assistance for the 1982 Winter symposium was received from
National Science Foundation
and
Abbott Laboratories
Beckman Instruments Inc
Becton Dickinso
Carnation Company
Chevron Research Company
E.I. du Pont de Nemours & Company, Inc.
Eastman Kodak Company
Exxon Research and Engineering Company
General Mills, Inc.
Gist-Brocades N V Research & Development
Glaxo Operations U K Limited
W. R. Grace & Company
Gulf Research and Development Company
Hoffmann-La Roche, Inc.
Kraft, Inc.
Merck Sharp and Dohme Research Laboratories
Monsanto Company
PPG Industries, Inc.
Pfizer, Inc.
Phillips Petroleum Company
The Procter and Gamble Company
Stauffer Chemical Company
Syntex Corporation
Weyerhaeuser Company

1
Models of Gene Function

General Methods of Kinetic Analysis and Specific Ecological Correlates
MICHAEL A. SAVAGEAU
University of Michigan, Department of Microbiology and Immunology,
Ann Arbor, MI 48109
Alternative molecular designs for gene control

involving positiv
related to the
that harbors them: positive elements are associated
with high demand for expression of the regulated
structural genes; while negative elements are
associated with low demand for their expression.
This is called the demand theory of gene regulation.
These results are based on a mathematical analysis
of the alternative models. The formalism used is a
general one involving power-law functions that has
been developed previously for the mathematical
analysis of complex biological systems. This
formalism, which is of interest in its own right, is
briefly reviewed, although details of the analysis
are not presented. The principal steps in the
development of the demand theory are presented and
the molecular, physiological and ecological
implications are made e x p l i c i t . This theory i s
supported by experimental evidence from more than 40
different systems and is used to make testable
experimental predictions for more than 14 additional
systems.
The primary purpose o f t h i s symposium i s t o have b i o l o g i s t s

and e n g i n e e r s examine the i n t e r d i s c i p l i n a r y area o f biotechnology
from the fundamental p e r s p e c t i v e o f k i n e t i c s and thermodynamics*
As w i t h any i n t e r d i s c i p l i n a r y area, p a r t i c u l a r l y one undergoing
r a p i d change and i n t e l l e c t u a l ferment, biotechnology means
d i f f e r e n t things t o d i f f e r e n t people* I t i s therefore appropriate
to give a thumb-nail sketch o f t h i s area as I see i t *
B i o t e c h n o l o g y i s as o l d as r e c o r d e d h i s t o r y . The e a r l y
domestication o f microorganisms f o r wine making, b r e w i n g , b a k i n g
and f o o d p r e s e r v a t i o n i s a prime example g r a p h i c a l l y depicted on
0097-6156/83/0207-0003$06.75/0
© 1983 American Chemical Society

4 BIOCHEMICAL ENGINEERING
Egyptian tombs (1)· Of course, t h i s was a very e m p i r i c a l a r t t h a t

advanced o n l y s l o w l y . Even i n more r e c e n t times, d u r i n g the p o s t -
war expansion o f modern i n d u s t r i a l t e c h n o l o g y , t h e r e has been
important biotechnology, e.g., as a sector of modern
pharmaceutical and chemical i n d u s t r i e s . Throughout t h i s p a s t , the
b i o l o g i c a l component has o f t e n been t h e weakest l i n k i n t h e
o v e r a l l bio-technology c h a i n . To be sure, there was mass balance
and the Monod growth law, which helped t o p r e d i c t the b e h a v i o r o f
t h e b i o l o g i c a l component, but t h a t was about a l l . One g e n e r a l l y
had t o l i v e w i t h t h e u n c e r t a i n t i e s i n h e r e n t i n t h e b i o l o g i c a l
component o f the o v e r a l l system.
Recent developments i n b i o l o g y have ushered i n a " r e v o l u t i o n "
i n o u r knowledge o f the c e l l , p a r t i c u l a r l y the m i c r o b i a l c e l l .
For the f a m i l i a r b a c i l l u s E s c h e r i c h i a c o l l 3/5 t o 4/5 of a l l i t s
molecular elements are known and new p r o t e i n s e p a r a t i o n techniques
are speeding t h i s i d e n t i f i c a t i o
we s h a l l soon have t h
However, we s t i l l know r e l a t i v e l y l i t t l e about t h e i n t e g r a t e d
b e h a v i o r o f t h e c e l l , how i t w i l l behave when i t s m o l e c u l a r
elements are changed or when i t f i n d s i t s e l f i n a novel
environment·
The f u l l p o t e n t i a l of biotechnology w i l l be r e a l i z e d , i n my
view, o n l y when (a) the b i o l o g i c a l component i s brought w i t h i n the
realm o f engineering design and (b) e n g i n e e r i n g methods of systems
a n a l y s i s and p r e d i c t i o n are brought w i t h i n the realm o f c e l l and
m o l e c u l a r b i o l o g y . O n l y t h e n w i l l t h e r e be a s t r o n g u n i t a r y
b i o t e c h n o l o g y . In the l a s t few years developments i n recombinant
DNA methodology have shown t h a t the f i r s t of t h e s e o b j e c t i v e s i s
now f e a s i b l e . T h e r e a l s o has been p r o g r e s s toward the second
o b j e c t i v e , but t h e r e i s s t i l l much t o be done here.
In t h i s paper I s h a l l f i r s t r e v i e w b r i e f l y some o f our own
a t t e m p t s t o d e v e l o p general methods o f k i n e t i c a n a l y s i s t h a t are
a p p r o p r i a t e f o r complex b i o l o g i c a l systems. Next I s h a l l d i s c u s s
t h e a p p l i c a t i o n o f t h e s e methods t o a l t e r n a t i v e models o f gene
r e g u l a t i o n . The r e s u l t o f such an a n a l y s i s i s t h e demand t h e o r y
of g e n e r e g u l a t i o n i n which t h e m o l e c u l a r n a t u r e o f g e n e t i c
r e g u l a t o r y s y s t e m s i s d i r e c t l y r e l a t e d t o t h e demand f o r gene
expression, as determined by the organism's e c o l o g i c a l n i c h e .
Power-law Formalism
E a r l y e f f o r t s t o a n a l y z e b i o l o g i c a l systems were b l i n d e d by
the success t h a t engineers have had in analyzing complex
t e c h n o l o g i c a l ( o f t e n e l e c t r i c a l and mechanical) systems and by the
power and elegance o f the mathematical methods a t t h e i r d i s p o s a l .
Over a p e r i o d o f years the e f f o r t t o make b i o l o g i c a l phenomena f i t
i n t o the same mold became i n c r e a s i n g l y f r u s t r a t e d . Biological
phenomena a r e h i g h l y n o n l i n e a r and cannot be made t o wear the
l i n e a r s t r a i g h t j a c k e t t h a t t e c h n o l o g i c a l systems o f t e n s u b m i t t o
willingly. F u r t h e r m o r e , t h e t y p e s o f n o n l i n e a r i t i e s t h a t are

1. SAVAGEAU Models of Gene Function
found i n t e c h n o l o g i c a l systems are very d i f f e r e n t from those found

i n b i o l o g i c a l systems. On the other hand, d i s e n c h a n t m e n t set i n
w i t h t h e t h e n f a s h i o n a b l e approach o f p i e c i n g together i n an ad
hoc manner complex computer models t h a t could be " a d j u s t e d " u n t i l
t h e y came t o mimic some aspect o f the b i o l o g i c a l phenomenon under
study. In f o r m u l a t i n g our approach t o these problems, we d e c i d e d
t o go back and s t a r t from s c r a t c h . T h i s r e q u i r e d going back t o
fundamental k i n e t i c s , i d e n t i f y i n g the e s s e n t i a l character of
b i o l o g i c a l n o n l i n e a r i t i e s , and a t t e m p t i n g the development o f a
mathematical formalism rooted i n b a s i c b i o l o g y and s p e c i f i c a l l y
designed t o d e a l with these types of systems.
The fundamental kinetic d e s c r i p t i o n o f the u n d e r l y i n g
mechanisms i n b i o l o g i c a l systems i n v o l v e s rational-function
n o n l i n e a r i t i e s ( 2 - 4 ) . Although i n p r i n c i p l e these f u n c t i o n s can
a c c u r a t e l y represent any mechanism of interest they are
mathematically much
b i o l o g i c a l systems compose
some form o f s i m p l i f y i n g a p p r o x i m a t i o n i s r e q u i r e d f o r t h e s e
systems; such methods must capture the e s s e n t i a l n o n l i n e a r
a t t r i b u t e s o f b i o l o g i c a l phenomena and yet be s u f f i c i e n t l y simple
so t h a t complex b i o l o g i c a l systems can be handled m a t h e m a t i c a l l y .
These methods a l s o s h o u l d y i e l d a standard or c a n o n i c a l form to
f a c i l i t a t e comparisons o f a l t e r n a t i v e models and t o a l l o w a n a l y s i s
of a p r i o r i as w e l l as ad hoc models. Although t h e s e a r e i d e a l s
and no c u r r e n t l y a v a i l a b l e method o f k i n e t i c a n a l y s i s f u l l y
s a t i s f i e s a l l of these requirements, a method based on power-law
f u n c t i o n s i s p a r t i c u l a r l y promising.
D e r i v a t i o n o f t h e B a s i c Equations. The b a s i c equations are

obtained by forming the T a y l o r s e r i e s e x p a n s i o n o f the r a t i o n a l
f u n c t i o n i n a l o g a r i t h m i c space and t h e n r e t a i n i n g o n l y t h e
constant and l i n e a r terms (2,5,6,3)· T h i s procedure y i e l d s a
product o f power f u n c t i o n s i n the corresponding C a r t e s i a n space.
The f u n c t i o n a l e q u a t i o n s d e s c r i b i n g a g e n e r a l system of η
elements d i f f e r e n t i n k i n d and/or l o c a t i o n a r e composed o f sums
and d i f f e r e n c e s o f r a t i o n a l f u n c t i o n s , but s i n c e the sum o f two
r a t i o n a l f u n c t i o n s i s a l s o a r a t i o n a l f u n c t i o n , the system of
e q u a t i o n s can be r e w r i t t e n as a s i m p l e d i f f e r e n c e between two
composite r a t i o n a l f u n c t i o n s that are each p o s i t i v e .
When these composite r a t i o n a l f u n c t i o n s a r e a p p r o x i m a t e d by
power-law f u n c t i o n s , the d e s c r i p t i o n o f the e n t i r e system can be
w r i t t e n (5,7,3,4):
(1)
i = 1, 2 , ···

The behavior of complex b i o l o g i c a l systems i s d e t e r m i n e d by

the nature o f the u n d e r l y i n g mechanisms and their abundant
i n t e r r e l a t i o n s h i p s , as expressed mathematically i n Eqn. ( 1 ) .
Exact Representation. Although the power-law f o r m a l i s m was

o r i g i n a l l y d e r i v e d as an a p p r o x i m a t i o n , one can o f t e n o b t a i n an
exact representation of rational-function nonlinearities by
i n t r o d u c i n g a d d i t i o n a l v a r i a b l e s ( 8 ) . T h i s o b v i o u s l y extends the
g e n e r a l i t y and utility of the power-law formalism. Any
disadvantage caused by the i n c r e a s e i n number of v a r i a b l e s i s more
t h a n o f f s e t by t h e decrease i n complexity o f the n o n l i n e a r i t i e s .
The number of parameter values plus i n i t i a l c o n d i t i o n s n e c e s s a r y
to characterize a system remains the same in either
representation.
T h i s procedure allows a set o f d i f f e r e n t i a l equations
involving rational functions
form, t o be transformed
functions, which do have a standard form. This greatly
f a c i l i t a t e s modeling and a n a l y s i s . One can e a s i l y a l t e r the model
or change c o n d i t i o n s without r e w r i t i n g e q u a t i o n s , f u n c t i o n s , o r
subroutines. One s i m p l y i n s e r t s an a l t e r n a t i v e s e t or subset o f
parameter values i n t o the standard format. T h i s has allowed us t o
develop a standardized s e t o f e f f i c i e n t computer a l g o r i t h m s f o r
g r a p h i c a l i n t e r a c t i v e modeling and a n a l y s i s of b i o l o g i c a l systems.
Justification. The power-law formalism i s j u s t i f i e d by f o u r

types of evidence. F i r s t , i t s v a l i d i t y r e s t s on t h e o r e t i c a l
grounds. Because t h e a p p r o x i m a t i o n i s d e r i v e d u s i n g T a y l o r ' s
theorem, i t i s g u a r a n t e e d t o be an a c c u r a t e r e p r e s e n t a t i o n a t
l e a s t o v e r a c e r t a i n range of values f o r the c o n c e n t r a t i o n
v a r i a b l e s . Because the approximation i s n o n l i n e a r , t h e r a n g e o f
v a l i d i t y i s c o n s i d e r a b l y g r e a t e r than t h a t f o r the corresponding
l i n e a r approximation. Second, the power-law f o r m a l i s m has been
j u s t i f i e d by d i r e c t experimental o b s e r v a t i o n o f r e a c t i o n s i n s i t u .
Kohen etal. (9) have examined the k i n e t i c s o f individual
biochemical r e a c t i o n s w i t h i n l i v i n g c e l l s and found t h a t these are
best described by power-law f u n c t i o n s of the reactant
concentrations. T h i r d , t h i s formalism p r e d i c t s t h a t i n a s e r i e s
of steady s t a t e s the c o n c e n t r a t i o n v a r i a b l e s should be r e l a t e d t o
one a n o t h e r by a s t r a i g h t l i n e i n a l o g - l o g p l o t . The range o f
concentration v a l u e s f o r which a s t r a i g h t - l i n e r e l a t i o n h o l d s
c o r r e s p o n d s t o t h e range o f v a l i d i t y f o r the approximation. A
number o f d i f f e r e n t systems have been examined p r e v i o u s l y i n t h i s
regard and the approximation was found t o be v a l i d f o r more than a
1 0 0 - f o l d change i n c o n c e n t r a t i o n v a r i a b l e s ( 3 ) . F i n a l l y , one can
d e r i v e a general growth law i n t h i s f o r m a l i s m and a s k t o
what e x t e n t r e a l systems adhere t o the law. As i t turns out, a l l
of the w e l l - e s t a b l i s h e d growth laws t h a t one finds i n the
b i o l o g i c a l l i t e r a t u r e are s p e c i a l cases o f t h i s general growth law
(**,10K There a r e a number o f other i m p l i c a t i o n s that have been

1. SAVAGEAU Models of Gene Function 7
deduced with t h i s formalism and t e s t e d a g a i n s t experimental

evidence, and agreement has been found (e.g., see Ref. Thus,
t h e agreement between t h e o r e t i c a l p r e d i c t i o n s and a l a r g e number
of independent e x p e r i m e n t a l o b s e r v a t i o n s p r o v i d e s a w e a l t h o f
evidence j u s t i f y i n g use o f the power-law formalism.
P r i n c i p a l Advantages. T h e r e a r e a number o f d i f f i c u l t i e s
a s s o c i a t e d with the m o d e l i n g and a n a l y s i s o f complex n o n l i n e a r
systems, ( a ) The f u n c t i o n a l form o f the n o n l i n e a r i t i e s i s o f t e n
unknown, as are the numbers o f i n t e r a c t i o n s and p a r a m e t e r s t h a t
must be s p e c i f i e d , ( b ) Once one h a s assumed a f u n c t i o n a l form
there i s s t i l l d i f f i c u l t y i n e x t r a c t i n g s t a t i s t i c a l e s t i m a t e s o f
the p a r a m e t e r v a l u e s from experimental data, (c) The amount o f
experimental data i t s e l f t h a t i s r e q u i r e d t o c h a r a c t e r i z e many
n o n l i n e a r mechanisms i n c r e a s e s e x p o n e n t i a l l y with the dimensions
of the problem, (d) Genera
system o f n o n l i n e a r equation
Linear analysis largely overcomes these difficulties.
However, there i s a c o n s i d e r a b l e c o s t . I t i s v a l i d f o r only small
v a r i a t i o n s i n t h e c o n c e n t r a t i o n v a r i a b l e s and t h i s i s u s u a l l y
u n r e a l i s t i c f o r most b i o l o g i c a l systems o f i n t e r e s t .
The power-law formalism provides a j u d i c i o u s compromise that
has many o f the advantages o f l i n e a r a n a l y s i s w i t h o u t i t s s e v e r e
limitations. ( a ) The f u n c t i o n a l form i s known and the
i n t e r a c t i o n s and p a r a m e t e r s a r e r e a d i l y s p e c i f i e d , ( b ) From
experimental data one can e x t r a c t parameter values f o r the
i n d i v i d u a l r a t e laws by l i n e a r r e g r e s s i o n i n a l o g a r i t h m i c s p a c e ,
(c) The d a t a n e c e s s a r y f o r such an a n a l y s i s has a p o l y n o m i a l
( r a t h e r than e x p o n e n t i a l ) i n c r e a s e w i t h t h e d i m e n s i o n s o f t h e
problem, (d) General methods f o r o b t a i n i n g s t e a d y - s t a t e s o l u t i o n s
a r e a v a i l a b l e by t r a n s f o r m i n g t h e p r o b l e m i n t o l i n e a r t e r m s .
General purpose computer algorithms have been developed f o r
dynamic s o l u t i o n s , and parameter e s t i m a t i o n from i n t e g r a t e d system
behavior i s also possible. (For more d e t a i l e d d i s c u s s i o n o f the
advantages and disadvantages o f t h i s and o t h e r a p p r o a c h e s t o t h e
modeling and a n a l y s i s o f n o n l i n e a r systems, see Ref. 3·)
T h i s concludes my b r i e f review o f the k i n e t i c methods t h a t we
have developed f o r d e a l i n g w i t h complex b i o l o g i c a l systems. These
methods a l r e a d y have been a p p l i e d t o a v a r i e t y o f biochemical and
g e n e t i c systems (e.g., see Réf. 3)· However, f o r the remainder o f
t h i s paper I s h a l l t r e a t one p a r t i c u l a r a p p l i c a t i o n i n some
detail·
Demand Theory o f Gene Regulation
The c e n t r a l theme t h a t I would l i k e t o emphasize i n t h i s

a p p l i c a t i o n i s summarized g r a p h i c a l l y as f o l l o w s :

Environment + Physiology
Demand f o r Gene E x p r e s s i o n
Molecular Design o f Gene C o n t r o l
By the c o n c l u s i o n o f t h i s paper you w i l l come t o a p p r e c i a t e t h e

l o g i c and i m p l i c a t i o n s of t h i s diagram, and, as you w i l l see, the
message i s a simple but general one.
A l t e r n a t i v e M o l e c u l a r Designs. Let us begin w i t h the

i n f l u e n t i a l model of Jacob and Monod (J2) shown i n F i g u r e 1 Note
t h a t c o n t r o l i s exercise
i n i t i a t i o n of t r a n s c r i p t i o n
paradigm of b i o l o g i s t s c o n c e r n e d w i t h t h e n o r m a l p r o c e s s e s o f
homeostasis, growth and d i f f e r e n t i a t i o n , as w e l l as their
pathological manifestations—infectious diseases, inborn g e n e t i c
e r r o r s and c a n c e r . By almost any c r i t e r i o n t h i s has been one o f
the most f r u i t f u l ideas i n modern b i o l o g y .
What i s the evidence f o r i t s v a l i d i t y ? B a c t e r i a l systems
were t h e f i r s t t o be understood i n s u f f i c i e n t molecular d e t a i l t o
t e s t these i d e a s . There i s now good evidence s u p p o r t i n g t h i s mode
o f r e g u l a t i o n i n many p r o k a r y o t i c s y s t e m s (V3) · However, i n
r e c e n t y e a r s e v i d e n c e f o r a l t e r n a t i v e molecular designs a l s o has
been accumulated.
In some systems the r e g u l a t o r y p r o t e i n i s not a r e p r e s s o r but
an activator that i s necessary for the initiation of
transcription. The example most o f t e n c i t e d i s t h e arabinose
c a t a b o l i c operon o f E. c o l i (14). A schematic model i s shown i n
F i g u r e 2. The N-gene product of bacteriophage lambda i s another
example o f a p o s i t i v e - a c t i n g r e g u l a t o r p r o t e i n , but i n t h i s c a s e
i t a c t s as an a n t i t e r m i n a t o r m o d u l a t i n g the t e r m i n a t i o n o f RNA
transcripts before they can be extended i n t o the r e g u l a t e d
s t r u c t u r a l genes (15-19) · A schematic model of t h i s mechanism i s
given i n F i g u r e 3· We can p r e d i c t a t h i r d a l t e r n a t i v e i n w h i c h
the regulatory protein i s a n e g a t i v e - a c t i n g proterminator
a f f e c t i n g t h e t e r m i n a t i o n o f RNA t r a n s c r i p t s a t a s i t e t h a t
precedes the r e g u l a t e d s t r u c t u r a l genes (see F i g u r e 4 ) .
D e t a i l s o f t h e s e a l t e r n a t i v e s , and t h e terminology t h a t I
s h a l l adopt i n d i s c u s s i n g them, a r e g i v e n i n t h e c a p t i o n s o f
F i g u r e s 1-4 and summarized i n T a b l e I . T h e r e a r e a number of
e s t a b l i s h e d v a r i a t i o n s on these themes. Although we are c u r r e n t l y
a t a l o s s t o r a t i o n a l i z e the e n t i r e v a r i e t y o f molecular d e s i g n s ,
some s u c c e s s i n u n d e r s t a n d i n g t h e r o l e o f p o s i t i v e vs negative
elements has been achieved with the demand theory o f gene
r e g u l a t i o n (20).

INDUCIBLE SYSTEMS
inducers.
REPRESSOR
SG| SG 2 |Q| SGi SG 2
• OPERON- RNA transcript ^
REPRESSIBLE SYSTEMS
corepressor^.
REPRESSOR
Ï1 IPIOJ SG| I S62 \T\j { |R J jf SGi SG 2
• OPERON- RNA transcript ^
Figure 1. Repressor control of transcription initiation. A regulatory gene, R, codes for a repressor
protein. Structural genes, SG, are preceded by a control region in the DNA consisting of a promoter
site, P, and a modulator site, O.

In an inducible system, the repressor protein normally binds to a modulator site called the operator, O, and
consequently blocks transcription of the adjacent structural genes. However, when the specific inducer for the
system is present in an appropriate environment, inducer binds to the repressor and alters its conformation so that

the repressor is released from the operator site. The promoter site is thereby freed, and the transcription machinery
can initiate synthesis of mRNA. The mRNA, in turn, is translated to yield the protein products of the operon. In
a repressible system, the operon is normally expressed because the repressor protein normally has a conformation
that prevents its binding to the operator site. However, when the specific corepressor for the system is present,
corepressor binds to the {apo)remessor protein and alters its conformation so that repressor now binds the operator
and blocks the initiation of transcription at the promoter site.
ο
INDUCIBLE SYSTEMS
inducer
ACTIVATOR
SGi SG 2 SGi SG 2
3C I I
OPERON- 1 RNA tronscript~*>
REPRESSIBLE SYSTEMS
compressor^.
ACTIVATOR
SGi SG 2 1 Π » Π ( SGi I SG 2 1τ|$

• OPERON• RNA transcript^
Ο
Ο
Figure 2. Activator control of transcription initiation. A regulatory gene, R, codes for an activator m
protein. Structural genes, SG, are preceded by a control region in the DNA consisting of a promoter

site, P, and a modulator site, I. ο
>
In an inducible system, the activator protein normally has a conformation that prevents it from interacting with
a modulator site called the initiator, I. Without this specific interaction, transcription is not initiated and there is w

no expression of the operon. However, when inducer is present in an appropriate environment, activator is con
verted to a conformation that can bind the initiator site and facilitate the initiation of transcription at the promoter.
In a repressible system, the activator protein normally interacts with the initiator site to facilitate initiation of w
transcription. However, when the specific corepressor (antagonist) is present, activator is no longer able to facilitate w
the initiation of transcription at the promoter site and expression of the operon is turned off. See also Figure I. δ
§
INDUCIBLE SYSTEMS
inducer
ANT I TERMINATOR
• LEADER-
LT SG,
REPRESSOR SYSTEMS
corepressor
ANTITERMINATOR
«—LEADER
<_>
SGi SG2 SG. SG 2
RNA tronscript
Figure 3. Antiterminator control of transcription termination. A regulatory gene, R, codes for an

antiterminator protein. The control region of DNA preceding the structural genes, SG, which in
Figures 1 and 2 consisted of modulator and promoter sites, has been expanded and redefined as

the "leader region," which begins with a promoter, P, and ends with a terminator, T. (For sim
plicity, initiation of transcription is assumed to occur constitutively at the promoter.)

In an inducible system, the antiterminator protein normally is not able to interact with a modulator site (called
the liberator, L) and transcripts initiated at Ρ terminate at T. However, under inducing conditions when the anti
terminator protein interacts with the liberator site, the transcription machinery is freed from the influence of the
terminator T, and the transcripts initiated at Ρ continue on through the adjacent structural genes. In a repressible
system, the antiterminator protein normally interacts with the liberator site and there is expression of the adjacent
structural genes. However, under repressing conditions when the antiterminator protein no longer interacts with
the liberator, transcripts initiated at the promoter, P, terminate at the terminator, T.
INDUCIBLE SYSTEMS
inducer
PROTERMINATOR
te SG, i m {
=>
REPRESSOR SYSTEMS
corepressor »...
PROTERMINATOR
1-
SGi
Figure 4. Proterminator control of transcription termination. A regulatory gene, R, codes for a

proterminator protein. The leader region preceding the structural genes, SG, extends from the pro-

moter, P, to the terminator, T, with its associated modulator site, A.
In an inducible system, the proterminator protein normally interacts with a modulator site called the arrestor, A ,
to facilitate termination of transcription at T. However, under inducing conditions the proterminator is unable to

interact with the arrestor site and transcripts initiated at the promoter, P, can be extended beyond the terminator,
T, through the adjacent structural genes. In a repressive system, the proterminator protein normally is unable to
interact with the arrestor site, and there is transcription through the structural genes resulting in expression of the
operon. However, under repressing conditions, when the proterminator protein interacts with the arrestor site to
facilitate termination of transcripts at T, expression of the operon is turned off. See also Figure 3.
Why are there p o s i t i v e and negative mechanisms o f r e g u l a t i n g

gene expression? Are these d i f f e r e n c e s simply historical
a c c i d e n t s r e p r e s e n t i n g e q u i v a l e n t e v o l u t i o n a r y s o l u t i o n s t o the
same r e g u l a t o r y problem, a s some have c l a i m e d ? Alternatively,
have t h e y been s e l e c t e d f o r t h e i r a b i l i t y t o p e r f o r m s p e c i f i c
f u n c t i o n s and, i f so, can we d i s c o v e r what these may be?
These are important b i o l o g i c a l questions t h a t a r e d i f f i c u l t
t o answer by u s i n g o n l y t h e d i r e c t e x p e r i m e n t a l approach. For
example, one can not s i m p l y compare r e p r e s e n t a t i v e examples o f
s y s t e m s r e g u l a t e d by p o s i t i v e ( e . g . , t h e m a l t o s e o p e r o n ) and
negative (e.g., the l a c t o s e operon) elements. T h i s i s not a w e l l -
c o n t r o l l e d experiment. In other cases, an a l t e r n a t i v e may not be
a v a i l a b l e f o r s u c h a comparative t e s t because i t may e x i s t i n an
undiscovered s t a t e . I d e a l l y one would l i k e a w e l l - c o n t r o l l e d
comparison. F o r some systems t h i s may soon be p o s s i b l e through
the imaginative use o f
a r e a l a r g e number o
g e n e r a l l y a r e , t h i s approach becomes i m p r a c t i c a l . At the p r e s e n t
t i m e , i t i s much more p r a c t i c a l t o simulate such experiments by
a p p r o p r i a t e mathematical a n a l y s i s and, u l t i m a t e l y , t h i s may be the
o n l y acceptable approach f o r d e a l i n g with more general q u e s t i o n s .
I have used s u c h an a p p r o a c h i n an a t t e m p t t o answer t h e
q u e s t i o n , why p o s i t i v e and negative mechanisms of gene r e g u l a t i o n ?
I n what f o l l o w s I s h a l l not be c o n c e r n e d with the mathematical
methods p e r s e . I n s t e a d , my a p p r o a c h w i l l be t o o u t l i n e t h e
principal steps i n the development o f the theory and then
emphasize the most important r e s u l t s .
Development o f the T h e o r y . What I have c a l l e d t h e demand

t h e o r y o f gene r e g u l a t i o n may be summarized as f o l l o w s . The
regulatory element will be positive (e.g., activator
a n t i t e r m i n a t o r ) when i n t h e n a t u r a l environment t h e r e i s a high
demand f o r expression o f the r e g u l a t e d s t r u c t u r a l genes; i t w i l l
be n e g a t i v e ( e . g . , r e p r e s s o r , proterminator) when t h e r e i s a low
demand (20-22). The development o f t h i s t h e o r y b e g i n s w i t h an
a s s e s s m e n t o f the f u n c t i o n a l d i f f e r e n c e s between p o s i t i v e and
n e g a t i v e mechanisms o f r e g u l a t i o n . D e t a i l e d a n a l y s i s o f t h e
a l t e r n a t i v e mechanisms has shown t h a t i n most r e s p e c t s they behave
identically. The i n h e r e n t d i f f e r e n c e s a p p e a r i n r e s p o n s e t o
common r e g u l a t o r y mutations (3) · To d i s c e r n t h e i m p l i c a t i o n s o f
these functional d i f f e r e n c e s one asks how f r e q u e n t l y these
mutations a r i s e and how the mutant organisms f a r e i n t h e i r n a t u r a l
environment when competing with the wild-type parent organism. An
a n a l y s i s of the r e l e v a n t p o p u l a t i o n dynamics y i e l d s t h e r e s u l t s
shown i n T a b l e I I . Thus, a c o r r e l a t i o n i s p r e d i c t e d between the
( p o s i t i v e or n e g a t i v e ) n a t u r e o f the r e g u l a t o r and t h e n o r m a l
demand f o r expression o f the r e g u l a t e d s t r u c t u r a l genes: p o s i t i v e
with high demand; negative w i t h low demand.

Table I . Terminology
Nature o f Regulator Modulator Transcript

control molecule site delimitor
Negative Repressor Operator Promoter
Positive Activato
Negative Proterminator Arrestor Terminator
Positive Antiterminator Liberator Terminator
Table II· P r e d i c t e d c o r r e l a t i o n between

r e g u l a t o r y mechanism and demand
Regulatory mechanism
Demand
Positive Negative
High Functional regulatory Regulation l o s t

mechanism s e l e c t e d through g e n e t i c d r i f t
Low Regulation l o s t Functional regulatory

through g e n e t i c d r i f t mechanism s e l e c t e d

1. SAVAGE AU Models of Gene Function 15
Physiology and Demand. In o r d e r t o make t h i s c o r r e l a t i o n

u s e f u l , we need t o know what high/low demand means i n d i f f e r e n t
p h y s i o l o g i c a l contexts. Table I I I l i s t s s e v e r a l d i f f e r e n t t y p e s
of physiological functions that have been i d e n t i f i e d in
microorganisms and t h e i r phages, and the a s s o c i a t e d e n v i r o n m e n t a l
c o n d i t i o n c o r r e s p o n d i n g t o h i g h demand. F o r example, when an
amino a c i d i s absent from an organism's environment, the o r g a n i s m
must s y n t h e s i z e t h a t amino a c i d endogenously. Thus, expression o f
t h e c o r r e s p o n d i n g amino a c i d b i o s y n t h e t i c o p e r o n i s i n h i g h
demand. When a given carbon source i s absent from an o r g a n i s m ' s
e n v i r o n m e n t and, t h e r e f o r e , cannot be u t i l i z e d , expression o f the
corresponding c a t a b o l i c operon i s i n low demand. I f gene exchange
between organisms o c c u r s i n f r e q u e n t l y , t h e n e x p r e s s i o n o f the
genes c o d i n g f o r t h e exchange m a c h i n e r y i t s e l f w i l l be i n low
demand·
Ecology and Demand

t o the n a t u r a l environment of the organism. S i n c e most w e l l -
s t u d i e d systems are found i n e n t e r i c b a c t e r i a and t h e i r phages, I
s h a l l f o c u s on t h e i r e n v i r o n m e n t . T h i s has t h e a d v a n t a g e o f
p r o v i d i n g a l a r g e number o f e x p e r i m e n t a l r e s u l t s w i t h w h i c h t o
t e s t the theory.
The l o w e r i n t e s t i n a l t r a c t o f warm-blooded animals i s the
p r i n c i p a l h a b i t a t of these microbes. I t i s a r a t h e r complex
ecosystem t h a t i s p o o r l y understood. The l o c a l environment to
which the microbes are exposed depends upon t h e d i e t , p h y s i o l o g y
and immunological s t a t e o f the host, the microorganisms t h a t share
t h e same g e n e r a l h a b i t a t , as w e l l as complex p h y s i c a l and
geometrical f a c t o r s . Nevertheless, one can estimate the l e v e l s of
c e r t a i n c o n s t i t u e n t s of t h i s microenvironment. I s h a l l describe
two types of estimates: those made i n d i r e c t l y from measurements of
a b s o r p t i o n by t h e h o s t and those made d i r e c t l y from measurements
of i n t e s t i n a l contents.
The host enzymes that cleave v a r i o u s d i e t a r y s u b s t a n c e s t o
a l l o w a b s o r p t i o n by the host are not uniformly d i s t r i b u t e d along
the i n t e s t i n a l t r a c t . Some are l o c a t e d i n the proximal p o r t i o n o f
the s m a l l i n t e s t i n e ( e . g . , t h e l a c t a s e enzymes); o t h e r s a r e
l o c a t e d i n t h e d i s t a l p o r t i o n o f the small i n t e s t i n e and i n the
colon i t s e l f (e.g., the maltase enzymes). T h i s l o c a l i z a t i o n alone
a l l o w s one t o make c e r t a i n i n f e r e n c e s about t h e p r e s e n c e or
a b s e n c e o f t h e s u b s t r a t e s f o r t h e s e c l e a v a g e and a b s o r p t i o n
enzymes. Furthermore, the d i f f e r e n t absorption systems w i t h i n the
same general r e g i o n o f t e n e x h i b i t very d i f f e r e n t a c t i v i t i e s . For
example, the host's t r a n s p o r t system f o r the a b s o r p t i o n o f
g a l a c t o s e i s very a c t i v e and n e a r l y impossible t o s a t u r a t e under
p h y s i o l o g i c a l c o n d i t i o n s ; whereas t h a t f o r t h e a b s o r p t i o n o f
arabinose i s r e l a t i v e l y i n e f f e c t i v e . Thus, from indirect
information concerning the relative abundance o f v a r i o u s
substances i n the d i e t , the l o c a l i z a t i o n o f h o s t enzyme s y s t e m s
that permit absorption o f these, and the r a t e s at which a b s o r p t i o n

Table I I I . Physiology and demand
Type o f C o n d i t i o n corresponding t o
system high demand f o r expression
Repress!ble scavenging Substrate seldom present

pathway i high concentration
R e p r e s s i b l e drug g f r e q u e n t l y presen
sensitivity i n high concentrations
Repressible biosynthetic End product seldom present

pathway i n high concentrations
Inducible biosynthetic End product seldom present

enzymes i n high concentrations
Inducible catabolic Substrate f r e q u e n t l y present

pathways i n high concentrations
Inducible detoxification Substrate f r e q u e n t l y present

pathway i n high c o n c e n t r a t i o n
Inducible genetic Genetic exchange f r e q u e n t l y

exchange occurs
Inducible repair Repair response f r e q u e n t l y

response required
Inducible toxin Toxin f r e q u e n t l y

production produced
Inducible biosynthesis of A n t i g e n i c determinants

s u r f a c e antigens frequently required

by the host occurs, one can estimate the r e l a t i v e c o n c e n t r a t i o n s

o f s u b s t a n c e s t o which the e n t e r i c microorganisms are exposed i n
the c o l o n (2^,3)· For example, the a f f i n i t i e s o f v a r i o u s s u g a r s
f o r t r a n s p o r t s y s t e m s i n the i n t e s t i n e can be ranked as f o l l o w s :
(High) D-glucose > D - g a l a c t o s e > g l y c e r o l ; (Low) D - x y l o s e > L -
g l u c o s e > L-mannose > L - f u c o s e > L-rhamnose > L-arabinose. I t
should be emphasized t h a t t h i s r a n k i n g i s o n l y s u f f i c i e n t ( a t
l e a s t i n the extreme cases) t o determine whether a given sugar has
a "high" o r "low" r e l a t i v e c o n c e n t r a t i o n i n t h e c o l o n .
Amino a c i d l e v e l s i n t h e c o l o n have been measured more
directly. Samples were removed from v a r i o u s l o c a t i o n s a l o n g t h e
i n t e s t i n a l t r a c t with a c a t h e t e r and t h e i r amino a c i d content was
analyzed (23). The p a t t e r n o f r e l a t i v e c o n c e n t r a t i o n s , w h i c h i s
i n d e p e n d e n t o f d i e t , can be summarized as f o l l o w s : (High) l y s i n e
> glutamate > a r g i n i n e > t y r o s i n e > t r y p t o p h a n ; (Low) g l y c i n e >
l e u c i n e > phenylalanin
aspartate > proline >
methionine. A g a i n , t h e r a n k i n g i s o n l y s u f f i c i e n t t o determine
w n
whether a g i v e n amino a c i d h a s a " h i g h " o r l o w relative
c o n c e n t r a t i o n i n the c o l o n .
Experimental Implications
The p r e d i c t i o n s o f demand t h e o r y ( T a b l e I I ) can be t e s t e d

with systems f o r which t h e p o s i t i v e o r n e g a t i v e n a t u r e o f t h e
r e g u l a t o r y element i s known a t the molecular l e v e l and f o r which
the p h y s i o l o g i c a l f u n c t i o n and e c o l o g i c a l context a l s o a r e known.
The r e s u l t s f o r 40 such systems r e p r e s e n t i n g 11 d i f f e r e n t types o f
physiological f u n c t i o n s a r e summarized i n Table IV. Five
p r e d i c t i o n s r e g a r d i n g the molecular n a t u r e o f t h e r e g u l a t o r and
nine p r e d i c t i o n s regarding the demand f o r gene expression a l s o are
given i n Table IV f o r cases i n which p a r t i a l experimental evidence
is available. E v i d e n c e f o r some o f t h e s e examples has been
presented elsewhere (24). Since a complete review o f the m a t e r i a l
(25) i s beyond the scope o f t h i s a r t i c l e , o n l y a few cases w i l l be
t r e a t e d here as examples.
R e p r e s s i b l e B i o s y n t h e t i c Systems. In e n t e r i c b a c t e r i a the
r e p r e s s i b l e s y s t e m s f o r h i s t i d i n e and t r y p t o p h a n b i o s y n t h e s i s
provide examples o f systems c o n t r o l l e d by p o s i t i v e and n e g a t i v e
elements, r e s p e c t i v e l y (see Table I V ) . Since the l e v e l o f
h i s t i d i n e i n the colon i s among t h e l o w e s t o f a l l amino a c i d s ,
e x p r e s s i o n o f t h e h i s t i d i n e b i o s y n t h e t i c operon i n the bacterium
w i l l be i n high demand (Table I I I ) . Thus, demand theory p r e d i c t s
that the h i s t i d i n e b i o s y n t h e t i c operon w i l l be p o s i t i v e l y
r e g u l a t e d (Table I I ) . I n f a c t , t h e r e i s good e v i d e n c e t o show
t h a t t h i s o p e r o n i s c o n t r o l l e d p r i m a r i l y by an a n t i t e r m i n a t o r
mechanism (26.27). On the other hand, the l e v e l o f t r y p t o p h a n i n
t h e c o l o n i s among the h i g h e s t o f the amino a c i d s and, t h e r e f o r e ,
expression o f the tryptophan b i o s y n t h e t i c operon i n the b a c t e r i u m

Table IV. Nature o f r e g u l a t o r

c o r r e l a t e s with demand f o r e x p r e s s i o n
Nature o f Demand f o r
System" " 1
regulator expression
R e p r e s s i b l e b i o s y n t h e t i c systems:
Arginine Negative Low
Cysteine Positive High
Histidine
Isoleucine-valine Positive High
Leucine Positive High
Lysine Negative Low
Phenylalanine Positive High
Threonine Positive High
Tryptophan Negative Low
Biotin Negative Low
Inducible catabolic systems:
Arabinose Positive High
Deoxyribonucleotides Negative Low
Galactose Negative Low
Glycerol Negative Low
Histidine Negative Low
Lactose Negative Low
Maltose Positive High
Nitrogen f i x a t i o n (K. pneumoniae) Positive High
Octopine (A. tumefaciens) Negative Low

Table IV. Continued.
System" "1
Rhamnose Positive High
Xylose Positive High
Tryptophanase • High
Positive
I n d u c i b l e g e n e t i c movement:
Prophage i n d u c t i o n
λ Negative Low
P1 Negative Low
P2 Negative Low
P22 Negative Low
Transposition
Tn3 Negative Low
Tn5 Negative Low
Tn10 Negative Low
Conjugation
Ti (A. tumefaciens) Negative Low
F Negative Low
R100 Negative Low
Inducible resistance/
d e t o x i f i c a t i o n systems:
Alcohol (yeast) Positive High
Mercury Positive High
Penicillinase (S. aureus) Negative Low
Continued on next page.

System" " 1
Tetracycline Negative Low
Chloramphenicol (S. aureus) » Low

Negative
Erythromycin (S. aureus) « Low
Negative
D-serine Positive High*
Toluene (P. p u t i d a )
Inducible r e p a i r systems:
SOS (lexA-recA) Negative Low
Adaptive response t o
* Low
Negative
a l k y l a t i n g agents
Inducible biosynthetic systems:
Isoleucine-valine Positive High
Tryptophan (P. p u t i d a ) Negative •

Low
R e p r e s s i b l e scavenging systems:
Alkaline phosphatase Positive High
General aromatic AA transport Negative Low
Tyrosine-specific transport Negative Low
Iron « High
Positive
Sulfate Positive High*
Inducible toxin production:
C o l i c i n e E1 Negative Low
Cholera (V. cholerae) Negative Low
D i p t h e r i a (C. d i p h t h e r i a e ) Negative «
Low

1. SAVAGE AU Models of Gene Function 21
System" "1
Inducible surface antigens:
Outer c e l l w a l l p r o t e i n 1b Positive High*
K88 (K) Positive High*
+
Enteric bacteria unless i n d i c a t e d otherwise
ft
Predicted

w i l l be i n low demand* Here demand t h e o r y p r e d i c t s n e g a t i v e

c o n t r o l , which i s i n agreement with the wealth o f evidence showing
t h a t r e g u l a t i o n o f t h i s operon i s accomplished p r i m a r i l y through
the c l a s s i c a l r e p r e s s o r mechanism ( 1 8 ) .
I n d u c i b l e C a t a b o l i c Systems* Among the i n d u c i b l e c a t a b o l i c

s y s t e m s i n e n t e r i c b a c t e r i a , the g a l a c t o s e and arabinose opérons
a r e examples o f systems c o n t r o l l e d by n e g a t i v e and p o s i t i v e
e l e m e n t s , r e s p e c t i v e l y ( s e e T a b l e IV)· G a l a c t o s e i s absorbed
e a r l y i n the s m a l l i n t e s t i n e a t a very r a p i d r a t e and i s u n l i k e l y
t o s u r v i v e t r a n s i t t h r o u g h t h e i n t e s t i n e t o t h e c o l o n (21)·
Expression o f the b a c t e r i a l g a l a c t o s e operon, t h e r e f o r e , w i l l be
i n low demand (Table I I I ) , and demand theory p r e d i c t s that i t w i l l
be n e g a t i v e l y c o n t r o l l e d . T h i s agrees with the f i n d i n g o f
classical repressor control f o r the g a l a c t o s e operon ( 28 )
A r a b i n o s e , on t h e o t h e
i n t e s t i n e and i s l i k e l
attenuation i n concentration (21). Arabinose i n the colon at
r e l a t i v e l y high l e v e l s i m p l i e s t h a t e x p r e s s i o n o f t h e a r a b i n o s e
c a t a b o l i c o p e r o n i n t h e b a c t e r i u m w i l l be i n high demand. The
p r e d i c t i o n by demand theory o f p o s i t i v e c o n t r o l f o r the a r a b i n o s e
o p e r o n i s c o n s i s t e n t with the experimental evidence f o r a c t i v a t o r
c o n t r o l o f t h i s system ( 1 4 ) .
I n d u c i b l e Gene Movement. Induction o f gene movement a p p e a r s

t o be a r e l a t i v e l y r a r e event i n nature. Demand f o r expression o f
the m a c h i n e r y f o r movement i s consequently i n low demand and i t s
r e g u l a t i o n w i l l be under the c o n t r o l o f a n e g a t i v e - a c t i n g e l e m e n t
(see T a b l e I V ) . F o r example, most n a t u r a l i s o l a t e s o f c o l i f o r m
organisms a r e l y s o g e n i c f o r one or more temperate phages (29) and
s p o n t a n e o u s i n d u c t i o n o f the prophage i s a r e l a t i v e l y i n f r e q u e n t
event i n nature (30). These f a c t s imply t h a t the l y t i c f u n c t i o n s
o f t h e prophage a r e n o r m a l l y i n low demand, which i s c o n s i s t e n t
with the c l a s s i c a l r e p r e s s o r c o n t r o l e s t a b l i s h e d f o r a number o f
prophages (31-34). T r a n s p o s i t i o n o f genes a l s o must occur
i n f r e q u e n t l y i n nature. Otherwise, the genome o f a bacterium l i k e
E. c o l i would have become s c r a m b l e d t o t h e p o i n t where g e n e t i c
maps as we know them would not e x i s t . The t r a n s p o s i t i o n machinery
w i l l be i n low demand and i t s i n d u c t i o n w i l l be under the c o n t r o l
of a n e g a t i v e - a c t i n g element. Again, t h i s i s c o n s i s t e n t w i t h t h e
f i n d i n g o f c l a s s i c a l r e p r e s s o r c o n t r o l f o r a number o f transposons
(35-38).
S i m i l a r a r g u m e n t s c a n be made w i t h r e g a r d t o b a c t e r i a l
c o n j u g a t i o n and, a g a i n , t h e r e i s evidence f o r c l a s s i c a l r e p r e s s o r
control i n these cases (39-41). The case o f Agrobacterium
tumefaciens i s p a r t i c u l a r l y i n t e r e s t i n g . T h i s organism i s w i d e l y
found i n n a t u r e . When present a t the s i t e o f a f r e s h wound i n a
dicotyledonous p l a n t under appropriate circumstances, the
bacterium can t r a n s f o r m t h e normal plant tissue into a
d i s o r g a n i z e d tumor. T h i s occurs when s p e c i f i c DNA l o c a t e d on a

b a c t e r i a l p l a s m i d becomes i n c o r p o r a t e d i n t o t h e p l a n t genome.
T h i s s p e c i f i c DNA a l s o codes f o r a b i o s y n t h e t i c system that causes
t h e p l a n t t o produce a r a r e amino a c i d , octopine i n t h i s example.
Octopine i n t u r n a c t s as a s p e c i f i c inducer upon the b a c t e r i a t h a t
h a r b o r t h e t u m o r - i n d u c i n g p l a s m i d t o c a u s e e x p r e s s i o n o f an
o c t o p i n e c a t a b o l i c pathway and e x p r e s s i o n o f a s p e c i f i c system of
b a c t e r i a l c o n j u g a t i o n f o r plasmid t r a n s f e r (see R e f s . 39,40).
S i n c e o c t o p i n e i s o n l y r a r e l y found i n nature, e x p r e s s i o n o f the
octopine c a t a b o l i c system and t h e b a c t e r i a l c o n j u g a t i o n s y s t e m
w i l l be i n low demand. Demand theory p r e d i c t s t h a t these systems
w i l l be under t h e c o n t r o l o f a n e g a t i v e - a c t i n g e l e m e n t . The
e x p e r i m e n t a l e v i d e n c e showing t h a t t h e s e systems are under the
c o n t r o l o f a r e p r e s s o r p r o t e i n (40) i s e n t i r e l y c o n s i s t e n t w i t h
these e x p e c t a t i o n s .
Discussion
Our i n t e r e s t i n the i n t e g r a t e d behavior o f complex b i o l o g i c a l

s y s t e m s has been p u r s u e d on two l e v e l s . On the f i r s t , we have
concentrated on the development of mathematical methods
s p e c i f i c a l l y a p p r o p r i a t e f o r complex b i o l o g i c a l systems. On the
s e c o n d , we have a p p l i e d t h e s e methods t o a number o f c a s e s
r e p r e s e n t i n g s e v e r a l c l a s s e s o f systems.
The development o f the power-law formalism, reviewed b r i e f l y
i n the f i r s t p o r t i o n o f t h i s paper, began w i t h a c o n s i d e r a t i o n o f
the fundamental n o n l i n e a r i t i e s t h a t c h a r a c t e r i z e the kinetic
behavior o f b i o l o g i c a l systems at the molecular l e v e l (2,5). From
t h i s m o l e c u l a r - b i o l o g i c a l context a "dynamical s y s t e m s " a p p r o a c h
to biochemical and g e n e t i c networks was developed (3-7,11,42).
The j u s t i f i c a t i o n f o r t h i s f o r m a l i s m i s based s o l i d l y on
t h e o r e t i c a l g r o u n d s (5 6 3)
9 f as w e l l as agreement w i t h d i r e c t
experimental observations (42,9,4)· An a p p r e c i a t i o n o f i t s
u t i l i t y i s perhaps best g a i n e d by an e x a m i n a t i o n o f s u c c e s s f u l
applications. Numerous examples can be f o u n d e l s e w h e r e (43-
Μ*.!£·6.·2· £ 4 ) , but the l i m i t a t i o n s o f space have p r e c l u d e d
d i s c u s s i n g more t h a n one example, the development o f the demand
theory o f gene r e g u l a t i o n .
T h i s demand theory i s now supported by experimental e v i d e n c e
from more than 40 systems r e p r e s e n t i n g more than a dozen d i f f e r e n t
t y p e s o f p h y s i o l o g i c a l f u n c t i o n s . T h i s theory provides a simple
u n i f i e d e x p l a n a t i o n f o r what o t h e r w i s e would be u n c o n n e c t e d and
d i s p a r a t e o b s e r v a t i o n s . F u r t h e r m o r e , i t p r o v i d e s a wealth o f
s p e c i f i c , t e s t a b l e p r e d i c t i o n s t h a t can s e r v e as a g u i d e f o r
experimental i n v e s t i g a t i o n (e.g., see R e f s . 49,50 and Table I V ) .
T h i s t h e o r y a l s o p r e d i c t s the n a t u r e o f the r e g u l a t o r y
mechanisms t h a t govern c e l l - s p e c i f i c f u n c t i o n s i n o r g a n i s m s w i t h
d i f f e r e n t i a t e d c e l l types and t h a t these mechanisms w i l l " s w i t c h "
i n a c c o r d a n c e w i t h t h e demand r e g i m e d u r i n g t h e p r o c e s s o f
d i f f e r e n t i a t i o n (22,5jO. E x p e r i m e n t a l evidence c o n s i s t e n t with
these p r e d i c t i o n s i s presented elsewhere (51).

In summary, the r e s u l t s p r e s e n t e d i n t h i s p a p e r i l l u s t r a t e
the use o f k i n e t i c methods t o achieve fundamental understanding o f
gene f u n c t i o n . T h e r e a l s o a r e p r a c t i c a l i m p l i c a t i o n s that have
yet t o be f u l l y e x p l o r e d . C l e a r l y , i f one w i s h e s t o o p t i m a l l y
e x p l o i t t h e p r o d u c t o f a p a r t i c u l a r gene, then one must know the
physiology and n a t u r a l ecosystem o f i t s h o s t organism as
i n t i m a t e l y as i t s molecular g e n e t i c s .
Acknowledgments
T h i s work was s u p p o r t e d i n part by grants from the N a t i o n a l

Science Foundation.
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RECEIVED June 1, 1982

2
Kinetics of Enzyme Systems
DAVID F. OLLIS
University of California, Chemical Engineering Department, Davis, CA 95616
The many circumstances leading to the Henri

equation for enzyme conversion of soluble
substrates are first noted, followed by some
kinetic forms fo
hydrolysis. Effect
enzyme systems are summarized. Illustrative
applications discussed include metabolic
kinetics, lipid hydrolysis, enzymatic cell
lysis, starch liquefaction, microenvironment
influences, colloidal forces, and enzyme
deactivation, a l l topics of interest to the
larger themes of kinetics and thermodynamics
of microbial systems.
Enzyme k i n e t i c s i s by now a c l a s s i c a l f i e l d , even i n the

arena o f immobilized enzymes, and d e t a i l e d t r a n s i e n t as w e l l as
steady s t a t e k i n e t i c models of c e l l u l a r and s u b c e l l u l a r
functions have been advanced f r e q u e n t l y which hinge h e a v i l y on
the k i n e t i c forms appropriate to s i n g l e enzyme systems. (See
l a t e r conference papers of Urabarger, Shuler and Domach, B a i l e y ,
Agathos and Demain, Marc and Engasser, Costa and Moreira, and
Frederickson, f o r examples.) We open t h i s d i s c u s s i o n with a
review of causes f o r success and u n c e r t a i n t y regarding the s i n g l e
enzyme r a t e equation, f o l l o w i n g which we w i l l touch upon r e s u l t s
f o r p a r t i c u l a t e substrates, and c l o s e with some r e s u l t s f o r
immobilized enzyme systems.
1. Soluble substrates
1
The Henri form f o r r e a c t i o n v e l o c i t y , v, of an enzyme
c a t a l y z e d r e a c t i o n was proposed i n 1902 as eqn (1.1):
v ( 1 υ
- W ï f s ï ·
0097-6156/83/0207-0027$07.50/0

where ν = maximum v e l o c i t y , Κ = constant, and S = concentra

t i o n of f r e e (uncomplexed) s u b s t r a t e . T h i s form was l a t e r
2
d e r i v e d by M i c h a e l i s and Menten i n 1913 using an e q u i l i b r i u m
assumption i n eqn (1.2a) and an assumed s i n g l e slow step, eqn
(1.2b), which y i e l d s form (1.1) above:
S + Ε «- ES (1.2a)
k 2
ES -> Ε + products (1.2b)
where
Michaelis-Menten: Κ Ξ
ν = k E3 where Ε = t o t a l (1.3b)
max ο ο
enzyme c o n c e n t r a t i o n
(E + ES)
3
The subsequent Briggs-Haldane treatment recognized that i n
general the r a t e constants ( k i , k ) and k could be of a r b i t r a r y
2 3
r e l a t i v e magnitudes, r a t h e r than only ( k i , k ) » k assumed 2 3
i m p l i c i t l y by eqns (1.2a,b), l e a d i n g to the form (1.1) again but

with Κ and ν ^ given by eqns. (1.4a,b):
χ
Briggs-Haldane: Κ = (1.4a)
= k E ν 3
3 (1.4b)
max ο
Thus, the constant Κ i s no longer a simple e q u i l i b r i u m constant^
Extensions of form (1.1) a r e apparently endless i n number.
For example, f o r a common case of a (two substrate)-(one enzyme)
system g i v e n below,
Reaction D i s s o c i a t i o n Equibrium Constant
Ε + S i / J ESi Ki (1.5a)
Ε + S 2 J ES 2 K 2 (1.5b)
ESi + S 2 J ESiS 2 Ki 2 (1.5c)
ESi + S ->ESiS 2 2 Κ (1.5d)
ES S £ Ρ + E (1.5e)
eqn (1.1) i s again recovered where ν and Κ are i n d i c a t e d i n

eqns (1.6a,b);

2. OLLis Kinetics of Enzyme Systems 29
kE S 2
ν = — - — (1.6a)
max Ki2 + S 2
Κ 2 1 l K l 2
substrate Κ = ^ + * (1.6b)
S2 + K i 2
Thus, i f S2 i s approximately constant as expected f o r S2 = s o l
vent or S2 = b u f f e r e d Η or OH , the Henri form of constant
parameters ν and Κ w i l l appear to f i t the data f o r S i .
a
S i m i l a r l y , i¥ ?he order of s u b s t r a t e b i n d i n g r e a c t i o n s (5a) and
(5b) i s o b l i g a t o r y , form (1.1) i s again obtained (e.g., by
e l i m i n a t i o n of eqn (1.5d)).
When the second bindin e n t i t y S2 i t s u b s t r a t e but
instead an enzyme a c t i v a t o
i s again regained with
X
i n h i b i t o r l e v e l as i n d i c a t e d 6y eqns (1.6a,b).
The extension of the network i n eqns (1.5 a-e) to m u l t i p l e
intermediates i s important i n d e v i s i n g p l a u s i b l e multi-enzyme
r a t e forms f o r m i c r o b i a l metabolism. The Henri form obtained i n
eqns (1.5a-e) i n v o l v e s three d i f f e r e n t enzyme complexes, a l l
e q u i l i b r a t e d with each other "upstream" to the k i n e t i c a l l y slow
step (eqn (1.3)). The n e g l i g i b l e f r e e energy changes evident
f o r manyôf the steps i n the Embden-Meyerhof g l y c o l y t i c
sequence (Figure 1) i n d i c a t e that the three r e a c t i o n sequences
(1.7a,b,c),
glucose-6-P £ fructose-6-P -> (1.7a)
f r u c t o s e - d i - P J · · · J 2(phosphoenolpyruvate) (1.7b)
and
pyruvate j l a c t a t e (1.7c)
w i l l behave k i n e t i c a l l y l i k e the simpler r e a c t i o n sequence

(1.7d):
A (glucose) •> B(fructose-6-phosphate)

•+ C(phosphoenolpyruvate)
-* D ( l a c t a t e ) (1.7d)
Moreover, the a c t u a l feedback i n h i b i t i o n on the conversion

of Β by the ( l a t e r ) products c i t r a t e and ATP (not shown) leads
to the yet f u r t h e r s i m p l i f i c a t i o n given by the network below:

BIOCHEMICAL ENGINEERING
50
40
<
CD
CD
30
Figure 1. Free energy of intermediates of Embden-Myerhoff glycolytic sequence

in human erythrocytes. Reproduced, with permission, from Ref. 6. Copyright 1970,
Worth Publishers.

A ( e x t r a c e l l u l a r glucose) membrane
0
permease ( E i )
B(fructoee-6-phoephate)(Si) - phosphofructokinase
products ( i n c l . ATP, citrate)

Thus, the k i n e t i c s of conversions i n metabolic c e l l u l a r
sequences, and even i n whole c e l l k i n e t i c s , at or near steady
s t a t e may be expected to resemble the k i n e t i c r a t e form appro
p r i a t e to one or a very small number of s e q u e n t i a l enzyme c a t a
l y z e d steps. The i m p l i c a t i o n s of t h i s point i n k i n e t i c models
of s t r u c t u r e d c e l l systems are r e f l e c t e d i n l a t e r c o n t r i b u t i o n s
i n t h i s conference.
The Henri form thu
u t i l i t y i n representin
Si. The i n t e r p r e t i v e ambiguity accompanying the success of t h i s
u n i v e r s a l " f i t t i n g " f u n c t i o n leads us to r e c a l l G i l e a d i ' s
5
c h a r a c t e r i z a t i o n of the Langmuir form, i d e n t i c a l to ( e q n ( l . ) ) i n
n o n - b i o l o g i c a l heterogeneous c a t a l y s i s :
The Langmuir a d s o r p t i o n isotherm may now be
regarded as a c l a s s i c a l law i n p h y s i c a l
chemistry. I t has a l l the i n g r e d i e n t s of a
c l a s s i c a l equation: i t i s based on a c l e a r
and simple model, can be d e r i v e d e a s i l y from
f i r s t p r i n c i p l e s , i s very u s e f u l now, about
50 (now 60) years a f t e r i t was f i r s t d e r i v e d
and w i l l probably be u s e f u l f o r many years
to come, and i s r a r e l y ever a p p l i c a b l e to
r e a l systems, except as a f i r s t approximation.
A l l r e a c t i o n s , c a t a l y z e d or otherwise, are r e v e r s i b l e at the
molecular l e v e l . T h i s f a c t i s most important i n k i n e t i c s when
o v e r a l l conversion i s l i m i t e d thermodynamically. For example,
25
the i s o m e r i z a t i o n of glucose to f r u c t o s e by glucose i s o m e r a s e
ki k
G + E J EXJE + F (1.8)
k-i k- 2
gives (glucose) ^ ( f r u c t o s e ) at e q u i l i b r i u m . Applying the

pseudo-steady s t a t e assumption to the s i n g l e intermediate gave
eqn (1.9a)
ν [G - G*]
m a x
Π Q) a
U , a ;
Κ + [G-G*] *
where the parameters ν and Κ are found i n eqns (1.9b,c)
max
K
Γ Κ*+1 Ί r F
(1.9b)

κ ] G + 9 K ( L E 9 C )
κ = [ ΐ ς ^ Π ν ν * ** *ρ G
and K* = g l u c o s e / f r u c t o s e e q u i l i b r i u m constant
Kp, K G = Briggs-Haldane constants f o r f r u c t o s e to glucose

and glucose to f r u c t o s e , i . e . ,
Kj, = ( k e l +k /k
2 - 2 and K Q = (k^+k^/^
G = glucose c o n c e n t r a t i o n a t e q u i l i b r i u m .
Here we note t h a t , even when s t a r t i n g with G » G * , the equation

(1.9a) w i l l s t i l l r e f l e c t a r e v e r s i b l e kinetîc form through eqn
(1.9c) where Κ = f ( G * )
s a t i s f y eqn (1.1), wit
apparent
2. P a r t i c u l a t e substrates
The use of enzymes to l y s e c e l l s , hydrolyze f a t emulsions,

s o l u b i l i z e proteinaceous c o l l o i d s , l i q u i f y or s a c c h a r i f y s t a r c h
g e l s and granules, and degrade v a r i o u s components o f c e l l u l o s i c
s u b s t r a t e s i n d i c a t e s that many s u b s t r a t e s a r e present i n a par
t i c u l a t e form. K i n e t i c forms f o r such enzyme c a t a l y z e d r e a c t i o n
r a t e s a r e here noted, and w i l l be r e v i s i t e d i n the subsequent
d i s c u s s i o n of immobilized enzyme k i n e t i c s .
A f i r s t example i s the h y d r o l y s i s of a uniform s u b s t r a t e ,
i n the form of a g i v e n t r i g l y c e r i d e emulsion, by a l i p a s e . Thus,
7 8
the data of Sarda and D e s n e u e l l e * ( F i g u r e 2a,b) i n d i c a t e
c l e a r l y that p u r i f i e d p a n c r e a t i c l i p a s e p r e p a r a t i o n s provide
a c t i v i t y only when a substrate:water i n t e r f a c e i s present. The
corresponding r e a c t i o n v e l o c i t y v s . s u b s t r a t e s u r f a c e area
f u n c t i o n (Figure 2a,b) can be d e s c r i b e d by the form (2.1), which
i s the analog to (1.1) f o r the case where the i n i t i a l enzyme
l e v e l Ε i s much l a r g e r than the i n i t i a l s u b s t r a t e l e v e l ( s u r f a c e
area).
ν = ν · bfzr]; ν
L J>
= kS (2.1)
max K+E max ο
where Ε = f r e e (uncomplexed) enzyme c o n c e n t r a t i o n and ν , Κ =
constants as f o r eqn. (1.1). In c o n t r a s t to s o l u b l e s u B s i r a t e s
where a s i n g l e r e a c t i o n product i s t y p i c a l l y i d e n t i f i a b l e ,
m u l t i p l e r e a c t i o n products r o u t i n e l y occur i n p a r t i c u l a t e degra
d a t i o n and/or enzymatic depolymerizations. A p a r a l l e l / s e q u e n t i a l
conversion network appears to be provided f o r t r i g l y c e r i d e s by
9
the data o f Constantin et a l . (Figure 3) which a r e c o n s i s t e n t
with the f o l l o w i n g network (2.2):

Interfacial Area Interfacial Area
5 2
5 2 (Methyl Butyrate) ,10 cm
(Triocatin), 10 cm
ι—ι—τ- ι ι—ι 1
— Ι 0.1 0.3 0.5 0.7
- Soluble ι Insoluble
S 4 - i /
-
1JA
I I 1
0.5S LOS 1.5S 2.0S 3.5S 0 0.5S LOS 1.5S 2.0S
(0.328 M) (0.153 M)
Saturation Saturation

Figure 2. Variation of the rate of lipase hydrolysis of triacetin and methyl butyrate with the
concentration of the substrate. The concentration is expressed as a fraction of the saturation con
centration (S) and the hydrolysis rate as a percentage of the rate of triolein hydrolysis under optimal

conditions. Key: O, impure lipase plus esterolytic activity; A, lipase purified by electrophoresis.
Reproduced, with permission, from Ref. 8. Copyright 1965, Academic Press.
Hydrolysis, %
Figure 3. Liberation of diglycerides, monoglycerides, and glycerol during hydrol-

ysis of a triglyceride by pancreatic lipase in the presence of sodium taurocholate and
calcium ions. Key: O, triglyceride; 0, diglyceride; • , monoglyceride; X, glycerol.
Reproduced, with permission, from Ref. 8. Copyright 1965, Academic Press

2. OLLIS Kinetics of Enzyme Systems 35
,triglyceride^
diglyceride monoglyceride (2.2)
glycerol'
The s i m p l i c i t y of k i n e t i c r e s u l t s (Figure 3) i s aided by the

presence of s u r f a c t a n t (deoxycholate) and d i v a l e n t c a t i o n Ca
which a s s i s t s i n remova^ of f a t t y a c i d product from the r e a c t i o n
i n t e r f a c e . Lacking Ca presence, f a t t y a c i d w i l l accumulate at
the i n t e r f a c e and compete with enzyme Ε f o r the l i m i t e d s u b s t r a t e
surface area; the r e a c t i o n w i l l consequently e x h i b i t product
inhibition. F u r t h e r , the extent of t r i g l y c e r i d e decomposition i s
s t r o n g l y dependent upo
only t r i - t o d i g l y c e r i d
present and high enzyme l e v e l s , monoglyceride i s not converted to
9
g l y c e r o l u n t i l the t r i g l y c e r i d e i s consumed.
A simple depolymerization of c e l l w a l l m a t e r i a l apparently
occurs i n l y s i s of Micrococcus l y s o d e i k t i c u s by lysozyme. The
homogenous r e a c t i o n k i n e t i c s of enzyme a t t a c k of using oligomers
of t h i s monopolymeric c e l l w a l l example are conveniently studied
by using oligomers of the monomer [disaccharide:(GlcNac-MurNac)
(N-acetyl-D-glucosamine-N-acetylmuramic a c i d ) ] . The endoenzyme
can cleave an oligomer of i u n i t s , at any of the s u s c e p t i b l e
bonds measured from a r e f e r e n c e (e.g., non-reducible) end. A
model r e a c t i o n network a l l o w i n g f o r such random i n t e r n a l cleavage
(but f o r b i d d i n g cleavage of the f i r s t s i t e from e i t h e r end) was
10
proposed by Chipman:
κ
«
Ε + S ± J (ES^j j = 0,1,1 1 1 i + i (2.3a)
(ES ).
± - C
Aj + S ( i - j ) j = 1, . . . , i (2.3b)
k
V
[(ES.). - A, + H 0 ? (2.3c)]
k.
1
3
Α..(+Η 0) V Ε + S
2 Jj
(2.3d)
*Τ
±A + S^ Ε + S ± + j (2.3e)
1
Here, the (i+2)-mer S^, i s complexed at the j * * s i t e ,
numbered 0 to i+1, but only b i n d i n g at s i t e s 1< j < i can r e s u l t
i n r e a c t i o n , as a s i t e at an end i s u n r e a c t i v e . The model f i t
11
k i n e t i c data from e x p e r i m e n t s s t a r t i n g with (uncleavable) dimer
only. Other measures of r e a c t i o n r a t e f o r c e l l l y s i s are t y p i
c a l l y more convenient: o p t i c a l d e n s i t y of p a r t i a l l y l y s e d c e l l s

i n a whole c e l l r e a c t i o n mixture may s u f f i c e to represent the

2 0 , 2 2
overall c o n v e r s i o n .
The h y d r o l y s i s of starches by amylases i s most conveniently
considered i n terms of sugar production ( f o r s a c c h a r i f i c a t i o n )
or v i s c o s i t y r e d u c t i o n ( f o r l i q u e f a c t i o n ) . Starch pastes are
c h a r a c t e r i s t i c a l l y p s e u d o p l a s t i c (shear t h i n n i n g ) , and thus
s a t i s f y , over an intermediate range of shear r a t e s , a power law
r e l a t i o n s h i p between shear s t r e s s , τ, and shear r a t e , γ, of the
form (2.4):
N
τ = K(^) (2.4)
where Κ = apparent u n i t shear v i s c o s i t y
XT - . x l pseudoplastic
Ν = power law inde
r
=
As s t a r c h pastes were hydrolyzed by α-amylase , the para
meters Κ and Ν were observed to change with r e a c t i o n progress i n
a coupled manner according to the equation (2.5):
log Κ - log - Ν log (2.5)
Here the p o i n t s (τ^,γ^) have the s i g n i f i c a n c e of being a l i m i t i n g

h i g h shear r a t e (and shear s t r e s s ) at which the s o l u t i o n i s pre
d i c t e d to achieve the Newtonian h i g h shear l i m i t .
Thus, i f reducing ends of product were measured to provide
an average molecular weight of remaining s t a r c h molecules, i t
would be p o s s i b l e to r e l a t e Κ to the extent of r e a c t i o n and,
through eqn (2.5), the consequent rheology of the p a r t i a l l y
l i q u e f i e d product. (See l a t e r paper of R o l l i n g s , Okos, and
Tsao f o r a d i s c u s s i o n of s t a r c h h y d r o l y s i s products.)
The a c t i o n of the enzyme rennet on milk, known to d e s t a b l i z e
a K-casein and t r i g g e r agglomeration of a m u l t i - c a s e i n m i c e l l a r
suspension (a,β,γ,ε,κ) to produce coagulated m i l k f o r cheese-
13
making, has been shown to produce a r e a c t i o n suspension with
power law behavior a l s o described by eqns (2.4-2.5)(Tucznicki
1 1
and Scott B l a i r ) . *
Where production of p a r t i c u l a r mono- or d i s a c c h a r i d e s i s
d e s i r e d , employment of an a p p r o p r i a t e exoenzyme, such as
3-amylase i s noted. The r a t e of depolymerization i s again
e s s e n t i a l l y an Henri form, unless modified by a product i n h i b i
t i o n term.
Non-productive b i n d i n g , l e a d i n g to apparent i n h i b i t i o n ,
occurs f r e q u e n t l y f o r endoenzymes. For example, lysozyme
h y d r o l y s i s of poly (GlcNac-MurNAc) y i e l d s dimer (GlcNac-MurNAc)2
which cannot be cleaved (being an end bond), j = 0 , i n eqn 2.3b
above), but i t s non-productive b i n d i n g decreases the number of
enzyme s i t e s a v a i l a b l e f o r a c q u i r i n g a c l e a v a b l e oligomer or
polymer. S i m i l a r l y , the h y d r o l y s i s of ( i n s o l u b l e ) c e l l u l o s e [G]

to c e l l o b i o s e , Gz i n eqns 2.6a-d and thence t o glucose, G,

9
i n v o l v e s a c e l l o b i o s e noncompetitive b i n d i n g to the c e l l u l a s e ;
t h i s i n h i b i t i o n i s r e l i e v e d by c e l l o b i o s e conversion to glucose
15
by β-glucosidase :
k
i
[G] + Ej^ ( c e l l u l a s e ) + E[G] (2.6a)
k
E [G]
1 -> 3
Ε χ + G 2 (2.6b)
E + G E G c e l l o b i o s e
l 2 * 1 2 ( binding) (2.6c)
E (B-glucosidase) + G
2 £ 2G + E £ (2.6d)
The form of the i n h i b i t i o
b i o s e , thus Κ i s unaffecte
same K) but the maximal v e l o c i t y i s diminished according t o eqn
(2.7):
k
i 3
1 + (
K^ k
N
max = k
3 o E [
l+î/Κ^ 1 ( 2
' 7 )
C e l l u l o s i c residues c h a r a c t e r i s t i c a l l y c o n s i s t of a mixture
of c e l l u l o s e , h e m i c e l l u l o s e , and l i g n i n . Even the i n d i v i d u a l
components are heterogeneous: the i n s o l u b l e c e l l u l o s e i t s e l f con
s i s t s of a s t r u c t u r e d m a t e r i a l of v a r y i n g degrees of c r y s t a l -
linity. An i l l u s t r a t i v e s t r u c t u r e d substrate d e s c r i p t i o n
i n c l u d i n g a k i n e t i c model and supporting data was provided by
16
Ryu, Lee, T a s s i n a r i and Macy. These i n v e s t i g a t o r s modelled the
substrate heterogeneity by assuming that the c e l l u l o s e c o n s i s t e d
p r i m a r i l y of two phases: "an impermeable, denser, and h i g h l y
ordered p a r a c r y s t a l l i n e or amorphous phase." Correspondingly,
each phase possesses a d i f f e r e n t r e a c t i v i t y . With S (amor
phous), S ( c r y s t a l l i n e ) and S ( r e s i d u a l i n e r t , i n c l u d i n g
l i g n i n ) making up the t o t a l ceïlulose mass, S , the k i n e t i c
model i n c l u d i n g binding of s o l u b l e product Ρ So the enzyme i s the
system of r e a c t i o n s given by eqns (2.8a-3):
k
(enzyme ad
Ε E* (2.8a)
adsorption)
des
k k
la M „ 3
(amorphous E* + S J E*S ·> E*+P (2.8b)
conversion) k 0
Za

(crystalline
E* +S E*+P (2.8c)
conversion)
(inert
E* + S (2.8d)
binding)
( s o l u b l e pro
duct b i n d i n g
Ε + Ρ EP (2.8e)
to s o l u b l e
enzyme)
These authors determine
x-ray d i f f r a c t i o n data; molecular a c c e s s i b i l i t y and surrace area
of v a r i o u s l y m i l l e d samples were obtained from i o d i n e adsorption
and BET measurements. Degree of c e l l u l o s e p o l y m e r i z a t i o n was
determined from viscometry of cadoxen-dissolved s o l u t i o n s , with
the s p e c i f i c v i s c o s i t y extrapolated to zero c o n c e n t r a t i o n to
obtained the i n t r i n s i c v i s c o s i t y , [η], from which i n turn the
v i s c o s i t y average molecular weight M was estimated from the Mark-
Houwink equation:
-5 0.76
W
[η] = 38.5 χ 10 J
M (2.9)
The experimental r e s u l t s supported both the p a r t i t i o n of sub

s t r a t e i n t o three sub-classes, and the product i n h i b i t i o n as
well.
Immobilized Enzymes
The attachment of c a t a l y t i c a l l y a c t i v e s i t e s to m a t e r i a l s
which may be e a s i l y recovered from a r e a c t i o n mixture has been
the s i n e qua non of most u s e f u l examples of c a t a l y s i s o u t s i d e of
enzymology, and t h i s l a t t e r area has begun to f o l l o w s u i t (see
17
f o r example, the s i x Enzyme Engineering C o n f e r e n c e s , as w e l l
as s e v e r a l summary t e x t s ). In a p l e a s a n t l y exhaustive review
1 9
of the k i n e t i c s of immobilized enzyme systems, G o l d s t e i n
s e v e r a l years ago assigned "the e f f e c t s of i m m o b i l i z a t i o n on the
k i n e t i c behavior of an enzyme" to four s i t u a t i o n s :
"1. Conformational and s t e r i c e f f e c t s : the enzyme may be

conformâtionally d i f f e r e n t when f i x e d on a support; a l t e r n a t i v e l y
i t may be attached to the s o l i d c a r r i e r i n a way that would
render c e r t a i n p a r t s of the enzyme molecule l e s s a c c e s s i b l e to
substrate or e f f e c t o r . These e f f e c t s are ( i n 1976) w e l l under-
stood."

"2. Partitioning effects: the e q u i l i b r i u m s u b s t r a t e , or

e f f e c t o r concentrations w i t h i n the support may be d i f f e r e n t from
those i n the bulk s o l u t i o n . Such e f f e c t s , r e l a t e d to the chemi
c a l nature of the support m a t e r i a l , may a r i s e from e l e c t r o s t a t i c
or hydrophobic i n t e r a c t i o n s between the matrix and low-molecular
weight species present i n the medium, l e a d i n g t o a modified
microenvironment, i . e . , to d i f f e r e n t concentrations of s u b s t r a t e ,
product or e f f e c t o r , hydrogen and hydroxyl ions, e t c . , i n the
domain of the immobilized enzyme p a r t i c l e .
"3. Microenvironmental e f f e c t s on the i n t r i n s i c c a t a l y t i c

parameters o f the enzyme: such e f f e c t s due t o the p e r t u r b a t i o n
of the c a t a l y t i c pathway of the enzymic r e a c t i o n would r e f l e c t
events a r i s i n g from the f a c t that enzyme-substrate i n t e r a c t i o n s
occur i n a d i f f e r e n t microenvironmen whe i immobi
l i z e d on a s o l i d support
"4. D i f f u s i o n a l or mass-transfer e f f e c t s : such e f f e c t s

would a r i s e from d i f f u s i o n a l r e s i s t a n c e s to the t r a n s l o c a t i o n of
s u b s t r a t e , product, or e f f e c t o r to or from the s i t e of the
enzymic r e a c t i o n and would be p a r t i c u l a r l y pronounced i n the case
of f a s t enzymic r e a c t i o n s and c o n f i g u r a t i o n s , where the p a r t i c l e
s i z e or membrane thickness are r e l a t i v e l y l a r g e . An immobilized
enzyme f u n c t i o n i n g under c o n d i t i o n s o f d i f f u s i o n a l r e s t r i c t i o n s
would hence be exposed, even i n the steady s t a t e , to l o c a l
concentrations of s u b s t r a t e product or e f f e c t o r d i f f e r e n t from
those i n the bulk s o l u t i o n . "
These i n f l u e n c e s have been evaluated s u c c e s s f u l l y , i n d i
c a t i n g a pleasant grasp of these fundamental i n f l u e n c e s on
enzyme k i n e t i c s . We consider s e v e r a l i l l u s t r a t i v e r e s u l t s . The
i n f l u e n c e of the number of covalent couplings t o an enzyme on i t s
a c t i v i t y i s shown f o r lysozyme, l i p a s e , and chymotrypsin f o r
2 0
s o l u b l e enzyme (Figure 4a) and immobilized enzyme (Figure 4 b ) .
Here, the s i m i l a r i t y i n a c t i v i t y p a t t e r n changes f o r a l l three
enzymes, as the r a t i o of s o l u b l e or i n s o l u b i l i z e d c o u p l i n g
groups (Figures 4a and 4b, r e s p e c t i v e l y ) to enzyme i s i n c r e a s e d ,
suggests c l e a r l y that a l t e r a t i o n of r e l a t i v e enzyme a c t i v i t y
upon immobilization (on only the e x t e r n a l surfaces of these
polyacrylamide supports) i s governed by conformation or s t e r i c
changes e f f e c t e d by the extent of enzyme covalent c o u p l i n g .
The microenvironment e f f e c t due to Donnan e q u i l i b r i a i s
shown by the now c l a s s i c r e s u l t s (Figure 5) f o r p H - a c t i v i t y
p r o f i l e s of chymotrypsin on p o l y c a t i o n i c , and p o l y a n i o n i c sup
p o r t s vs. that of the s o l u b l e enzyme.
Here, the charged s u b s t r a t e p a r t i t i o n i n g i n bulk s o l u t i o n
(S ) and i n the porous support (S ) i s given by
ο
(3.1a)
where ze = s u b s t r a t e charge, ψ = e l e c t r o s t a t i c p o t e n t i a l of

8;^
>—
E S
MOLA
ENZYME LOADING) χ Ι Ο - 2
1.0
>- I ^
α- -Chymotrysin
>
ι
ο
± 0.1
V \
χ
\ MAcylated)
ο.
Lysozyme^ \
L( Diazotized)
°\
^ Lipase
3 0.01 ο
Κ ο ο \ >
1.0 10 100 1000
MOLAR RATIO (REGENT/ENZYME)
Figure 4. Top, relative specific activity of immobilized enzyme versus the molar
ratio (ratio of immobilized surface coupling groups to enzyme immobilized). Key:
· , diazotized lysozyme; X, diazotized lipase; Δ, acylated a-chymotrypsin. Bottom,
relative specific activity of modified soluble enzymes versus the molar ratio (ratio of
soluble coupling reagent to enzyme). Key: O, diazobenzenesulfonic acid lysozyme;
X, diazobenzenesulfonic acid-lipase; | , diazobenzenesulfonic acid-chymotrypsin;
Δ, acetic anhydride-chymotrypsin. Reproduced, with permission, from Ref. 20.

ι — ι — ι — ι — ι — ι — ι — Γ
Figure 5. Activity-pH curves. Key: O, chymotrypsin; · , a polyanionic ethylene-

maleic acid (EMA) copolymer derivative of chymotrypsin, and A, a polycationic
derivative, polyornithyl chymotrypsin. Substrate is acetyl-L-tryrosine ethyl ester.
Ionic strength is 0.01 mol/L. Reproduced, with permission, from Ref. 19. Copyright
1976, Academic Press, Inc.

support, and k = Boltzman constant. With Henri's eqn (1.1)

assumed t o apply f o r the a c t u a l i n t e r n a l c o n c e n t r a t i o n S.^
adjacent t o the enzyme, the observed r a t e expressed i n terms of
the measurable S value i s s t i l l i n agreement with the Henri
f
form, but the apparent K i s given by eqn (3.1b).
f
K = Kexp(zei|;/kT) (3.1b)
D
as seen from using eqn (3.1a) i n eqn (1.1):
ν ν S
max i _ max ο ,~ , ν
V
K+S ± Kexp(zei|;/k T) + S B q U-ic;
Donnan e q u i l i b r i u m , being an example of charge-charge i n t e r

a c t i o n , i s a l s o i n f l u e n c e d b i o n i s t r e n g t h th t
M i c h a e l i s constant K' o
with i o n i c s t r e n g t h according eq (3.2)
f , l$
K = γΚ[1 - K Zm /KI] c (3.2)
i n n e r b u l
where the a c t i v i t y c o e f f i c i e n t r a t i o i s γ = y /y k^ Z m
i s the e f f e c t i v e f i x e d charge c o n c e n t r a t i o n i n s i d e the support,

and I the bulk s o l u t i o n i o n i c s t r e n g t h .
The i n f l u e n c e of c o l l o i d a l f o r c e s on r e a c t i o n s i n v o l v i n g
immobilized enzymes a c t i n g on i n s o l u b l e s u b s t r a t e s has r e c e i v e d
l e s s a t t e n t i o n , y e t i t appears to o f f e r some c l e a r examples of
2 2
fundamental phenomena important i n enzyme k i n e t i c s . Datta
examined l y s i s of Micrococcus l y s o d e i k t i c u s by s o l u b l e and
(polyacrylamide) immobilized lysozyme. He noted that the
decrease i n s o l u b l e enzyme a c t i v i t y with decreasing i o n i c
strength (Table 1) p a r a l l e l e d the measured decrease i n c e l l l y s i s
measured i n flow through a packed bed r e a c t o r of immobilized
enzyme.
2 2
Table l
Soluble Enzyme Immobilized C e l l Surface

I o n i c Strength
Activity Enzyme Rela Potential
[M]
tive activity
ψ (mV)
exp't (theory)
ΙΟ" 4
1700 ^0 +83.5mV
ΙΟ" 3
4000 2.3 (^4.3) 81.0mV
ΙΟ" 2
12000 13.3 (^8.3) -36mV
icf 1
12000 22.6 (^8.3) -37.3mV
* ( f i r s t numbers a r e experimental r e s u l t s , second ( i n parentheses)
are values p r e d i c t e d by p a r t i c l e c o l l e c t o r theory, i n c l u d i n g i n
fluence of i n t e r c e p t i o n ( p a r t i c l e s i z e ) and Brownian motion on
mass t r a n s f e r of p a r t i c l e s to immobilized enzyme surface.)

The d e c l i n e i n r a t e with decreasing i o n i c strength i s e v i

dently a s s o c i a t e d with a s i g n r e v e r s a l i n net c e l l charge, from
a t t r a c t i v e (ψ s u r - +44.5mV, ψ -, - -(36 to 37.3mV)) to
f a c e
a
r e p u l s i v e (ψ = ¥feï to 83.5mV) as determined from e l e c t r o -
p h o r e t i c m o b i l i t y measurements. A M i c h a e l i s constant (K) modi-
f i c a t i o n may a l s o be involved (eqn 3.2 above). The agreement
between observed r a t e and the c a l c u l a t e d mass t r a n s f e r l i m i t e d
r a t e ( l a s t column i n parenthesis) means that the f l u i d mechanical
and c o l l o i d a l f o r c e s which a c t to a t t r a c t / r e p e l enzyme and c e l l
p a r t i c l e s are, a t the highest i o n i c strengths, dominating the
slow step k i n e t i c s , i . e . , that every c e l l which encountered the
22
immobilized enzyme surface was subsequently h y d r o l y z e d . Simi-
l a r l y , the d e c l i n e i n r a t e f o r s o l u b l e enzyme i s to be a s s o c i a -
ted with a more d i f f i c u l t binding o r lessened substrate a f f i n i t y
occasioned i n part by the s h i f t from a t t r a c t i v e to r e p u l s i v e
c o l l o i d a l f o r c e betwee
The mass t r a n s f e r enzym p a r t i
c l e brings a r e l a t i v e l y modest number of hydrolyzable bonds per
c e l l p a r t i c l e to the c a t a l y s t . The above lysozyme data i n d i c a t e
that the r e a c t i o n r a t e i s e s s e n t i a l l y mass t r a n s f e r c o n t r o l l e d ,
i . e . , the enzyme can cleave the small number of c e l l w a l l bonds
per u n i t volume of c e l l rather r a p i d l y . S o l i d and emulsion
p a r t i c l e s of s i m i l a r s i z e , such as c e l l u l a s e p a r t i c l e s or l i p i d
emulsion d r o p l e t s , b r i n g a much l a r g e r number of c l e a v a b l e bonds
per p a r t i c l e to the immobilized enzyme. Immobilized p a n c r e a t i c
2 3
lipase shows an observed r e a c t i o n r a t e which i s 10 to 100
times l e s s than that p r e d i c t e d from c o l l o i d a l p a r t i c l e t r a n s -
port (Figure 6 ) . The p a r t i c l e c o l l e c t i o n e f f i c i e n c y u t i l i z e d i n
c a l c u l a t i o n of the p a r t i c l e mass t r a n s f e r c o e f f i c i e n t i s composed
of only two important terms, i n t e r c e p t i o n and Brownian motion
( g r a v i t a t i o n a l s e t t l i n g and i n e r t i a l impactions a r e n e g l i g i -
2 3
ble). Thus, the p a r t i c l e mass e f f i c i e n c y of c o l l e c t i o n , η, i s
23 2
the simple sum of two terms: * **
η +
overall ^Brownian înterception
9( ) 2 / 3 + x 5 ( 2 (3 3
• °· ΐ Τ Τ Ί Γ - d^> ' >
e enz ο enz
where d , d ^ e are substrate and immobilized enzyme p a r t i c l e d i a
n
meter, r e s p e c l i v e l y , and Ν i s the f l u i d v e l o c i t y e n t e r i n g the

enzyme r e a c t o r .
The observed r a t e of r e a c t i o n (Figure 6) i s a l s o orders of
magnitude higher than the r a t e c a l c u l a t e d by assuming that only
a few molecules of product are formed per emulsion p a r t i c l e
c o l l i s i o n with the enzyme surface. Thus, p a r t i c l e - c a t a l y s t
encounters r e s u l t , on the average, i n the cleavage of thousands
to m i l l i o n s of l i p i d bonds per p a r t i c l e - c a t a l y s t c o l l i s i o n event.
The above p a r t i c u l a t e examples i n v o l v i n g lysozyme and pan
c r e a t i c l i p a s e concerned substrates with uniform reactant

M
^ ^ 2 0 0 - 2 5 0 Mesh 3
s ι , - —325 Mesh
CD
i 10 •
# —50-100 Mesh^
/ r 325 Mesh 3
£
1 ^ « *
50-100 Mesh
Figure 6. Comparison of calculated mass

transfer rate and experimentally observed
reaction rates at various substrate concen 200-250 Mesh
trations. Broken lines are calculated values.
Experimentally observed values for stain
less steel are: X, 50-100 mesh; O, 200- 0.1 1
* ' ' ' ' •' ' ' 1 É 1 1
250 mesh; and Δ, 325 mesh. Reproduced, 43 0 0.4 0.8 1.2 1.6 2.0 2.4 2.8
with permission, from Ref. 23. Copyright
1975, John Wiley and Sons, Inc. Substrate Concentration (Volume %)

s t r u c t u r e : Micrococcus l y s o d e i k t i c u s ( p r o c a r y o t i c ) and t r i b u t y -
r i n , r e s p e c t i v e l y . More complex p a r t i c u l a t e s t r u c t u r e s provide
a v a r i e t y of hydrolysable bonds per p a r t i c l e , l e a d i n g to a
greater d i f f i c u l t y i n reaching a d e s c r i p t i o n of enzyme h y d r o l y t i c
kinetics. Examples here i n c l u d e e u c a r y o t i c c e l l w a l l s (e.g.,
yeast, with c r o s s l i n k e d mannan and glucan components), and the
c e l l u l o s i c substrates discussed i n the e a r l i e r s e c t i o n .
Enzyme immobilization leads always to the need to consider
d i f f u s i o n a l l i m i t a t i o n s , as the l a t t e r can give r i s e to r a t e
l i m i t a t i o n s and d i s g u i s e s . The general k i n e t i c r e s u l t s are w e l l
e s t a b l i s h e d , and we touch on only a few i l l u s t r a t i v e examples i n
t h i s short paper; d i f f u s i o n a l l i m i t a t i o n s , enzyme e l e c t r o d e
design, and polystep conversions.
Internal d i f f u s i o n a l r e s i s t a n c e i s widely appreciated now,
and t e s t s f o r freedom fro
used i n k i n e t i c s t u d i e s
i n t r u s i o n s i s i l l u s t r a t e d with c o n s i d e r a t i o n of an immobilized
dual system: glucose oxidase (E^) and c a t a l a s e (E«) a r e used to
o x i d i z e glucose (G) to o x i d i z e d glucose ( G ^ ) , and to decompose v
hydrogen peroxide ( H 0 ) : ox
2 2
G + 0 G + H ( 3 4 a )
2 Î ox 2°2 -
E
2 1
( 3 , 4 b )
H
2°2 t H
2° +
2°2
(In a developing commercial process, the o x i d i z e d glucose i s

reduced subsequently to f r u c t o s e using a t h i r d c a t a l y s t . ) The
enzymes E^ and E« are both d e a c t i v a t e d by hydrogen peroxide,
l e a d i n g to a d i f f u s i o n - r e a c t i o n s i t u a t i o n which changes slowly
i n time. K i n e t i c equations f o r t h i s system have been widely
2 6
studied; the equations (3.5 a,b,c,d,e,f) used r e c e n t l y by
27
Chang a r e c h a r a c t e r i s t i c of the l i t e r a t u r e phenomena examined:
[G = glucose, A = oxygen, Β = i ^ C ^ , and Ρ = o x i d i z e d glucose]
Glucose consumption r a t e (by oxidase (ox)) :
ν
- V
l• Γ E
ox / ( 1 + K
G / C
G +
V a>
c ( 3
- 5 a )
Peroxide conversion r a t e (by c a t a l a s e ( c a ) ) :
= V - k Ε C (3.5b)
z ca ca D
Oxidase d e a c t i v a t i o n :
dE
O X
- - k 0 C E_ B (3.5c)
dt 3 Β ox

Catalase activity:
dE
= -k.CÊ (3.5d)
dt 4 Β ca
Product formation (at pseudo-steady s t a t e ) :
dP = ^dG
(3.5e)
dt dt
In commercial l a r g e s c a l e c a t a l y t i c conversions, a c t i v i t y
27
maintenance i s important. C a l c u l a t i o n s by C h a n g i n d i c a t i n g the
glucose oxidase a c t i v i t y p r o f i l e s vs. d i s t a n c e i n the c a t a l y s t
p e l l e t are shown i n Figure 7 f o r v a r i o u s c a t a l a s e enzyme loadings
l 9
20
0 = / R k
Ε /D
ca ca A
i n F i g s . 7a (t=0), 7b (t=100), 7d(t=150), 73(t=20C0, and
7f(t=300). Lack of any c a t a l a s e (φ = 0, F i g s . 7a-3, curve a)
2
r e s u l t s i n the most r a p i d d e a c t i v a t i o n of Ε , while the highest

E^ l o a d i n g , case <i, r e s u l t s i n longest maintenance of E^ c a t a
l y s t . A d d i t i o n a l l y , i n the absence of E«, the H^C^ concentra
t i o n i s l a r g e s t i n the center of the p e l l e t , g i v i n g most r a p i d
d e a c t i v a t i o n at r = 0, while with appreciable E l e v e l s , the 2
i s maximal at the l o c a t i o n where the r a t e i s l a r g e s t , which

i s near r = .75 to .9, and f a l l s toward zero near the p e l l e t cen
ter s i n c e most of the o r i g i n a l d i s s o l v e d oxygen has been consumed
by r = 0.5 and the inner part of the p e l l e t acts p r i m a r i l y as a
peroxide sink (and oxygen source).
The glucose -> glucose oxidase system i s probably the best
studied example of c a t a l y s t a c t i v i t y maintenance. E v a l u a t i o n of
system behavior, based upon H e n r i - r e l a t e d r e a c t i o n s (3.5a, 3.5b)
and d e a c t i v a t i o n s (eqns 3.5c, 3.5d) o c c u r r i n g simultaneously with
i n t e r n a l (eqns 3.5a,b) d i f f u s i o n and e x t e r n a l mass t r a n s f e r pre
sents as complete a k i n e t i c a n a l y s i s as i s c o n v e n t i o n a l l y a v a i l
able i n other w e l l studied commercial examples of c a t a l y s i s .
A second a p p l i c a t i o n of immobilized enzymes i n v o l v e s enzyme
e l e c t r o d e s . An i l l u s t r a t i v e example of k i n e t i c fundamentals i s
provided by the urease electrode,which uses a urease membrane
to cleave urea i n t o bicarbonate and ammonium i o n , coupled to an
ammonium i o n s p e c i f i c pontentiometric e l e c t r o d e . As with the
glucose oxidase/catalase example above, the homogeneous phase
k i n e t i c s can be a p p l i e d to describe the response of the immobi
2 8
lized urease:
i n "planar" membrane:
d i f f u s i o n + r e a c t i o n of urea s u b s t r a t e :

Figure 7. Activity profiles for glucose oxidase (E ) coimmobilized with catalase

t
(Eg) in an initially uniform concentration. The glucose oxidase loading is constant;

the catalase loading increases as reflected by increased Thiele modulus, <f> = s
R V Ε,ο/DSo, from Curve a (E = 0) to Curve d (E (max)). Dimensionless time

M0 t0
appears at the lower right-hand corner of each graph (27).
American Chemical
Society Library
1155 16th St., N-W.
Washington,
ACS Symposium Series; D.C. 20036
American Chemical Society: Washington, DC, 1983.
d 2 g k kE S3 Q
P = ( 3 6 a )
s ^ 2 " (1+KS) (1+K- [NH +] 4 ° '
production of ammonium i o n product

,2 +, 2k-KE S
r M H
v; +
( n«)(i«'[HHî) = 0 ( 3
· 6 b )
with boundary c o n d i t i o n s (at steady state)
d[NH+] d s
—j = 0 =— at ζ = L (ammonium i o n (3.6c)
d z d z
electrode)
V ~ = L( ) ζ (externa (3.6d)
s αζ Z D - ν
surface)
. dNHT
V 4 -d^ N H4 = k
^ " îlb> A
( [ N H l M H
· ( 3 6 β )
2 9
Using publisheddata of L a i d l e r et a l . , the c a l c u l a t e d ammonium
ion concentration p r o f i l e i s presented i n Figure 8 f o r v a r i o u s
urease loadings i n the t h i n "planar" membrane g e l surrounding the
t i p of the ammonium i o n e l e c t r o d e . These r e s u l t s c o n t a i n u s e f u l
design information. I f ê wish to use the e l e c t r o d e to measure
substrate (urea), the NH^ s i g n a l a t the ammonium e l e c t r o d e sur
face (z/L = 1.0, F i g . 8) should be independent of Ε , thus we
should produce an e l e c t r o d e with Ε > 20-30 mg urease per ml
of g e l . I f we wish t o detect an i n h i b i t o r , then we should load
the enzyme membrane e l e c t r o d e very l i g h t l y (e.g., Ε = 2 mg/ml)
so that a 50% a c t i v i t y inhibition,corresponding to E^ = Ε /2,
produces a considerable drop i n NH^ s i g n a l . The experimental
voltage response o f an urease enzyme e l e c t r o d e with v a r i o u s
30
urease loadings was determined by G u i l b a u l t and M o n t a i v o ;
F i g . 9 i n d i c a t e s that the i n i t i a l l o a d i n g of 20-30 mg c a l c u l a t e d
above i s i n reasonable agreemen£ with the experimental r e s u l t s
(note that c a l c u l a t i o n gives NH^ (Z=0) while measurement i s
reported as ammonium i o n e l e c t r o d e voltage.) Many other sub
stances have been analyzed experimentally with s i m i l a r enzyme
3 2
e l e c t r o d e probes ( G u i b a u l t ) , i n d i c a t i n g major p o t e n t i a l f o r
analogs of eqns (3.6a-e).
Summary
The k i n e t i c s of s o l u b l e and immobilized enzymes, involved i n

r e a c t i o n s of s o l u b l e and i n s o l u b l e substrates appears to be
s u f f i c i e n t l y w e l l studied over the l a s t 20 years that r e a c t o r
design procedures based on fundamental k i n e t i c s r a t e equations
may be executed with considerable confidence. The a p p l i c a t i o n o f
such emzyme k i n e t i c s forms to s t r u c t u r e d models of m i c r o b i a l
metabolism has a l s o progressed, as t h i s book documents.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
DIMENSIONLESS THICKNESS
Figure 8. Influence of enzyme loading (mg enzyme/cc gel) on product profiles for
50μ membrane; bulk urea concentration, 0.0833 M; bulk ammonium ion, negligible
1
(10 M). Reproduced, with permission, from Ref. 28. Copyright 1972, Plenum
Publishing Corp.

τ 1 1 1 1 Γ
UREASE (mg//tgel)
Figure 9. Comparison of experimental and calculated electrode steady-state

responses versus enzyme loading. Key: Δ, experimental data (L = 350μ, bulk
urea = 0.0833 M, bulk ammonium ion negligible); O, calculation from model given
1
in text (L = 50μ, bulk urea = 0.0833 M, bulk ammonium ion negligible (10 M).
Reproduced, with permission, from Ref. 28. Copyright 1972, Plenum Publishing
Corp.

2. OLLIS Kinetics of Enzyme Systems 51
Literature Cited
1. Henri, V. Comptes Rendus Acad. Sci., Paris, 1902, 135, 916.
2. Michaelis, L; Menten, M. L., Biochem. Ζ., 1913, 333, 49.
3. Briggs, G. E.; Haldane, J. B. S. Biochem. J., 1925, 19, 338.
4. Dixon, M.; Webb, E. C. "Enzymes", Academic Press, 1964;
Chapter IV).
5. Gileadi, E. (ed), "Electrosorption", Plenum Press, New York,
1967.
6. Lehninger, A. L. "Biochemistry", Worth Publishers, New York,
1970, p. 327.
7. Sarda, L.; Desnuelle, P. Biochim. Biophys. Acta, 1958, 30,
513.
8. Wills, E. D. Adv. in Lipid Research, 1965, 3, 219.
9. Constantin, M. J.; Pasero L.; Desnuelle P Biochim
Biophys. Acta, 1960
10. Chipman, D. M. Biochemistry, , ,
11. Chipman, D. M.; Pollock, J . J.; Sharon, N. J. Biol. Chem.,
1968, 243, 481.
12. Cruz, Α., Russel; W. B.; Ollis, D. F. AIChE J., 1976, 22,
832.
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(eds), Plenum Press, New York; V, Plenum Press, New York,
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Kodansha Press, Tokyo, 1978.
19. Goldstein, L. "Adv. In Enzymology, XLIV, (K. Mosbach (ed.)),
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20. Datta, R.; Ollis, D. F., "Immobilized Biochemicals and
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mentals", McGraw H i l l , 1977, p. 469.

25. Fratzke, A. R.; Lee, Y. Y.; Tsao, G. T. G.V.C./AIChE - Joint

Meeting, Munich, 1974, 4, F2-1.
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RECEIVED July 7, 1982

3
Optimization of Fermentation Processes Through
the Control of In Vivo Inactivation of Microbial
Biosynthetic Enzymes
SPYRIDON N. AGATHOS and ARNOLD L. DEMAIN

Massachusetts Institute of Technology, Department of Nutrition and Food Science,
Fermentation Microbiology Laboratory, Cambridge, MA 02139
Prolonged productio
by in vivo stabilizatio
As a model system we have focused on the gramicidin S
synthetase complex in Bacillus brevis. Our group
previously reported an O -dependent loss of total bio-
2
synthetic activity preventable by anaerobiosis. We

further found this inactivation to be independent of
aerobic energy-yielding metabolism and of de novo pro-
tein synthesis through inhibitor studies. However,
retardation of inactivation was achieved upon addition
of utilizable carbon sources to aerated cell suspen-
sions, the degree of stabilization being proportional
to the ease of uptake and to the concentration of
each C-source. Dithiothreitol contributed to retard-
ation and partial reversal of the inactivation. These
results suggest that the in vivo inactivation may be
due to an energy deficiency at the end of exponential
growth with a concomitant exposure of sulfhydryl groups
on the synthetases to autoxidation.
Important commodity chemicals are c u r r e n t l y being produced by

microorganisms i n a v a r i e t y of fermentation and bioconversion pro-
cesses. The e f f i c i e n t production of these substances r e q u i r e s
considerable enzyme l e v e l s c a t a l y z i n g the b i o s y n t h e s i s of metabo-
l i t e s , as w e l l as good c a t a l y t i c a c t i v i t i e s and adequate opera-
t i o n a l s t a b i l i t i e s . T h i s i s c e r t a i n l y true of both primary and
secondary metabolites.
U n t i l r e c e n t l y , most work aimed a t o p t i m i z i n g production of
valuable secondary metabolites such as a n t i b i o t i c s has c o n s i s t e d
of environmental and genetic approaches a f f e c t i n g almost e x c l u -
s i v e l y the l e v e l s and c a t a l y t i c a c t i v i t i e s of the relevant
b i o s y n t h e t i c enzymes, rather than t h e i r o p e r a t i o n a l s t a b i l i t y .
Environmental manipulations have included: (a) the a d d i t i o n o f
appropriate precursors; (b) the d i s r u p t i o n of r e g u l a t o r y mechanisms
0097-6156/83/0207-0053$06.00/0

c o n t r o l l i n g the i n i t i a t i o n of a n t i b i o t i c s y n t h e s i s , such as r e -
p r e s s i o n and i n h i b i t i o n of t h e i r synthetases. For example, carbon
c a t a b o l i t e r e g u l a t i o n i s r e l i e v e d through a d d i t i o n of a slowly
u t i l i z e d carbon source at the beginning of a batch fermentation or
through the slow a d d i t i o n of the otherwise r e a d i l y consumed carbon
source i n various fed-batch type o p e r a t i o n s . S i m i l a r l y , n i t r o g e n
and phosphate r e g u l a t i o n are bypassed e i t h e r through r e s t r i c t i o n
of the r e s p e c t i v e N- or P- source or through a d d i t i o n of a slowly
metabolized a l t e r n a t i v e source of those n u t r i e n t s . Feedback
i n h i b i t i o n of a n t i b i o t i c synthetases has been minimized by appro-
p r i a t e b i o r e a c t o r design, e.g., a d i a l y s i s c u l t u r e i n which the
end product i s continuously removed; (c) f i n a l l y , the s p e c i f i c
growth r a t e of the a n t i b i o t i c - p r o d u c i n g microorganism i s optimized
during the idiophase (e.g., through l i m i t a t i o n of a key n u t r i e n t ,
pH c o n t r o l , etc.) s i n c e i n i t i a t i o n and maintenance of i d i o l i t e
production i s favored a
Genetic s o l u t i o n s t problem regu
l a t o r y mechanisms have a l s o been a p p l i e d s u c c e s s f u l l y f o r the
e f f i c i e n t production of secondary m e t a b o l i t e s . Mutation and
s e l e c t i o n f o r s u p e r i o r producing mutants can account f o r much of
the success of the modern a n t i b i o t i c i n d u s t r y .
However, even with s u p e r i o r producer s t r a i n s and r a t i o n a l
environmental s t r a t e g i e s f o r adequate expression and a c t i v i t y of
the synthetases, these b i o s y n t h e t i c enzymes u s u a l l y decay q u i c k l y
during the idiophase. Thus, the i n v i v o i n a c t i v a t i o n of
b i o s y n t h e t i c enzymes i s widespread and appears to be a l i m i t i n g
f a c t o r i n the prolonged production of a n t i b i o t i c s and r e l a t e d
secondary m e t a b o l i t e s . Despite i t s importance, i n v i v o enzyme
i n a c t i v a t i o n of secondary metabolism has not received any a t t e n -
t i o n as a d i s t i n c t b i o l o g i c a l phenomenon, although a fundamental
understanding of the process(s) could hold the key to i t s success-
f u l circumvention as i s the case with other types of c e l l u l a r
enzyme r e g u l a t i o n .
We have reasoned that a novel approach to prolong the produc-
t i o n phase of secondary metabolites should be based on an attempt
to prevent or r e t a r d the process of in v i v o i n a c t i v a t i o n of the
enzymes (synthetases) c a t a l y z i n g t h e i r formation i n fermentations.
Such an attempt would r e q u i r e an understanding of the chemical
nature of the i n a c t i v a t i o n . T h i s knowledge could be subsequently
t r a n s l a t e d i n t o process development and c o n t r o l i n a c t u a l fermen-
t a t i o n s , which, f o r the most p a r t , are c a r r i e d out i n batch
r e a c t o r s . If the object of the fermentation i s the recovery of
the enzymes f o r f u r t h e r use i n a c e l l - f r e e system or the a c q u i s i -
t i o n of a c t i v e whole c e l l s f o r repeated use i n a f i x e d bed-type
b i o r e a c t o r , the prevention or r e t a r d a t i o n of the i n a c t i v a t i o n
process would ensure both an adequate margin of time for primary
h a r v e s t i n g and a longer h a l f - l i f e of the a c t i v i t y f o r the b i o -
c a t a l y s t s i n the c e l l s .
The purpose of t h i s communication i s to i l l u s t r a t e the
p o t e n t i a l of the above-mentioned approach by f o c u s i n g on our

3. AGATHOS AND DEMAIN Optimizing Fermentation 55
i n v e s t i g a t i o n of the in v i v o i n a c t i v a t i o n of the enzyme (synthe-

tase) complex r e s p o n s i b l e f o r the formation of g r a m i c i d i n S (GS),
a c y c l i c peptide a n t i b i o t i c i n B a c i l l u s b r e v i s ATCC 9999. This
i n v e s t i g a t i o n represents the f i r s t attempt, to our knowledge, t o
understand and to c o n t r o l in v i v o i n a c t i v a t i o n of a synthetase
i n v o l v e d i n the production of a secondary metabolite. The choice
of GS s y n t h e s i s , as our model system, was i n f l u e n c e d by the r e l a -
t i v e l y l a r g e amount of i n f o r m a t i o n i n the l i t e r a t u r e about the
enzymology of GS formation, the p a r t i c i p a t i o n of only two polyen-
zymes i n i t s b i o s y n t h e s i s and the existence of a c e l l - f r e e
b i o s y n t h e t i c system. Also the p a t t e r n of appearance and disap-
pearance of the enzyme complex (GS synthetase) i s t y p i c a l of a
l a r g e number of a n t i b i o t i c fermentations. In a d d i t i o n , the pro-
ducing organism, B a c i l l u s b r e v i s , i s a non-filamentous, r a p i d l y
growing prokaryote and only a s i n g l e a n t i b i o t i c i s produced i n
t h i s fermentation.
I n v e s t i g a t o r s have appearanc
s o l u b l e GS synthetases at the end of exponential growth, they
r a p i d l y disappear as the c e l l p o p u l a t i o n moves i n t o s t a t i o n a r y
phase (12,L3) ( F i g - 1)· These changes have beeen observed using
the assay which measures the t o t a l synthetase a c t i v i t y as w e l l as
both L - o r n i t h i n e and L-phenylalanine-dependent ATP-PPi exchange
r e a c t i o n s (assays f o r the i n d i v i d u a l heavy and l i g h t GS syntheta-
ses r e s p e c t i v e l y ) .
F r i e b e l and Demain (.4,5.) found that i n a c t i v a t i o n of s o l u b l e GS
synthetase i n v i v o i s oxygen-dependent and i r r e v e r s i b l e . They
a l s o found that N gas prevented in v i v o i n a c t i v a t i o n of the
2
enzyme complex under otherwise fermentation c o n d i t i o n s . However,

under these c o n d i t i o n s no GS was produced, presumably due to the
need of oxygen f o r ATP production i n t h i s aerobic organism.
I t i s u s e f u l to review b r i e f l y some of the current motions on
i n v i v o i n a c t i v a t i o n of m i c r o b i a l enzymes:
In Vivo Enzyme I n a c t i v a t i o n . Enzyme l e v e l s i n microorganisms

are known to be c o n t r o l l e d through r e g u l a t i o n of the r a t e of t h e i r
s y n t h e s i s ( i n d u c t i o n / r e p r e s s i o n ) whereas enzyme a c t i v i t i e s are
c o n t r o l l e d by non-covalent b i n d i n g of v a r i o u s ligands ( a c t i v a t i o n /
i n h i b i t i o n ) . However, through s t u d i e s that have focused almost
e x c l u s i v e l y on the disappearance of enzymes of primary metabolism,
i t i s now apparent that an a d d i t i o n a l type of enzyme r e g u l a t i o n i s
o p e r a t i v e i n microorganisms, i . e . , the c o n t r o l of enzymatic a c t i -
v i t y by s e l e c t i v e i n a c t i v a t i o n . This i n a c t i v a t i o n of p a r t i c u l a r
enzymes i s widespread among microorganisms, i s brought about by
s e v e r a l mechanisms, and i s observed under s p e c i f i c p h y s i o l o g i c a l
c o n d i t i o n s (2,LI). In v i v o i n a c t i v a t i o n i s defined as the i r r e -
v e r s i b l e l o s s of an enzyme's c a t a l y t i c a c t i v i t y i n the c e l l . This
d e f i n i t i o n i s designed to c o n t r a s t i n a c t i v a t i o n from i n h i b i t i o n ,
which i s the r e v e r s i b l e l o s s of enzyme a c t i v i t y through u s u a l l y
non-covalent b i n d i n g of an i n h i b i t o r that can be d i a l y z e d o r
d i l u t e d away to r e s t o r e a c t i v i t y . I n v i v o enzyme i n a c t i v a t i o n can

GROWTH (Klett)
4 5 6 7 8 9
FERMENTATION TIKE (hours)
Figure 1. Dynamics of appearance and disappearence of GS synthetases. Key: O,

growth; GS; and A, total GS biosynthetic activity (Synthetase scale).

be classified into modification inactivation, which involves

usually a covalent modification of the protein molecule and dégra-
dât ive inactivation, which involves cleavage of at least one pep-
tide bond of the enzyme protein. The in vivo inactivation of
microbial enzymes i s usually observed as the cells approach or
enter stationary phase i n a batch reactor. It occurs mainly at a
point i n which the microbe's nutritional/energetic state changes
upon exhaustion or nutrients or a shift in carbon or nitrogen
source. Possibly, i t i s a general regulatory mechanism aimed at
phasing out a particular enzyme when a metabolic shift renders i t
wasteful of metabolites and/or directly harmful to the c e l l .
Materials and Methods
The culture used i thes experiment Bacillu brevi ATCC

9999. The preparation o
tion of growth were as previously (9)
determined by the biuret method (6).
In order to study the i n vivo disappearance of GS synthetase,
we have used a system that exhibits inactivation kinetics i n
short-term experiments. The system u t i l i z e s frozen and thawed
c e l l s of IJ. brevis harvested after growth i n yeast extract-peptone
(YP) medium in a 180-1 fermentor and frozen at -20°C immediately
after harvesting. Small batches of cells were thawed just before
performing i n vivo inactivation studies according to the methodo-
logy of Friebel and Demain (4). In brief, these cells are agi-
tated under a i r i n buffer for various periods of time in the
presence of appropriate compound vs. nitrogen-covered controls,
crude cell-free extracts are subsequently prepared using lysozyme,
and the extracts are assayed by the overall GS synthetase assay
3
(incorporation of L-[ H]ornithine into GS).
Results and Discussion
A typical kinetic pattern of the in vivo loss of GS synthetase

in our frozen-thawed c e l l system i s shown i n Figure 2. The enzyme
survives over 8 hours under nitrogen gas, but i s usually lost at
the end of 2-3 hours under conditions of agitation at 250 rpm,
37°C i n a i r (simulated aerobic fermentation conditions). The time
scale should not be taken as an absolute measure of the enzyme's
survival, because the rate of the inactivation depends upon the
previous growth history of the c e l l s . Therefore, comparisons are
made only between experiments performed with c e l l s from the same
batch of c e l l s .
Lack of Inactivation by Energy-yielding Cellular Metabolism. We

f i r s t examined whether the dependence of synthetase inactivation
on oxygen reflects a requirement for aerobic energy-yielding meta-
bolism. Some enzymes are inactivated i n vivo i n the presence of
0 due to the operation of energy-yielding circuits i n the c e l l ,
2

Figure 2. Typical kinetic pattern of in vivo inactivation of GS synthetase complex

in frozen-thawed cells of B. brevis. Standard conditions for inactivation: agitation
at 250 rpm, 37°C under air, vs. N -covered control.
t

such as glycosis, the c i t r i c acid cycle, electron transport, etc

(11)· Especially i n Bacillus s u b t l l i s , i t has been demonstrated
that inhibitors of such energy-yielding pathways prevent the i n
vivo inactivation of aspartase transcarbamylase, a process
requiring oxygen and entry into the stationary phase (15). In our
case, inhibitors of glycolysis (NaF), the c i t r i c acid cycle
(malonate) and oxidative phosphorylation (DNP), i n concentrations
known to inhibit their respective metabolic c i r c u i t s , were not
effective i n preventing enzyme inactivation (Table I)·. Therefore,
i t appears as i f the inactivation i s not due to energy-yielding
metabolic c e l l a c t i v i t y .
Table I.
Effect of Energy Metabolism Inhibitors on

in vivo Stabilit
Atmosphere Additive Activity %
Nitrogen None 100

Air None 4
Air 50 mM NaF 2
Air 50 mM Malonate 0
Air 0.2 mM DNP 6
Nitrogen 50 mM NaF 77
Nitrogen 50 mM Malonate 85
Nitrogen 0.2 mM DNP 103
a
Incubation with shaking for 90 min at 250 rpm, 37°C.
Independence of Inactivation from Protein Synthesis. A similar

experiment was carried out to determine whether the inactivation
was dependent upon protein synthesis. Conceivably the process of
in vivo synthetase inactivation could be enzymatic, involving the
mediation of an enzyme that i s derepressed at some point during
late exponential growth i n actively growing cultures of 1J. brevis,
e.g., a protease with an unusual requirement for oxygen or an oxy-
genase or an unknown "inactivase". Nevertheless, when a protein
synthesis inhibitor (chloramphenicol) was included i n the aerated
c e l l suspensions of our system i n a concentration known to inhibit
de novo protein synthesis i n IJ. brevis, no prevention or slow-
down of the inactivation process was noted, thus suggesting that
the process i s not protein-synthesis dependent.
Carbon Source-mediated Retardation of Inactivation. As mentioned

previously, a shift in C-metabolism i s one of the physiological
conditions most commonly associated with in vivo inactivation of

m i c r o b i a l enzymes. I t might be assumed that as B. b r e v i s moves

i n t o the s t a t i o n a r y phase, the o p e r a t i o n of the ITS synthetase f o r
continued formation of the a n t i b i o t i c from i t s c o n s t i t u e n t amino
a c i d s becomes wasteful of p r o t e i n p r e c u r s o r s . Thus enzyme i n a c
t i v a t i o n may spare b u i l d i n g blocks and energy and may be brought
about by s i g n a l s of p r o g r e s s i v e d e p l e t i o n of carbon and energy
source and/or the i n t r a c e l l u l a r ATP p o o l . To determine whether
carbon sources have any e f f e c t on i n a c t i v a t i o n of GS synthetase,
v a r i o u s u t i l i z a b l e ( g l y c e r o l , f r u c t o s e , i n o s i t o l ) and n o n u t i l i z
able (glucose, s o r b i t o l ) carbon sources were added to f r o z e n -
thawed c e l l suspensions a g i t a t e d under a i r . As shown i n Table I I ,
s e v e r a l C-sources were able to p a r t i a l l y s t a b i l i z e the enzyme.
Table I I .
Effect o
Stabilit
O v e r a l l synthetase
Atmosphere Additive specific activity Activity
Γ "g GS 1 (%)
Lmg-hr J
Nitrogen None 7.8 100

Air None 0.5 6
Air 1% G l y c e r o l 7.0 90
Air 1% Fructose 5.4 69
Air 1% Glucose 3.6 46
Air 1% I n o s i t o l 3.1 40
Air 1% S o r b i t o l 0.4 5
a
I n c u b a t i o n with shaking f o r 90 min at 250 rpm, 37°C.
The best s t a b i l i z a t i o n was obtained with g l y c e r o l , followed by

f r u c t o s e , glucose, and i n o s i t o l i n decreasing order of e f f e c t .
S o r b i t o l was i n c l u d e d i n t h i s experiment s i n c e i t i s not a source
of carbon f o r growth but i s known to s t a b i l i z e at high concentra
t i o n s ("20%) some c e l l - f r e e enzymes. In our case i t was i n a c t i v e .
The r e s u l t s suggest that in v i v o synthetase s t a b i l i z a t i o n by c a r
bon sources i s l i n k e d with the u t i l i z a t i o n of these compounds by
the c e l l s under the c o n d i t i o n s of a e r a t i o n used here. Studies by
A s a t a n i and Kurahashi ÇI) have revealed that there e x i s t systems
of a c t i v e uptake f o r both g l y c e r o l and f r u c t o s e i n Β· b r e v i s . The
uptake of g l y c e r o l i s c o n s i d e r a b l y f a s t e r than that of f r u c t o s e
and they are both c a t a b o l i z e d through the g l y c o l y t i c (EMP) path
way. The same workers have reported that glucose, while not able
to support growth as sole C-source f o r B^. b r e v i s , can d i f f u s e

p a s s i v e l y and very slowly i n t o the c e l l s and, once i n s i d e , i t can

be c a t a b o l i z e d through the EMP pathway. In our frozen-thawed
c e l l s we are e v i d e n t l y experiencing increased p e r m e a b i l i t y of the
c e l l membrane to glucose as a r e s u l t of the freeze-thaw treatment.
I n o s i t o l has been found by Vandamme and Demain (14) to be a poor
source of carbon f o r t h i s organism. Our r e s u l t s i n d i c a t e that
there i s a d i r e c t r e l a t i o n s h i p between magnitude of the s t a b i l i
z a t i o n e f f e c t brought about by the C-sources i n question and t h e i r
u t i l i z a t i o n . This c o r r e l a t i o n favors the idea of a metabolic
b a s i s f o r the observed s t a b i l i z a t i o n in v i v o . I t i s p o s s i b l e that
the c e l l s of B^. b e v i s are starved f o r a C- (and energy) source at
the point of the in v i v o i n a c t i v a t i o n and that such n u t r i e n t s must
be added to r e t a r d the i n a c t i v a t i o n process o c c u r r i n g under a i r .
An experiment was c a r r i e d out i n which 1% g l y c e r o l was added to a
c e l l suspension a f t e r i t had been subjected to i n a c t i v a t i n g con
d i t i o n s ( i . e . , shaking
i n a c t i v a t e d synthetase
g l y c e r o l , demonstrating that g l y c e r o l i s e f f e c t i v e i n r e t a r d i n g
synthetase i n a c t i v a t i o n i n a i r but cannot restore a c t i v i t y a f t e r
i t has been l o s t .
Table I I I .
F a i l u r e of Carbon Source to R e - a c t i v a t e
i n a c t i v a t e d GS Synthetases
O v e r a l l Synthetase
Atmosphere Additive specific activity
Γ Ug GSΤ
L mg'hr J
Nitrogen None 10.3

Air None 0.4
Air None, i n i t i a l l y ; 0.4
1% g l y c e r o l added
after inactivation
a
I n c u b a t i o n with shaking f o r 90 min at 250 rpm, 37°C.
Given the immediate i m p l i c a t i o n s of a simple step, such an

a d d i t i o n or p u l s i n g of carbon source f o r prolonged p r e s e r v a t i o n of
the synthetase a c t i v i t y i n whole c e l l s , f u r t h e r experiments were
conducted over s e v e r a l hours, to examine the time course of the
enzyme a c t i v i t y i n the presence of s e v e r a l l e v e l s of g l y c e r o l .
Figure 3 shows that p r o g r e s s i v e l y higher l e v e l s of carbon source
from 1 to 4% are able to p r o p o r t i o n a t e l y r e t a r d the i n a c t i v a t i o n

process. In the same experiment, the d i s s o l v e d oxygen (D.O.) con-

c e n t r a t i o n s were a l s o monitored i n t e r m i t t e n t l y i n the v e s s e l s con-
t a i n i n g v a r i o u s g l y c e r o l l e v e l s . I t can be seen ( F i g . 3) that
there e x i s t s a c o r r e l a t i o n between enzyme a c t i v i t y , l e v e l s of C-
source and D.O. concentration. The r e t e n t i o n of enzyme a c t i v i t y
i s g e n e r a l l y compatible with "lower" D.O. l e v e l s . T h i s could mean
that the lower D.O. l e v e l s are maintained, at l e a s t p a r t l y ,
through increased r e s p i r a t o r y a c t i v i t y i n the e a r l i e r domains of
such time-course experiments, and t h a t , i n the presence of C-source
such as g l y c e r o l , r e s p i r a t o r y a c t i v i t y d e p l e t i n g the D.O. i n the
c e l l suspension i s maintained f o r a longer period of time thus
preventing premature 0 -mediated i n a c t i v a t i o n . I t i s a l s o p o s s i -
2
b l e that the catabolism of the C-source i s required to continue

f u r n i s h i n g both ATP (a co-substrate and p o s s i b l e p o s i t i v e a l l o s -
t e r i c e f f e c t o r of the enzyme' s t r u c t u r a l i n t e g r i t y ) well
reduced p y r i d i n e n u c l e o t i d e s
retarding i n a c t i v a t i o n below) ,
c o r r e l a t i o n of D.O. with remaining l e v e l of enzyme a c t i v i t y i s a
s i g n i f i c a n t r e s u l t not only because i t immediately suggests ways
of engineering i n t e r v e n t i o n (e.g., through a c o n t r o l l e d regime of
D.O. during production phase) i n a batch a n t i b i o t i c fermentation
to achieve optimal GS production, but a l s o because i t might sug-
gest a general model of regulatory a c t i o n of oxygen upon enzymes
i n aerobic microorganisms producing secondary metabolites. A case
i n point i s the secondary metabolic process of cyanogènesis (pro-
ducion of HCN) by Pseudomonas species described by C a s t r i c and h i s
colleagues £ 2 , 3 2 which i s a l s o subject to oxygen-mediated i n a c t i v a -
t i o n . I t i s p o s s i b l e that oxygen becomes harmful to b i o s y n t h e t i c
enzymes of secondary metabolism i n o b l i g a t e aerobes at the point of
C- and energy source d e p l e t i o n p a r t l y by v i r t u e of the f a c t that
D.O. l e v e l s are kept low only during aerobic C-source catabolism.
A p o s s i b l e mechanistic scheme that would l i n k d i r e c t l y the
C-source catabolism with the molecular events c o n s t i t u t i n g the
i n a c t i v a t i o n of the sytnthetase v i a oxygen, could be formulated as
f o l l o w s : the a c t u a l target of the i n a c t i v a t i o n could w e l l be
l a b i l e s u l f h y d r y l (SH-) a c t i v e groups on the enzyme s u r f a c e .
According to Laland and Zimmer (8) i t seems that at l e a s t seven
SH- groups are d i r e c t l y required f o r a c t i v i t y and p o s s i b l y some
more are involved i n ensuring the s t r u c t u r a l i n t e g r i t y of the
enzyme macromolecule. I t i s then p l a u s i b l e to assume that while
such SH- groups are s u s c e p t i b l e to a u t o x i d a t i o n by D.O. the pre-
sence of an a c t i v e l y metabolized C-source could lead to a low
i n t r a c e l l u l a r redox p o t e n t i a l which i s conducive to maintaining
the i n t r a c e l l u l a r p r o t e i n c y s t e i n e residues i n the SH- ( i . e . ,
reduced) s t a t e . Conceivably NAD(P)H produced under such con-
d i t i o n s may be coupled to regeneration of SH- groups on the enzyme
v i a t h i o l interchange ( n u c l e o p h i l i c s u b s t i t u t i o n ) with g l u t a t h i o n e
(reduced) or lipoamide or t h i o r e d o x i n . For example, g l u t a t h i o n e ,
the most abundant i n t r a c e l l u l a r t h i o l i n most types of c e l l s may
interchange with the enzyme as shown i n Figure 4.

AGATHOS A N DDEMAIN Optimizing Fermentation
0 1 2 3 4 5
time [ hr ]
Figure 3. Time course of specific activity of G S synthetase under air in the presence
of various levels of utilizable carbon source (glycerol). Shown also are the corre
sponding levels of dissolved oxygen (DO). Standard conditions for inactivation:
agitation at 250 rpm, 3TC. Glycerol levels: M, 4%; A, 2%; Δ, 1%; O, 0%.
S SH
Enz'i + GSH ^ Enz' -h GSSG
S ^ ^SH
GSH reductase
n a d p* nad ρ H
Stabilizer compound —> —> —> —> Τ
Figure 4. Hypothetical scheme illustrating the action of a stabilizer compound

(e.g., utilizable carbon source) on an enzyme containing O -sensitive SH—
t
groups. GSH, reduced glutathione; GSSG, oxidized glutathione.

I n a c t i v a t i o n Through Autoxidation o f S u l f h y d r y l Groups. To

examine f u r t h e r the p o s s i b i l i t y that SH- groups are o x i d i z e d and
i n a c t i v a t e d by 0 i n v i v o we checked the e f f e c t of an organic
2
t h i o l (DTT) on the a c t i v i t y of the synthetase i n long-term e x p e r i -

ments. Because hydrogen peroxide and superoxide f r e e r a d i c a l s are
generated during most o x i d a t i o n s of organic t h i o l s , such as DTT, i n
a i r (10), such reagents cannot be used as a d d i t i v e s i n f o r c i b l y
aerated c e l l suspensions, but only under N gas. 2 Results from
long-term incubations are shown i n Figure 5. The experiment was
c a r r i e d out i n a way which only assumes a l a c k of forced 0 2 trans-
f e r to a l l c e l l suspensions [both experimental (with DTT) and
c o n t r o l s (without DTT)]. This was achieved through a p a r t i a l
atmosphere of N gas i n rubber-capped v i a l s , which, however, per-
2
mit the gradual entry of a i r l a t e r i n the i n c u b a t i o n . DTT at 15

mM preserved p r a c t i c a l l y the e n t i r e i n i t i a l a c t i v i t y of the enzyme
during 16 hours of incubation
v e s s e l (no DTT) had l o s
the slow r e t u r n to a e r o b i o s i s i n the c o n t r o l v e s s e l s (no DTT) lead
to a u t o x i d a t i o n and i n a c t i v a t i o n f a s t e r than i n the v e s s e l s con-
t a i n i n g DTT.
S t i l l another approach to determine the p o s s i b l e involvement
of SH- groups as t a r g e t s of o x i d a t i v e i n a c t i v a t i o n of the enzyme
was to incubate c e l l s , having already been exposed to various
degrees of 0 ~dependent i n a c t i v a t i o n , under N i n the presence of
2 2
d i t h i o t h r e i t o l (DTT). I f the hypothesis i s c o r r e c t then the

synthetase a c t i v i t y should increase under these c o n d i t i o n s . I t i s
of course assumed that the postulated SH- a u t o x i d a t i o n has led to
s u l f u r d e r i v a t i v e s of the enzyme, which are amenable to r e d u c t i o n
back to the t h i o l s t a t e (SH-) i n the presence of excess
d i t h i o t h r e i t o l . An i n i t i a l experiment along these l i n e s i s shown
i n Figure 6. I t can be noted that moderate r e a c t i v a t i o n was
achieved even from s t a t e s that assume t o t a l l o s s of a c t i v i t y under
air. In t h i s experiment a 45-minute a d d i t i o n a l incubation of the
p r e v i o u s l y exposed c e l l suspensions was c a r r i e d out under N i n 2
the presence of 15 mM DTT. The r e s u l t s of t h i s experiment

s t r o n g l y i m p l i c a t e SH- groups on the synthetase as the a c t u a l
t a r g e t s of the 0 ~dependent i n a c t i v a t i o n .
2
Conclusions
I t seems that u t i l i z a b l e C-sources are able to r e t a r d i n v i v o

i n a c t i v a t i o n of the synthetases of GS and i t a l s o appears as i f
the i n a c t i v a t i o n i s brought about by a u t o x i d a t i o n of SH- groups on
the enzyme complex. Moreover the l o s s of synthetase a c t i v i t y i s ,
at l e a s t p a r t i a l l y , r e v e r s i b l e through e x t e r n a l l y added d i t h i o -
threitol. These r e s u l t s throw considerable l i g h t on the process
of i n v i v o i n a c t i v a t i o n of the b i o s y n t h e t i c enzyme complex c a t a -
l y z i n g the formation of GS i n B^. b r e v i s . We b e l i e v e that these
f i n d i n g s not only suggest f r e s h ways of optimizing the GS fermen-
t a t i o n by c o n t r o l l i n g the r a t e of synthetase i n a c t i v a t i o n , but

3. AGATHOS A N DDEMAIN Optimizing Fermentation 65
0 4 8 12 16 20 24
time [ hr J
Figure 5. Long-term incubation of frozen-thawed cells of B . brevis under N in t
the presence and absence of an organic thiol (DTT). Standard conditions: agitation
at 250 rpm, 37°C. Key: O, N present; Δ, N + 15 mM DTT present.
t a
%
S.a.
Figure 6. Effect of DDT on specific activity of GS synthetase at various degrees

of prior exposure to standard inactivating conditions (air, 250 rpm, 37°C). The
subsequent incubation of the cell suspensions was under N in the presence of 15
g
mm DTT at 37°C for 45 min. Key: O, air only; Δ, air, then N, + 15 mM DTT.

a l s o that such knowledge should be d i r e c t l y a p p l i c a b l e to the

e f f i c i e n t production of c l o s e l y r e l a t e d a n t i b i o t o c s of commerical
s i g n i f i c a n c e , such as b a c i t r a c i n . The methodology o u t l i n e d i n
t h i s i n v e s t i g a t i o n and p o s s i b l y s p e c i f i c f i n d i n g s may a l s o be
exendable to d i f f e r e n t processes c a l l i n g f o r i n v i v o s t a b i l i z a t i o n
of b i o s y n t h e t i c enzymes, p a r t i c u l a r l y o f oxygen-labile enzymes i n
a e r o b i c organisms. Furthermore a fundamental understanding o f the
i n v i v o i n a c t i v a t i o n of such synthetases appears to hold the pro
mise f o r a more r a t i o n a l i n t e r v e n t i o n on the part of the biochemi
c a l engineer i n making e f f i c i e n t use o f the b i o s y n t h e t i c p o t e n t i a l
of a wide v a r i e t y of i n d u s t r i a l l y s i g n i f i c a n t microorganisms.
While the immediate b e n e f i t from such an understanding would be an
extended production phase i n batch fermentations, t h i s knowledge
should a l s o be c e n t r a l i n designing a l t e r n a t i v e w h o l e - c e l l pro
cesses (e.g., immobilized c e l l r e a c t o r s ) i n which keeping the
enzymes " a l i v e " i n t h e i
could be the optimal approac
batch t o a continuous process.
Acknowledgements
We thank the Greek M i n i s t r y of Coordination and Planning f o r a

NATO Science Fellowship f o r S. N. Agathos.
Literature Cited
1. Asatani, M.; Kurahashi, K. J. Biochem. (Tokyo) 1977, 81

813-822.
2. Castric, P.Α.; Ebert, R.F., Castric K.F. Current Microbiol.
1979, 287-292.
3. Castric, P.Α.; Castric K.F.; Meganathan, R. "Hydrogen Cyanide
Metabolism," Conn, E . E . , Knowles, C . J . , Vennesland, B.,
Wissing F . , Eds.; Academic Press: New York, in press.
4. Friebel, T . E . ; Demain, A.L. J. Bacteriol. 1977a, 130,
1010-1016.
5. Friebel, T . E . ; Demain, A.L. FEMS Microbiol. Lett. 1977b, 1,
215-218.
6. Gornall, S.G.; Bardawill, C . J . , David, M.M. J. Biol. Chem.
1949, 177, 751-766.
7. Holzer, H. Trends Biochem. Sci. 1976, 1, 178.
8. Laland, S.;, Zimmer, T. Essays Biochem. 1973, 9, 31.
9. Matteo, C.C.; Glade, M.; Tanaka, Α.; Piret, J.: Demain, A.L.
Biotechnol. Bioeng. 1975 17, 129-142.
10. Misra, H.P. J. Biol. Chem. 1974, 249, 2151.
11. Switzer, R.L. Ann. Rev. Microbiol. 1977, 31, 135.
12. Tomino, S.; Yamada, M.; Itoh, H.; Kurahashl, K. Biochemistry
1967, 6, 2552.
13. Tzeng, C H . ; Thrasher, K.D.; Montgomery, J.P.; Hamilton,
B.K.; Wang, D.I.C. Biotechnol. Bioeng., 1975, 17, 143.

3. AGATHOS A N D D E M A I N Optimizing Fermentation
14. Vandamme, E . J . ; Demain, A.L. Antimicrob. Agents Chemother.

1976, 10, 265-273.
15. Waindle, L.M.; Switzer, R.L. J. Bacteriol. 1973, 114, 517.

4
Microbial Regulatory Mechanisms:

Obstacles and Tools for the Biochemical Engineer
H. E. UMBARGER
Purdue University, Department of Biological Sciences, West Lafayette, IN 47907
The integration of metabolic activity of bacte-

rial cells with
extent to which
for industrial purposes. Since the mechanisms under-
lying this integration are genetically controlled,
they can be often bypassed or compensated by
judicious selection of mutants. The general physio-
logical and molecular patterns of regulatory mecha-
nisms that should be considered in the use of mutant
methodology are briefly reviewed. Examples of the
way such knowledge can be exploited to obtain valine-
-and isoleucine-producing strains are described.
There i s today a widespread a p p r e c i a t i o n of the f a c t that

metabolic pathways i n microorganisms are remarkably w e l l i n t e -
grated with the growth process. I t i s f u r t h e r remarkable that
t h i s i n t e g r a t i o n i s p o s s i b l e under a broad range o f environmental
conditions — c o n d i t i o n s that vary both chemically and p h y s i c a l l y .
This i n t e g r a t i o n r e s u l t s from c o n t r o l s exerted i n two ways:
one, the c o n t r o l of enzyme a c t i v i t y , and the other, the c o n t r o l
of enzyme amount. ( S t r i c t l y speaking, t h i s l a t t e r category should
a l s o i n c l u d e c o n t r o l of the amount o f other macromolecules, such
as the ribosomal p r o t e i n s , ribosomal and t r a n s f e r RNA, and mem-
brane components.) L e t me consider the p h y s i o l o g i c a l patterns
found to a f f e c t enzyme a c t i v i t y , which I l i k e to c a l l the c o n t r o l
of metabolite flow. (Throughout t h i s a r t i c l e , the words " c o n t r o l "
or " r e g u l a t i o n " are meant to connote f a c t o r s a f f e c t i n g b i o l o g i c a l
functions that can be modulated, i n contrast to a f a c t o r that has
consequences on b i o l o g i c a l f u n c t i o n . An analogy with a domestic
water supply would be an adjustable valve that can be manipulated,
i n contrast to an o b s t r u c t i o n i n the supply l i n e . )
Broadly speaking, there are three b a s i c patterns a f f e c t i n g
the c o n t r o l o f metabolite flow (Table I ) . The simplest i s end-
product i n h i b i t i o n i n which the endproduct o f a pathway i s an
0097-6156/ 83/0207-0071 $ 0 6 . 5 0 / 0

Table I
P h y s i o l o g i c a l Patterns o f C o n t r o l o f Metabolite Flow
Pattern Example Reference

En dp r ο duct I n h i b i t i o n of threonine (1)
inhibition deaminase by i s o l e u c i n e
(E. Qoli)
Precursor S t i m u l a t i o n o f l a c t a t e dehydro (2)

activation genase by f r u c t o s e - l , 6 - d i p h o s -
phate (Streptococcus fecalis)
Compensatory S t i m u l a t i o n o f carbamylphosphate (1)

modulation synthetase by o r n i t h i n e
(E. coli)
i n h i b i t o r of the i n i t i a l enzyme o f the pathway. The i n h i b i t i o n

of threonine deaminase i s a w e l l known example to which I r e f e r
l a t e r i n t h i s a r t i c l e (1). There are v a r i a t i o n s on t h i s general
theme i n which m u l t i p l e endproducts may act i n concert or syner-
g i s t i c a l l y upon a s i n g l e enzyme c a t a l y z i n g the f i r s t step i n a
common pathway o r i n which a segment of a s i n g l e pathway i s con
t r o l l e d by an intermediate i n h i b i t i n g an e a r l i e r step [e.g., the
i n h i b i t i o n o f phosphofructokinase of Escherichia coli by phospho-
enolpyruvate ( 2 ) ] .
The reverse of endproduct i n h i b i t i o n i s precursor a c t i v a t i o n .
Although i t i s more d i f f i c u l t to c i t e examples i n b i o s y n t h e t i c
pathways, an example i s found i n the a c t i v a t i o n of c e r t a i n micro
b i a l l a c t a t e dehydrogenases by fructose-l,6-diphosphate. Finally,
there i s another p a t t e r n o f enzyme a c t i v a t i o n that I would c a l l
compensatory modulation of a c t i v i t y . The example that i s p a r t i c u
l a r l y w e l l s t u d i e d i s the a c t i v a t i o n of carbamyl phosphate synthe
tase o f E. coli by o r n i t h i n e (3). This enzyme forms carbamyl
phosphate needed f o r both pyrimidine n u c l e o t i d e b i o s y n t h e s i s and
a r g i n i n e b i o s y n t h e s i s . The enzyme i s i n h i b i t e d by UMP, an i n d i
cator of the g l o b a l pyrimidine p o o l . However, an ample supply o f
carbamylphosphate f o r a r g i n i n e b i o s y n t h e s i s i s assured by o r n i
thine s t i m u l a t i n g the enzyme and antagonizing the UMP-mediated
i n h i b i t i o n . O r n i t h i n e , o f course, i s an intermediate that would
appear only when the a r g i n i n e supply was low.
This c l a s s i f i c a t i o n o f r e g u l a t o r y patterns implies nothing
with respect to the mechanisms by which r e g u l a t i o n was achieved.
The common mechanisms that have been recognized i n c o n t r o l l i n g
carbon flow are c l a s s i f i e d i n Table I I . We can recognize mecha
nisms that are s p e c i f i c i n that they are r e l a t e d to the s p e c i f i c
r o l e the modulated enzyme plays and mechanisms that are general i n
that they may a f f e c t many enzymes o r pathways that are not func
t i o n a l l y r e l a t e d o r have l i t t l e to do with the p h y s i o l o g i c a l r o l e
of the enzyme.

4. UMBARGER Microbial Regulatory Mechanisms
Table I I
Enzymic Mechanisms Underlying C o n t r o l of Metabolite Flow
Mechanism Example i n E. coli Référer

S p e c i f i c mechanisms:
Binding at Inhibitio o asparagine (4)
catalytic site synthetase by asparagine
Binding at I n h i b i t i o n of threonine (
regulatory site deaminase by i s o l e u c i n e
and i t s antagonism by
valine
Enzyme A d e n y l y l a t i o n of glutamine (
modification synthetase
Protein-protein S t i m u l a t i o n of α-subunit (
interaction of tryptophan synthase by
β-subunit
General mechanisms:
Energy charge Retardation of asparto- (
kinase I I I a c t i v i t y at
low energy charge
Reducing I n h i b i t i o n of pyruvate (
potential dehydrogenase a t low
NAuVNADH r a t i o s
Substrate None known
availability

The simplest of these mechanisms would be the a c t i o n of an

i n h i b i t o r b i n d i n g at the a c t i v e s i t e . The i n h i b i t i o n of succinate
dehydrogenase by oxaloacetate can be looked upon as such an exam
p l e (10). It may be that t i g h t b i n d i n g of a product may a l s o play
a r o l e i n r e g u l a t i n g the a c t i v i t y of an enzyme (11). The i n h i b i
t i o n of asparagine synthetase by asparagine i n microorganisms
provides a l i k e l y candidate f o r r e g u l a t i o n by t i g h t binding of
product (4).
Obviously more complex would be those enzymes on which a
separate regulatory s i t e has been developed f o r b i n d i n g of e i t h e r
a s t i m u l a t o r y or an i n h i b i t o r y l i g a n d that i s p h y s i o l o g i c a l l y
r e l a t e d to the f u n c t i o n but not i t s e l f s t o i c h i o m e t r i c a l l y r e l a t e d
to the c a t a l y t i c events. When such a l i g a n d i s an i n h i b i t o r , i t
would be an example of what Monod and Jacob (12) had defined as
an a l l o s t e r i c i n h i b i t o r i n contrast to the previous example
which they defined as i s o s t e r i c
regulatory process hav
s y n t h e t i c pathways. However, i n r e f e r r i n g to binding of a r e g u l a
tory l i g a n d to a d i s t i n c t r e g u l a t o r y s i t e , I would not wish to
imply a common mechanism by which the i n h i b i t i o n or the s t i m u l a
t i o n occurs. There probably are s e v e r a l mechanisms i n v o l v e d , and
at the l e v e l of enzyme s t r u c t u r e there i s seldom complete agree
ment among a l l i n v e s t i g a t o r s regarding the p r e c i s e d e t a i l s .
At the present time, we can c i t e more examples of enzyme
m o d i f i c a t i o n o c c u r r i n g as r e g u l a t o r y mechanisms i n animal metabo
l i s m than we can i n m i c r o b i a l metabolism. In animal metabolism,
phosphorylation by p r o t e i n kinases a f f e c t s many enzymes (13).
There are examples of phosphorylation of b a c t e r i a l p r o t e i n s , but,
with few exceptions, the r o l e or even the i d e n t i t y of the p r o t e i n
i s unknown. One example that has been s t u d i e d i s the i s o c i t r a t e
dehydrogenase of E. coli, which i s phosphorylated when the c e l l
i s s h i f t e d from glucose to acetate as the carbon source and which
thereby undergoes a very r a p i d decrease i n a c t i v i t y (14).
The b e s t - s t u d i e d example of m o d i f i c a t i o n of an enzyme i s that
of the a d e n y l y l a t i o n and deadenylylation of glutamine synthase of
E. coli, which, i n e f f e c t , serves to i n h i b i t and to a c t i v a t e the
enzyme (6).
To some extent, the idea of p r o t e i n - p r o t e i n i n t e r a c t i o n s
a f f e c t i n g the a c t i v i t y of b a c t e r i a l pathways may s t i l l be hypo
thetical. In yeast, we can c i t e at l e a s t one c l e a r l y important
example. That i s the i n h i b i t i o n of o r n i t h i n e transcarbamylase
upon binding to arginase, an enzyme induced by adding a r g i n i n e to
the medium (15). This p r o t e i n - p r o t e i n i n t e r a c t i o n thus prevents
a f u t i l e c y c l e i n which o r n i t h i n e , formed from exogenous a r g i n i n e ,
would be converted back to a r g i n i n e . There are i n v i t r o models
that can be c i t e d i n b a c t e r i a l metabolism that demonstrate the
f e a s i b i l i t y of such a mechanism, i n which an a c t i v i t y of one pro
t e i n i s markedly enhanced upon b i n d i n g to another. For example,
the conversion of s e r i n e and i n d o l e to tryptophan i s slowly c a t a
l y z e d by the α subunit of tryptophan synthase alone but i s

4. UMBARGER Microbial Regulatory Mechanisms 75
stimulated some 30-fold upon b i n d i n g to 3-subunit dimers.

Under the category o f general c o n t r o l s , I have l i s t e d energy
charge and reducing p o t e n t i a l , which could be looked upon as spe
c i a l cases of substrate a v a i l a b i l i t y . The energy charge e x p e r i
ments of Atkinson (16) have demonstrated s e v e r a l examples of i n
v i t r o response to energy charge, but i t i s s t i l l d i f f i c u l t to
assess the i n v i t r o s i g n i f i c a n c e of such a response to energy
charge, s i n c e c e l l s do have a remarkable c a p a c i t y f o r maintaining
the energy charge w i t h i n rather narrow l i m i t s (17). Similarly,
the extent to which a p y r i d i n e n u c l e o t i d e - l i n k e d r e a c t i o n proceeds
can be modulated by the NAD/NADH (or NADP/NADPH) r a t i o (9). The
question i s whether these r a t i o s can be so changed i n the growing
c e l l to be e x p l o i t e d as a r e g u l a t o r y mechanism.
One can a n t i c i p a t e that the f l u x through a pathway might be
l i m i t e d not by some r a t e - l i m i t i n th beginnin withi
the pathway but by the
the pathway i s s u p p l i e d supply
s t r a t e i s modulated, such a mechanism might play a r e g u l a t o r y
r o l e . That a l i m i t a t i o n i n supply can have important consequences
on the extent that a pathway might f u n c t i o n w i l l become c l e a r i n
the d e s c r i p t i o n of the development o f an i s o l e u c i n e - p r o d u c i n g
strain later i n this a r t i c l e . However, i n the sense of the word
" r e g u l a t i o n " as used here, t h i s example would not be considered a
r e g u l a t o r y mechanism.
Just as p h y s i o l o g i c a l patterns of c o n t r o l over metabolite
flow can be recognized, so can p h y s i o l o g i c a l patterns of c o n t r o l
over the amount of enzyme. Table I I I l i s t s a convenient c l a s s i f i
cation. C l e a r l y , the amount o f enzyme a t any time i s a summation
of the amount formed and the amount destroyed. Two s p e c i f i c
patterns c o n t r o l l i n g formation might be recognized: repression
by endproducts and i n d u c t i o n by substrate or a precursor. I t
should be s t r e s s e d that the terms " r e p r e s s i o n " and " i n d u c t i o n "
are o p e r a t i o n a l and should carry no connotations regarding the
mechanisms.
In a d d i t i o n , there are general mechanisms c o n t r o l l i n g l a r g e r
groups o f enzymes or groups o f pathways. One general mechanism
i s involved i n r e p r e s s i n g the formation of carbon- and energy-
y i e l d i n g pathways when there i s a good carbon and energy source
( t h i s i s b e t t e r known as c a t a b o l i t e repression) (27). An anala-
gous c o n t r o l mechanism i s r e l a t e d to the n i t r o g e n supply, and
many i n d u c i b l e c a t a b o l i c pathways y i e l d i n g ammonia are repressed
when there i s already an adequate n i t r o g e n supply (21). Another
important c o n t r o l mechanism i s that r e l a t e d to growth r a t e . At
high growth rates i n a r i c h medium, the ρrotein-forming apparatus
(ribosomes, tRNA, etc.) i s high, whereas the enzymes forming the
small molecule b u i l d i n g blocks are repressed to an extent much
greater than that accounted f o r by the presence o f the endproducts
i n the medium (22).
Recent s t u d i e s by Neidhardt and h i s colleagues (23) have
revealed a newly recognized c l a s s of enzymes i n E. coli that are
induced at e l e v a t e d temperatures.

Table I I I
P h y s i o l o g i c a l Patterns o f Regulation of P r o t e i n Amount
Pattern T y p i c a l examples Reference

E f f e c t on Enzyme Formation
Specific:
Repression by endproducts Repression o f (18)
arginine biosyn
t h e t i c enzymes
Induction by substrates Induction of lac (19)
or precursors operon
General:
Related to carbon suppl
Related to n i t r o g e n Histidase induction (21)

supply i n glucose-containing,
N ^ - f r e e media
(K. aerogenes)
Related to growth r a t e Derepression of the (22)

protein synthesizing
system at f a s t
growth rates
Related to temperature "Thermometer" p r o t e i n s , (23)
which increase o r
decrease i n amount
with growth
temperature
E f f e c t on Enzyme D e s t r u c t i o n
Specific:
S t a b i l i z a t i o n by l i g a n d s The s t a b i l i z a t i o n of (24)
glutamine phospho-
ribosylpyrophosphate
amidotransferase of
B. subtilis by
substrates
General:
Starvation of c e l l s f o r Increased p r o t e i n turn (25)
carbon or n i t r o g e n over with onset o f
starvation
I n h i b i t i o n of protease Serine protease i n h i b i (26)
activity ±. υ·
t o r of B. subtilis Ό * S ^ ^ »
*The examples c i t e d are found i n E. coli, except where i n d i c a t e d .

F i n a l l y , the degradation of an enzyme by p r o t e o l y s i s can be

modulated i n s p e c i f i c ways through substrate o r product s t a b i l i z a -
t i o n (24). These would c o n s t i t u t e s p e c i f i c c o n t r o l s over enzyme
destruction. I t i s a l s o known that, when c e l l s are starved f o r
amino a c i d s , n i t r o g e n , o r energy, there i s increased turnover o f
many c e l l p r o t e i n s (25). Such a c o n t r o l might be considered a
general one.
Some of the mechanisms underlying the p h y s i o l o g i c a l responses
l i s t e d i n Table I I I are given i n Table IV. Enzyme d e s t r u c t i o n ,
which w i l l not be considered f u r t h e r , occurs o f course by proteo-
l y t i c breakdown and, i n that i t can be impeded by ligands ( e i t h e r
substrates o r i n h i b i t o r s ) that bind to the p r o t e i n , could be sub-
j e c t to a s p e c i f i c c o n t r o l . P r o t e o l y s i s might be enhanced by
f a c t o r s that a c t i v a t e proteases o r r e l e a s e protease i n h i b i t o r s .
Such an enhancement might be considered a general c o n t r o l over
p r o t e o l y t i c breakdown.
The formation o f p r o t e i n regulate ,
e i t h e r by a f f e c t i n g formation o f mRNA ( t r a n s c r i p t i o n ) or i t s u t i -
l i z a t i o n as template ( t r a n s l a t i o n ) . In turn, t r a n s c r i p t i o n can be
c o n t r o l l e d i n two ways. One i s a c o n t r o l o f i n i t i a t i o n of t r a n -
s c r i p t i o n ( i . e . , whether the RNA polymerase forms an i n i t i a t i o n
complex). The other i s a c o n t r o l over how f a r a polymerase
t r a v e l s and whether i t proceeds i n t o a s t r u c t u r a l gene or not
( i . e . , whether a t t e n u a t i o n o f t r a n s c r i p t i o n o c c u r s ) . T r a n s l a -
t i o n a l c o n t r o l would more l i k e l y be achieved by c o n t r o l l i n g r i b o -
some b i n d i n g to the mRNA, although mechanisms a f f e c t i n g ribosome
t r a v e l are conceivable.
Among the molecular mechanisms c o n t r o l l i n g the i n i t i a t i o n of
t r a n s c r i p t i o n i n b a c t e r i a that might be considered s p e c i f i c mecha-
nisms o f c o n t r o l are those that i n v o l v e a negative c o n t r o l element,
or the repressor of the o r i g i n a l model of Jacob and Monod (44).
Two kinds o f c o n t r o l were envisioned i n that model. One i n v o l v e d
a repressor p r o t e i n that was a c t i v e only i n the presence o f the
endproduct of the pathway. The b e s t - s t u d i e d example i s the pro-
duct of the trpR gene, which becomes a c t i v e only a f t e r b i n d i n g to
tryptophan. The b i n d i n g o f the a c t i v a t e d repressor t o the opera-
tor s i t e s o f the trp operon, of the aroH gene, o r o f the trpR
gene and the binding o f RNA polymerase to the corresponding pro-
moters are mutually e x c l u s i v e (28). The inverse o f t h i s p a t t e r n
i s the i n a c t i v a t i o n of the repressor p r o t e i n by the substrate^ (or
a d e r i v a t i v e ) . The b e s t - s t u d i e d example o f t h i s p a t t e r n i s the
i n a c t i v a t i o n o f the lac repressor by i t s b i n d i n g o f a l l o l a c t o s e ,
a d e r i v a t i v e o f l a c t o s e formed by the a c t i o n o f 8-galactosidase
on l a c t o s e (29).
Although i t was considered as one p o s s i b i l i t y by Jacob and
Monod (44) i n t h e i r formulation o f a model f o r c o n t r o l o f gene
expression, r e g u l a t i o n by p o s i t i v e c o n t r o l elements was considered
a l e s s l i k e l y mechanism f o r c o n t r o l of gene expression. There
have been s e v e r a l examples described i n which p o s i t i v e c o n t r o l

Table IV
Molecular Basis of Regulation of P r o t e i n Formation
Mode o f C o n t r o l T y p i c a l Examples Reference
Transcriptional Control
C o n t r o l of T r a n s c r i p t i o n I n i t i a t i o n
Specific:
Negative C o n t r o l Elements
A c t i v a t e d by endproducts Repression of the trp (28)
I n a c t i v a t e d by substrat
or precursor operon by a l l o l a c t o s e
P o s i t i v e C o n t r o l Elements
A c t i v a t e d by s u b s t r a t e Induction of acetohydroxy (30)
or precursor isomeroreductase by
acetohydroxy acids
( I n a c t i v a t i o n by None known
endproduct)
General:
P o s i t i v e C o n t r o l Elements
A c t i v a t e d by cAMP Dependence of lac (31)
operon i n d u c t i o n on
cAMP-CRP i n t e r a c t i o n
A c t i v a t e d by temperature Dependence of s e v e r a l (32)
heat-induced p r o t e i n s
on the htp gene product
σ-Like p r o t e i n s S h i f t o f RNA polymerase (33)
s p e c i f i c i t y by B a c i l l i
during s p o r u l a t i o n
[ppGpp-mediated I n h i b i t i o n of RNA (34)
regulation] polymerase b i n d i n g to
rrn promoters.
Enhancement of polymerase (35)
pausing i n rrn opérons
[Nitrogen-mediated
regulation] The glnG-dependent a c t i v a - (36)
t i o n of genes i n v o l v e d i n
catabolism of n i t r o g e n -
c o n t a i n i n g compounds

Table IV, cont'd.
Control of Transcription Termination
General:
p-Dependent termination trpt* terminator a t end (37)
of trp operon
t R 1 terminator of λ (38)
p-Independent termination Termination o f t r a n - (39)
s c r i p t i o n at the end
of leader sequence
f o r amino a c i d
b i o s y n t h e t i c operon
Transient terminâtion
polymerase pausing i n rmB operon
Specific:
A n t i t e r m i n a t i o n by λ Ν gene-dependent (41)
s p e c i f i c proteins t r a n s c r i p t i o n beyond
the tp2 terminator
A n t i t e r m i n a t i o n by Attenuation o f ilv, trp, (39)
ribosome h a l t at his, and other amino
s p e c i f i c codon a c i d b i o s y n t h e t i c codons
Control of T r a n s l a t i o n
Specific-
Masking of ribosomal Binding o f small r i b o - (42)
b i n d i n g s i t e s by p r o t e i n somal p r o t e i n s a t r i b o
binding some b i n d i n g s i t e f o r
the rbsL (str) operon
By p e r t u r b a t i o n of The masking o f erm (43)
secondary s t r u c t u r e (erythromycin r e s i s t a n c e )
of message ribosome b i n d i n g s i t e by
retarded t r a n s l a t i o n o f
the erm leader t r a n s c r i p t
General:
(Amino a c i d supply) None known
(Inhibition) None known
Mechanisms enclosed i n ( ) may be h y p o t h e t i c a l , s i n c e no examples

have been reported. Mechanisms enclosed i n [ ] remain unexplained,
i . e . , p r e c i s e mechanism i s unknown.

provides the primary c o n t r o l over expression of a gene. One ex

ample i s the p o s i t i v e c o n t r o l element ( u p s i l o n , the product of
the ilvY gene) required f o r the i n d u c t i o n of acetohydroxy a c i d
isomeroreductase, the i n d u c i b l e enzyme i n the i s o l e u c i n e and va
l i n e b i o s y n t h e t i c pathway of E. coti (30). ( I t should be pointed
out that some regulatory elements can play both a p o s i t i v e and a
negative c o n t r o l r o l e . An example i s the araC gene product r e
q u i r e d f o r i n d u c t i o n of the a r a b i n o s e - u t i l i z i n g pathway (45). In
the absence of arabinose, i t acts as a negative c o n t r o l element
or repressor, and i n i t s presence i t acts as a p o s i t i v e c o n t r o l
element.) Again, the inverse of t h i s p a t t e r n , i n a c t i v a t i o n of a
p o s i t i v e c o n t r o l element by the endproduct of a pathway, might
provide a mechanism f o r r e p r e s s i o n . However, t h i s w r i t e r i s
aware of no example of such a mechanism.
Several of the general c o n t r o l formatio *
to be achieved by a f f e c t i n
no reason to assume tha genera
diated v i a p o s i t i v e or negative c o n t r o l elements of broad s p e c i f i
c i t y , and i t may be that many are. A w e l l - s t u d i e d example of a
p o s i t i v e c o n t r o l element a f f e c t i n g many c a t a b o l i c systems i n bac
t e r i a i s the i n t e r a c t i o n between cAMP and i t s binding p r o t e i n
(CAP), which allows binding of CAP to the DNA near the promoters
of c e r t a i n c a t a b o l i c enzymes, a f t e r which RNA polymerase can more
r e a d i l y bind (31). The heat-induced p r o t e i n s r e c e n t l y demon
s t r a t e d i n E. coli by Neidhardt's group may be considered another
example (23). The i n d u c t i o n of these enzymes appears to be depen
dent upon a p o s i t i v e c o n t r o l element s p e c i f i e d by the htp gene
(32).
Another kind of c o n t r o l has been found to be important i n
the B a c i l l i and might be important i n other organisms as w e l l .
This i s a c o n t r o l of l a r g e numbers of genes by a l t e r i n g the s p e c i
f i c i t y of RNA polymerase by the binding of d i f f e r e n t sigma (σ)
f a c t o r s (33). Thus, during the s p o r u l a t i o n phase, the 55-kd σ
f a c t o r that i s predominantly bound to RNA polymerase i s replaced
by a 29-kd σ f a c t o r which allows some of the s p o r e - s p e c i f i c genes
to be expressed. To date, at l e a s t four d i f f e r e n t σ f a c t o r s have
been i d e n t i f i e d i n B. siibtiUs.
Two other general regulatory mechanisms have been s t u d i e d
that appear to a f f e c t t r a n s c r i p t i o n i n i t i a t i o n . Neither, however,
are w e l l enough understood to decide whether they might c o n s t i t u t e
unique mechanisms or are s p e c i a l cases of p o s i t i v e c o n t r o l e l e
ments that exert a g e n e r a l i z e d c o n t r o l r o l e . One i s the ppGpp-
mediated r e g u l a t i o n that impedes s t a b l e (ribosomal and t r a n s f e r )
RNA formation and, at l e a s t i n v i t r o , stimulates t r a n s c r i p t i o n of
many b i o s y n t h e t i c and c a t a b o l i c opérons. While ppGpp does pre-
vent RNA polymerase binding to s t a b l e RNA promoters, the pro-
nounced e f f e c t of ppGpp on s t a b l e RNA formation cannot s o l e l y be
explained on t h i s b a s i s alone (see below) (34,35). The other i s
the c o n t r o l that i s an e f f e c t of n i t r o g e n l i m i t a t i o n and leads to
the expression of genes involved i n r e l e a s i n g NH3 from n i t r o g e n -

containing compounds (36). The glnG gene i s somehow i n v o l v e d , but

whether that product i s a p o s i t i v e c o n t r o l element a c t i n g i n a way
analogous to that by which cAMP bound to CAP acts on carbon and
energy producing pathways i s not c l e a r .
Table IV a l s o l i s t s some o f the f a c t o r s that c o n t r o l t r a n -
s c r i p t i o n termination. T r a n s c r i p t i o n termination plays two r o l e s .
One, of course, i s the termination that occurs a short distance
beyond the end of a given s t r u c t u r a l gene or beyond the end of the
operon. The other i s a termination that serves to attenuate the
t r a n s c r i p t or to terminate t r a n s c r i p t i o n at a s i t e upstream of
that at which the maximal-sized t r a n s c r i p t i s terminated (39). I t
i s the modulation of the l a t t e r kind o f t r a n s c r i p t i o n termination
that provides an important mechanism of t r a n s c r i p t i o n a l c o n t r o l .
T r a n s c r i p t i o n termination i s s i g n a l l e d by e i t h e r p-dependent
or p-independent sequence i th (and RNA) Th l a t t e
r e a d i l y recognized as region
y i e l d stem and loop s t r u c t u r e y sequenc
of s e v e r a l u r i d i n e residues i n which t r a n s c r i p t i o n i s terminated
(46). p-Dependent termination s i t e s seem to be c h a r a c t e r i z e d by
t r a n s c r i p t s with more weakly base-paired stem and loop s t r u c t u r e s
near the termination s i t e i n the t r a n s c r i p t s and with l e s s p r e c i s e
p o i n t s of termination than those found i n p-independent termina-
t i o n s i t e s (47,48).
C l o s e l y r e l a t e d to the s i t e s of p-dependent and p-independent
termination s i t e s are s i t e s at which pausing o f RNA polymerase
occurs. Pausing i s a l s o apparently c h a r a c t e r i s t i c of termination
s i t e s , but the " r u l e s " f o r polymerase pausing are even l e s s c l e a r
than those f o r termination (40,49).
Polymerase pausing and t r a n s c r i p t i o n termination can thus
a f f e c t the extent to which the DNA downstream of the pausing or
termination s i t e are t r a n s c r i b e d independently of the t r a n s c r i p -
t i o n i n i t i a t i o n event. That pausing or termination can be modu-
l a t e d allows a c o n t r o l of the process. I t i s an enhancement by
ppGpp o f polymerase pausing at a s i t e e a r l y i n ribosomal RNA
opérons that may account f o r the s t r i k i n g e f f e c t that t h i s nucleo-
t i d e has on the s y n t h e s i s of s t a b l e RNA over and above that which
occurs a t the polymerase b i n d i n g step (40). The prolonged pausing
of polymerase progress at a s p e c i f i c p o i n t has been termed " t u r n -
s t i l e " attenuation.
S i m i l a r l y , when termination at a s i t e w i t h i n an operon or a t
the end o f a leader region i s a f f e c t e d by a s p e c i f i c r e g u l a t o r y
s i g n a l , an e f f i c i e n t c o n t r o l can be achieved. One of the most
s t u d i e d c o n t r o l s over a p-dependent termination i s that which
occurs at the XtRl s i t e and i s prevented by the λ Ν gene product
(41). L i k e the a c t i v i t y of ρ i t s e l f , the a c t i v i t y of the Ν gene
product i s dependent on the elongation subunit of RNA polymerase
s p e c i f i e d by the E. coli, nusA gene. I t might be a n t i c i p a t e d that
a t t e n u a t i o n of t r a n s c r i p t i o n by a s p e c i f i c a n t i t e r m i n a t i n g p r o t e i n
i s a general p a t t e r n that w i l l be found to occur frequently i n
m i c r o b i a l systems.

Another c o n t r o l over termination that has been e x t e n s i v e l y

s t u d i e d i s the attenuation of t r a n s c r i p t i o n at the end of the
leader regions of s e v e r a l amino a c i d b i o s y n t h e t i c opérons (or
genes) i n E. coli and r e l a t e d organisms (39). The primary i n d i c a -
t i o n that such a mechanism i s involved i n the c o n t r o l of an amino
a c i d b i o s y n t h e t i c pathway i s an endproduct r e p r e s s i o n that i s de-
pendent on the amino a c i d being t r a n s f e r r e d to i t s cognate tRNA
at an ample r a t e . Thus, derepression of the h i s t i d i n e biosynthe-
t i c pathway can be achieved by any mechanism that reduces the
i n t r a c e l l u l a r l e v e l of h i s t i d y l tRNA, f o r example, by l i m i t i n g the
supply of h i s t i d i n e i t s e l f or by l i m i t i n g the a c t i v i t y of the h i s -
t i d y l tRNA synthetase (51).
Thus f a r , the amino a c i d b i o s y n t h e t i c genes c o n t r o l l e d i n
t h i s way are c h a r a c t e r i z e d by the presence of a leader region be-
tween the promoter and the s t r u c t u r a l gene (52,53) The leader i s
t r a n s c r i b e d at a frequenc
polymerase a v a i l a b i l i t
t i o n i n i t i a t i o n i t s e l f i s c o n t r o l l e d by the trp repressor). The
leader t r a n s c r i p t c a r r i e s the information f o r a short peptide that
i s unusually r i c h i n the amino a c i d ( s ) f o r which the pathway i s
concerned. Whether RNA polymerase stops at the end of the leader
or proceeds i n t o the s t r u c t u r a l gene i s dependent upon whether a
ribosome can t r a n s l a t e the leader t r a n s c r i p t to the stop codon
(amino a c i d excess) or i s s t a l l e d at a codon f o r the amino a c i d
for which the pathway i s concerned (amino a c i d l i m i t a t i o n ) . The
mechanism underlying termination i s that competing secondary
s t r u c t u r e s of the leader t r a n s c r i p t are s e l e c t e d by the extent of
ribosomal t r a v e l and that one of these a l t e r n a t i v e secondary
s t r u c t u r e s contains a t y p i c a l ρ (rho)-independent termination s i g
nal. The general features have been studied best i n the amino
a c i d b i o s y n t h e t i c opérons of s e v e r a l Enterobacteriaceae (39).
Although the nature of the b a c t e r i a l c e l l makes p o s s i b l e spe-
c i f i c c o n t r o l over t r a n s c r i p t i o n i n ways that cannot be achieved
i n eukaryotic systems, c o n t r o l of gene expression at the l e v e l of
t r a n s l a t i o n i s a l s o important i n b a c t e r i a . Control of t r a n s l a t i o n
i s achieved p r i m a r i l y by the prevention of ribosomal binding.
Such a c o n t r o l might be achieved by masking the binding s i t e
e i t h e r by s p e c i f i c b i n d i n g by p r o t e i n s or by the secondary s t r u c -
ture of the message i t s e l f . The b e s t - s t u d i e d examples are the
ribosomal p r o t e i n s (54). Although the genes f o r a l l ribosomal
p r o t e i n s have not yet been examined, the general pattern that
seems to be emerging i s that one of the ribosomal p r o t e i n s s p e c i -
f i e d by a m u l t i c i s t r o n i c , ribosomal p r o t e i n operon prevents mes-
sage t r a n s l a t i o n by binding at the t r a n s l a t i o n a l s t a r t s i t e on the
message. The b a s i s for the s p e c i f i c binding i n the case of r i b o -
somal p r o t e i n s S4 and S7 i s that the RNA i n the v i c i n i t y of the
ribosomal s t a r t s i t e has a s t r u c t u r e s t r o n g l y homologous to that
of the region on 16S RNA that binds to S4 (or to S7) during r i b o -
some assembly (42).
The c o n t r o l over the erythromycin r e s i s t a n c e gene, which

s p e c i f i e s a ribosomal methylase, i s a l s o achieved by i n t e r f e r i n g

with t r a n s l a t i o n of the message (43). Masking of the ribosome
masking s i t e i s not by a p r o t e i n binding to i t but by a secondary
s t r u c t u r e o c c u r r i n g i n the message whenever a t r a n s l a t i n g ribosome
i s able to t r a n s l a t e the peptide s p e c i f i e d by the leader sequence
(as could occur only i f already modified ribosomes were f u n c t i o n -
ing i n the presence of erythromycin or i f normal ribosomes were
f u n c t i o n i n g i n the absence of erythromycin). The ribosome binding
s i t e of the methylase message would be opened when the t r a n s l a t i n g
ribosome s t a l l e d i n the e a r l y part of the leader (as could occur
i f unmodified, s e n s i t i v e ribosomes were f u n c t i o n i n g i n the pre-
sence of erythromycin). This mechanism, a f f e c t i n g a c c e s s i b i l i t y
of a ribosome b i n d i n g s i t e i s thus reminiscent of the c o n t r o l of
formation of a t r a n s c r i p t i o n termination s i t e by the t r a n s l a t i o n
of the leader sequence of l amin a c i d b i o s y n t h e t i
described above.
These examples are a small sample of many that could be c i t e d
that make i t q u i t e c l e a r that m i c r o b i a l metabolism i s r a t h e r
r i g i d l y c o n t r o l l e d f o r the primary f u n c t i o n of the microbe: to
grow and reproduce i t s e l f as e f f i c i e n t l y as p o s s i b l e . These are
what I would term obstacles to the biochemical engineer who may
wish to e x p l o i t a p a r t i c u l a r enzyme r e a c t i o n or a p a r t i c u l a r path-
way to a f u n c t i o n that may w e l l be detrimental to the primary
f u n c t i o n of the organism. However, i n every case, the c o n t r o l
mechanisms that c o n s t i t u t e these o b s t a c l e s are g e n e t i c a l l y con-
t r o l l e d and, t h e r e f o r e , are subject to mutation. To the extent
that mutations can be s e l e c t e d that are advantageous to the en-
gineer without being l e t h a l to the organism, these c o n t r o l mecha-
11
nisms can be considered " t o o l s to be manipulated and c a r e f u l l y
honed.
It i s , however, sometimes d i f f i c u l t to e x p l o i t the capacity
of an organism to perform a u s e f u l biochemical transformation.
Often Nature has s e l e c t e d organisms i n which the s t r i c t u r e s found
i n most organisms are modified, and the organism occupies a unique
niche. Such organisms, i f discovered, can serve u s e f u l f u n c t i o n s .
Just such organisms are those that are c u r r e n t l y being used i n the
Japanese amino a c i d fermentation i n d u s t r y . In most cases, the
organisms have been i s o l a t e d and i d e n t i f i e d by e m p i r i c a l research
and screening procedures. In some cases, the b a s i s f o r the non-
normal formation of amino a c i d s can be explained post facto, but
seldom does i t appear that these organisms were developed by a
preconceived, r a t i o n a l approach. I would t h e r e f o r e l i k e to c i t e
and analyze what I think provides an exception.
The e x c e p t i o n a l case i s one i n which the i n v e s t i g a t o r s j u d i -
c i o u s l y combined e m p i r i c a l and r a t i o n a l approaches to prepare an
organism capable of producing l a r g e amounts of the amino a c i d i s o -
l e u c i n e . The p r o j e c t was performed by i n v e s t i g a t i o n at the Tanabe
Seiyeku Company i n Osaka, Japan, and i n v o l v e d the organism Serra-
tia marcescens (55). Before review of t h i s work, i t i s necessary
to consider b r i e f l y s e v e r a l aspects of the pathways by which

i s o l e u c i n e and the c l o s e l y r e l a t e d amino a c i d , v a l i n e , are formed.

Figure 1 shows these pathways. An important feature i s that
the four steps l e a d i n g to v a l i n e are c a t a l y z e d by enzymes that
are a l s o needed f o r i s o l e u c i n e formation. In a d d i t i o n , a f i f t h
enzyme, threonine deaminase, i s r e q u i r e d i n most p l a n t s and micro
organisms f o r the formation of α-ketobutyrate. Several d e t a i l s
of the pathways are given i n the legend.
Also shown i n Figure 1 i s the arrangement of the s t r u c t u r a l
genes found i n E. coli. I t should be pointed out that a l l of the
genes, except those f o r the two v a l i n e - i n s e n s i t i v e acetohydroxy
a c i d synthases, are contained i n the ilv gene c l u s t e r at the 84-
minute region of the E. coli chromosome. I t might t h e r e f o r e be
a n t i c i p a t e d that an E. coli s t r a i n c a r r y i n g a plasmid with the
ilv gene c l u s t e r would be an e x c e l l e n t overproducer of v a l i n e .
(If the chromosomal ilv fragment derived fro th K-12 s t r a i
of E. coli y i t would hav
would be expressed. Se
more e f f e c t i v e i f the i n s e r t e d DNA c a r r i e d an ilvA lesion, i . e . ,
was threonine deaminase negative. Overproduction of v a l i n e would
be expected, s i n c e the v a l i n e pathway derives i t s carbon e x c l u
s i v e l y from a key intermediate i n the c e n t r a l metabolic route,
pyruvate. Flow i n t o the pathway would then never be r e s t r i c t e d
as long as ample carbon source was present.
An analogous c o n d i t i o n f o r i s o l e u c i n e overproduction would
not be expected to a r i s e i f the corresponding plasmid c o n t a i n i n g
an ilvA s t r u c t u r a l gene mutation l e a d i n g to a feedback negative
threonine deaminase were to be employed. As can be seen i n Figure
2, carbon flow i n t o the i s o l e u c i n e pathway i s dependent not only
on the supply of pyruvate but a l s o on threonine, an endproduct
i t s e l f , and the s y n t h e s i s of which i s a l s o s t r i n g e n t l y r e g u l a t e d .
It i s doubtful whether threonine could be formed f a s t enough i n
an otherwise normal c e l l to e x p l o i t f u l l y the high l e v e l expres
s i o n of the ilv genes c a r r i e d on a plasmid as d e s c r i b e d here. The
procedures followed by Komatsubara and h i s coworkers i l l u s t r a t e s
w e l l how t h i s k i n d of o b s t a c l e can be circumvented.
The sequence of manipulations employed by Komatsubara et a l .
(55) i s summarized i n Figure 3. The simplest step was to i s o l a t e
a d e r i v a t i v e o f the parent S. maroeeoens s t r a i n , 8000, that was
derepressed f o r the ilvGEDA operon and c a r r i e d a l e s i o n i n the ilvA
gene that allowed the formation of an i s o l e u c i n e - i n s e n s i t i v e
r
(feedback negative) threonine deaminase ( T D ) . This kind of i s o
l a t i o n had e a r l i e r been accomplished by the s e q u e n t i a l s e l e c t i o n
of an aminobutyrate-resistant s t r a i n (derepressed) followed by
s e l e c t i o n of an i s o l e u c i n e hydroxamate-resistant d e r i v a t i v e (feed
back r e s i s t a n t ) (58). The i n v e s t i g a t o r s were more fortunate i n
the s e l e c t i o n of s t r a i n GIHVL-r6426, which arose as an i s o l e u c i n e
hydroxamate-resistant s t r a i n a f t e r mutagenesis by n i t r o s o g u a n i -
dine, a mutagen known f o r causing c l o s e l y l i n k e d mutations i n the
same c e l l . The p r e c i s e nature of the mutation a l l o w i n g derepres
s i o n was not e s t a b l i s h e d , but, i n analogy with what has been

Η Η
Ο OH Ο Ο Ο NH 3
AHSU TrC
CH -C-COO
3 CH -C-C-C00~
3 CH -C-CH-COO~
3
CH 3 -CH-C-COO" CH, -CH-CH-COO"
IR DH ι TrB ι
Pyruvate CH 3 CH 3
CH, CH 3
a-Acetolactate α,β-Dihydroxy- a-Ketoisovalerate L- Valine

isovalerate
CH -C-COO'
3
Pyruvote
Ο Ο Ο Ο NH 3
IR OH TrB
CH -CH-C-COO
3
CH -C-C-COO"
3 CH -C-CH-COO"
3 CH -CH-C-COO"
3 CH -CH-CH-COO"
3
AHSU
a-Ketobutyrote CH 2 CH 2 ÇH 2 CH 2
ι ι
CH 3 CH 3
CH 3
CH 3
a - Aceto - a - α,β-Dihydroxy- a-Keio-β-
hydroxybutyrate /3-methylvolerote methy I valerate L- Isoleucine
TD-*--
NH 3
CH -CH - CH-COO"
3
L-Threonjne
H I Β C Y A D E G G G Gp
a e
I—; 1 q
—ι—-pi ι—:—ι—;—ι—;—ι ; ι—;
I I ι
—ι—I, t i *ι "i
AHSm AHSI ι TD DH TrB AHSU
IR
Figure 1. Biosynthesis of isoleucine and valine. The enzymes catalyzing the indicated steps are
abbreviated, and the corresponding structural genes (where known) are indicated in parentheses
as follows.

TD (ilvA), threonine deaminase; AHS I (ilvB) and AHS III (ilvHI), valine-inhibited acetohydroxy acid jyn-
thases; AHS II (ilvG), valine-insensitive acetohydroxy acid synthase; IR (ilvC), acetohydroxy acid isomeroreduc~'
tase; DH (ilvD), dihydroxy acid dehydrase; TrB (ilvE), transaminase B; TrC, transaminase C. In E. coli strain

K-12, ilvG exhibits no activity owing to a polar lesion in the ilvO region of the gene. Certain frameshift mutations
in ilvO result in ilvG expression and increased downstream expression. Control of the ilvGEDA operon occurs by
attenuation at ilvGa of transcripts initiated at the promoter ilvGp. A putative 32-residue leader peptide, rich in
isoleucirte, leucine, and valine is specified by ilvGe. The ilvY product, μ, is a positive control element needed
for induction of ilvC by the substrates of its product, IR.
thrA *>'' ι
L-Aspartate r> Aspartyl 2 Aspartic wVAjr Homoserine thvC I
h o s h a t e β
metLM "~f"* P P ' semialdehyde • Homoserine phosphate
L-Threonine
\metA
• ' i: ilvA
Dihydrodipicolinic O-succinyl
acid homoserine a-Oxobutyrate
BJdapB \metB
I i| i/t/B
Tetrahydrodipicolinic Cystathionine δ
acid Acetohydroxy-
\metC butyrate
C JdapCorD \ ilvC
Homocysteine Dihydroxy-
N-succinyl- <-OXO-L-
ι
^metE, metF isoieucine
a-aminopimelate
•
DjcfapC or D jj iluD
' L-Methionine
N-succinyl-LL- a-Oxoisoleucine
diaminopimelate
ilvE
Ε I dapE
-L-Isoleucine
LL-Diaminopimelate
meso-diaminopimelate
cJ/ysA
i ! L-Lysine

Figure 2. The biosynthesis of lysine, methionine, threonine, and isoleucine in E . coli and S.
marcescens. Solid arrows, steps catalyzed by enzymes repressed by lysine. Broken arrows, steps

catalyzed by enzymes repressed by methionine. Open arrows, steps catalyzed by enzymes repressed
by threonine plus isoleucine. Open, dashed arrows, steps catalyzed by enzymes controlled as de
scribed in Figure 1. Structural genes indicated in italics. Dashed lines indicate reactions controlled
by endproduct inhibition. Reproduced, with permission, jrom Ref. 57. Copyright 1975, American
Society for Microbiology.
THREONINE AND ISOLEUCINE PRODUCING STRAINS

(Komatsubara et a l . )
AECrl74
N-ll
uth
KNr31
ilvA uth
uth ilvA
?(Met-leaky)
ilvA r ? (Met-leaky)
lysC (AK ) r
?(Met-leaky) lysC (AK )
0.3 g T h r / 1
T-570
HNr21 T-693
uth uth uth
ilvA ilvA ilvA
r r
r
thrA (AK HD ) r thrA (AK HD )
?(Itet-leaky)
11 g T h r / 1
ileS lysC (AK ) r
8 g Thr/1
lyeCo ?
r r
thrA (AK HD )
île S
25 g T h r / 1
HNr59 E-60
uth uth
ilvA ilvA
r
thrA (HD ) thrA (HD ) r
T-803
ileS ileS
5 g Thr/1 thrB/C uth
r
ilvA (TD )
GIHVL-r6426 ilvGa ?
r
ilvA (TD ) ?(Met-leaky)
r
ilvGa ? lysC (AK )
Mu-910 3.5 g I l e / 1 lysCo ?
r r
uth 8000 thrA (AK HD )

ileS
25 g I l e / 1
Figure 3. The development of an isoleucine-producing strain from S. marcescens

strain 8000. Key: φ, phage-mediated transduction; M , nitrosoguanidine mutagenesis.
See text for details.

learned about the c o n t r o l of the ilvGEDA operon i n E. coli, i t is

most l i k e l y a mutation a f f e c t i n g the attenuator (ilvGa), thereby
a b o l i s h i n g the m u l t i v a l e n t c o n t r o l of the p a r e n t a l s t r a i n . That
ilvGy the s t r u c t u r a l gene f o r the v a l i n e - i n s e n s i t i v e acetohydroxy
a c i d synthase, was derepressed assured that acetohydroxy a c i d i s o -
meroreductase would be h i g h l y induced by s u b s t r a t e , even though
i t s s t r u c t u r a l gene, ilvC 9 i s not part of the derepressed operon.
The r e s i s t a n t mutant was shown to excrete up to 3.5 gm of i s o l e u -
cine per l i t e r i n a minimal medium c o n t a i n i n g 150 gm of sucrose
as carbon source. That t h i s l e v e l was l i m i t e d by a v a i l a b i l i t y of
threonine was l a t e r demonstrated by the f a c t that t r a n s f e r of the
two l i n k e d ilv mutations to a s t r a i n overproducing l a r g e q u a n t i -
t i e s of threonine r e s u l t e d i n a much b e t t e r i s o l e u c i n e producer.
The steps used to develop the threonine producer were complex
and are a l s o o u t l i n e d i F i g u r 3 Th f i r s t f thes step
the i s o l a t i o n of n i t r o s o g u a n i d i n
l i z e threonine as a n i t r o g e (59)
was i n the uth gene, which s p e c i f i e s threonine dehydrogenase. To
obtain an organism even l e s s able to break down exogenous t h r e o -
nine, mutagenesis to y i e l d an i s o l e u c i n e - r e q u i r i n g s t r a i n l a c k i n g
threonine deaminase was employed. When preformed threonine was
added to f u l l y grown c u l t u r e s of the r e s u l t i n g s t r a i n , D-60, the
r a p i d disappearance noted with the o r i g i n a l parent s t r a i n was no
longer observed (59).
S t r a i n D-60 was then used to s e l e c t mutants r e s i s t a n t to the
threonine analog, 3-hydroxynorvaline, f o l l o w i n g n i t r o s o g u a n i d i n e
mutagenesis (60,61). Three kinds of r e s i s t a n t s t r a i n were saved
for subsequent use. One of these (HNr31) had an incomplete but
undefined b l o c k i n methionine b i o s y n t h e s i s , which may have ac-
counted f o r the s m a l l amount o f threonine that was accumulated
from homoserine being funneled i n t o the threonine pathway.
The other two mutants produced much g r e a t e r amounts of threo-
nine. One, s t r a i n HNr21, appeared to contain a s i n g l e mutation i n
the thrA gene, which s p e c i f i e s the b i c e p h a l i c enzyme, a s p a r t o -
kinase I-homoserine dehydrogenase I (Figure 2). Normally, both of
these a c t i v i t i e s are i n h i b i t e d by threonine. The mutation i n
s t r a i n HNr21 a b o l i s h e d the threonine s e n s i t i v i t y f o r both a c t i v i -
ties. This l o s s of feedback c o n t r o l r e s u l t e d i n an overproduction
of threonine to an extent of 11 gm of threonine per l i t e r . The
other s t r a i n , HNr59, a l s o had a thrA l e s i o n , but t h i s one appeared
to a b o l i s h only the threonine s e n s i t i v i t y of the homoserine dehy-
drogenase. There was another mutation i n the same s t r a i n which
appeared to be i n the ileS gene. I f so, i t undoubtedly caused the
corresponding gene product, i s o l e u c y l tRNA synthetase, to have a
reduced a f f i n i t y f o r i t s s u b s t r a t e . Such an enzyme would l e a d to
reduced l e v e l s o f i s o l e u c y l tRNA and derepressed l e v e l s of the thr
operon and the ilvGEDA operon. Since the mutant was an ilvA mu-
tant, the concomitant derepression of the ilvGEDA operon was of no
consequence. However, the iVoA mutation made i t necessary f o r the
s t r a i n to be grown i n the presence of exogenous i s o l e u c i n e and may

have reduced the e f f e c t i v e n e s s of the l o w - a f f i n i t y i s o l e u c y l tRNA

synthetase i n causing a derepression of the threonine b i o s y n t h e t i c
enzymes.
In an attempt to enhance threonine production, steps were
taken to r e p l a c e the thrA gene mutation a f f e c t i n g only homoserine
dehydrogenase I a c t i v i t y i n s t r a i n HNr59 with that of s t r a i n
HNr21, which a f f e c t e d both aspartokinase I and homoserine dehydro-
genase I a c t i v i t i e s . This t r a n s f e r was accomplished by i n t r o -
ducing a thrB (or C) mutation i n HNr59, to y i e l d s t r a i n E-60 (62).
This s t r a i n then served as r e c i p i e n t f o r phage-mediated transduc-
t i o n with s t r a i n HNr21 as donor. The r e s u l t i n g s t r a i n , T-570,
contained the doubly i n s e n s i t i v e aspartokinase I-homoserine dehy-
drogenase I a c t i v i t i e s of s t r a i n HNr21 but had not l o s t the c l o s e -
l y l i n k e d ileS l e s i o n of s t r a i n E-60 (or i t s parent, HNr59). Sur-
prisingly, this strain did t produc q u i t h threonin
did s t r a i n HNr21.
Attempts were t h e r e f o r
threonine pathway by enhancing the a c t i v i t y of the l y s i n e - s e n s i -
t i v e aspartokinase. This was accomplished by s e l e c t i n g n i t r o s o -
guanidine- induced mutants r e s i s t a n t to aminoethylcysteine (61).
One of these s t r a i n s , AECrl74, was probably another doubly mutated
s t r a i n with a l y s i n e - i n s e n s i t i v e aspartokinase that was a l s o de-
repressed. The derepression might have been e i t h e r an "up" pro-
moter or an operator mutation i n the lysC gene (as i s suggested i n
Figure 3). This s t r a i n accumulated 7 gm of threonine per l i t e r .
Mutagenesis to threonine auxotrophy y i e l d e d s t r a i n N - l l , which
allowed i n t r o d u c t i o n of the thrA gene and ileS genes of s t r a i n
T-570 by phage-mediated t r a n s d u c t i o n (62).
The r e s u l t a n t s t r a i n , T-693, produced up to 25 gm of threo-
nine per l i t e r and provided e x a c t l y the k i n d of background that
might feed carbon i n t o an i s o l e u c i n e pathway that had l o s t both
feedback and r e p r e s s i o n c o n t r o l . This i n t r o d u c t i o n was p o s s i b l e
because s t r a i n T-693 c a r r i e d the o r i g i n a l ilvA l e s i o n of s t r a i n
D-60. Phage grown on s t r a i n G1HVL-6426 was t h e r e f o r e used to ren-
der s t r a i n T-693 i s o l e u c i n e independent. In so doing, both the
feedback r e s i s t a n c e of threonine deaminase and the derepression of
the ilvGEDA operon were introduced. The r e s u l t i n g s t r a i n , T-803,
produced up to 25 gm of i s o l e u c i n e per l i t e r . The carbon flow
i n t o the u n c o n t r o l l e d threonine pathway had thus been almost com-
p l e t e l y d i v e r t e d to i s o l e u c i n e production with only about 3 gm of
threonine now being formed by s t r a i n T-803.
This r a t h e r i n v o l v e d example serves perhaps as an i n t e r e s t i n g
model f o r the way e m p i r i c a l l y generated mutations can be r a t i o n -
a l l y manipulated to y i e l d u s e f u l organisms. In the example c i t e d ,
the u s e f u l t o o l of using analogs to s e l e c t s t r a i n s with a l t e r e d
c o n t r o l c i r c u i t s was i n v a l u a b l e . There were r e a l l y no unexpected
mutations, yet there was no way to p r e d i c t which combination of
l e s i o n s would have supplemented each other to have y i e l d e d such
p r o l i f i c overproducers. I t may be that the maximally e f f e c t i v e
combination was not obtained i n s t r a i n T-803, and i t i s l i k e l y

that the i n v e s t i g a t o r s t r i e d many more combinations than those

that were reported.
I n v e s t i g a t o r s are undoubtedly now being tempted to e x p l o i t
the use o f recombinant DNA technology to produce s t r a i n s with cer-
t a i n biochemical a c t i v i t i e s enhanced. The lesson the model system
described here has f o r such i n v e s t i g a t o r s i s t h a t , i n a d d i t i o n to
r e q u i r i n g elevated enzyme a c t i v i t i e s , i t i s a l s o necessary to
assure that the c e l l can provide the substrates o r the carbon r e -
quired f o r the enhanced pathway to f u n c t i o n . The other lesson i s
that s t r a i n development w i l l remain l a r g e l y an e m p i r i c a l process
but that empiricism w i l l be more r e a d i l y harnessed i f the i n v e s -
t i g a t o r has a thorough knowledge o f the physiology and biochemis-
t r y o f the process.
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5
Mathematical Models of the Growth of

Individual Cells
Tools for Testing Biochemical Mechanisms
M. L. SHULER and M. M. DOMACH

Cornell University, School of Chemical Engineering, Ithaca, NY 14853
The rationale for and development of mathe-

matical models fo
potential use o
coli in ascertaining the plausibility of basic
biological hypotheses is illustrated with respect
to the control of the initiation of DNA synthesis
and with respect to ammonium ion assimilation.
Mechanisms postulated on the basis of in vitro
enzymology can be tested for in vivo compatibility
using the model. The transient behavior of single-
-cells to step-up and step-down in glucose or
ammonium ion is shown to result in oscillatory
responses and hysterisis. Methods to construct
population models from single-cell models are
discussed. Population models are important to
engineering analysis and in relating data for
experiments with large populations to the responses
of a "typical cell".
Why S i n g l e - C e l l Models?
The b a s i c conceptual u n i t i n microbiology i s the s i n g l e c e l l .

Although m i c r o b i o l o g i s t s and biochemists work with l a r g e popula-
t i o n s of c e l l s , the goal i s g e n e r a l l y to understand the behavior
of a " t y p i c a l " c e l l . There i s very l i t t l e d i r e c t data on the
growth of i n d i v i d u a l c e l l s - and what data e x i s t s i s o f t e n of*
questionable value because the c o n d i t i o n s r e q u i r e d f o r observa-
t i o n s may lead to "unnatural responses". Thus, the behavior o f
the " t y p i c a l " c e l l must be i n f e r r e d from the aggregated behavior
of the t o t a l p o p u l a t i o n . B i o l o g i s t s u s u a l l y are i n t e r e s t e d i n a
s i n g l e aspect of c e l l growth (e.g. p r o t e i n s y n t h e s i s ) . Cells
c o n t a i n a complex, n o n l i n e a r , h i g h l y regulated s e r i e s of chemical
r e a c t i o n s . Human l o g i c c o n s i s t s of a few l i n e a r s t e p s . I t i s
e s s e n t i a l l y impossible f o r the unaided mind to i n t e r r e l a t e p r o t e i n
s y n t h e s i s , DNA r e p l i c a t i o n , n u t r i e n t t r a n s p o r t , e t c . i n t o a coher-
ent conceptual model of a c e l l .
0 0 9 7 - 6 1 5 6 / 8 3 / 0 2 0 7 - 0 0 9 3 $ l 1.50/0

Computer models of s i n g l e c e l l s act as an a i d In b u i l d i n g

such conceptual models. Because such models make q u a n t i t a t i v e
p r e d i c t i o n s - p r e d i c t i o n s which are experimentally v e r i f i a b l e -
they serve as good v e h i c l e s to r e v e a l e r r o r s i n e i t h e r b a s i c
mechanisms or the manner i n which they are i n t e g r a t e d i n the con-
ceptual model. Population models can be constructed from ensem-
b l e s of s i n g l e - c e l l models. Such models may be u s e f u l a i d s i n
r e l a t i n g the behavior of whole populations to biochemical mechan-
isms w i t h i n a t y p i c a l c e l l . S i n g l e - c e l l models are p a r t i c u l a r l y
w e l l s u i t e d to the conceptual and experimental needs of b i o l o -
gists.
S i n g l e - c e l l models are a l s o of importance to biochemical en-
gineers. The motivation i s d i f f e r e n t from b i o l o g i s t s . Engineers
are i n t e r e s t e d i n manipulating a population of c e l l s ; such manip-
u l a t i o n i s f a c i l i t a t e d b mathematical models that can p r e d i c t the
response of the populatio
ronment. H i s t o r i c a l l y
i n biosystems has sprung from a need to model populations of
cells. Population models have inherent l i m i t a t i o n s - l i m i t a t i o n s
that can be circumvented by using ensembles of s i n g l e - c e l l models.
Population Models. To understand why s i n g l e - c e l l models are

u s e f u l i t i s important to review previous attempts at modeling
populations. The h i s t o r y and philosophy of modeling as w e l l as
the v i r t u e s and f a u l t s inherent i n previous models has been w e l l
described i n the l i t e r a t u r e . Among these a r t i c l e s are those by
Tsuchiya, F r e d r i c k s o n , & A r i s (1); P a i n t e r & Marr (2);
Van Uden (3); G a r f i n k e l , eit a l . (4); F r e d r i c k s o n , Megee, &
Tsuchiya (5); N y i r i (6); Boyle & Berthouex (7) ( f o r waste t r e a t -
ment) ; F r e d r i c k s o n (8) ( f o r s t r u c t u r e d models o n l y ) ; and
Bailey (9).
One method of c l a s s i f y i n g models i n v o l v e s the concept of
" s t r u c t u r e " . Structured models have the inherent a b i l i t y to de-
s c r i b e the p h y s i o l o g i c a l s t a t e of a microorganism or a c u l t u r e of
such c e l l s . Unless the p h y s i o l o g i c a l s t a t e can be s p e c i f i e d , the
dependence of a c u l t u r e on i t s previous h i s t o r y cannot be ac-
counted f o r . Such dependence on h i s t o r y i s known to be important
C5). T y p i c a l l y , s t r u c t u r e i s added to a model by c o n s i d e r i n g the
c e l l or c u l t u r e to c o n s i s t of two or more components (e.g. n u c l e i c
a c i d s and p r o t e i n , etc.) or to d i s t i n g u i s h between separate c e l l s
on the b a s i s of s i z e or age.
Any model not i n c o r p o r a t i n g the foregoing p r i n c i p l e i s
u n s t r u c t u r e d — f o r example, the well-known Monod model. I t has
been shown that unstructured models are a p p l i c a b l e only i n b a l -
anced growth s i t u a t i o n s (10), which, according to Campbell's (11)
d e f i n i t i o n , r e q u i r e s that each component of the c u l t u r e be accu-
mulated at the same r a t e . Unstructured models are never general,
give very l i t t l e i n s i g h t i n t o c e l l u l a r mechanisms, and cannot be
used to describe l a g and d e c l i n e phases i n batch growth, t r a n s i e n t

5. SHULER AND DOMACH Models of Cell Growth 95
response i n chemostats, or growth i n m u l t i s t a g e continuous c u l -

t u r e . I f the formation of a product i s dependent on c e l l h i s t o r y
and i s not growth a s s o c i a t e d , then an u n s t r u c t u r e d model w i l l be
u n s u i t a b l e . Unstructured models have been widely used, however,
and have been s u c c e s s f u l when a p p l i e d to the commonly o c c u r r i n g
balanced growth s i t u a t i o n s of e x p o n e n t i a l phase batch c u l t u r e and
steady-state s i n g l e - s t a g e d continuous c u l t u r e .
Two of the f i r s t s t r u c t u r e d models proposed were those by
Williams (_L2, 13) and Ramkrishna, F r e d r i c k s o n , & Tsuchiya (14).
Both models were d e t e r m i n i s t i c and d e a l t with a nonsegregated b i o -
mass. A nonsegregated model i s one which does not recognize ex-
p l i c i t l y the e x i s t e n c e of i n d i v i d u a l c e l l s . For example, the con-
tents of a fermenter can be thought of as c o n s i s t i n g of two
p h a s e s — o n e b i o t i c and the other a b i o t i c . The b i o t i c phase can
then be t r e a t e d as a homogeneous e n t i t y Nonsegregated models of
course, cannot make any
the e f f e c t s of c e l l geometr
easy to handle mathematically, and the o v e r a l l biomass and i t s
major components can be determined e x p e r i m e n t a l l y with reasonable
accuracy.
I t i s obvious to anyone who has m i c r o s c o p i c a l l y examined mi-
c r o b i a l c u l t u r e s that c e l l s are i n d i v i d u a l s and can vary g r e a t l y
i n observable p r o p e r t i e s such as c e l l s i z e . Only segregated mod-
e l s can capture t h i s p r o p e r t y . Such models c o n t a i n s t r u c t u r e i n
the sense that changes are allowed i n the d i s t r i b u t i o n of s t a t e s
among the p o p u l a t i o n i n response to the e x t e r n a l environment.
Examples of models u s i n g age as a measure of the index of a c e l l ' s
stage i n c l u d e those by Von F o e r s t e r (15); Trucco (16); Yakovlev,
et a l . (17); F r e d r i c k s o n & Tsuchiya (18); Kozesnik (19); and
Lebowitz & Rubinow (20). C e l l s i z e can a l s o be u t i l i z e d as sug-
gested by Koch & Schaechter (21) and Eakman, et a l . (22). In the
above examples only one parameter was used as an index of s t a t e ;
i t would be impossible to i n s e r t any i n f o r m a t i o n about biochemical
mechanisms i n t o the model.
Models which i n c l u d e both s t r u c t u r e and segregation lead to
mathematical equations which are extremely d i f f i c u l t to s o l v e even
with the a i d of modern high-speed computers. B a i l e y and co-work-
ers (23, 24, 25) have made progress i n circumventing some of these
problems. Nonetheless i t appears impossible to prepare p o p u l a t i o n
models which would c o n t a i n a h i g h l e v e l of s t r u c t u r e (say more
than f i v e components) and s e g r e g a t i o n , and which s t i l l are mathe-
matically tractable.
Population models which c o n t a i n both a h i g h - l e v e l of s t r u c -
ture and segregation are d e s i r a b l e . Such models have the poten-
t i a l to make accurate p r e d i c t i o n s of t r a n s i e n t responses - such
p r e d i c t i o n s are important not only f o r process c o n t r o l but may be
u s e f u l i n d e s i g n i n g c y c l e d - r e a c t o r s which can have product y i e l d s
greater than comparable s t e a d y - s t a t e c u l t u r e s (26, 27). There i s
a l s o p r e l i m i n a r y evidence that under some circumstances only a
small sub-population of c e l l s produce most of the product (24);

thus the manipulation of the d i s t r i b u t i o n of sub-populations can

be p r e d i c t e d only i f a structured-segregated model i s a v a i l a b l e .
Tanner (28) has made persuasive arguments f o r the need f o r
more s o p h i s t i c a t e d models t o optimize the design of commercial
fermentation processes. He has s t a t e d that commercial fermenta-
t i o n processes have not been s i g n i f i c a n t l y optimized "because
there a r e no simple, g e n e r a l , and accurate mathematical models f o r
p r e d i c t i n g the c a s u a l e f f e c t s of c o n t r o l v a r i a b l e changes". Fur-
t h e r , he claims that "the establishment of j u s t one of these pro-
f i l e s [pH, temperature, n u t r i e n t , e t c . ] t o optimize a batch f e r -
mentation i s a n e a r l y impossible task t o perform d i r e c t l y on a
process". The use of mathematical models i n c o n j u n c t i o n with mod-
ern q u a n t i t a t i v e o p t i m i z a t i o n procedures could circumvent much of
the experimental work. The need f o r a good model of b a c t e r i a l
growth which could accommodate product formation has been w e l l
recognized (e.g. 6^, 23
s u f f i c i e n t l y general t
A way to generate h i g h l y structured-segregated models which
are mathematically t r a c t a b l e i s to b u i l d p o p u l a t i o n models u s i n g
an ensemble of s i n g l e - c e l l models. Such an approach can avoid
the generation of i n t e g r a l - d i f f e r e n t i a l equations which are so
computationally d i f f i c u l t to s o l v e . Thus, s i n g l e - c e l l models
f i l l c e r t a i n r e a l needs of both b i o l o g i s t s and engineers. The
advantages of s i n g l e - c e l l models compared to normal p o p u l a t i o n
models a r e :
1. e x p l i c i t accounting of c e l l geometry and i t s p o t e n t i a l
e f f e c t s of n u t r i e n t t r a n s p o r t ;
2. the a b i l i t y to p r e d i c t temporal events during the d i v i -
sion cycle;
3. the a b i l i t y to consider the e f f e c t s of s p a t i a l arrange-
ments w i t h i n a c e l l ;
4. and the ease i n which d e t a i l s about biochemical pathways
and t h e i r i n t e g r a t i o n and metabolic c o n t r o l can be i n c l u d e d .
These advantages make s i n g l e - c e l l models p a r t i c u l a r l y w e l l - s u i t e d
to t e s t i n g the p l a u s i b i l i t y of hypotheses about metabolic mechan-
isms.
S i n g l e - c e l l models i n v i t e complexity. T h e i r main disadvan-
tage i s that s i n g l e - c e l l models represent only a " t y p i c a l " c e l l
and are adequate r e p r e s e n t a t i o n s of the growth of c e l l populations
only i f the moments o f d i s t r i b u t i o n of c e l l u l a r p r o p e r t i e s higher
than the f i r s t - o r d e r can be ignored. The l a s t disadvantage i s
circumvented by u s i n g an ensemble of s i n g l e - c e l l models t o b u i l d a
p o p u l a t i o n model ( i . e . each s i n g l e - c e l l represents some small
f r a c t i o n of the t o t a l p o p u l a t i o n ) .
I d e a l Model. Having discussed why s i n g l e - c e l l models may be

u s e f u l , what c h a r a c t e r i s t i c s should such models have? The f o l l o w -
ing f e a t u r e s are important:
1. the model must be c o n s i s t e n t with experimentally con-
firmed observations over a wide v a r i e t y of growth c o n d i t i o n s ,

5. SHULER A N D DOMACH Models of Cell Growth 97
2. the model must have a s t r u c t u r e which can e a s i l y allow

the i n c o r p o r a t i o n o f p o s t u l a t e d biochemical mechanisms,
3. the parameters o f the model must be determined d i r e c t l y
from independent experiments o r estimated by an o b j e c t i v e s e r i e s
of r u l e s (no a d j u s t a b l e parameters),
4. the e f f e c t of c e l l geometry and shape on n u t r i e n t uptake
must be included (the e x t e r n a l environment must be e x p l i c i t l y
accounted f o r ) ,
5. the model must allow f o r the i n c l u s i o n o f randomness i n
key metabolic systems,
6. the model must not i n c l u d e a r t i f i c i a l c o n s t r a i n t s such as
growth must be e x p o n e n t i a l , the c e l l must maintain a given shape,
the c e l l w i l l d i v i d e when the amount of given component doubles,
etc.,
7. the only s i g n a l
t r a t i o n s of v a r i o u s biochemica
8. the model must be mathematically t r a c t a b l e .
The model should be a model - not a c o l l e c t i o n of phenomenological
equations based on curve f i t s .
Examples of S i n g l e - C e l l Models
One of the f i r s t examples of i n d i v i d u a l - c e 1 1 models i s that

suggested by Von B e r t a l a n f f y (see 1). In h i s model growth was a
r e s u l t of competition between the process o f n u t r i e n t a s s i m i l a t i o n
and endogenous metabolism. N u t r i e n t uptake was p o s t u l a t e d t o be
p r o p o r t i o n a l t o the c e l l ' s surface area and the c o n c e n t r a t i o n of
n u t r i e n t i n the a b i o t i c environment and the r a t e of endogenous
metabolism was p o s t u l a t e d to be p r o p o r t i o n a l t o c e l l mass.
Heinmets (35) suggested i n 1966 a model f o r a s i n g l e c e l l (or the
nucleus of an e u c a r y o t i c c e l l ) that incorporated 19 d i f f e r e n t i a l
equations. The model contained an amino a c i d p o o l , a n u c l e o t i d e
pool f o r RNA s y n t h e s i s , a general i n t r a c e l l u l a r metabolic p o o l ,
t o t a l p r o t e i n , RNA polymerase, genes f o r s y n t h e s i s of v a r i o u s
RNA's, m-RNA (2 t y p e s ) , r-RNA, and t-RNA. The model could respond
to step changes i n the e x t r a c e l l u l a r n u t r i e n t p o o l s . The main
purpose o f the model was t o examine how a c e l l would change from
normal t o abnormal growth. The mechanistic scheme r e f l e c t s the
general understanding of c e l l u l a r biochemistry i n 1965 but d i f f e r s
somewhat from the present conception of the c e l l . The constants
chosen were a r b i t r a r y , and the model was not formulated t o any
s p e c i f i c organism, and no attempt was made t o compare the p r e d i c -
t i o n s t o a c t u a l experimental data. E f f e c t s of c e l l geometry and
s i z e on n u t r i e n t uptake were not considered. The c e l l d i v i s i o n
hypothesis depends on an imposed c r i t e r i o n to do with the concen-
t r a t i o n s of i n t r a c e l l u l a r components. C e l l d i v i s i o n i s not a
" n a t u r a l " response of the model. Davison (36) has solved
Heinmets' model with only the d i g i t a l computer and has compared
the r e s u l t s with two experimental s i t u a t i o n s . The f i r s t was the

recovery of an IS. c o l i f o r Mg s t a r v a t i o n , and the second was the

e f f e c t of s p l i t r a d i a t i o n doses on Chinese Hamster c e l l s . In both
cases reasonable q u a l i t a t i v e p r e d i c t i o n s were obtained from the
model.
Another example of s i n g l e - c e l l model i s that proposed by
Simon (37) i n 1973. He considered only steady-state balanced
growth. His model c e l l contained enzymes involved i n d i v i s i o n
i n i t i a t i o n , i n the formation of DNA p r e c u r s o r s , and i n RNA synthe-
sis. DNA s y n t h e s i s was i n i t i a t e d when a c r i t i c a l m a t e r i a l accu-
mulated to a t h r e s h o l d value; DNA i n i t i a t i o n caused a l l the t h r e s -
hold m a t e r i a l to be consumed. Each p r o t e i n s y n t h e s i s r a t e was
made p r o p o r t i o n a l to the degree of genome a c t i v a t i o n . The r a t i o
of c e l l volume to p r o t e i n was considered constant, and volume
growth was forced to be e x p o n e n t i a l . The surface to volume r a t i o
was considered constant (a c y l i n d e r without ends) The e f f e c t s of
the a b i o t i c environmen
value of the growth r a t
required to d i v i d e when the amount of each component i s doubled.
I n i t i a l c e l l s i z e could be p r e d i c t e d . A short general q u a l i t a t i v e
comparison of the model to experimental observations was given;
the model was found to be s a t i s f a c t o r y f o r the parameters com-
pared.
Weinberg, Z e i g l e r , & L a i n g (38) and Z e i g l e r & Weinberg (39)
have attempted to construct a model of an IS. c o l i c e l l . They have
considered how the v a r i o u s components of the c e l l might be aggre-
gated. T h e i r c e l l contains e x p l i c i t concentrations f o r c e l l w a l l ,
DNA, m-RNA, t-RNA, ribosomes, p r o t e i n , amino a c i d s , w a l l pre-
c u r s o r s , n u c l e o t i d e s , ATP, and ADP are shown (38, 39) along with
a pool c a l l e d glucose which i n c l u d e s glucose and other small
metabolites made from glucose. They c l a i m that t h e i r model of a
c e l l d i f f e r s from others i n that the a b s t r a c t i o n i n v o l v e d i n model
formulation a r i s e s from the aggregation of v a r i a b l e s r a t h e r than
the s e l e c t i o n of subsystems. D i f f e r e n c e and Boolean equations
were used to d e s c r i b e the system. Hyperbolic ("saturation k i n e t -
i c s " ) r a t e forms were not used; f o r example, the r a t e of produc-
t i o n of amino a c i d s from glucose was made p r o p o r t i o n a l to the
product of glucose, ATP, and enzyme 2 c o n c e n t r a t i o n s . Enzyme
a c t i v i t y was modified to simulate an a l l o s t e r i c enzyme by use of
Boolean equations. T h e i r c e l l was constrained to grow exponen-
t i a l l y i n volume. A mechanism f o r i n i t i a t i o n of DNA r e p l i c a t i o n
was included but none f o r c e l l d i v i s i o n . From the above papers
(38, 39) i t i s not c l e a r whether n u t r i e n t uptake was e x p l i c i t l y
accounted f o r or not. They (39) d e s c r i b e how they evaluated r a t e
constants from the data i n the l i t e r a t u r e . A d i r e c t comparison to
experimental data was made (38) f o r s h i f t experiments between
v a r i o u s media; r-RNA and DNA concentrations were s a t i s f a c t o r i l y
p r e d i c t e d by the model i f feedback c o n t r o l s were i n c l u d e d .
Another approach to p r e d i c t i n g the response of a " t y p i c a l "
c e l l to a s h i f t i n media has been suggested by Bremer and co-work-

5. SHULER AND DOM ACT Models of Cell Growth 99
ers (40, 41, 42)· They have made extensive measurements of RNA
metabolism and p r o t e i n s y n t h e s i s i n IS. c o l i and have c o r r e l a t e d
t h e i r r e s u l t s with phenomenological expressions. These expres-
sions coupled with s i m i l a r r e l a t i o n s h i p s suggested by other
workers (e.g. Cooper and Helmstetter (43)) allow r a t h e r accurate
c a l c u l a t i o n of c e l l s i z e and composition f o r u n r e s t r i c t e d growth
i n media of v a r i o u s compositions. These expressions appear to be
u n s a t i s f a c t o r y f o r growth r e s t r i c t e d by a l i m i t i n g n u t r i e n t as
glucose (41). The above expressions do not e x p l i c i t l y depend on
the e x t e r n a l environment; the growth r a t e which a media w i l l
support must be known beforehand. These expressions are r e a l l y
c o r r e l a t i o n s r a t h e r than a t r u e model. They are l i m i t e d to a
narrow range of growth c o n d i t i o n s and are not p o t e n t i a l l y as
general as the other models d i s c u s s e d .
Another type of s i n g l e - c e l
Nishimura and B a i l e y (24)
c e l l mass and DNA content are e x p l i c i t l y recognized. Rules con
cerning DNA r e p l i c a t i o n and the timing of i n i t i a t i o n DNA s y n t h e s i s
are imposed on the c e l l ; the r u l e s are d e r i v e d from the observa-
t i o n s of Cooper and Helmstetter (43) and Donachie (44). The
s i n g l e - c e l l model was e s s e n t i a l l y the b a s i s f o r the c o n s t r u c t i o n
of a p o p u l a t i o n model - a formulation among the most general
c u r r e n t l y a v a i l a b l e . The main l i m i t a t i o n of the model i s i t s
i n a b i l i t y to permit an e x p l i c i t c a l c u l a t i o n of each c e l l ' s growth
r a t e i n terms of the e x t e r n a l environment and the c e l l ' s p h y s i o -
logical state.
Ho and Shuler (45) proposed a mathematical model f o r the
growth of an i n d i v i d u a l bacterium i n c o r p o r a t i n g feedback c o n t r o l
of n u t r i e n t uptake. T h i s simple model could p r e d i c t the growth
p a t t e r n f o r a c e l l of a given shape (filamentous, b a c i l l u s , or
s p h e r i c a l ) . The model c e l l contained four components (ammonium
ion, glucose, p r e c u r s o r s , and macromolecules). An a n a l y t i c a l
s o l u t i o n was p o s s i b l e f o r filamentous c e l l s , but numerical s o l u -
t i o n s were r e q u i r e d f o r other c e l l shapes.t
More r e c e n t l y Shuler, Leung, and Dick (46) have presented a
more complete model f o r the growth of a s i n g l e - c e l l of E s c h e r i c h i a
c o l i B/r A. The model contained 14 components. A l l of the c e l l ' s
components were included i n one of the model components. The
model c e l l r e l i e d on chemical concentrations as c o n t r o l s i g n a l s so
that the growth p a t t e r n , timing of DNA s y n t h e s i s , c e l l shape, c e l l
s i z e , c e l l composition, and c e l l d i v i s i o n could be considered
n a t u r a l responses to e x p l i c i t changes (e.g. glucose concentration)
i n the e x t e r n a l environment. An attempt was made to evaluate
k i n e t i c parameters from independent measurements on e x p o n e n t i a l l y
growing c e l l s . Four parameters, having to do with c e l l envelope
tThe example c i t e d i n Equation B9-B14 of Ho and Shuler (45) i s

f a u l t y . However, i t i s easy to demonstrate that the c o n c l u s i o n i s
c o r r e c t . D e t a i l s of the c o r r e c t e d example are a v a i l a b l e upon
request.

and c r o s s - w a l l formation were taken as a d j u s t a b l e . T h i s prototype

model i s being f u r t h e r developed.
The C o r n e l l S i n g l e - C e l l Model
The prototype model has been revamped. The base model has
20 components; the a d d i t i o n a l components are necessary to allow
an accurate d e s c r i p t i o n of the systems a l l o w i n g the i n c o r p o r a t i o n
of ammonium i o n i n t o amino a c i d s , to allow more accurate estimates
of c e l l u l a r energy expenditures, and to allow a more complete
s i m u l a t i o n of systems c o n t r o l l i n g t r a n s c r i p t i o n and t r a n s l a t i o n .
Some of the parameters i n the prototype model (46) were c a l c u
l a t e d based on c e l l dimensions obtained f o r c e l l s f i x e d i n osmium
t e t r o x i d e ; these have been r e c a l c u l a t e d u s i n g s i z e parameters
obtained from g l u c o s e - l i m i t e d chemostat c u l t u r e with g l u t e r
aldehyde-fixed c e l l s .
F i g u r e 1 and Tables I to IV d e s c r i b e the current model.
J u s t i f i c a t i o n of parameter values have been given elsewhere (46-
50). Almost a l l parameters were estimated from independent
measurements on e x p o n e n t i a l l y growing c e l l s or c e l l - f r e e systems.
Values f o r η2 and η (parameters a s s o c i a t e d with the r a t i o of
3
envelope used f o r extension and c r o s s - w a l l formation) were r e

quired to be p o s i t i v e but were otherwise considered a d j u s t a b l e .
Values f o r and K p ^ were adjusted w i t h i n the range of 0 to
A2
0.05 gm/cc of c e l l volume. Values f o r η 2 , r\

39 , and K
p | > A 2
were estimated by comparing data to model p r e d i c t i o n s at y =

1 1
0.95 hr and μ = 0.5 h r " . A parameter f o r u n i d e n t i f i e d energy
consumption, 6^ was i n c l u d e d to make the p r e d i c t e d growth y i e l d
9
1
at μ = 0.95 hr match experimental measurements. Values f o r
K P Z a n < K r e ( u i r e <
iIF* * * * ZMi» l * experimental growth data f o r μ <
1
0.95 hr but were evaluated independently of the model's p r e
d i c t i o n s . A l l parameters were set based on g l u c o s e - l i m i t e d
growth o n l y . No f u r t h e r adjustments were made f o r p r e d i c t i o n of
ammonium i o n - l i m i t e d growth.
The model makes reasonable p r e d i c t i o n s of the dependence of
c e l l s i z e , c e l l shape, c e l l composition, growth r a t e , and the
timing of c e l l u l a r events on e x t e r n a l concentrations of glucose.
Results f o r g l u c o s e - l i m i t e d growth are given elsewhere (47, 50).
The c u r r e n t model makes use of recent observations of
F r a l i c k (51), Messer, et a l . (52) , and Fayet & Louarn (53) to
construct a mechanism f o r the c o n t r o l of the i n i t i a t i o n of DNA
s y n t h e s i s . The scheme i s i l l u s t r a t e d i n Figure 2. The dnaA gene
which i s l o c a t e d near the o r i g i n makes a gene product (RP) which
represses the t r a n s c r i p t i o n of the 0-RNA gene. I n i t i a t i o n r e
q u i r e s 0-RNA as a primer. An a n t i - r e p r e s s o r (ARP) i s made which
i n a c t i v a t e s RP. Experimental evidence f o r ARP e x i s t s (51), and
there are i n d i c a t i o n s that ARP production i s r e l a t e d to c e l l
envelope formation (54). In the model we have made the r a t e of

Figure 1. An idealized sketch of the model for E . coli B/r A growing in a glucose-ammonium
salts medium with glucose or ammonia as the limiting nutrient. A t the time shown the cell has just
completed a round of DNA replication and initiated cross-wall formation and a new round of DNA
replication. Solid lines indicate the flow of material, while dashed lines indicate flow of information.
At = ammonium ion M , = DNA
At = glucose (and associated compounds in the Mi = non-protein part of cell envelope (assume
cell) 16.7% peptidoglycan, 47.6% lipid, and
W = waste products (CO H O, and acetate)
it t 35.7% polysaccharide)
formed from energy metabolism during Ms = glycogen
aerobic growth PG = ppGpp

P, = amino acids E, = enzymes in the conversion of P, to P t
P, = ribonucleotides Et, Es = molecules involved in directing cross-wall
P, = deoxy ribonucleotides
formation and cell envelope synthesis—the
Pt = cell envelope precursors

Mi = protein (both cytoplasmic and envelope) approach used in the prototype model was*
M = immature "stable" RNA used here but more recent experimental sup-
tRTI
M tRTM= mature "stable" RNA (r-RNA and t-RNA— port is available

assume 85% r-RNA throughout) G L N = glutamine
Ms t r messenger RNA
M
Ei = glutamine synthetase
*—the material is present in the external environment.
TABLE I : S t o i c h i o m e t r i c r e l a t i o n s f o r the lumped energy, mass,

and reductant consumptive processes represented i n the
c e l l model.
The f o l l o w i n g symbols are used:

a,3 = a n a b o l i c use of ammonium i o n or glucose,
respectively
γ = conversion of precursor i n t o macromolecule
ε = conversion of one precursor i n t o another
ω = d i r e c t use of reducing e q u i v a l e n t s f o r
e n e r g e t i c s or f o r b i o s y n t h e s i s
δ = A T P r e c u i r e m e n t s t o
ΑΤΡ l d r i v e given r e a c t i o n
X = t o t a l p o o l of reducing power
The r e s t of the symbols are d e f i n e d i n Figure 1.
Transport *Couple
(1) Αχ* t r a n s p o r t s i n & becomes Αχ ω. Αι + ω ^ 0 2 •> ω
χ
Η2Ο Α Α
Αι Αχ Α χ
(2) A 2 * t r a n s p o r t s i n & becomes A 2 6 ΑΤΡ -* δ (ADP + Ρ.)

Α2 Α2
Α Α
i
Precursor Formation
(3) otiAi + 6 ι Α + ... •+ P i
2
+ ... δ ΑΤΡ
ρ ι
-V δ (ΑΟΡ
ρι
+
(4) ε Ρ ι + 32A2 + . . .
2 •> P 2 + ... δ Α Τ Ρ -> δ
ρ 2 ρ (ADP + ν
(5) ε Ρ2 + ω
3 Ό
r
Χ + ... + Ρ 3 + .. . δ Α Τ Ρ -> δ ( Α 0 Ρ
ρ 3 ρ 3 +
3
(6) ει»Ρι + $ι*Α + ... -> Ρι» 2 + ... δ ρΐ( ΑΤΡ -> δ ρ (ADP + p
i>
Macromolecule Formation
->
(7) ΎιΡι
J
Μι + ... V ATP
y (ADp +
(8) Ύ2?2 £ Μ 2 + ... δ Μ ΑΤΡ ->-
V ( A D P
+
(9) Ύ3Ρ3 J Μ 3 + ... ΑΤΡ -+ δ (ADP + p
i>
Μ Μ3
3
(10) Ύ^Ρ* δ Τ Ρ
-> δ (ADP + p
i>
Μ/ Mit
(11) γ 5A2 J Μ + ... r (ADP +

->
V
5 p
δ
Μ 5
Α Τ Ρ
i>
A d d i t i o n a l D i r e c t Reductant Use
Transport of ions + ω W H 2
W ΐ Ο Ν *>* - I0N °
+
Membrane recharge to o f f s e t H
leakage

Table I , continued.
Additional Biosyntheti
=
SO" -> S ω 8 0 ι + Χ + SO -> [ S ]
Mass use ω Β Ι ( ) Χ + M J * - M^* D
A d d i t i o n a l ATP Coupling
PG formation ό ATP -> 6 „ ( A D P + P.)
PG PG i
Non-Specified ATP Use

Size linked oyATP -* δ (ΑΟΡ + P )
γ ±
*
Coupled to e i t h e r phosphate bond energy or o x i d a t i v e r e a c t i o n s .
Products of o x i d a t i o n i n c l u d e pmf and reducing e q u i v a l e n t s .
**
M = u n r e c u c e ( a n c
0X* **RED * l * reduced c e l l mass, r e s p e c t i v e l y .

TABLE I I : Equations D e s c r i b i n g the Rate of Change of Each Compo

nent i n the C e l l .
The symbols are the same as l i s t e d i n Figure 1 and Table I .

A d d i t i o n a l symbols used a r e :
μ, k, η = maximum r a t e s of synthesis of the macromolecules, p r e
c u r s o r s , and enzymes, r e s p e c t i v e l y
V = maximum transport r a t e
Κ = s a t u r a t i o n constants
k
= decomposition r a t e s
i
R = r a t e of transport
F = number of f o r k s i n DNA molecule
Ν = number of DNA o r i g i n s
ο
S c e l l u l a r surfac
V = volume
RI = mass of i d l i n g ribosomes
7 moles acetate formed
L moles glucose d i s s i m i l a t e d
GD = number of genes coding f o r s t a b l e RNA synthesis
P = density
f = p r o p o r t i o n a l i t y constant between Mi* and c e l l surface
area
SL = septum length
CL = length of c y l i n d r i c s e c t i o n of the c e l l
W = c e l l width
SEPF = mass of septum
IF = i n i t i a t i o n f a c t o r s f o r p r o t e i n synthesis
RP = repressor p r o t e i n
ARP = a n t i - r e p r e s s o r p r o t e i n
(P/0) = maximum P/0 r a t i o
max
When S and D are used as s u b s c r i p t s , they denote r a t e of

synthesis and r a t e of degradation r e s p e c t i v e l y . A and C as sub
s c r i p t s r e f e r to anabolism and catabolism.
d P l d P l
dAi _ c r/ \ / N ι ,dGLN N / 1 Λ
it " V s
- α ι [ (
ΐΓ> 5
(
- -dT> D
] a (
- i,GLN ^r> s
( 1 )
NET
,dM . 5
(2)

Table I I , continued.
.dATP ,dP!
K ;
dt
rf d M 2
s
ν
i RTI
dt
[1 »
,dPG
l d t >
s
(3)
GLN ^ t -
g +
W~dt> s
s + < ω + ω
f - "a!'V ΐΟΝ Ο
dP 3
δ + ω (4)
ν Ρ <1ΠΓ>
3
(dATP/dt). + dX/dt(P/0)
(dA /dt),2
S max •180 (5)
4 + (12 - 4·Ζ)(Ρ/0)
max
/ (dMWdt) s
Ζ =μ (6)
^<d*/dt) g + K z M i >
dA 2
(7)
A L»
GLN/V A /V
2
(8a)
+ A l / V
SxGUI + G L N / V
«ΡχΑ,
dt " "A 2
Ai/V Pi/V GLN

. W + A l / V
W + P l / V
J L K G L N + (GLN/V)^
(8b)
,dGLN.
s
( d t )
ι · r.
Term i s equivalent t o glutamine d r i v e n Glutamate Synthase

N E T
dp
* Term i s equal to net Ρ χ formation, ("^~") » where the f i r s t
term r e f e r s to Αχ i n c o r p o r a t i o n v i a glutamate dehydrogenase,

( d t
V
** T h i s term i s equivalent t o : e [ ( ^ f ) 2 - 1
S D
ει
t T h i s term i s equivalent t o : *(~ΠΓ*)
S

Table II, continued. y
dMi
(12)
dM 2
RTI P /V iPG
= P2
2
dt P + PG/
^ P ^ * ^ 1 P G 7
\LIF
•GD -
rr dMi/dtv Mi l S ι
K + [ (
iIF Μ
rdM 2
RTI RTM
k
dt (13)
TM 2 (dMi/dt) s | M 2
RTI
RTI1
RTI
dM 2
RTM RTM
(14)
dt RTM <RTI T M 2 R U + A 2 / V
RTM
dM 2
"*M /dM \ . X
(15)
ίγ ' μ 2
Μ ^ - ν δ Μ * Μ 2
Μ
dM 3
= „ / Pa/V V A 2 / V
(16)
dt
(17)
dM 5
(18)
dt
(19)

Table II, continued.

dPG * ( K i P
> }
RT
R I
-k k — ± l £ L — •PG (20)
dt 7V| TPG A /V 2 +
C
A* __Aj7y__ A /V 2
Αχ Ai K. . + A /V 2
K
A L
+ C
A J ' K
A X + A L / V AiA 2
Κ
Ai A /V2 ^
V +
(21)
^K c
Ul iAiK + A i / v
A A K
I A 2
+
A2/V
J
/ c* 2 \/ K i A 2 \
(22)
*A 2
dEi dMi
niN (23)
dt ο dt
dE dMi
2
n (24)
dt 2
dt
dE 2
TI3M1
dt (25)
dEij_
ni»
dt (26)
Μι»
V =
(27)
cyto
S = f ΜΗ and dS = f dMt» (28)

s s
2
S • nW + πΝ·α,+ 2πW·SL (29)
V ÏÏW »CL + h irW2 * S L - - | T T - S L

2
= χ 3
+ πΝ (30)
SEPF
SL (31)
2ÏÏW
SEPFι = SEPF, + _ E _ - dS 3
(32)
|tH-At 11 E + E 2 3
(33)

Table I I , continued.
/dARP\ „ k .(W| (34a)
At (34b)
ÎRP
RP (35)
burst
/dM l / d t\ («i/^) "
8
K +
iRP
RP„ . « RP + RP, (36)

Total burst

TABLE I I I : Values of Parameters A s s o c i a t e d with Rate Equations
ammonium i o n gm Κ, = 2.3 χ 1 θ " ^ 3

v A = 9.5 χ 10 , A
Αχ h r cm iAi cc
6
K * = 3.6 χ 10"
Αχ cc
2.056 χ 1 0 - 7
JES- κ - 1.0 χ 10~ 5
Β»
ce h r Αι ce
Κ Α
1
= 1.0 χ Ι Ο " 7
&* Κ Α = 1.0 χ ΙΟ" 5
Β&.
Αι ce Αχ ce
glucose ο = 1.7 χ ΙΟ" . 8* 5

Κ * = 1.2 χ ΙΟ" 5 5 5 1
Α2
κ 2
hr cm Α ce
~iA 2
cc'
gms
amino a c i d s k i = 0.390
hr cc *P I A i -· *2 5 1 0 - 5
S
IL, = 5.0 χ ΙΟ" -SB 2
Τι ce
-5 gm
= 0.025 h r " 1
= 1.1 χ 10
^Pi
k i = 475 hr»gm Et» GLNAi 3. 6 x 1 0 -6 2»

cc
K 9.9 χ ÎO"*
GLNPi " ' " cc
β
q 3
k '' β 0.39 J = S - 3 3 1 0 1 2
hr cc ^GLN = · * "
m
ribonucleotides k 2 - 0.19 , S
hr cc ce
3
K- = 9.0 χ 10" S E K
= 2.5 χ Î O " -
«
r 2 CC P A 2 2 c c
V = 0-03 h r " 1
^ f - l . l x l O - « a
CC
deoxyribonu- k 3 = 40 - — ^ IL, „ = 9.9 x 10-" M

h r g m E l
t3p 2 cc
cleotides
IL, - 2.2 χ 10~ SS. %
7 2 1 0 5
P3 ce *P A 3 2 ' ' * ' f!

Table III, continued.
c e l l envelope ki, = 0.060 — 2 2 — κ = 9.9 χ 10~* ^
h r c c P l c c
precursors ^"
5 x10-3 10
ν •·° S V A - ^ * " I?2
VA, * 7
· 2 x 1 0
" 5
f? NM 1 ° ° · 0 2 5 H R _ 1
K 1 x 1 0 - 5 = 0 2 5h r _ 1
V • f! ν °·
"te "
3
K i p G - 2.0 χ 1 0 - i ^ K i I F = 0.009 ( ^ )
1 1
μ 2 = 14 h r " k =0.07 h r "
RTM ™2 R T M
τ> ·» Λ οο gm RNA
Ρ 3 μ 2 3 2
" Μ " °· gmProtein
5 1
Κ™ - 1.1 χ ΙΟ" « k = 21.0 h r "
2 C C 2
™ RTM ™ M
1S 8 6
DNA u = 6.0 x 10" . S ? .
3 1C, _ = 4.0 χ 10" f §
hr fork M P 3 3 CC
s
IL, . = 2.0 x 10"
M A 3 2 CC
c e l l envelope μ„ - 100.0 h r g = 5.0 x 10"* f f
VA, " · 1 8 x 10
~* f ? Si, " °' 2 3h r _ 1
1 8X 1 0
W a = · "S
2 Uh r _ 1
glycogen μ - 2.0 χ 10"
5 ^TMs " ° '
2 x10 3
VA. =·° " S ^ 1
- -° * ' f ! l 0 3

Table I I I , continued* 3
enzymes = 1.0 χ 10 Π ι η 2 == 3Q. 2ο χ 1iθn" 3 v
2 1
η13 - 1.6 χ 10" h r "
3
2
10
IL, = 2 . 0 χ ΙΟ" (SE)' n i f = 0.01
£ι|» ce
β 2 k 0 5 h r X
* TE, - ° · '
P p G - 6 . 4 χ ΙΟ" - J g L - 3
k ± p i - 1 2 . 7 χ ΙΟ"» £
k 1 2 5 h r X k 7 2 X 1 0 5
TPG « ' TPGA = · 2 ' S
acetate u = 0.9 Κ 1.0 ΙΟ" * 1

^
ζ ζΜι
ρ
oxidation —. =1.5
0
'max
6
c e l l shape ρ = 0 . 2 5 8 *E* f = 259 χ 1 0 —
cyto ce s gm
ο = 0.553 BS.
env cc
3
DNA i n i t i a t i o n k = 2.6 χ 1 θ " - ^ r - IT = 0.22 - S * —
ARP gm Mi* iRP cc-hr
3 6 5 x 1 0 - 1 7
*** · · S r
Stoichiometric Coefficients:
«i - 0.179 ε 2 =1.149 γ χ - 1.167

α l.GLN =
°· 1 2 2 2 Ε
3 - 1·049 γ 2 = 1.057
β ι = 1 > 1 2 8 ε„ = 0.128 γ 3 = 1.053
1 1 0
Β 2 = -0.456 ° ·
βι, - 1.28 Υ5 = 1.11

Table I I I , continued*
2 - 1
glycogen y = 2.0 χ 1 0 "
5 = .14 h r
0
5
K
M A 5 2
= 2
-° x 1 0
" f 3 K
T M « 1-0 x 10"
5
3
g
3 3
enzymes n i = 1.0 χ 1 0 " n = 3.2 χ 1 0 "
2
2 1
n = 1.6 x 1 0 " h r "
3
Κ Ει> = 2.0 χ ΙΟ"» φ " „„ . 0 > 0 1
%= 2 1
k TEit = 0.05 h r '
PpGpp P p G = 6.4 χ 1 0 " H ? -gl r a o f K i P i - 12.7 χ 10"» j g
k T p G = 125hr- 1
K TpGft2 - 7.2 χ 1(Γ« g
acetate P = 0.9 Z = 1.0 χ 1 0 " 3 2 l k
oxidation Pi
^| =1.5
I
max
6
c e l l shape . = 0.258 = 5 9 χ 10 ^
P /
cyto ce f
s 2
gm
env = 0.553 c3 c
P p n v 2i
DNA i n i t i a t i o n k f t R p - 2.6 χ 10"· ^ K ( R p = 0.22 j ^ r
' . • ' • « • " ' " i Î

0
Stoichiometric Coefficients:
oi = 0.179 e = 1.149
2 Y i = 1.167
a = 1 2 2 2 Ε = Ί Μ 9
l 6LN ° · 3 Ύ2 = 1-057
β ' = 1 > 1 2 8 e« = 0.128 γ = 1.053

3
Ύ = 1 Λ 0
β 2 = -0.456 "
3H. 1.28 Ύ5 = 1 . U

TABLE IV: Values of Parameters f o r C a l c u l a t i o n of Energy Require

ments
Mole ATP Mole 'H^

<5_, — ω,
Compound or Process i gm i gm
A , NH^+,
x transport 0.028
A2, glucose transport 0.0056

+P ,
X Amino Acids 0.0025
P, 2 Ribonucleotides 0.022
P, 3 Deoxyribonucleotides 0.0031
P^, Non-Protein Envelo
M, x protein 0.03
M,
2 RNA 0.0067
M, 3 DNA 0.0071
M^, Non-Protein Envelope 0.0081
M, 5 Glycogen 0.0124
3
2 χ 10" mole
Ion Transport
gm c e l l
5.8 χ 10"* moles

Membrane Energy Recharge
2
cm -hr
3 moles
PG Formation
mole
0.0633 moles
Uncoupled ATP Use
3
cm
3
1.21 χ 1Q- mole
SO^ S
gm
2
1.39 χ I P " mole
Biosynthesis Reduction
gm
+Glutamate dehydrogenase pathway f o r carbon and energy limited

conditions.
For glutamine synthetase, assume 1 mole ATP i s used f o r every
mole glutamine formed which r e s u l t s i n a value of 0.007 f o r δ
GLN
i n Equation 3, Table I I .

5. SHULER AND DOMACH Models of Cell Growth
Figure 2. A potential scheme for the

control of initiation of DNA synthesis.
The dnaA gene product, RP, is a repres-
sor protein. If a molecule of repressor
protein combines with anti-repressor
protein, ARP, it is inactivated. Active RP
prevents the transcription of O-RNA,
which is required for initiation.

ARP formation to be a constant f r a c t i o n of the r a t e of c e l l enve

lope formation. When the l e v e l of unbound RP approaches zero,
0-RNA t r a n s c r i p t i o n i s allowed. The combining of RP with ARP i s
s i m i l a r to a mechanism proposed by Fantes, et a l . (55). The
model mechanism r e q u i r e s that the dnaA gene be t r a n s c r i b e d only
for a short p e r i o d of time so as to o b t a i n a " b u r s t " . P r i t c h a r d ,
Barth, & C o l l i n s (56) have p r e v i o u s l y p o s t u l a t e d the production
of an i n i t i a t i o n - i n h i b i t o r as a " b u r s t " by a gene l o c a t e d near
the o r i g i n .
The p l a u s i b i l i t y of the above mechanism was e s t a b l i s h e d by
comparing to data the model's p r e d i c t i o n s of the length of the C
p e r i o d (the time to r e p l i c a t e a whole new DNA molecule) and D
p e r i o d (the time from the termination of the chromosome s y n t h e s i s
to the next c e l l d i v i s i o n ) and the timing of i n i t i a t i o n . The
r e s u l t s are given i n Table V d VI Othe mechanism (e.g
p o s i t i v e c o n t r o l due t
of c e l l envelope s y n t h e s i s
to the t o t a l c e l l mass) have been shown to be i m p l a u s i b l e when
tested f o r growth under glucose l i m i t a t i o n (46). The form of the
equations f o r the g l u c o s e - l i m i t e d c e l l have been j u s t i f i e d (46,
47, 50). For the case of ammonium i o n l i m i t a t i o n the equation
for ammonium i o n transport (Equation 21) can be j u s t i f i e d based
on the observations of Stevenson and S i l v e r (57). They presented
evidence f o r a d u a l - t r a n s p o r t system i n 12. c o l i . The scheme f o r
i n c o r p o r a t i o n of ammonium i o n i n t o c e l l u l a r metabolites can be
described by the f o l l o w i n g equations:
(1) L-glutamate + ATP + NHi/ + L-glutamine + ADP + Ρ
+
(2) α-Ketoglutarate + L-glutamine + NADPH -> 2 glutamate + NADP
+ +
(3) α-Ketoglutarate + NHi» + NADPH -> L-glutamate + H 0 2 + NADP
Reaction 1 i s mediated by glutamine synthetase while Reactions 2

and 3 are c a t a l y z e d by glutamate synthase and glutamate dehydro
genase r e s p e c t i v e l y .
In Table I I the s e r i e s process described by Reactions (1) and
(2) are represented by Equations (8a) and (8b) and Reaction (3) by
the f i r s t part of Equation (8e).
Glutamate i s considered a key compound because i t represents
the f i r s t major transformation of inorganic-N to organic; hence,
n i t r o g e n f l u x i n t o the amino a c i d pool cannot occur any f a s t e r
than glutamate-N production. T h i s f o l l o w s from glutamate's high
a c t i v i t y with transaminases.
(4) L-glutamate + RC0C00H + RCHNH C00H + α-Ketoglutarate

2
Presumably two p o t e n t i a l routes of ammonia i n c o r p o r a t i o n

e x i s t (58, 59). The high a f f i n i t y route c o n s i s t s of glutamine
synthetase a c t i v i t y working i n conjunction with glutamate

TABLE V: Comparison of Model and Experimental Estimates of C&D

Periods
y C* _ D* ^
Snodel ^model expt. expt
1
hr" min. min. min. min.
0.95 41 21 42 22
0.68 47 28 42 22
0.51 59 35 56 28
0.39 75 42 73 39
0.29 99
0.24 120 60 ^115 -57
0.16 189 81 -178 -89
Values from Helmstetter & P i e r u c c i (71) . No measurements were

1 1
made f o r μ < 0.35 h r " . Values f o r μ < 0.35 hr were estimated
from the suggestion (26) that C = 2/3 τ and D = 1/3 τ.
TABLE VI: Comparison of Model and Experimental Estimates of the

Time of I n i t i a t i o n of Chromosome Synthesis
t/τ *
t / T
Model expt.
1
hr"
0.95 0.59 0.58 ± 0.1

0.68 0.78 0.91 ± 0.1
0.51 0.86 0.96 ± 0.1
0.39 0.90 0.98 ± 0.1
0.29 0.94 0.99 ± 0.1
0.24 0.95 0.99 ± 0.1
0.16 0.99 1.0 ± 0.1
From Helmstetter & P i e r u c c i (71). Note that t/τ = 0 = 1.0 or

i n i t i a t i o n at c e l l d i v i s i o n i s e s s e n t i a l l y the same as i n i t i a t i o n
at c e l l b i r t h .

synthase* Glutamate dehydrogenase a c t i v i t y i s r e s p o n s i b l e f o r

the low a f f i n i t y system which predominates under c o n d i t i o n s of
high i n t r a c e l l u l a r ammonium i o n l e v e l s . There i s evidence (60,
61\ 62) that the high a f f i n i t y system i s subject to ammonium i o n
r e p r e s s i o n and modulation of i t s a c t i v i t y by a number of mechan
isms, i n c l u d i n g covalent m o d i f i c a t i o n s through a cascade enzyme
system. In c o n t r a s t the low a f f i n i t y system i s modulated only by
l a r g e concentrations of glutamate (61, 63). Data to support t h i s
scheme has been obtained p r i m a r i l y with c e l l - f r e e systems.
Whether t h i s scheme can f u n c t i o n i n a complete organism can be
v e r i f i e d by comparing the computer p r e d i c t i o n s with data.
In Figure 3 the model's p r e d i c t i o n s of growth r a t e dependence
on ammonium i o n c o n c e n t r a t i o n i s compared with chemostat data.
Generally the model's p r e d i c t i o n s are q u i t e reasonable. The
model, however, suggest that th changeove fro th lo t h i g h
a f f i n i t y r e g i o n occur a
break appears to occur y ζ
p r e d i c t s a lower e f f e c t i v e s a t u r a t i o n constant than i n d i c a t e d by
the data. Nonetheless, the general c h a r a c t e r of the c e l l r e
sponse i s w e l l captured by the model.
In F i g u r e 4 the model's p r e d i c t i o n s of c e l l s i z e f o r both
glucose- and ammonium-limited growth are compared, to chemostat
data. In both cases the model's p r e d i c t i o n s are i n c l o s e accord
with the observed responses. The reason the n i t r o g e n - l i m i t e d
c e l l s are l a r g e r than the g l u c o s e - l i m i t e d c e l l s i s probably due
to increased storage of carbon as glycogen. The model's p r e
d i c t i o n s of glycogen content of ammonium-limited c e l l s i s com
pared to the data of Holme (65) i n F i g u r e 5.
The p r e d i c t i o n s of the model f o r n i t r o g e n - l i m i t e d growth are
i n remarkable agreement with experiment when c o n s i d e r i n g that no
new a d j u s t a b l e parameters were introduced to d e s c r i b e n i t r o g e n -
l i m i t e d growth. A l l parameter values were set with respect to
g l u c o s e - l i m i t e d growth. The mechanisms p o s t u l a t e d on the b a s i s
of i n v i t r o enzymology appear to be q u i t e p l a u s i b l e upon i n t e
g r a t i o n i n t o the whole c e l l .
T r a n s i e n t Response of S i n g l e - C e l l Models. With the base

model w e l l - e s t a b l i s h e d by comparison to steady-state data, t r a n
s i e n t response p r e d i c t i o n s can be made with some minimum degree
of confidence. The h i g h l y s t r u c t u r e d nature of the C o r n e l l
s i n g l e - c e l l model should endow the s i n g l e - c e l l model with the
p o t e n t i a l to a c c u r a t e l y p r e d i c t dynamic i n t e r n a l changes i n com
p o s i t i o n . A computer simulated experiment i s shown i n F i g u r e 6
to t e s t t r a n s i e n t changes i n growth r a t e to a step change i n
glucose c o n c e n t r a t i o n . In the experiment the glucose concentra
t i o n i s _ a l t e r e d suddenly from a high value p e r m i t t i n g y =
1
0.95 hr at steady-state to a glucose c o n c e n t r a t i o n p e r m i t t i n g
1
growth at y = 0.50 h r " at s t e a d y - s t a t e . The change i n c e l l -
growth r a t e drops d r a m a t i c a l l y and o s c i l l a t e s s l i g h t l y . E i g h t
c e l l generations are r e q u i r e d before the new steady-state growth

SHULER AND DOMACH Models of Cell Growth 119
0.4 0.6 0.8 1.0 .8

l/S (mg Ν / J ) - 1
Figure 3. The model's prediction of growth-rate (solid line) dependence on

ammonium-ion concentration is compared to experimental data. The data and the
model predictions indicate that more than a single system is responsible for
ammonium-ion uptake, assimilation, or both.

0.8 h
Figure 4. Variation of cell volume with growth rate. For glucose-limited cells:
O, data from Cornell; data from Ref. 64. For ammonium-limited cells: Δ,
data from Cornell. Solid line, the model's prediction for glucose-limited cells;
dashed line, model's prediction for ammonium-limited cells.

Figure 5. Model's predictions of the effects of ammonia limitation on glycogen

content. Data points from Ref. 65.

l.2r
1.0
UJ
0.8
<
tr
UJ 0.6
u 0.4
0.2
0.0
4 6 8 10 12
GENERATIONS
Figure 6. Transient responses of the computer cell: O, response to step-decrease

in external glucose concentration (1000 mg/L to 4.5 mg/L); Δ, response to a
step-increase (4.5 mg/L to 1000 mg/L). Relative rate corresponds to the average
growth rate during that cell generation.

r a t e i s approximated. I f the experiment i s reversed and glucose

i s suddenly changed from the lower to the higher v a l u e , the c e l l
responds by s t e a d i l y i n c r e a s i n g i t s growth r a t e . Again i t takes
about e i g h t c e l l generations to get a new s t e a d y - s t a t e . The
important f e a t u r e here i s that the c e l l returns to the o r i g i n a l
growth-rate v i a a d i f f e r e n t path. There i s s u b s t a n t i a l h y s t e r e -
s i s i n the computed p r e d i c t e d response to these p e r t u r b a t i o n s .
The explanation f o r the r a p i d response of the c e l l to a step
down i n energy a v a i l a b i l i t y i s shown i n Figure 7. The decrease
i n glucose causes a r a p i d i n c r e a s e i n ppGpp which d r a s t i c a l l y
reduces RNA s y n t h e s i s and consequently reduces other c e l l u l a r
processes such as p r o t e i n s y n t h e s i s . Such responses have been
observed experimentally (66, 67).
N i t r o g e n - l i m i t e d c e l l s a l s o show h y s t e r e s i s when subjected
to an experiment analogou
The biggest d i f f e r e n c
sponse and overshoot during the step-up part of the experiment.
The overshoot i s probably due to the high l e v e l of both low and
high a f f i n i t y systems f o r c o n v e r t i n g ammonium i n t o amino a c i d s .
The growth r a t e of JE. c o l i i n glucose-minimal medium can be
enhanced by the a d d i t i o n of amino a c i d s . The l a r g e decrease i n
growth r a t e during step-down i s l i k e l y due to the i n a b i l i t y of
the low a f f i n i t y glutamate dehydrogenase system to produce
s u f f i c i e n t amino a c i d s by i t s e l f with low ammonium i o n concen-
t r a t i o n s and a moderately long l a g i n the production of the
enzymes f o r the high a f f i n i t y system.
Population Models From S i n g l e - C e l l Models
The s i n g l e - c e l l model i s l i m i t e d i n i t s u s e f u l n e s s f o r
engineering c a l c u l a t i o n s unless a p o p u l a t i o n model can be con-
s t r u c t e d from the information i n the s i n g l e - c e l l model. I t i s
p o s s i b l e to b u i l d a p o p u l a t i o n model u s i n g an ensemble of s i n g l e -
c e l l models to mimic the response of a l a r g e p o p u l a t i o n of c e l l s
(24, 46, 68). Since computer c a p a c i t y i s f i n i t e , the question i s
r e a l l y , "how few c e l l models can be included i n a p o p u l a t i o n and
s t i l l allow reasonable p r e d i c t i o n s of the behavior of a n a t u r a l
p o p u l a t i o n of c e l l s ? "
F i g u r e 9 presents a sketch of an " i d e a l " s i z e d i s t r i b u t i o n
and a " t y p i c a l " s i z e d i s t r i b u t i o n . The " i d e a l " d i s t r i b u t i o n
would occur i f there were no v a r i a t i o n producing processes w i t h i n
the c e l l ( i . e . a l l c e l l s have i d e n t i c a l c e l l c y c l e s ) . In the
i d e a l d i s t r i b u t i o n c e l l s a u t o m a t i c a l l y d i v i d e at twice the b i r t h
volume, and there are two times as many c e l l s of b i r t h s i z e as
d i v i s i o n s i z e . The frequency f u n c t i o n of c e l l s of intermediate
s i z e would decrease approximately e x p o n e n t i a l l y from b i r t h to
fission-ready c e l l s .
However, v a r i a t i o n causing processes e x i s t . H y p o t h e t i c a l
p r o b a b i l i t y f u n c t i o n s r e l a t i n g b i r t h and d i v i s i o n to c e l l s i z e
are shown. Such f u n c t i o n s w i l l r e s u l t i n a " t y p i c a l " s i z e

8r
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

TIME (hrs)
Figure 7. Changes in the relative magnitude of internal ppGpp concentration (Ci)

and in the rate of RNA synthesis (A) are given for the first division cycle after the
step-down in glucose concentration (see Figure 6). The decrease in energy-avail-
ability causes a rapid increase in ppGpp that significantly reduces RNA synthesis
and consequently, reduces cell metabolism and growth.

I4r
0.2l 1 1 1 1 L
0 2 4 6 8 10 12
GENERATIONS
Figure 8. Transient response of the computer cell to a step-change in external
ammonium-ion concentration: O, response to step-decrease (2.5 mg/L to 1.4
mg/L); Δ, response to step-increase (lΛ mg/L to 2.5 mg/L). The relative rate
corresponds to the average growth rate during that cell generation.
Figure 9. Sketches of ideal size distribution ( - ' - · - ) and typical size distribu
tion ( ). The typical distribution results from random processes that cause the
cell to have a significant probability of fission over a range of sizes.

d i s t r i b u t i o n p l o t which e x h i b i t s a s l i g h t p o s i t i v e skewing. For

each s i z e c l a s s i n the d i s t r i b u t i o n c e l l s can enter by growth or
b i r t h and may e x i t the s i z e c l a s s due to f u r t h e r growth or
division.
C o n s i d e r a t i o n of " i d e a l " and " t y p i c a l " s i z e d i s t r i b u t i o n s
suggests c e r t a i n c h a r a c t e r i s t i c s r e q u i r e d to simulate a l a r g e
p o p u l a t i o n with a f i n i t e number of c e l l s . The ensemble to be
constructed w i l l have some number of d i s c r e e t s i z e c l a s s e s and
some other number of c e l l models w i t h i n each s i z e c l a s s . Require
ments f o r an accurate s i m u l a t i o n are:
1. S u f f i c i e n t number of s i z e c l a s s e s so that growth through
the i n t e r v a l s i s smooth. I f the c e l l s are spaced f u r t h e r apart
than the mesh s i z e of the s i z e analyzer i n use, then gaps i n the
frequency curve w i l l p e r i o d i c a l l y occur.
2. Mechanisms causin
identified.
3. Once the v a r i a t i o n causing processes are q u a n t i f i e d ,
then s u f f i c i e n t c e l l s must be put i n t o each s i z e c l a s s to
a c c u r a t e l y mimic the random processes w i t h i n each s i z e c l a s s ( i . e .
the sample must be large enough that the d i s t r i b u t i o n of prop
e r t i e s can reproduce i t s e l f f o r the case of s t e a d y - n u t r i e n t
supply).
F a c t o r s that could c o n t r i b u t e to v a r i a b i l i t y w i t h i n the
c e l l can e a s i l y be imagined. Any time a small number of copies
e x i s t the process d e s c r i p t i o n becomes l e s s d e t e r m i n i s t i c and more
stochastic. Some p o s s i b l e v a r i a t i o n producing processes are:
1. RP " b u r s t " s i z e
2. RP p a r t i t i o n upon f i s s i o n
3. ARP v a r i a t i o n
4. R e p l i c a t i o n v e l o c i t y - p o s s i b l y r e l a t e d to the number of
unwinding enzymes
5. R a t i o of E2 to Ε3 and consequently r a t i o of c r o s s - w a l l
formation to c e l l extension
6. Asymmetric d i v i s i o n .
To more e f f i c i e n t l y t e s t ways of c o n s t r u c t i n g p o p u l a t i o n
models we have simulated the s i m u l a t i o n by using a number of
s t r u c t u r e l e s s c e l l s . The c e l l s were assigned a mode of c e l l i n
crease, and t h e i r v a r i a t i o n of f i s s i o n s i z e was accomplished by
s e l e c t i n g the next termination s i z e on a random b a s i s and s t o r i n g
it. The range and frequency of f i s s i o n s i z e was comparable to
observed patterns (e.g. 10% c o e f f i c i e n t of v a r i a t i o n and Gaussian
frequency d i s t r i b u t i o n (22, 69).
F i g u r e 10 shows the r e s u l t s of a s i m u l a t i o n with 200 c e l l s -
20 c e l l c l a s s e s and two c e l l s per c l a s s . A steady-state e n v i r o n
ment was imposed. Even a f t e r 30 generations the s i z e d i s t r i b u t i o n
exhibits significant i n s t a b i l i t i e s . By going to 2000 c e l l s -
20 c e l l c l a s s e s and 100 c e l l s per c l a s s (Figure 11) - the s t a b i l
i t y of the s i z e d i s t r i b u t i o n i s g r e a t l y improved. In the above
two cases equal c e l l s i z e s among daughters at d i v i s i o n was r e
q u i r e d . L i t t l e d i f f e r e n c e s i n d i s t r i b u t i o n s were observed when

Ο
c
q>
3
σ-
0.5 1.0 0.5 1.0

Size (/x.m )3
Size (/im )
3
ο
c
3
σ
0)
0.5 1.0 1.5 0.5 1.0

Size ( f t m )
3
Size l/xm ) 3
Figure 10. Computer simulation of a population using 200 unstructured cell

models. There were 20 size classes with 10 cells per class in the simulation. Even
at steady state the size distribution shows significant instabilities in shape.

>% >*
ο ο
c c
0) α>
3 3
σ
α) σ
α)
0.5 1.0 1.5 0.5 1.0 1.5

Size (/im ) Size (ftm )
3
Figure 11. Computer simulation of a population using 2000 unstructured cell

models. There were 20 size classes with 100 cells per class in the simulation. The
steady state size distributions are considerably more stable than the simulation with
200 cells (See Figure 10).

5. SHULER A N DDOMACH Models of Cell Growth 129
unequal f i s s i o n was allowed. The p r e c i s i o n of f i s s i o n i s h i g h

(70), so i t s i n c l u s i o n with r e a l i s t i c p r o b a b i l i t y values was not
expected t o g r e a t l y a l t e r the r e s u l t s . The bulk of v a r i a t i o n i n
b i r t h s i z e (> 75%) i s l i n k e d t o the v a r i a t i o n i n o v e r a l l f i s s i o n
s i z e and not the l o c a t i o n of the c r o s s - w a l l plane. Since the
assumption of equal f i s s i o n g r e a t l y s i m p l i f i e s programming and
reduces demands on computer memory, i t i s prudent t o assume equal
fission.
F i g u r e 12 d i s p l a y s the s t e a d y - s t a t e s t a b i l i t y of s i z e -
d i s t r i b u t i o n s generated with the s t r u c t u r e d s i n g l e - c e l l model.
Sixteen c e l l c l a s s e s with f i f t e e n c e l l s per c l a s s were used.
Random processes allowed were v a r i a t i o n i n " b u r s t " s i z e of RP
(20%) and i n r e p l i c a t i o n v e l o c i t y (10%). V a r i a t i o n with respect
to other c r i t e r i a are c u r r e n t l y being i n v e s t i g a t e d . The s t a b i l -
i t y d i s p l a y e d by the d i s t r i b u t i o n w i l l b acceptabl i t
potential applications
(e.g. RNA, DNA, p r o t e i n , ) generate popu
l a t i o n runs.
Summary
S i n g l e - c e l l models can be w r i t t e n that mimic very c l o s e l y

the responses of l i v i n g c e l l s . Such models are convenient t o o l s
f o r t e s t i n g the p l a u s i b i l i t y of b a s i c biochemical mechanisms.
They may be p a r t i c u l a r l y a t t r a c t i v e i n t e s t i n g the p o t e n t i a l i n
v i v o c o m p a t i b i l i t y of mechanisms p o s t u l a t e d on the b a s i s of i n
v i t r o enzymology. The s i n g l e - c e l l models make reasonable p r e -
d i c t i o n s during t r a n s i e n t c o n d i t i o n s and are n u m e r i c a l l y s t a b l e .
Since s t a b l e d i s t r i b u t i o n s of a p o p u l a t i o n can be constructed by
using an ensemble of s i n g l e - c e l l models, i t should be p o s s i b l e to
develop p o p u l a t i o n models with both high l e v e l s of segregation
and s t r u c t u r e d . Such models need t o be f u r t h e r developed and
t e s t e d f o r t h e i r a b i l i t y t o p r e d i c t the t r a n s i e n t behavior o f c e l l
populations to p e r t u r b a t i o n s i n the a b i o t i c environment.
Acknowledgment
T h i s work was supported, i n p a r t , by NSF Grant CPE-7921259.

φ
3
σ
α)
0.5 1.0 1.5 0.5 1.0 1.5

Size (/Ltm) 3
Size (/xm ) 3
0.5 1.03 0.5 1.0

Size (/im ) Size ζ
(μπ\ )
Figure 12. Computer simulation of a population using 240 structured cells (the
Cornell single-cell model). There were 16 size classes and 15 cells per class in the
simulation. The steady-state size distributions display a level of stability acceptable
for most applications.

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6
Single-Cell Metabolic Model Determination

by Analysis of Microbial Populations
JAMES E. BAILEY
California Institute of Technology, Department of Chemical Engineering,
Pasadena, CA 91125
The properties of microbial populations result from

the characteristic
the level of the
tions of population behavior may therefore be con-
structed based on understanding of single-cell
metabolism, and, conversely, studies of population
properties may be employed to extract information
about single-cell operation. Population balance
equations provide the primary mathematical tool for
these purposes, and flow cytometry allows experi-
mental access to the distribution of cell states in
the population. Application of these methods to bac-
teria and yeasts is illustrated using three example
systems.
The m i c r o b i a l populations propagated i n r e a c t o r s ranging from

P e t r i dishes to fermentors to n a t u r a l ecosystems are t y p i c a l l y
heterogeneous with respect to age, s i z e , and biochemical a c t i v i t y .
The o v e r a l l r a t e s of substrate u t i l i z a t i o n , b i o s y n t h e s i s and prod-
uct formation i n these r e a c t o r s a r e sums, taken over the c e l l pop-
u l a t i o n , o f the r a t e s o f the corresponding processes i n the i n d i -
v i d u a l c e l l s present (1,2). Viewed from t h i s p e r s p e c t i v e , o p t i m i z -
ing the performance o f a m i c r o b i a l process r e q u i r e s determination
of the c e l l s ' genotype and o f a r e a c t o r design such that the optim-
a l d i s t r i b u t i o n o f s t a t e s i s achieved i n the process's m i c r o b i a l
population.
Adoption o f t h i s approach to m i c r o b i a l process development
cannot occur u n t i l methods e x i s t f o r determining the i n f l u e n c e o f
r e a c t o r design and o p e r a t i n g parameters on s i n g l e - c e l l metabolic
c o n t r o l a c t i o n s and r e a c t i o n r a t e s . I f t h i s i n f o r m a t i o n i s a v a i l -
a b l e , p o p u l a t i o n balance equations and a s s o c i a t e d medium conserva-
t i o n equations provide the r e q u i r e d bases f o r r e a c t o r a n a l y s i s
(_1,2). For example, f o r a well-mixed, continuous-flow isothermal
m i c r o b i a l r e a c t o r a t s t e a d y - s t a t e , the p o p u l a t i o n balance equation
may be w r i t t e n :
0097-6156/83/0207-0135$07.50/0

^ [ r ( p , s ) f ( p , s ) ] = D[4<K2p,s)-<f>(p,s)-f(p,s)]
f (1.1)
f(0,s) = 0 (1.2)
where here i t has been assumed that the s t a t e o f a s i n g l e c e l l can

be c h a r a c t e r i z e d by a s i n g l e v a r i a b l e ρ (e.g., c e l l age, c e l l pro
t e i n content, o r c e l l volume) and that the e f f e c t o f the e n v i r o n
ment on c e l l k i n e t i c s and c o n t r o l s can be represented by a s i n g l e
environmental v a r i a b l e s (e.g., l i m i t i n g n u t r i e n t c o n c e n t r a t i o n ,
temperature). Even i n t h i s r e l a t i v e l y simple case, the above
equation shows the connection among r e a c t o r o p e r a t i n g c o n d i t i o n s ,
represented here by the d i l u t i o n r a t e D (= mean residence time-1),
s i n g l e - c e l l k i n e t i c s an
(<Kp,s)dp = the f r a c t i o
ρ and p+dp) and r ( p , s ) ( t h e time r a t e o f change o f ρ i n a c e l l
f
with a c e r t a i n p-value i n a c e r t a i n environment s ) , and the d i s

t r i b u t i o n o f s t a t e s i n the c e l l p o p u l a t i o n , here w r i t t e n as a f r e
quency f u n c t i o n f ( p , s ) f o r s i n g l e - c e l l p-values (f(p,s)dp = the
f r a c t i o n o f c e l l s i n the p o p u l a t i o n with p-values between ρ and
P+dp) .
The p o p u l a t i o n balance equation f o r the process has an ex
tremely important a l t e r n a t i v e a p p l i c a t i o n . I f the frequency func
t i o n o r subpopulation f r a c t i o n s evaluated using the frequency
f u n c t i o n can be measured experimentally, the p o p u l a t i o n balance
equations provide a l i n k between a l t e r n a t i v e models f o r r e g u l a t i o n
and s i n g l e - c e l l k i n e t i c s and parameter values employed i n those
models on the one hand, and experiment on the o t h e r . That i s , one
may c a l c u l a t e , u s i n g the p o p u l a t i o n balance equations, frequency
f u n c t i o n s corresponding to v a r i o u s s i n g l e c e l l models and then
compare the frequency f u n c t i o n s so c a l c u l a t e d with the experimental
ones. Through t h i s s o r t o f comparison, q u a l i t a t i v e f e a t u r e s o f
presumed s i n g l e c e l l models can be t e s t e d and, furthermore, param
e t e r s i n those models may be evaluated. Use o f t h i s s t r a t e g y w i l l
be i l l u s t r a t e d below i n s e v e r a l examples.
While c l e v e r experimental design may make i t p o s s i b l e to t e s t
p r e d i c t i o n s o f s i n g l e - c e l l models without recourse to measurements
of f , d i r e c t experimental access to the frequency f u n c t i o n i s o f t e n
d e s i r a b l e and sometimes necessary i n order to evaluate s i n g l e - c e l l
c o n t r o l and k i n e t i c p r o p e r t i e s using the s t r a t e g y j u s t o u t l i n e d .
A v a r i e t y o f methods a r e a v a i l a b l e f o r a c h i e v i n g frequency func
t i o n measurements. The p a i n s t a k i n g i n v e s t i g a t i o n s o f H e n r i c i (_3)
and l a t e r workers demonstrate the f e a s i b i l i t y o f measuring d i s t r i
butions o f c e l l s i z e s and shapes from microscopic o b s e r v a t i o n o f
c e l l p o p u l a t i o n s . T h i s approach i s u s u a l l y l i m i t e d by d i f f i c u l t i e s
i n o b t a i n i n g f i n e q u a n t i t a t i v e r e s o l u t i o n and i n examining a s u f
f i c i e n t l y l a r g e sample p o p u l a t i o n to o b t a i n good s t a t i s t i c s .

6. BAILEY Single-Cell Metabolic Model 137
The advent o f s i n g l e - c e l l measurements i n flowing c e l l sus-

pensions, f i r s t accomplished i n the C o u l t e r p a r t i c l e s i z i n g i n -
strument (4), has r e v o l u t i o n i z e d the experimental c h a r a c t e r i z a t i o n
of d i s t r i b u t i o n s o f s t a t e s i n c e l l and other p a r t i c u l a t e popula-
t i o n s . O p t i c a l techniques, i n c l u d i n g l i g h t s c a t t e r i n g , l i g h t ab-
s o r p t i o n , and fluorescence measurements from i n d i v i d u a l c e l l s i n
flow systems, have broadened the scope of c e l l u l a r parameters
which can be measured on an i n d i v i d u a l c e l l b a s i s i n a m i l l i -
second o r l e s s i n a flowing c e l l suspension ( 5 ) . As i l l u s t r a t e d
schematically i n Figure 1, such instrumentation allows r a p i d de-
termination, from a p r o p e r l y prepared and l a b e l e d sample o f the
c e l l population, o f an experimental approximation t o the frequency
f u n c t i o n f o r s i n g l e - c e l l content o f the l a b e l e d component. The
sample preparation process shown i n Figure 1 may be s i m p l i f i e d
g r e a t l y i n cases where l i g h t s c a t t e r i n g o r l i g h t absorption a r e
used to measure the c e l
now been a p p l i e d s u c c e s s f u l l
and f l u o r e s c e n t s t a i n s a r e a v a i l a b l e which can be used t o achieve
measurements o f s i n g l e - c e l l p r o t e i n , DNA, double-stranded n u c l e i c
a c i d content and v i a b i l i t y o f some s t r a i n s (e.g., 6-9).
A framework f o r formulating models o f s i n g l e - c e l l c o n t r o l s
and k i n e t i c s i s provided by c o n s i d e r a t i o n o f the c e l l c y c l e o f the
organism (10). The c y c l e o f chemical and morphological changes
which occurs as a daughter c e l l ages, grows, and u l t i m a t e l y d i v i d e s
to y i e l d new daughter c e l l s may be decomposed i n t o s e v e r a l i n t e r -
l o c k i n g c y c l e s , such as the c e l l d i v i s i o n c y c l e , which describes
the c y c l e o f growth and d i v i s i o n , and the n u c l e a r c y c l e , which con-
cerns the processes o f DNA synthesis and segregation o f DNA mole-
cules upon c e l l d i v i s i o n . Previous i n v e s t i g a t i o n s by g e n e t i c i s t s ,
m i c r o b i o l o g i s t s and biochemists provide guidance i n t o the b a s i c
features o f these c y c l e s and i n t o t h e i r i n t e r r e l a t i o n s h i p s and
coordination.
In the examples discussed below, c e l l c y c l e p r o p e r t i e s r e -
ported i n the b i o l o g i c a l sciences l i t e r a t u r e comprise the s t a r t i n g
point o f e i t h e r mathematical o r experimental analyses which r e l a t e
s i n g l e - c e l l operation with the r e s u l t i n g p r o p e r t i e s o f a growing
c e l l population. The t o o l s o f mathematical modeling o f c e l l popu-
l a t i o n s and o f flow cytometry allow p r e d i c t i o n o f m i c r o b i a l popula-
t i o n behavior under circumstances d i f f e r e n t from those used i n the
o r i g i n a l e x p o s i t i o n o f the c e l l c y c l e models. Consequently, such
simulations and experiments provide a means f o r e v a l u a t i n g the gen-
e r a l i t y o f the proposed c e l l c y c l e models and, i n some cases, f o r
q u a n t i t a t i v e determinations o f parameters o r k i n e t i c forms which
are part o f the c e l l c y c l e model. This i s an extremely important
aspect o f the s t u d i e s to be described here, s i n c e , given the d i v e r -
s i t y and complexity o f c o n t r o l systems i n microorganisms and other
c e l l s , the observation o f a p a r t i c u l a r phenotypic c o n t r o l behavior
i n one o r a s e t o f c e l l environments i n no way guarantees that the
same c o n t r o l s o r r e s u l t s w i l l occur when growth conditions are
changed. An area o f s p e c i a l i n t e r e s t here i s the a p p l i c a t i o n o f

c e l l c y c l e models based upon t r a n s i e n t , unbalanced growth e x p e r i -

ments to steady-state balanced growth s i t u a t i o n s and v i c e - v e r s a .
The value of combinations of steady-state and t r a n s i e n t experiments
i n formulating and t e s t i n g models of s i n g l e c e l l k i n e t i c s and con-
t r o l s w i l l be apparent i n the examples which f o l l o w .
Three example organisms are considered i n the f o l l o w i n g sec-
t i o n s . These have been chosen to i l l u s t r a t e a v a r i e t y of b i o l o g i -
c a l f e a t u r e s , mathematical modeling approaches and r e s u l t s , and
a l t e r n a t i v e experimental approaches and s t r a t e g i e s f o r i d e n t i f y i n g
and t e s t i n g models. Escherichia coli, the w i d e l y - s t u d i e d i n t e s -
t i n a l bacterium, i s examined f i r s t . This simple procaryote d i v i d e s
by binary f i s s i o n and e x h i b i t s a p a t t e r n of DNA synthesis during
the c e l l c y c l e which i s r a t h e r complex and q u i t e d i f f e r e n t from
that observed i n e u c a r y o t i c microorganisms. Furthermore, i t w i l l
be seen that, f o r t h i s microorganism accurate c a l c u l a t i o n of c e l l
numbers r e q u i r e s a t t e n t i o
c e l l and i t s c o n t r o l (her
term "DNA" r e f e r s only to DNA i n the chromosome(s) of the c e l l ) .
The next example considers the f i s s i o n yeast, Schizosaccharomyces
pombe, which d i v i d e s by b i n a r y f i s s i o n and which e x h i b i t s a nu-
c l e a r c y c l e - c e l l s e p a r a t i o n c o o r d i n a t i o n which i s s i g n i f i c a n t l y
d i f f e r e n t from that normally a s c r i b e d to eucaryotes. The f i n a l
example w i l l consider growth of Saccharomyces cerevisiae popula-
t i o n s . This y e a s t , used widely i n baking and brewing, provides an
i n t e r e s t i n g t h i r d type of s i n g l e - c e l l behavior with DNA synthesis
o c c u r r i n g i n the c l a s s i c a l e u c a r y o t i c p a t t e r n , but with asymmetric
cell division.
A Mathematical Model f o r Growth of E. coli Populations
Cooper and Helmstetter (11) have shown t h a t , under a v a r i e t y

of growth c o n d i t i o n s , many experimental observations are c o n s i s t -
ent w i t h a model i n which the time f o r r e p l i c a t i o n of the e n t i r e
chromosome of E. coli B/r i s approximately f o r t y minutes. During
t h i s time, the r a t e of r e p l i c a t i o n fork advance appears to be con-
s t a n t . Furthermore, c e l l d i v i s i o n c o n t r o l i s r e l a t e d to DNA syn-
t h e s i s : the b a c t e r i a l c e l l d i v i d e s approximately twenty minutes
a f t e r a r e p l i c a t i o n fork reaches the terminus of the chromosome.
These features i n combination imply i n t e r e s t i n g patterns of DNA
synthesis i n i n d i v i d u a l b a c t e r i a l c e l l s .
As shown i n Figure 2, the s i t u a t i o n i s q u i t e simple when the
organism has a doubling time of s i x t y minutes. DNA s y n t h e s i s oc-
curs at a constant r a t e f o r the f i r s t f o r t y minutes of the l i f e of
a s i n g l e c e l l (the times shown i n t h i s diagram are times remaining
u n t i l d i v i s i o n ) , and the c e l l synthesizes no DNA during i t s f i n a l
twenty minutes of growth before d i v i s i o n . However, the growth of
an E. coli c e l l w i t h a doubling time of f i f t y minutes implies a
more complicated p a t t e r n of DNA s y n t h e s i s , i n which a new s e t of
r e p l i c a t i o n forks begins s y n t h e s i z i n g DNA ten minutes before the
c e l l divides.

STAIN PROTEIN
0
WITH
FLUORESCENT SU
COMPOUND
STAINED
SAMPLE FLOW
SAMPLE
PHOTOMULTIPLIER
TUBE
ANALYZE
PULSES
Figure 1. Schematic diagram of flow cytometry measurement of the frequency

function, f, for single-cell protein content, p, in a cell population. After cellular pro
tein is stained with a specific fluorochrome, the dilute cell suspension is analyzed in
the flow cytometer. Laser radiation excites stain fluorescence in a single cell; that is
measured by a photomultiplier tube. The resulting voltage pulses are sorted and
stored to obtain the frequency function.
DOUBLING TIME = 60 MIN
<
60 20 20-0
J I I I L
60 50 40 30 20 10 0
30 20
<
ζ *ΞΕ=
ο
>
<
UJ DOUBLING TIME = 50 MIN
or 50 40 30 20 10 0
TIME UNTIL
DIVISION (MIN)
Figure 2. DNA synthesis rates and chromosome configurations according to the
Cooper-Helmstetter (11) model for E . coli growing at different rates. (The circular
chromosome is shown here in linear form for schematic convenience.)

Thus, during the f i n a l ten minutes of c e l l growth, DNA i s synthe

s i z e d a t double the r a t e observed at any time during the slower
growth s i t u a t i o n . This model i n d i c a t e s that DNA s y n t h e s i s i n bac
t e r i a , f a r from being a continuous process, i s a l s o one that pro
ceeds at v a r i o u s r a t e s , with d i s c o n t i n u i t i e s i n r a t e depending
upon time elapsed i n the c e l l c y c l e and the doubling time of the
cell. T h i s f e a t u r e of t h i s simple bacterium provides a dramatic
example of v a r i a t i o n i n n e t a b o l i c a c t i v i t i e s of c e l l s during t h e i r
l i f e cycle.
The Cooper-Helmstetter model does not i n i t s e l f provide a
complete model f o r growth and d i v i s i o n of a s i n g l e bacterium, but
such a model may be obtained by adding two a d d i t i o n a l elements.
Donachie (12) has shown that DNA s y n t h e s i s - growth r a t e - c e l l
s i z e data i s c o n s i s t e n t with the hypothesis that r e p l i c a t i o n f o r k s
n
begin at every chromosome o r i g i n whenever m = 2 m* η = 1,2,...
where m* i s the mass of
assuming that the s p e c i f i
i s independent of c e l l mass or p h y s i o l o g i c a l s t a t e and i s a func
t i o n of time that i s e i t h e r s p e c i f i e d or determined by c a l c u l a t i o n
using an a u x i l i a r y model (13). Figure 3 summarizes the d i f f e r e n t
p a r t s of the model and the way i n which they i n t e r l o c k i n order to
provide a complete d e s c r i p t i o n of s i n g l e - c e l l mass s y n t h e s i s , DNA
s y n t h e s i s , and c e l l d i v i s i o n . As noted i n the f i g u r e , the Cooper-
Helmstetter model allows determination not only of the amount of
DNA i n the i n d i v i d u a l c e l l , but a l s o of the chromosome c o n f i g u r a
t i o n ; that i s , p o s i t i o n of the r e p l i c a t i o n f o r k s along the chromo
some. Thus, by reference to a map of the chromosome, one may de
termine the numbers of a l l chromosomal genes i n the c e l l at any
i n s t a n t of time under any growth environment and growth h i s t o r y .
By means of a c l e v e r transformation of v a r i a b l e s and some i n
genious reasoning, Nishimura was able to o b t a i n a n a l y t i c a l s o l u
t i o n s f o r the d i s t r i b u t i o n s of mass, DNA content, chromosome con
f i g u r a t i o n , and t o t a l c e l l numbers i n populations of b a c t e r i a de
s c r i b e d by the above model (14). F i g u r e 4 shows the model r e s u l t s
f o r s t e a d y - s t a t e balanced growth of E. coli at a v a r i e t y of doub
l i n g times. The r e a r panel, which shows the c a l c u l a t e d r a t i o of
mean DNA content to mean c e l l mass, e x h i b i t s a decreasing depend
ence on doubling time which i s c o n s i s t e n t with previous experiment
a l observations (15). However, there are many simpler models which
presumably could be derived which would a l s o reproduce t h i s par
t i c u l a r r e s u l t . The present model a l s o gives the e f f e c t s of popu
l a t i o n growth r a t e on DNA and mass frequency f u n c t i o n s as shown i n
the sequences along the f l o o r of the f i g u r e . However, experimental
data on these frequency f u n c t i o n s are not p r e s e n t l y a v a i l a b l e .
Consequently, one must f i n d a l t e r n a t i v e s i t u a t i o n s to t e s t the
model i n which the model i s i n some sense more s e v e r e l y and c r i t i c
a l l y exposed.
Several l i n e s of research i n the p h y s i c a l sciences and engin
e e r i n g suggest that unsteady-state experiments are o f t e n extremely
u s e f u l i n e x p l o r i n g g e n e r a l i t y of mathematical models. For example,

CELL
ENVIRONMENT
μ (t)
CELL
GROWTH
ι DONACHIE
INITIATION OF
DNA
ONA
COOPER- ^ CONFIGURATION
REPLICATION HELMSTETTER AND UtLL
m DIVISION
"m =
M m = 2 m" TIMING
DAUGHTER
t BINARY FISSION
CELL MASS
Figure 3. Summary of a model for coordinated mass and DNA synthesis and cell
division for individual cells of E . coli. Reproduced, with permission, from Ref. 14.
Copyright 1980, Elsevier North Holland, Inc.
Figure 4. Calculated properties of E . coli populations in steady state balanced

growth at different rates. Frequency functions for cell mass and DNA content are
shown on the bottom panels; the rear panel shows the calculated ratio of mean cell
DNA content to mean cell mass. Reproduced, with permission, from Ref. 13.
Copyright 1981, American Institute of Chemical Engineers.

t r a n s i e n t experiments were a p p l i e d i n the s t u d i e s of Kjeldgaard

et ai. (16) i n s t u d i e s of E. eoli macromolecular s y n t h e s i s . In
p a r t i c u l a r , the time dependence of t o t a l c u l t u r e c e l l mass, DNA,
RNA, and c e l l numbers were followed a f t e r a s h i f t increase i n
growth r a t e . These experiments show c l e a r l y that c e l l mass, DNA,
and c e l l numbers f o l l o w d i f f e r e n t t r a j e c t o r i e s with c e l l mass r e -
sponding f i r s t to the new environment, DNA second, and c e l l num-
bers l a s t . The number of c e l l s i n the p o p u l a t i o n increases f o l -
lowing the s h i f t at a r a t e c h a r a c t e r i s t i c of the o l d medium u n t i l
roughly one hour a f t e r the s h i f t . The model d e s c r i b e d , when used
to c a l c u l a t e the r e s u l t s of the same experiment, y i e l d s the be-
h a v i o r shown i n Figure 5. The t i m e - v a r i a t i o n s of t o t a l c u l t u r e
c e l l mass, DNA content, and c e l l numbers shown on the r e a r panel
mimic very c l o s e l y the experimental r e s u l t s . Consequently the
model e a s i l y passes t h i t e s t I t would b i n t e r e s t i n t desig
new t r a n s i e n t experiment
of the model j u s t o u t l i n e
l a t i o n behavior.
This example shows how b a s i c information from the b i o l o g i c a l
sciences l i t e r a t u r e combined with f a c i l e mathematical a n a l y s i s can
provide u s e f u l working r e l a t i o n s h i p s which connect b a s i c s i n g l e -
c e l l c y c l e parameters with p r o p e r t i e s of c e l l populations growing
under d i f f e r e n t c o n d i t i o n s . Related models of E. coli population
dynamics have been developed e x t e n s i v e l y by Bleecken (17-19), who
has s t r u c t u r e d h i s model and a n a l y s i s to allow d i r e c t determina-
t i o n of the number of any p a r t i c u l a r gene present i n the c u l t u r e
during balanced growth and during the t r a n s i e n t f o l l o w i n g a growth
r a t e change.
Also of i n t e r e s t i n t h i s connection i s the elaborate model of
the b a c t e r i a l c e l l developed by Shuler and colleagues which i s de-
s c r i b e d i n d e t a i l elsewhere i n t h i s volume. That model o f f e r s the
advantages of more complete r e p r e s e n t a t i o n of b a s i c c e l l u l a r func-
t i o n s and, thereby, a b i l i t y to simulate c o r r e c t l y a wider v a r i e t y
of responses to growth environment. Furthermore, that model does
not r e q u i r e any supplementary or a u x i l i a r y c a l c u l a t i o n s to deter-
mine growth r a t e of the organism as does the model o u t l i n e d above.
On the other hand, the s t r u c t u r e d model of Shuler and coworkers
r e q u i r e s considerably more computational e f f o r t f o r i t s s o l u t i o n
and l e s s p o s s i b i l i t y of i d e n t i f i c a t i o n of c e r t a i n q u a l i t a t i v e f e a -
tures than does the approach o u t l i n e d above. I t i s expected t h a t ,
i n the coming years, through lumping procedures guided by s e n s i -
t i v i t y analyses, a spectrum of models f o r t h i s organism w i l l be-
come a v a i l a b l e which w i l l allow the e x p e r i m e n t a l i s t or r e a c t o r de-
s i g n e r to s e l e c t the l e v e l of model complexity r e q u i r e d f o r the
problem under c o n s i d e r a t i o n .
C e l l Cycle Operation and S i n g l e - C e l l P r o t e i n Synthesis Kinetics

f o r S. pombe
Flow cytometry provides d i r e c t q u a l i t a t i v e i n s i g h t s i n t o the

w

Figure 5. Calculated results of a shift in specific growth rate for an E . coli population (μ =
1 1

1.038 hr for t < 0, μ = 2.076 hr for t > 0). Curves A , B , and C on the rear panel are the mass,
DNA content, and cell numbers in the culture, each normalized by their respective values at t =
0. Reproduced, with permission, from Ref. 14. Copyright 1980, Elsevier North Holland, Inc.
c o o r d i n a t i o n of DNA synthesis and c e l l d i v i s i o n i n the f i s s i o n

yeast S. pombe. To put the experimental r e s u l t s i n t o p e r s p e c t i v e ,
consider the composite diagram showing the i n t e r r e l a t e d c y c l e s of
DNA s y n t h e s i s and s e p a r a t i o n , c e l l shape, and c e l l volume i n d i -
cated s c h e m a t i c a l l y i n Figure 6 (20-24). Here the events which
occur during the c e l l c y c l e are d i s p l a y e d i n r e l a t i v e p o s i t i o n and
d u r a t i o n along arcs of a c i r c l e , the top of which i s taken a r b i -
t r a r i l y to be c o i n c i d e n t with the c e l l s e p a r a t i o n event. In t h i s
d i s c u s s i o n , the r e l a t i o n s h i p of the i n t e r v a l of DNA s y n t h e s i s ,
which i n t h i s eucaryote provides d u p l i c a t i o n of a l l of the chromo-
somes i n the mother c e l l , r e l a t i v e to the time of p h y s i c a l separ-
a t i o n of daughter c e l l s i s of s p e c i a l i n t e r e s t .
For t h i s organism, as i t i other eucaryotes, the DNA content
of the c e l l e x h i b i t s l e s s v a r i a t i o n than i n the b a c t e r i a l case,
with the content f o r an i n d i v i d u a l c e l l ranging between one com
p l e t e set of genetic information
e q u i v a l e n t (1C), and two
organism a r a t h e r unusual f e a t u r e occurs i n which p h y s i o l o g i c a l
s e p a r a t i o n of the daughter c e l l s takes place before p h y s i c a l sep-
a r a t i o n , so that DNA s y n t h e s i s may begin i n the p h y s i o l o g i c a l l y
p a r t i t i o n e d daughter c e l l s before those c e l l s a c t u a l l y p h y s i c a l l y
separate. Thus, depending upon the d i s p o s i t i o n of the i n t e r v a l of
DNA synthesis r e l a t i v e to c e l l s e p a r a t i o n , c e l l s w i l l e x i s t i n the
p o p u l a t i o n c o n t a i n i n g up to 4C DNA content i f DNA s y n t h e s i s i s
complete before the c e l l s separate and as l i t t l e as 1C DNA i f the
c e l l s separate before DNA s y n t h e s i s begins (here and below, the
term " c e l l " r e f e r s to the p h y s i c a l l y separated u n i t s which are ob-
served i n a flow cytometry measurement) . Previous experiments on
the c o o r d i n a t i o n between DNA s y n t h e s i s and c e l l s e p a r a t i o n i n t h i s
organism are somewhat ambiguous (20-24), so that the S-phase has
been shown i n the above diagram as s t r a d d l i n g the p o i n t of c e l l
separation. Notice that, independent of the d i s p o s i t i o n of t h i s
r e l a t i v e l y b r i e f i n t e r v a l of the c e l l c y c l e , the m a j o r i t y of c e l l s
i n a f i s s i o n yeast p o p u l a t i o n w i l l possess a DNA content of 2C.
The upper four frames of Figure 7 show flow cytometer mea-
surements of the frequency functions of i n d i v i d u a l c e l l DNA con-
tent i n S. pombe (972 h"") populations grown i n steady-state chemo-
s t a t c u l t u r e . The parameters i n d i c a t e d i n the f i g u r e frames are
1
the s p e c i f i c growth r a t e s i n u n i t s of h r " . The dominant peak i n
each of these measurements corresponds, as explained p r e v i o u s l y ,
to a DNA content of two genome e q u i v a l e n t s (2C). An i n t e r e s t i n g
f e a t u r e i s observed i n the data obtained at lower growth r a t e s .
The frequency f u n c t i o n f o r an o v e r a l l p o p u l a t i o n s p e c i f i c growth
1
r a t e of 0.088 hr"" e x h i b i t s three modes, the f i r s t corresponding
to a DNA content of 1C, the second to the most prevalent 2C popu-
l a t i o n , and the f i n a l one to a p o p u l a t i o n of c e l l s c o n t a i n i n g a
DNA content of 4C. The l a s t mode i s not b e l i e v e d to be the r e s u l t
of c e l l clumping, as a l l samples i n these analyses were t r e a t e d by
s o n i c a t i o n immediately p r i o r to flow cytometric a n a l y s i s . (The
s u i t a b i l i t y of t h i s procedure f o r p r o v i d i n g accurate c e l l counts,

Cell diameter 3.5pm Cell diameter 3.5pm

Cell length ^ 14pm Cell length ^ 8pm
1 i near
mass i n -
crease ,
constant
volume
1 i n e a r mass
increase , exp-
onential volume
increase
Cell size
® Nucleus with 2 C DNA content
® Nucleus with DNA content between 1C& 2 C
® Nucleus with 1 C DNA content
CS - c e l l separation ; ND - n u c l e a r division ;
CP - c e l l plate formation
Figure 6. Schematic diagram of the nuclear and cell division cycles of fission
yeast S. pombe.

and thereby minimum clumping, was v e r i f i e d i n separate c o n t r o l

experiments.)
Consequently, these data i n d i c a t e that the c o o r d i n a t i o n be-
tween DNA s y n t h e s i s and c e l l s e p a r a t i o n becomes imprecise i n t h i s
organism at low growth r a t e s , with some c e l l s d i v i d i n g before i n -
i t i a t i n g DNA s y n t h e s i s and others completing DNA s y n t h e s i s before
dividing. This interesting imprecision i n c e l l cycle operation
would not have been evident by any population-average measurements
upon t h i s system. Only measurement of the frequency f u n c t i o n pro-
v i d e s the s e n s i t i v i t y and d e t a i l of i n f o r m a t i o n r e q u i r e d to ob-
serve t h i s f e a t u r e .
The two frequency f u n c t i o n s shown i n the bottom two frames of
Figure 7 correspond to d i f f e r e n t s t e a d y - s t a t e populations growing
at approximately the same o v e r a l l p o p u l a t i o n growth r a t e s as those
shown i n the two middl frames Thes d i f f e r e n t stead state
were achieved by d i f f e r e n
seem to be connected w i t clumping phenomeno
when r e a c t o r s t a r t - u p i n i t i a t e s with a s i g n i f i c a n t glucose l i m i t a -
t i o n of growth. A d d i t i o n a l i n f o r m a t i o n on t h i s m u l t i p l e steady-
s t a t e phenomenon and on i t s m a n i f e s t a t i o n i n other types o f mea-
surements i s a v a i l a b l e elsewhere ( 9 ) .
Figure 8 shows the frequency f u n c t i o n s f o r s i n g l e c e l l pro-
t e i n content obtained by the same c u l t i v a t i o n methods as j u s t out-
l i n e d . Considering the s e t of steady-state r e s u l t s obtained by
the same s t a r t - u p procedure (the top f o u r frames i n F i g u r e 8), i t
i s apparent that there i s a s i g n i f i c a n t s h i f t o f the frequency
f u n c t i o n towards higher p r o t e i n contents and a l s o i n c r e a s e d skew-
ing to the r i g h t as the o v e r a l l p o p u l a t i o n s p e c i f i c growth r a t e
increases.
Q u a n t i t a t i v e a n a l y s i s of these measurements has been under-
taken by two d i f f e r e n t s t r a t e g i e s based upon the governing popu-
l a t i o n balance equations which d e s c r i b e the system (25). One
method employed the Collins-Richmond equation (26), the i n t e g r a t e d
form o f the p o p u l a t i o n balance equation, with r e s u l t s which are
summarized i n d e t a i l elsewhere (25). In the second approach, a
simple f u n c t i o n a l form was chosen f o r the f i s s i o n frequency func-
t i o n <b which contained two a d j u s t a b l e parameters (the r e s u l t s of
Harvey, Marr and P a i n t e r (27) i n d i c a t e that the r e s u l t s o f popu-
l a t i o n balance model s o l u t i o n s are not h i g h l y s e n s i t i v e to the
f u n c t i o n a l form o f t h i s f u n c t i o n i n the model). Then, four d i f -
f e r e n t f u n c t i o n forms f o r the s i n g l e - c e l l r a t e o f p r o t e i n synthe-
s i s f u n c t i o n r ^ were examined. For the f o l l o w i n g four f u n c t i o n a l
forms,
r^(p,s) = k ( s ) ,
Q zero order (2.1)
r (p»s) = k ( s ) p ,
f 1 f i r s t order (2.2)

0.06
0.04 - 11 0.249 1 0.176
^ 0.02
ΰ 0 J \ L . I I I I \—ι I I I I
0 0.06
£0.04 " I 0.132 - Λ 0.088
LU 0.02
F ο λ V-M I I I I /| ι ι ι ι
<0.06
- Λ 0.131 0.088
ω 0.04
λ
Ο,
Ο 6
C E L L DNA C O N T E N T
(CHANNEL N U M B E R )
Figure 7. Frequency functions for cellular DNA content for S. pombe populations
propagated in a chemostat at different dilution rates (specific growth rates shown in
4
each frame). Channel number, one division = 6.8 X 10' pg DNA/cell. Repro
duced, with permission, from Ref. 9. Copyright 1981, John Wiley & Sons, Inc.
0.03
0.249 0.176
Ζ 0.01
rî
3 ο
ι J ι ι ι 1 / ι ι \ - ι ι
Ο 0.03
- ι^ν 0.132 - Λ 0.088
uj 0.01
Ê ο ι J\ ι j ι Il 1 N-i- ι ι ι
< 0.03
- /V 0.131 - 0.088
UJ 0.02
0.01
. 1/1 J*. 1
6 4 128 192 0 6 4 128 192 2 5 6
C E L L PROTEIN CONTENT
(CHANNEL NUMBER)
Figure 8. Protein content frequency functions for S. pombe populations grown in

a chemostat. Channel number, one division = 0.078 pg protein/cell. Reproduced,
with permission, from Ref. 9. Copyright 1981, John Wiley & Sons, Inc.
American Chemical
Society Library
1155 16th St., N.W.
ACS Symposium Series; Washington, U.C.
American Chemical 20036
Society: Washington, DC, 1983.
r
f(p»s) = k ( ) Q
s +
k^(s)p, two term (2.3)
2
r (p,s) = k (s) + k (s)p + k (s)p ,
f Q 1 2 three term (2.4)
a parameter search u s i n g the Marquardt a l g o r i t h m was conducted to

minimize i n an i n t e g r a l l e a s t squares sense the d i f f e r e n c e between
the p o p u l a t i o n balance model s o l u t i o n and measured frequency func-
t i o n . The best f i t c a l c u l a t i o n s f o r each of the four d i f f e r e n t
f u n c t i o n a l forms o f p r o t e i n s y n t h e s i s k i n e t i c s a r e shown by s o l i d
l i n e s i n F i g u r e 9 along with the experimental frequency f u n c t i o n
1
data f o r an o v e r a l l p o p u l a t i o n s p e c i f i c growth r a t e of 0.246 h r " .
C l e a r l y , even with o p t i m a l l y adjusted parameters, the zero order,
f i r s t order, and two-term models cannot provide a s a t i s f a c t o r y f i t
of the experimental data
second-order dependence
represents the data.
S i m i l a r c a l c u l a t i o n s seeking best f i t s between v a r i o u s models
and experimental data corresponding to d i f f e r e n t o v e r a l l s p e c i f i c
growth r a t e s provided estimates o f the k i n e t i c s o f s i n g l e c e l l pro-
t e i n s y n t h e s i s at each o f the growth r a t e s considered. These r e -
s u l t s a r e shown i n F i g u r e 10. The q u a l i t a t i v e features o f these
r e s u l t s are r a t h e r s u r p r i s i n g , i n d i c a t i n g that the b a s i c f u n c t i o n -
a l form o f s i n g l e c e l l p r o t e i n s y n t h e s i s k i n e t i c s changes s i g n i f -
i c a n t l y from approximately l i n e a r a t the lowest growth r a t e con-
s i d e r e d to d i s t i n c t l y p a r a b o l i c a t the more r a p i d growth r a t e s
investigated.
These trends may r e f l e c t the need f o r i n c l u s i o n o f a d d i t i o n a l
v a r i a b l e s i n modeling the k i n e t i c s o f s i n g l e c e l l p r o t e i n synthe-
sis. I t i s p o s s i b l e that the great d i f f e r e n c e i n p r o t e i n synthe-
s i s r a t e s observed a t small p r o t e i n contents a t d i f f e r e n t o v e r a l l
growth r a t e s i s because o f some change i n another aspect o f the
c e l l ' s metabolism which i s not represented i n t h i s model. The
minima i n p r o t e i n s y n t h e s i s rates d i s p l a y e d i n F i g u r e 10 may r e -
s u l t from a requirement that the c e l l engage i n other b i o s y n t h e t i c
a c t i v i t i e s during t h i s p a r t o f the c e l l c y c l e a t h i g h growth rates.
I t i s s i g n i f i c a n t to note that an independent a n a l y s i s o f the same
data u s i n g the Collins-Richmond equation i n d i c a t e d the same q u a l i -
t a t i v e r e l a t i o n s h i p between r a t e form and o v e r a l l growth r a t e as
i s i n d i c a t e d i n Figure 10 (25).
A comparison o f the outcomes o f t h i s approach to s i n g l e - c e l l
p r o t e i n s y n t h e s i s k i n e t i c s determination w i t h the a l t e r n a t i v e
method which has been widely used i n many previous s t u d i e s o f
c e l l c y c l e and c e l l k i n e t i c behavior o f many organisms i s i n s t r u c -
tive. In the l a t t e r methods, synchronous c u l t u r e o r an e q u i v a l e n t
experimental technique are used to make measurements of the time
v a r i a t i o n o f p r o t e i n content o r some other c e l l u l a r v a r i a b l e v e r -
sus time as the c e l l grows from a newborn daughter c e l l to a
mature and d i v i d i n g mother c e l l . Then, by e s t i m a t i n g the slope of

> 0.03
ZERO ORDER FIRST ORDER
S 0.02
0 1
S °·
Ê Ο
TWO TERM THREE TERM
LU
> 0.02
LU —ι ι^^^^ J ι J^^^^^NS^ 1
64 128 192 0 64 128 192 256
C E L L PROTEIN CONTENT
Figure 9. Comparison of experimentally measured protein content frequency

1
function (dots) for S. pombe, grown in a chemostat with D = 0.246 hr , with best
fit simulations (lines) using the kinetic forms of Equation 2. Reproduced, with
permission, from Ref. 25. Copyright 1981, John Wiley & Sons, Inc.
π r
0 64 128 192 256
CELL PROTEIN CONTENT
(CHANNEL NUMBER)
Figure 10. Rate functions for single-cell protein synthesis as determined by

analysis of protein content frequency functions measured in steady state chemostat
1
growth. Parameters near each curve indicate dilution rates employed (hr' ). Repro
duced, with permission, from Ref. 25. Copyright 1981, John Wiley & Sons, Inc.

t h i s c e l l v a r i a b l e versus time t r a j e c t o r y , one estimates s y n t h e s i s

r a t e s and e s t a b l i s h e d k i n e t i c s . The d i f f i c u l t y with t h i s procedure
i s the l i m i t e d range of the c e l l v a r i a b l e over which the measurement
can be made, namely from a value corresponding to a newborn c e l l to a
value corresponding to a d i v i d i n g c e l l . Over t h i s l i m i t e d range of
change of any v a r i a b l e , f o r a v a r i a b l e that i n c r e a s e s monotonically
during the c e l l c y c l e , i t i s very d i f f i c u l t to d i s c r i m i n a t e among
a l t e r n a t i v e k i n e t i c forms unless they are extremely d i f f e r e n t .
T h i s i n s e n s i t i v i t y of the t r a j e c t o r y - t r a c k i n g k i n e t i c s de
termination approach can be seen by i n v e r t i n g the procedure: as
sume d i f f e r e n t f u n c t i o n a l forms f o r the k i n e t i c s on a s i n g l e - c e l l
b a s i s and c a l c u l a t e , using these k i n e t i c s , the t r a j e c t o r y of the
c e l l u l a r v a r i a b l e versus time. The r e s u l t s w i l l show t h a t , f o r
q u i t e d i f f e r e n t k i n e t i c forms, the t r a j e c t o r i e s are very c l o s e
and, c o n s i d e r i n g unavoidabl
very d i f f i c u l t to d i s t i n g u i s h
use of a l i n e a r k i n e t i c form and the p a r a b o l i c one shown i n
1
F i g u r e 10 f o r the u = 0.246 h r " case gives very s i m i l a r ρ versus
time t r a j e c t o r i e s ; on the other hand, when these k i n e t i c forms are
used to c a l c u l a t e frequency f u n c t i o n s i n balanced growth, the r e
s u l t s are q u i t e d i f f e r e n t (compare frames Β and D o f F i g u r e 9 ) .
Therefore, i n t h i s case and presumably i n many o t h e r s , a n a l y s i s
of the r e l a t i o n s h i p between s i n g l e - c e l l k i n e t i c form and the c o r
responding frequency f u n c t i o n i n steady-state balanced growth pro
v i d e s a more s e n s i t i v e t o o l f o r d i s c r i m i n a t i o n and i d e n t i f i c a t i o n
of s i n g l e - c e l l k i n e t i c s than do c l a s s i c a l t r a j e c t o r y - t r a c k i n g pro
cedures of v a r i o u s k i n d s .
As a t e s t o f the range of a p p l i c a b i l i t y o f the k i n e t i c s de
termined i n the s t e a d y - s t a t e measurements, the t r a n s i e n t popula
t i o n balance equation has been s o l v e d , using the k i n e t i c s d e t e r
mined from steady s t a t e , to simulate the sequence of p r o t e i n con
tent frequency f u n c t i o n s obtained i n synchronous growth of t h i s
organism. The s i m u l a t i o n r e s u l t s are i n very good q u a l i t a t i v e
agreement with the experimental measurements o f the corresponding
q u a n t i t i e s (28).
Growth and Nuclear Cycle Operation i n S i n g l e C e l l s

of S. oerevisiae
The budding yeast S. oerevisiae i s c u r r e n t l y employed i n a

v a r i e t y of a p p l i c a t i o n s , i n c l u d i n g brewing, baking, and g e n e t i c
engineering. T h i s organism grows on a s i n g l e c e l l b a s i s by, at a
p a r t i c u l a r p o i n t i n i t s growth, producing a bud which i n c r e a s e s i n
s i z e and mass as the c e l l grows (29-34). In f a c t , i t has been
suggested that, to a good approximation, a l l of the new c e l l mass
synthesized between bud emergence and s e p a r a t i o n of the bud as a
daughter c e l l goes i n t o the growing bud e n t i r e l y (29). The mother
c e l l mass, at l e a s t i n the simplest form of t h i s model, i s not
presumed to i n c r e a s e at a l l during t h i s stage of the c e l l d i v i s i o n
c y c l e . These f e a t u r e s are summarized q u a l i t a t i v e l y i n the sketch

of the c e l l d i v i s i o n c y c l e f o r 5. oerevisiae provided i n F i g u r e 1L

As i n d i c a t e d i n t h i s diagram, upon c e l l d i v i s i o n , the mother c e l l
r e c y c l e s to a standard p o i n t , l a b e l e d " s t a r t " , and the bud, being
g e n e r a l l y smaller than the " s t a r t " c e l l mass, must grow f o r some
time before i t a t t a i n s the " s t a r t " s t a t e and before i t i s p r e -
pared to form a bud and i t s e l f become a mother c e l l .
T h i s view of the growth and d i v i s i o n of i n d i v i d u a l c e l l s o f
t h i s organism i s based upon a v a r i e t y o f experimental s t u d i e s
which have i n v o l v e d growth r a t e s h i f t experiments, s h i f t s o f grow-
ing c e l l s i n t o s t a r v a t i o n media, and growth o f yeast populations
i n d i f f e r e n t media supporting d i f f e r e n t growth r a t e s (29-34).
Other measurements, o f t e n made i n concert with the above e x p e r i -
ments, suggest that the n u c l e a r c y c l e f o r t h i s organism i s coor-
dinated w i t h the c e l l d i v i s i o n c y c l e i n the manner shown i n
F i g u r e 11. The G\ or f i r s t
9 i n t e r v a l extend fro c e l l " b i r t h "
u n t i l the p o i n t of bud emergenc
s y n t h e s i s occurs. The d u r a t i o S-phas reporte
r e l a t i v e l y i n s e n s i t i v e to o v e r a l l p o p u l a t i o n growth r a t e i n aerobic
s i t u a t i o n s i n shake f l a s k c u l t u r e s (aerobic i s used here i n a
somewhat generous sense, s i n c e shake f l a s k c u l t u r e s cannot gener-
a l l y be assumed to be s u f f i c i e n t l y aerated to e l i m i n a t e oxygen
l i m i t a t i o n s of growth and r e s u l t i n g responses of c e l l u l a r con-
t r o l s ) . F o l l o w i n g the DNA s y n t h e s i s i n t e r v a l , the second gap and
n u c l e a r d i v i s i o n phases occur, a f t e r which c e l l s e p a r a t i o n takes
p l a c e . I n t e r e s t i n g l y , the experiments c i t e d above suggest that
the t o t a l time spent i n the G 2 and M phases are a l s o r e l a t i v e l y
i n s e n s i t i v e to o v e r a l l growth r a t e o f the p o p u l a t i o n . Thus, the
e f f e c t o f o v e r a l l growth r a t e suggested by t h i s model i s to a f f e c t
p r i m a r i l y the length o f G\ and correspondingly, the f r a c t i o n o f
the p o p u l a t i o n which c o n s i s t s of unbudded c e l l s and the c o r r e s -
ponding c e l l mass and c e l l DNA frequency f u n c t i o n s .
Flow cytometry and mathematical a n a l y s i s of the r e s u l t s has
been a p p l i e d to study the o p e r a t i o n of the n u c l e a r c y c l e i n
5. oerevisiae grown i n steady-state chemostat c u l t u r e under anaer-
o b i c c o n d i t i o n s (36); a s i m i l a r s t r a t e g y was employed i n the i n -
v e s t i g a t i o n s o f S l a t e r et al. (30). I t i s worth n o t i n g here that
experiments under these c o n d i t i o n s have not been reported p r e v i -
o u s l y , and consequently these r e s u l t s c o n t r i b u t e to determination
of the extent of general a p p l i c a b i l i t y of the model suggested by
experiments under d i f f e r e n t growth c o n d i t i o n s .
Assuming that an i n d i v i d u a l c e l l n u c l e a r c y c l e operates q u a l -
i t a t i v e l y as shown i n F i g u r e 11, i t f o l l o w s that the expected form
of the frequency f u n c t i o n of i n d i v i d u a l c e l l DNA content i n a
growing p o p u l a t i o n of t h i s budding yeast w i l l have the f o l l o w i n g
f e a t u r e s : At a DNA content corresponding to one genome equiva-
l e n t (1C), there should be an impulse which has magnitude equal to
the f r a c t i o n of the p o p u l a t i o n i n the G1-phase o f the n u c l e a r
cycle. S i m i l a r l y , a second impulse l o c a t e d a t a fluoresence l e v e l
corresponding to a DNA content of two genome e q u i v a l e n t s (2C) w i l l
appear with a magnitude equal to the f r a c t i o n of the p o p u l a t i o n i n

CELL DIVISION CYCLE
bud emergence cell

separation
second,
first DNA gap (62) 61
gap (G|) synthesis - and ~
nuclear
(S) division (M)
I NUCLEAR CYCLE
Figure 11. Schematic diagram of the nuclear and cell division cycles for the
budding yeast S. cerevisiae. Reproduced, with permission, from Ref. 35. Copyright
1980, Pergamon Press Ltd.

the G2+M-phases. Between these two impulses w i l l be a connecting

curve which corresponds to c e l l s i n S-phase and with DNA contents
between 1C and 2C. I t must be recognized, however, that a c t u a l
experimental measurements o f the frequency f u n c t i o n u s i n g flow
cytometry w i l l not have t h i s appearance due to n o i s e both i n the
organism metabolism and i n the measurement procedures. Thus, one
expects to see something approximating normal d i s t r i b u t i o n s cen-
t e r e d where the two impulse f u n c t i o n s are a n t i c i p a t e d .
The r e s u l t s o f flow cytometry frequency f u n c t i o n measurements
at d i f f e r e n t continuous c u l t u r e d i l u t i o n r a t e s are i l l u s t r a t e d i n
F i g u r e 12. These possess the general forms j u s t suggested. Un-
f o r t u n a t e l y , the magnitude o f the d i s p e r s i o n i n these data i s s u f -
f i c i e n t l y great as to obscure the c o n t r i b u t i o n o f the S-phase
c e l l s . A v a i l a b i l i t y o f t h i s i n f o r m a t i o n f o r t h i s organism i s
hampered by the r e l a t i v e l
phase and by the r e s o l u t i o
t i o n s o f ways to improv analysi
niques i n t h i s regard are i n progress.
However, by adding to the expected i d e a l i z e d DNA frequency
f u n c t i o n a n o i s e model, i t i s p o s s i b l e through a parameter optim-
i z a t i o n procedure to e x t r a c t from the data shown i n F i g u r e 12 e s -
timates of the f r a c t i o n s o f the c e l l p o p u l a t i o n a t a given o v e r a l l
p o p u l a t i o n growth r a t e (= D) which are i n the Gi,S, and G2+M-
phases o f the nuclear c y c l e . Combining t h i s i n f o r m a t i o n with the
c a l c u l a t e d age d i s t r i b u t i o n i n the c u l t u r e , i t i s p o s s i b l e to e s -
timate the d u r a t i o n o f the nuclear c y c l e i n t e r v a l s under d i f f e r e n t
o v e r a l l growth r a t e s o f the yeast p o p u l a t i o n . The r e s u l t s o f t h i s
a n a l y s i s are i l l u s t r a t e d i n Figure 1 2 . These f i n d i n g s show that
the n u c l e a r c y c l e o p e r a t i o n , a t l e a s t as manifested by the dura-
t i o n o f the d i f f e r e n t phases, i s q u a l i t a t i v e l y i d e n t i c a l under
these c u l t u r e c o n d i t i o n s as under the d i f f e r e n t growth e n v i r o n -
ments i n v e s t i g a t e d i n formulating the model d i s c u s s e d above. That
i s , the d u r a t i o n o f the S-phase and the t o t a l time an i n d i v i d u a l
c e l l spends i n the G2+M-phases i s r e l a t i v e l y i n s e n s i t i v e to over-
a l l growth r a t e o f the c u l t u r e . On the other hand, the time spent
i n the Gi-phase changes s i g n i f i c a n t l y with respect to o v e r a l l pop-
u l a t i o n growth r a t e , decreasing d r a m a t i c a l l y as the c u l t u r e growth
rate i s increased.
The diagram o f budding yeast c e l l c y c l e o p e r a t i o n shown i n
F i g u r e 11 above i s somewhat o v e r s i m p l i f i e d . Important aging
phenomena occur as a c e l l c y c l e s around the i n n e r loop producing
buds and r e s u l t i n g daughter c e l l s i n each pass through the i n n e r
loop o f the c e l l d i v i s i o n c y c l e diagram. As a mother c e l l pro-
duces repeated o f f s p r i n g , i t accumulates bud s c a r s . A l s o , i t may,
through increased age and opportunity to conduct b i o s y n t h e s i s and
c o n s t r u c t i o n o f l a r g e o r g a n e l l e s , gain metabolic c a p c i t y as i t
ages. Therefore, i n formulating mathematical models o f s i n g l e -
c e l l growth o f t h i s a n d r e l a t e d organisms, the p o s s i b l e p o s i t i v e
e f f e c t of aging due to accumulation o f o r g a n e l l e s may need to be
considered, as may the p o s s i b l y i n h i b i t o r y e f f e c t o f the accumula-

Figure 12. Nuclear cycle time intervals

estimated by analysis of DNA content fre-
quency functions for S. cerevisiae ATCC
18790 populations grown at steady state
at different dilution rates. Reproduced,
with permission, from Ref. 36. Copyright
1981. DILUTION RATE <h")

t i o n on the outer c e l l surface o f bud scar m a t e r i a l which i s i n -

a c t i v e f o r m a t e r i a l exchange with the c e l l environment.
Discussion
The examples above i l l u s t r a t e how measurements o f c e l l popu-

l a t i o n frequency f u n c t i o n s , f r a c t i o n a l amounts o f v a r i o u s c e l l sub-
populations, and o v e r a l l behavior o f m i c r o b i a l c u l t u r e s can be
used to evaluate i n q u a l i t a t i v e and q u a n t i t a t i v e terms some o f the
fundamental processes o f c o n t r o l and b i o s y n t h e s i s a t the s i n g l e -
c e l l l e v e l . Population balance equations, o r equivalent computer
s i m u l a t i o n techniques and mathematical analyses, connecting s i n g l e -
c e l l behavior w i t h p o p u l a t i o n p r o p e r t i e s provide the r e q u i r e d
working connection between experiments and s i n g l e - c e l l p r o p e r t i e s .
This approach appears t
f o r some o f the cases considered
c e l l o p e r a t i o n than do attemp
s c r i b e s i n g l e - c e l l behavior d i r e c t l y . Furthermore, the flow cy-
tometry experimental methods i l l u s t r a t e d do not i n general r e q u i r e
s p e c i a l manipulation o f c u l t u r e c o n d i t i o n s o r c e l l s f o r the pur-
poses o f conducting the d e s i r e d experiment. C e l l s may be c u l t i -
vated under any c o n d i t i o n s o f i n t e r e s t , i n c l u d i n g t i m e - i n v a r i a n t
environments which give r i s e to balanced growth, and the methods
described i n t h i s paper a p p l i e d . Combinations o f flow cytometry
measurements and computer a n a l y s i s based on c e l l c y c l e models
provide r a p i d access t o q u a l i t a t i v e and q u a n t i t a t i v e information
on c e l l c y c l e operation and c o n t r o l .
Obviously, more research i n t h i s d i r e c t i o n i s r e q u i r e d to en-
able these s t r a t e g i e s to be a p p l i e d f o r the design purposes de-
s c r i b e d a t the beginning o f the paper. This w i l l r e q u i r e a
broader r e p e r t o i r e o f experimental c a p a b i l i t i e s , i n c l u d i n g the
a b i l i t y to measure more d e t a i l e d information about c e l l composi-
t i o n using flow cytometry. Furthermore, the mathematical modeling
approaches described i n t h i s work must be enhanced and expanded to
i n c l u d e more c e l l components and to consider r e l a t i o n s h i p s among
substrate u t i l i z a t i o n , c e l l growth and the s y n t h e s i s o f c e l l com-
ponents, and product formation on an i n d i v i d u a l c e l l b a s i s . This
general l i n e o f research i s an important avenue f o r r e l a t i n g r e -
a c t o r design and c h a r a c t e r i z a t i o n o b j e c t i v e s o f biochemical reac-
t i o n engineering with b a s i c metabolism and c e l l u l a r c o n t r o l i n f o r -
mation i n the b i o l o g i c a l s c i e n c e s .
I t i s i n t e r e s t i n g to note t h a t , i n some o f the examples
above, a k i n e t i c approach i s appropriate which d i f f e r s from the
t r a d i t i o n a l s t r a t e g y o f d e f i n i n g composition and temperature de-
pendence o f r e a c t i o n r a t e s f u n c t i o n s . C e r t a i n assemblies o f com-
p l i c a t e d c e l l u l a r r e a c t i o n and r e g u l a t i o n processes may be repre-
sented reasonably a c c u r a t e l y under a v a r i e t y o f growth c o n d i t i o n s
i n terms o f timers, the i n i t i a t i o n p o i n t s and durations o f which
may be dependent upon growth c o n d i t i o n s . However, the above ex-
amples show that c e r t a i n parameters a s s o c i a t e d with s t a r t i n g

p o i n t s and running time o f these timers are i n v a r i a n t s and thus

provide a u s e f u l foundation upon which to d e s c r i b e the features of
c e l l o p e r a t i o n and c e l l p o p u l a t i o n p r o p e r t i e s . More a t t e n t i o n to
mathematical modeling of systems which feature such c l o c k s and
which focus upon the s t a r t i n g and running time o f these clocks i s
an i n t e r e s t i n g avenue f o r f u t u r e work.
Acknowledgements
The author i s extremely indebted to h i s colleagues and s t u

dents, D. McQuitty, Y. Nishimura, D. Agar, M. H j o r t s o , J . F a z e l -
M a d j l e s s i , M. G i l b e r t , A. B a r t e l , L. Y. Lee, and J . Oro, f o r
t h e i r c e n t r a l r o l e i n the research described i n t h i s paper and to
the N a t i o n a l Science Foundation f o r f i n a n c i a l support o f t h i s r e
search.
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7
A C y b e r n e t i c P e r s p e c t i v e of Microbial Growth
DORAISWAMI RAMKRISHNA
Purdue University, School of Chemical Engineering, West Lafayette, IN 47907
What goes by cellular metabolism is an immense

class of chemica
and without the cel
diversity and specificity. It forms the basis of the
response of microorganisms to their environment, the
modelling of which must of necessity suffer some over-
simplification if it is to be of any value. In this
context, kinetic models of microbial growth within the
framework of interaction between lumped biochemical
species and the environment have had considerable
appeal in the past. However, the subtle facilities
which derive from the elaborate internal machinery
of the cells pose a challenge that no meager expan-
sion of the kinetic framework will ever meet.
We examine here a cybernetic perspective of
microbial growth which contends that the net asset
of the cell's internal machinery is the facility to
make 'rational' (optimal) decisions in responding to
its environment, one that seems markedly manifest in
the situation of diauxic growth. The growth of micro-
bial cells in the presence of multiple substrates is
addressed in this work within a cybernetic framework
which lays emphasis on the optmial allocation of exist-
ing 'resources' among parallel enzyme-synthesis
systems. The work to be presented will discuss the
main issues connected with this outlook, what the
chief assets of the framework might be and the inter-
pretation of several experimental results within the
cybernetic scheme.
0097-6156/83/0207-0161 $06.00/0

Mathematical models of v a r y i n g degrees of s o p h i s t i c a t i o n

are the main instruments with which q u a n t i t a t i v e understanding
has been secured of engineering processes both i n a n a l y s i s and
design. In d e a l i n g with m i c r o b i a l systems, an important develop-
ment i n the past has been of the adoption of the chemical k i n e t i c
framework viewing the c e l l mass as lumped biophase (the so-
c a l l e d non-segregated models) and i n t e r a c t i n g as a whole with i t s
environment. Such models have been the b a s i s f o r design and
a n a l y s i s of i n d u s t r i a l fermentations. Several i n t e r e s t i n g
features of the methodology of chemical r e a c t i o n engineering have
found t h e i r way i n t o biochemical engineering. Whether or not
p r e c i s e l y q u a n t i t a t i v e understanding f o l l o w s from modelling, an
e n t i r e l y u s e f u l aspect of i t has been the e f f i c i e n t assessment of
the i m p l i c a t i o n s of s p e c i f i c hypotheses concerning the behavior
of a given system thus formulating new r e v e a l i n g experiments It
i s perhaps i n t h i s sens
e l l i n g has been a v a l u a b l
systems, and i t i s i n t h i s sense too that the ideas to be pre-
sented here must be i n t e r p r e t e d .
In the adoption of a s t r i c t l y chemical k i n e t i c framework f o r
modelling m i c r o b i a l systems, an important d i s t i n c t i o n a r i s e s .
Microorganisms a r e not a "dead bag" of biochemical c o n s t i t u e n t s
responding to t h e i r environment i n s t r i c t conformity with a
k i n e t i c p r e s c r i p t i o n that i s c h a r a c t e r i s t i c of r e a c t i o n systems.
(In t h i s connection, i t i s i n t e r e s t i n g to a l l u d e to the remark of
F r e d r i c k s o n and Tsuchiya [1] that "... organisms ... are not t i n
s o l d i e r s ! " ) . I t i s germane that we quote Demain [ 2 ] i n t h i s
regard, "Microorganisms have evolved over the years, developing
b e t t e r and b e t t e r mechanisms to prevent overproduction of t h e i r
metabolites. Yet we m i c r o b i o l o g i s t s and bioengineers are
dedicated to i n c r e a s i n g the i n e f f i c i e n c y of fermentation organ-
isms as we continue to work toward the goal of almost complete
conversion of n u t r i e n t i n t o product with as l i t t l e as p o s s i b l e
going i n t o the m i c r o b i a l protoplasm (except, of course, i f we are
i n the s i n g l e c e l l p r o t e i n b u s i n e s s ) . " F u r t h e r , Demain observes
t h a t , " A l l microorganisms must possess r e g u l a t o r y ( c o n t r o l )
mechanisms i n order to s u r v i v e . Very e f f i c i e n t organisms are
tightly controlled. In fermentation organisms, c o n t r o l s are l e s s
r i g i d but nevertheless present." The i m p l i c a t i o n s of the f o r e -
going a s s e r t i o n s a r e deep and f a r - r e a c h i n g . The biochemical
engineer's o b j e c t i v e i s i n c o n f l i c t with that of the organism
1
and an attempt to ' c o n t r o l the organism without f a m i l i a r i t y with
i t s i n t e r n a l c o n t r o l f e a t u r e s could lead one away from p r o j e c t e d
optimal goals. ( I t must of course be mentioned here that there
e x i s t s e v e r a l instances of dramatic improvements i n the formation
of fermentation products through e f f o r t s based on a q u a l i t a t i v e
understanding of the r e g u l a t o r y processes w i t h i n the c e l l s . Some
of these i n v o l v e genetic v a r i a t i o n s while others do not.)
Demain p o i n t s out that c o o r d i n a t i o n of m i c r o b i a l metabolism
i s a n e c e s s i t y born out of the tremendous d i v e r s i t y which per-

7. RAMKRISHNA Cybernetic View of Growth 163
vades c e l l u l a r c o n s t i t u t i o n and a c t i v i t y . The genetic c a p a c i t y

of a b a c t e r i a l c e l l can accommodate over 1000 enzymes and that
only under some s t r i c t s u p e r v i s i o n can the resources of the c e l l
be invested f r u g a l l y . Such a c o o r d i n a t i o n i s accomplished
c h i e f l y by ( i ) i n d u c t i o n , ( i i ) c a t a b o l i t e r e g u l a t i o n and ( i i i )
feedback r e g u l a t i o n . Induction provides the c e l l with the
mechanism to form enzymes r a p i d l y when needed. C a t a b o l i t e
r e g u l a t i o n becomes u s e f u l , f o r example, i n the i n h i b i t i o n of
formation of c e r t a i n enzymes ( c a t a b o l i t e r e p r e s s i o n ) . Feedback
r e g u l a t i o n i n c l u d e s feedback i n h i b i t i o n and feedback r e p r e s s i o n .
Feedback i n h i b i t i o n i s d i s t i n g u i s h e d from r e p r e s s i o n i n that the
former involves an end product which i n h i b i t s the a c t i o n of an
enzyme somewhere "upstream" the pathway, while the l a t t e r implies
the prevention of the formation of one o r more upstream enzymes
by a d e r i v a t i v e of the
these r e g u l a t o r y processe
elaborate genetic mechanism.
The b i o s y n t h e s i s of a p a r t i c u l a r enzyme i s i t s e l f an
elaborate and complex process i n v o l v i n g s e v e r a l c e l l u l a r com
ponents. The genetic information f o r any p a r t i c u l a r enzyme i s
c a r r i e d i n a s t r e t c h of DNA which i s the ' s t r u c t u r a l gene' f o r
that p r o t e i n . The 'pattern' i s t r a n s c r i b e d i n a s t r i p of the
messenger RNA that d i c t a t e s the proper sequence of amino acids i n
the synthesis of the enzyme. Van Dedem and Moo-Young have made
an i n t e r e s t i n g beginning i n t o i n c o r p o r a t i o n of the operon theory
of Jacob and Monod i n t o a k i n e t i c model f o r enzyme syntheses [ 3 ] .
Indeed even a r e l a t i v e l y simple model leads to many u n i d e n t i f i
able k i n e t i c constants.
Our o b j e c t i v e i n t h i s work i s to present an approach a t
variance from modelling of m i c r o b i a l response based purely on
k i n e t i c considerations. We base t h i s approach on the viewpoint
that while the d e t a i l e d modelling of r e g u l a t o r y processes
(accounting f o r t h e i r underlying genetic mechanisms) i s i n t r a c t
ably complicated, i t may be p o s s i b l e to i n t e r p r e t them as being
i n s p i r e d by an optimal motive. I t would seem that such f a c i l i t y
f o r an optimal response of an organism to i t s environment would
be an a c q u i s i t i o n c o n s i s t e n t with the theory of e v o l u t i o n . While
t h i s viewpoint i s evident i n the statement of Demain quoted
e a r l i e r , i t has been a popular aspect of contemporary b i o l o g y
(see f o r example [ 4 ] ) . This approach based on p o s t u l a t i n g the
existence of an o p t i m a l i t y c r i t e r i o n i s what we have termed as
the 'cybernetic' p e r s p e c t i v e . (Cybernetics, which a r i s e s from
the Greek word χ υ β ε ρ ν η τ η Β meaning steersman, has been defined by
Wiener as c o n t r o l and communication i n the animal and the machine
[5]). The b a s i c merit of the cybernetic approach i s that i t
adopts a mathematically simple d e s c r i p t i o n of a complex organism
but compensates f o r the o v e r s i m p l i f i c a t i o n by a s s i g n i n g an o p t i
mal c o n t r o l motive to i t s response. The i m p l i c a t i o n i s that the
elaborate i n t e r n a l machinery of the c e l l provides the organism
with the f a c i l i t y to implement the c a l c u l a t e d c o n t r o l p o l i c y .

While a t r u l y d e t a i l e d model would a c t u a l l y be concerned with

d e t a i l s of t h i s implementation (even i f i t i s not viewed as such)
they are of no concern to the cybernetic model. There i s of
course no d i r e c t way of confirming an optimal s t r a t e g y so that
i t s a c c e p t a b i l i t y must be based on experimental evidence p e r t a i n -
ing to i t s i m p l i c a t i o n s . T h i s has been the b a s i s of some c r i t i c -
ism of the c y b e r n e t i c approach [6], I t i s emphasized here that
formulations of optimal p o l i c i e s must be construed merely as
i n n o v a t i v e d e s c r i p t i o n ( p r e d i c t i o n ) of observed phenomena r a t h e r
than as " u l t i m a t e " explanations. In t h i s connection, i t i s im-
portant to recognize that the approach could lead to many new and
i n t e r e s t i n g experiments. An example of the cybernetic approach
to m i c r o b i a l growth may be found i n the work of Swanson, A r i s
F r e d r i c k s o n and Tsuchiya [7,8] , who were s p e c i a l l y concerned with
the l a g phase i n s i n g l e s u b s t r a t e systems
I t may be seen tha
invariant strategy rathe
i n the framework of k i n e t i c models. Thus thaapproach envisaged
here i s more l i k e l y to d e s c r i b e t r a n s i e n t semi-continuous e x p e r i -
1
ments, i n which the c e l l s environment may be v a r i e d at w i l l , t h a n
a purely k i n e t i c approach. There are however important con-
s t r a i n t s only w i t h i n which the framework suggested here may be
considered. Since the organisms are deemed to respond based on
an i n v a r i a n t optimal s t r a t e g y ( i . e . , the c e l l s never l o s e s i g h t
of t h e i r optimal goal while experiencing the v a r y i n g environment)
we are not addressing s i t u a t i o n s i n which any a b e r r a t i o n i n the
r e g u l a t o r y mechanisms (leading to non-optimal goals) are encount-
ered. Thus only environmental manipulations i n which no genetic
changes are brought about i n the organisms can be l e g i t i m a t e l y
considered, s i n c e the mutants cannot be expected to r e t a i n the
optimal framework of the o r i g i n a l genotype. Having s a i d t h i s , we
wish to emphasize the d i s t i n c t i o n between s t r i v i n g f o r an optimal
goal and a c t u a l l y accomplishing i t . I f we grant that the f a c i l -
i t y f o r optimal behavior has been acquired f o r c e r t a i n types of
environmental v a r i a t i o n s , then one may expect that as long as the
organisms are subjected to the same c l a s s of environmental
changes, they would not only s t r i v e f o r an optimal goal but a l s o
accomplish i t . On the other hand, i f we subject the micro-
organisms to environmental changes of a type e n t i r e l y d i f f e r e n t
from those with which they can cope i n an optimal way, then the
r e s u l t could w e l l be a non-optimal behavior even i f there have
occurred no genetic changes. Such a b e r r a t i o n s from optimal be-
h a v i o r would seem to come w i t h i n the purview of the c y b e r n e t i c
approach. In f a c t t h i s i s an important i s s u e which we w i l l
address again at a l a t e r stage.
A c l a s s i c example i n which the i n t e r n a l r e g u l a t o r y processes
of the c e l l s play a very important r o l e i s the phenomenon of
d i a u x i c growth discovered by Monod [ 9 ] i n m u l t i p l e s u b s t r a t e
systems. In d i a u x i c growth there i s p r e f e r e n t i a l u t i l i z a t i o n of
c e r t a i n s u b s t r a t e s over o t h e r s , although each s u b s t r a t e by i t s e l f

would have been acceptable to the organism. The s i t u a t i o n

appears to f i t the c y b e r n e t i c approach r a t h e r w e l l s i n c e p r e f e r -
ence f o r a p a r t i c u l a r s u b s t r a t e could w e l l be the r e s u l t of an
optimal s t r a t e g y . We quote again from Demain [2]. "The c e l l
faces a problem when more than one u t i l i z a b l e growth s u b s t r a t e i s
present. Enzymes could be formed to c a t a b o l i z e a l l s u b s t r a t e s ,
but t h i s would be w a s t e f u l . Instead, enzymes are made which
u t i l i z e the best s u b s t r a t e ( u s u a l l y glucose) and only a f t e r ex-
haustion of the primary s u b s t r a t e are enzymes formed which
c a t a b o l i z e the poorer carbon source." That one s u b s t r a t e i s
" b e t t e r " than another i s unmistakably implied although a q u a l i -
f i c a t i o n as to what makes one b e t t e r than the other i s con-
s p i c u o u s l y absent.
THE CYBERNETI
Viewing the c e l l as a c o n t r o l system r a i s e s s e v e r a l impor

tant i s s u e s . We assume that the c o n t r o l system i s much l i k e that
conceived f o r c o n t r o l l i n g i n d u s t r i a l systems. F i r s t , an essen-
t i a l part of c o n t r o l of a process i s measurement of one or more
process v a r i a b l e s . Thus the transformation of n u t r i e n t m a t e r i a l
to protoplasmic mass and a l s o products which are released i n t o
the environment provides a spectrum of q u a n t i t i e s f o r "measure-
ment". I f c o n t r o l i s based on sensing concentrations of en-
vironmental components p r i o r to (such as n u t r i e n t s ) or a t e a r l y
stages of t r a n s f o r m a t i o n , i t comes w i t h i n the category of feed-
forward c o n t r o l . In c o n s t r a s t i f c o n t r o l i s based on c o r r e c t i o n
of performance by measuring v a r i a b l e s a t the downstream end of
the transformation process then we have feedback c o n t r o l . I n -
deed we could have both feedforward and feedback s t r a t e g i e s .
Feedback c o n t r o l i s p o s s i b l e , f o r example, i n muscular a c t i o n
( i n higher organisms) to accomplish a p a r t i c u l a r g o a l . In the
s i t u a t i o n s of i n t e r e s t to us here one i s i n c l i n e d towards a
feedforward s t r a t e g y because an e f f i c i e n t system would gear i t s
i n t e r n a l machinery to prepare ahead i n a complex process of
breakdown of n u t r i e n t s . I t i s recognized however that the p o s s i -
b i l i t y of feedback strategies cannot be e n t i r e l y overlooked.
The next i s s u e i s that of the c o n t r o l o b j e c t i v e . Prior
d i s c u s s i o n s i n t h i s paper and r e f e r e n c e to other works such as
Demain [2] g r a v i t a t e towards the concept of maximizing biomass
p r o d u c t i v i t y . Again t h i s suggestion i s more of an i n t e r e s t i n g
p o s s i b i l i t y than with a view to exclude others that may appear
more a t t r a c t i v e i n subsequent stages of t h i s work. Suppose we
grant that the c e l l ' s goal i s i n maximizing c e l l mass. Two b a s i c
questions a r i s e . I s the d e s i r e d maximization of the c e l l mass
p r o d u c t i v i t y at every i n s t a n t , or over a f i n i t e time i n t e r v a l i n
which case the average p r o d u c t i v i t y i s maximized? We may r e f e r
to the f i r s t case as a "short term" (or instantaneous) perspec-
tive. In the second case, we have a "long term" p e r s p e c t i v e . In
examining the r e l a t i v e p l a u s i b i l i t y of the two schemes we must

bear i n mind that growth represents a complex sequence of reac-

t i o n s o c c u r r i n g n e c e s s a r i l y over a f i n i t e time i n t e r v a l . Thus
an organism e v o l v i n g i n c e r t a i n patterns of environmental v a r -
i a t i o n s could conceivably account f o r t h i s i n formulating i t s
optimal p o l i c y much i n the s p i r i t of "saving ( i t s resources) f o r
1
a r a i n y day". This argument i s i n favor of a 'long term objec-
t i v e . Of course the organism's p r o j e c t i o n of i t s f u t u r e could
prove to be i n e r r o r (since the experimenter can vary i t s e n v i r -
onment at w i l l ) the r e s u l t of which would then be a non-optimal
response. D h u r j a t i [10] has i n v e s t i g a t e d a 'long term' model
f o r d i a u x i c growth which w i l l be discussed subsequently.
A 'short term' p e r s p e c t i v e i s r e f l e c t i v e of a d e c i s i o n to
make the best use of the environment at the i n s t a n t (without
concern f o r the f u t u r e ) . Here one has the option of assuming
that there may or may not b tim dela i th implementatio
of that p o l i c y . I f a smal
lay does not r e f l e c t a organism) y greatly
a f f e c t the organism's response i n slowly v a r y i n g environment.
However, f o r r a p i d environmental changes, s u b s t a n t i a l l y non-
optimal behavior may be expected from such models. Another i n -
t e r e s t i n g s i t u a t i o n to i n v e s t i g a t e here i s the e f f e c t of how
l i m i t a t i o n s on c o n t r o l v a r i a b l e s might a f f e c t a p o l i c y based on
a short term p e r s p e c t i v e . To make t h i s c l e a r e r , suppose the
c o n t r o l o b j e c t i v e i s to be implemented by a l l o c a t i n g e x i s t i n g
resources of the c e l l . Suppose f u r t h e r that resources are con-
s t a n t l y r e p l e n i s h e d by growth processes. A short term perspec-
t i v e makes no a n t i c i p a t i o n about subsequent replenishments so
that i t i s p o s s i b l e to conceive of s i t u a t i o n s where, i n the i n -
t e r e s t of an immediate optimal o b j e c t i v e , an impending c r i s i s
(such as inadequate resources to u t i l i z e a l a t e r f a v o r a b l e en-
vironment) i s not accounted f o r .
Another important i s s u e i s that of the proper c o n t r o l v a r -
i a b l e s . We have j u s t a l l u d e d to the p o s s i b i l i t y of viewing
m i c r o b i a l response as a resource a l l o c a t i o n problem. The r e -
sources might, f o r example, c o n s t i t u t e only a c r i t i c a l resource
such as ATP that i s the primary source of energy f o r metabolic
purposes. I t i s d i f f i c u l t to be any more s p e c i f i c about t h i s
i s s u e at t h i s i n c e p t i v e stage of our work.
In what f o l l o w s we w i l l present two models, one based on a
'long term' p e r s p e c t i v e and another based on a 'short term' per-
s p e c t i v e both addressing the phenomenon of d i a u x i c growth i n
m u l t i p l e substrate systems.
Growth i n M u l t i p l e Substrate Systems
Monod's pioneering work on d i a u x i c growth [ 9 ] i s an i n t e r -

e s t i n g s i t u a t i o n i n which i n t e r n a l r e g u l a t o r y processes appear
to play an important r o l e . Here, the c e l l s are confronted with
a mixture of two substrates ( g e n e r a l l y sugars) such as, say g l u -
cose and xylose. While growth could occur f o l l o w i n g a normal

lag phase with e i t h e r of the substrates i n the absence of the

other, i n the mixed substrate environment the c e l l s show an ex
c l u s i v e preference f o r glucose u n t i l a l l of i t i s v i r t u a l l y ex
hausted before xylose i s u t i l i z e d . This preference f o r glucose
could be i n t e r p r e t e d as the r e s u l t of an optimal d e c i s i o n .
D h u r j a t i [10] has considered a cybernetic model based on a long
term p e r s p e c t i v e of m i c r o b i a l response to a m u l t i s u b s t r a t e en
vironment. Thus he proposes that the c e l l s i n the inoculum, on
exposure to a mixture of two substrates (such as glucose) and
(such as x y l o s e ) , a t some i n i t i a l i n s t a n t (t=0) must decide
on a program of u t i l i z a t i o n of the two substrates such that a l l
the obtainable biomass i s r e a l i z e d i n the s h o r t e s t time. The
optimal o b j e c t i v e i s t h e r e f o r e the maximization of the average
biomass p r o d u c t i v i t y over the e n t i r e period of growth. The
t a c i t i m p l i c a t i o n here i tha batch h i d
the outset. T h i s " p r o j e c t i o
long term p o l i c y . Thu y
presumed to commit i t s e l f to a p o l i c y of a l l o c a t i o n of i t s r e
sources based on the assumption that no subsequent replenishment
of e i t h e r substrate would be a v a i l a b l e . Of course a c t u a l ex
perience could be otherwise s i n c e the experimenter could vary
the environment a t w i l l . The question a r i s e s then as to how the
model would view the growth of the c u l t u r e i n an environment
thus manipulated. Could the c e l l s not review the s i t u a t i o n a t
any i n s t a n t when there i s an e x t e r n a l manipulation and make a
r e a l l o c a t i o n of i t s resources ( i . e . , r e v i s e i t s c o n t r o l p o l i c y ) ?
If so could t h i s occur c o n t i n u a l l y r e g a r d l e s s of the speed with
1
which the substrate environment i s varied? D h u r j a t i s model i s
predicated on a long term p o l i c y which i s provoked only when a
step change i n s u b s t r a t e concentration occurs. While there a r e
some conceptual d i f f i c u l t i e s with t h i s model connected with the
questions j u s t r a i s e d , i t does lead to the d i a u x i c growth curve.
To h i g h l i g h t on the model, the c e l l growth process i s viewed as
comprising the growth r e a c t i o n
Β + S + E + 2B
± ± i = 1, 2,...
t h
where Β i s the biomass, S i i s the i substrate, E i i s consider
t n
ed as a 'key enzyme' i n the uptake of the i s u b s t r a t e . The
enzyme E i i s synthesized a t a r a t e dependent on the a l l o c a t i o n
of i t s resources and D h u r j a t i ' s [11] equation i s reproduced here
de.
1
- r - = ot.Ru. - B.e. (1)
dt i l i l
where e . i s the enzyme concentration, a i and $^ are r a t e con
#
s t a n t s , R i s a f i x e d r a t e of t o t a l resource a v a i l a b i l i t y the
f r a c t i o n of which a l l o c a t e d f o r enzyme E i i s given by U i . The
#
resource a v a i l a b i l i t y r a t e R i s assumed to be f i x e d here a l

though i n a more elaborate model i t i s p o s s i b l e to i n c l u d e r e
plenishment of resources during growth. Note that E q . ( l ) im
p l i e s a maximum l e v e l of enzyme concentration because of the
first order breakdown r a t e . The substrate consumption r a t e i s

assumed to i n v o l v e the enzyme c o n c e n t r a t i o n i n the f o l l o w i n g man

ner ds. V e.s
dt^ =
" b
K~+~s7 i - l . 2.·.. (2)
1 1
while growth on the substrates i s w r i t t e n as
J = - b E Y . ^ (3)
dt i ι dt
The biomass, whose concentration i s represented by b, i s viewed
as comprising key enzymes and the r e s t of the biomass. For the
sake of s i m p l i c i t y no m a t e r i a l resources have been e x p l i c i t l y i n
cluded i n the biomass f o r the present.
D h u r j a t i [10] based the optimal a l l o c a t i o n of resources on
minimizing the time r e q u i r e d f o r r e a l i z i n g a l l of the biomass
from the d i f f e r e n t s u b s t r a t e s . Thus the c o n t r o l o b j e c t i v e i s
given by
Mi
l 1
i î
where t f i s the time required f o r the substrate l e v e l s to drop to
some preassigned values (since a r b i t r a r i l y small values would
lead to a r b i t r a r i l y l a r g e times mathematically).
1
This completes D h u r j a t i s model. The mathematical techniaue
of computing the optimal a l l o c a t i o n r a t e s to accomplish the objec-
t i v e (4) i s contained i n the well-known minimum p r i n c i p l e of
Pontryagin [11] which w i l l not be discussed here. The l i n e a r
f u n c t i o n a l i t y of the c o n t r o l v a r i a b l e s u^ leads to what i s r e f e r -
red to as a "bang-bang" p o l i c y which i n the present context im-
p l i e s e x c l u s i v e u t i l i z a t i o n of one of the s u b s t r a t e s . ( D h u r j a t i
[10] has i n v e s t i g a t e d the p o s s i b i l i t y of s i n g u l a r c o n t r o l which
could accommodate simultaneous consumption of d i f f e r e n t sub-
s t r a t e s and f i n d s the c o n s t r a i n t s to be i m p l a u s i b l e ) . The
d i a u x i c growth s i t u a t i o n i s thus described by t h i s model.
D h u r j a t i was able to adapt model constants to d e s c r i b e growth
data obtained by him on K l e b s i e l l a pneumoniae grown on a mixture
of glucose and xylose. The preference f o r glucose with a higher
growth r a t e on i t i s c o n s i s t e n t with the i n t e r p r e t a t i o n s of the
model. The r e s u l t i s d i s p l a y e d i n F i g u r e 1. The question a r i s e s
as to whether the organism i n f a c t consumes both glucose and
xylose a f t e r the former has dropped to s u f f i c i e n t l y low l e v e l s .
While t h i s i s not r e a d i l y v e r i f i a b l e because of the d i f f i c u l t i e s
inherent i n measuring small d i f f e r e n c e s i n small substrate con-
1
c e n t r a t i o n s some a s s e r t i o n s can be made i n regard to D h u r j a t i s
model. The l i n e a r i t y i n the c o n t r o l v a r i a b l e w i l l permit only
e x c l u s i v e u t i l i z a t i o n of one substrate or the other. I f f o r ex-
ample 'more p r e f e r r e d * s u b s t r a t e , say drops to s u f f i c i e n t l y
low l e v e l s , the preference would switch to the 'less p r e f e r r e d '
substrate ( S ) a f t e r which e x c l u s i v e u t i l i z a t i o n of S w i l l
2 2
f o l l o w u n t i l again i t i s p r o f i t a b l e to switch to Si and so on.

This d i d not occur i n the c a l c u l a t i o n s presented because of the
high discrepancy between growth r a t e s on the two substrates and

Figure 1. The diauxic growth curve predicted by Dhurjati's model for Klebsiella
pneumoniae grown on a glucose-xylose mixture. Dashed lines, model; solid line,
experimental.

the preassigned l e v e l s of substrate concentrations at t f . Un-

doubtedly, f o r s u b s t r a t e p a i r s on which the d i f f e r e n c e i n the
growth r a t e i s not very great, the model would p r e d i c t a l t e r n a t e
switching between substrates u n t i l t f i s reached. Unless non-
l i n e a r r e l a t i o n s h i p s are entertained i n the c o n t r o l v a r i a b l e s
one i s constrained to t h i s s o r t of p r e d i c t i o n . I t w i l l be of i n -
t e r e s t to i n v e s t i g a t e experimentally whether such a l t e r n a t e
switching i s i n f a c t encountered. I t i s i n t h i s connection that
the measurement of d i s s o l v e d oxygen becomes a very i n t e r e s t i n g
diagnostic tool. For example, a change i n p o l i c y (such as
switching substrates) i s r e a d i l y detected by a r e l a t i v e l y abrupt
change i n d i s s o l v e d oxygen while no corresponding change i s l i k e -
l y to be apparent i n e i t h e r the biomass or the substrate concen-
trations. In Figure 2, are presented some r e s u l t s from
1
D h u r j a t i s experiments
terrupted by v a r y i n g amount
time. The d i s s o l v e d oxyge abrup p oxyge
c e n t r a t i o n at the i n s t a n t of glucose a d d i t i o n s i n c e the growth
r a t e i s higher on glucose and c a l l s f o r increased oxygen con-
sumption. The oxygen concentration shoots up again when the
added glucose i s exhausted. The concentration to which t h i s up-
shoot occurs depends on time elapsed f o l l o w i n g glucose a d d i t i o n
before the the organism i s ready to r e v e r t to xylose again. If
t h i s time i s short, growth resumes on xylose at the same l e v e l
at which i t was i n t e r r u p t e d . I f the time span i s longer the r e -
sumption occurs at a somewhat reduced l e v e l p o s s i b l y i n d i c a t i n g
that some breakdown of the key enzyme f o r xylose may have occur-
ed i n the i n t e r i m . There appears to be l i t t l e doubt that the
measurement of d i s s o l v e d oxygen w i l l play a key r o l e i n the
diagnosis of switching p o l i c i e s of the c e l l ' s i n t e r n a l machinery.
Kompala [12] has considered a model based on a short term
p e r s p e c t i v e f o r the growth s i t u a t i o n with which D h u r j a t i d e a l t .
This model adopts the viewpoint that the c e l l o p t i m a l l y a l l o c a t e s
i t s resources at every i n s t a n t . Thus the s t a t e of the c e l l and
i t s environment r e s u l t s i n an immediate resource a l l o c a t i o n with
the o b j e c t i v e of maximizing the a c c e l e r a t i o n ( or minimizing the
d e c e l e r a t i o n ) i n growth r a t e . The i m p l i c a t i o n i s that the c e l l
i s ever a l e r t to environmental changes and always does the best.
Some p o s s i b l e o b j e c t i o n s may a r i s e . F i r s t , i t i s not c l e a r ,
whether i t i s reasonable to expect that regardless of the r a t e
at which environmental changes occur, the c e l l s would o p t i m a l l y
gear i t s enzyme synthesis r a t e unhampered by any metabolic
i n e r t i a ; f o r example, could c o n t r o l a c t i o n taken at a p a r t i c u l a r
i n s t a n t r e s u l t i n an instantaneous change i n enzyme synthesis
rate? An attempt to cure t h i s would i n essence be a renewed
quest f o r a 'long term' model. Second, with the imposition of a
proper penalty f o r d e c i s i o n , i t does not n e c e s s a r i l y f o l l o w that
what i s best f o r each i n s t a n t i s best on the whole, an i s s u e
that has been r a i s e d before. Kompala's model constants do adapt
to produce the d i a u x i c curve. His model equations are v i r t u a l l y

TIME (HOURS)
Figure 2. Dissolved oxygen curve for Klebsiella pneumoniae grown on xylose
with periodic addition of glucose. G, 250 mg/mL glucose; X, 250 mg/mL xylose
(t = 0, 50 mLX).

the same a s t h o s e o f D h u r j a t i [10] e x c e p t f o r t h e e n z y m e s y n t h e

sis and breakdown r a t e s i n Eq.(1) w h i c h must be r e p l a c e d by
de. α!s.
dT - φ τ γ ι " 6
i i e ( 5 )
At any i n s t a n t t , the s t a t e o f t h e biomass i s determined by t h e

c o n c e n t r a t i o n s b , a n d {e^} w h i l e t h a t o f t h e e n v i r o n m e n t b y { s ^ } .
The growth r a t e a t t h i s i n s t a n t i s d e t e r m i n e d by t h e above v a r i
ables. However, t h e o r g a n i s m g e a r s t h e enzyme s y n t h e s i s r a t e a t
t h a t i n s t a n t t o maximize d^b/dt by m a n i p u l a t i n g the c o n t r o l v a r
i a b l e s {u^}. Because u ^ occurs l i n e a r l y i n Eq.(5) t h e o p t i m a l
decision is readily arrived at. By l e t t i n g
V.a's. 2
j = { i : m a x
(K'Vs.) 2 } ( 6 )
we find the optimal polic

"ι = < 7)
This remarkably simple r e s u l t as against the use of the

Pontryagin p r i n c i p l e r e q u i r e d f o r D h u r j a t i ' s model i s indeed a
great asset f o r t h i s model. ( P a r a l l e l to the p o s s i b i l i t y of
s i n g u l a r s o l u t i o n s i n D h u r j a t i ' s f o r m u l a t i o n , one needs to i n
v e s t i g a t e s i m u l t a n e o u s u t i l i z a t i o n h e r e w h e n Vjxl^s^ /^2
+ s^ 2
versus intersect for different i ) .

F i g u r e 3 shows t h e r e s u l t s o f c a l c u l a t i o n s f o r b a t c h g r o w t h
w i t h two s u b s t r a t e s . The s e q u e n t i a l u t i l i z a t i o n of t h e sub
s t r a t e s c h a r a c t e r i s t i c of the d i a u x i c growth s i t u a t i o n i s r e
produced by the model. T h i s by i t s e l f i s no more t h a n e s t a b l i s h
i n g t h a t t h e model p r e m i s e s a r e n o t i m p l a u s i b l e and i s thus more
a b e g i n n i n g t h a n t h e e n d . I n F i g u r e 4, t h e m o d e l ' s perspective
of an experiment i n w h i c h t h e growth on t h e ' l e s s preferred'
s u b s t r a t e i s i n t e r r u p t e d b y a d d i t i o n o f t h e more p r e f e r r e d s u b
strate. The immediate s w i t c h t o glucose observed i n D h u r j a t i ' s
e x p e r i m e n t s i s accommodated b y t h i s m o d e l . I f i t i s agreed that
a model based on a long term p e r s p e c t i v e such as that of D h u r j a t i
w i l l ignore environmental changes other than those on which t h e
o p t i m a l p o l i c y was b a s e d , t h e n t h e p e r t u r b e d b a t c h e x p e r i m e n t
j u s t r e f e r r e d to cannot be described by D h u r j a t i ' s model. In
t h i s sense, Kompala's model i s an improvement on D h u r j a t i ' s
model.
N e i t h e r o f t h e above models e x p l i c i t l y i n c l u d e t h e o r i g i n a l
c l a i m that the resources a r e a part of the biomass and t h e i r
a v a i l a b i l i t y may t h u s b e c o n s t r a i n e d . This feature i s readily
b u i l t into either model. F o r e x a m p l e , we may d e n o t e t h e r e
sources by R, t h e i r c o n c e n t r a t i o n by r , and p o s t u l a t e that t h e i r
formation occurs by growth, and consumption by t h e i r a l l o c a t i o n
f o r enzyme s y n t h e s i s . A d m i t t e d l y t h i s i s somewhat o f a n a r r o w
v i e w o f how r e s o u r c e s may b e u s e d i n m e t a b o l i c p r o c e s s e s b u t t h i s
s i m p l i f i c a t i o n i s v i r t u a l l y a t t h e same l e v e l a s t h a t i n h e r e n t i n
the lumped k i n e t i c models o f t h e p a s t . The d i f f e r e n t i a l equation
f o r r may b e w r i t t e n a s

UNDISTURBED BATCH EXPERIMENT
0.000 1.000 2.000 3.000 4.000

TIME
Figure 3. The diauxic growth curve predicted by Kompala's short term model.
AG (glucose) = 90.0; AX (xylose) = 60.0.

PERTURBED BATCH EXPERIMENT
0.000 1.000 2.000 3.000 4.000

TIME
Figure 4. Growth on xylose interrupted by glucose addition as predicted by

Kompala. AG = 90.0; AX = 60.0.

β
where ^Λ ^9 u r
i ) i s the r a t e of consumption of resources f o r
the synthesis of i enzyme E^; i t i s r e l a t e d to the r a t e of
t n
synthesis of E-^ to w i t h i n a s t o i c h i o m e t r i c m u l t i p l e . In Eq.(8)

γ i s a s t o i c h i o m e t r i c constant. In p a r t i c u l a r i t i s of i n t e r e s t
to note that
Σ u. < 1 (9)
1
i -
where the i n e q u a l i t y s i g n i s to account f o r the p o s s i b i l i t y that
not a l l of the a v a i l a b l e resources need n e c e s s a r i l y be invested
at any i n s t a n t . A model based on a long term p e r s p e c t i v e could
i n f a c t c a l l f o r a p o l i c y of u n d e r u t i l i z a t i o n of resources at a
given i n s t a n t i f a subsequent stage c a l l s f o r increased r e
sources. Propose, f o
f ( s U r ) = 6 ( 1 0 )
i i ' i ί(Κ| + S i ) (κ. + u.r)
with Eq.(5) modified by
de. a. S i u r
±
- 6!e. (11)
dt (iq + S i ) (κ ± + u.r) M
i ic
where the δ.^ i n Eq.(lO) represents a r e a d i l y i n t e r p r e t e d s t o i

chiometric constant. In E q . ( l l ) i s a M i c h a e l i s constant.
The n o n l i n e a r i t y with respect to u^ i n E q . ( l l ) i s to be noted
p a r t i c u l a r l y , which may e l i m i n a t e the e x c l u s i v e bang-bang optim-
a l i t y r e s u l t . Thus simultaneous u t i l i z a t i o n of substrates could
a l s o be p r e d i c t e d by such models. I f ample resources are pre
sent, then the above Michaelis-Menten k i n e t i c expression i n Eq.
(11) prevents o v e r - a l l o c a t i o n f o r any p a r t i c u l a r enzyme.
Whether the modelling be done based on a short or long term
p e r s p e c t i v e , the issue r a i s e d e a r l i e r i n regard to provoking
non-optimal responses by s u i t a b l e environmental v a r i a t i o n s i s an
important one. The short term models would not n e c e s s a r i l y pre
d i c t , f o r example, the maximum average p r o d u c t i v i t y of biomass
over the e n t i r e period of growth. S i m i l a r l y the long term
models would not p r e d i c t the maximum average p r o d u c t i v i t y i f
changes o c c u r r i n g i n the environment are other than those which
were accounted f o r i n the o p t i m i z a t i o n . Thus an i n t e r e s t i n g
method of e v a l u a t i n g the responses p r e d i c t e d by the models i s to
compare them with those i n which " p e r f e c t optimal behavior" i s
considered. Obviously p e r f e c t optimal behavior may be p r e d i c t e d
by using the Pontryagin p r i n c i p l e on the s t a t e equations i n
which the imposed environmental v a r i a t i o n s are b u i l t i n . One
need hardly point out that the s u p e r p o s i t i o n of the a c t u a l
m i c r o b i a l response r e l a t i v e to that of the model and that based
on p e r f e c t o p t i m a l i t y would then be of the most v i t a l i n t e r e s t .

Concluding Remarks
Our attempt here has been to f i n d a mathematical framework

for c e r t a i n well-expressed ideas concerning m i c r o b i a l behavior
i n regard to the r o l e of i n t e r n a l r e g u l a t o r y processes. This
framework e s s e n t i a l l y draws on the formulation of k i n e t i c models
i n that biomass i s lumped i n t o a small number of component
masses, and growth being regarded as increase i n t h e i r q u a n t i t i e s
by i n t e r a c t i o n with n u t r i e n t s . The component masses i n c l u d e key
enzymes whose s y n t h e s i s i s c o n t r o l l e d by optimal a l l o c a t i o n of
c e r t a i n c r i t i c a l resources. Various important i s s u e s have been
r a i s e d connected with t h i s c y b e r n e t i c p e r s p e c t i v e . The main
m o t i v a t i o n i s to evaluate the consequences of hypotheses concern
ing i n t e r n a l r e g u l a t o r y processes, and to f i n d experimental
s i t u a t i o n s i n which they manifest i n the most unambiguous ways
It i s not e s s e n t i a l tha
cise quantitative f i t s
data. Rather the f u n c t i o n of such model b u i l d i n g i s simply to
develop b e t t e r understanding of r e a l systems. Unfortunately, i t
i s not o f t e n that t h i s aspect of modelling i s perceived as such.
Ackrawledgnents
The work presented here owes much to the p a r t i c i p a t i o n of

graduate students Prasad D h u r j a t i and Dhinakar Kompala, and to
my colleagues Michael C. F l i c k i n g e r and George T. Tsao, and i s
the subject of a f u t u r e j o i n t p u b l i c a t i o n under p r e p a r a t i o n .
P a r t i a l support by the N a t i o n a l Science Foundation under Grant
No. Eng 7820964 i s g r a t e f u l l y acknowledged.
Nomenclature
Β Biomass
b Concentration of biomass
Ε Key enzyme
e Concentration of key enzyme
f Rate of comsumption of resources
for s y n t h e s i s of key enzyme.
Κ M i c h a e l i s constant f o r growth
Κ» M i c h a e l i s constant of enzyme syn
thesis
R T o t a l resource a b a i l a b i l i t y r a t e
r Concentration of l i m i t i n g resource
S Substrate
s Concentration of s u b s t r a t e
t Time
u Fractional allocation
V Rate constant i n growth

Yield coefficient
Greek symbols
α,a1
Rate constants i n enzyme s y n t h e s i s
β Rate constant f o r enzyme breakdown
Ύ S t o i c h i o m e t r i c constant f o r r e
plenishment of resources by growth
S t o i c h i o m e t r i c constant f o r con
sumption of resources f o r enzyme
synthesis
Kroenecker d e l t a equals u n i t y when
i=j and vanishes otherwise
M i c h a e l i s constant f o r consumption
of resources f o r enzyme s y n t h e s i s
Subscripts
Refers to i substrate
Refers to f i n a l s t a t e of growth
when s u b s t r a t e s drop to p r e a s s i g n -
ed l e v e l s
Literature Cited
1. Fredrickson, A.G., and H.M. Tsuchiya, in "Chemical Reactor

Theory," E. L. Lapidus and N.R. Amundson, Prenstice-
Hall, N . J . , 1975.
2. Demain, A . L . , Adv. Biochem. Eng. Ed. T.K. Ghose, A. Fiechter;
N. Blakebrough Springer Verlag, Heidelberg, 1971.
3. Van Dedem, G., and M. Moo-Young, Biotech, and Bioeng., (17),
927, 1975.
4. Rosen, R., "Foundations of Mathematical Biology," Ed. R.
Rosen, Academic Press, Vol II, 1972.
5. Wiener, Ν., "Cybernetics or Control and Communication in the
Animal and the Machine," M.I.T. Press, Mass. 1975.
6. Oster, G. and E.O. Wilson, Monographs in Population Biology
12, ch. 8. "A critique of optimization theory in
evolutionary biology," Princeton U. Press, 1970.
7. Swanson, C.H., R. Aris, A.G. Fredrickson and H.M. Tsuchiya,
J. Theor. Biol., (12), 228, 1966.
8. Tsuchiya, H.M., Fredrickson, A.G., and Aris, R., Adv. Chem.
Eng., (6), 125, 1966.
9. Monod, J., "Recherches sur la croissance des cultures
bacteriennes," Herman & Cie. Paris, 1942.

10. Dhurjati, P., Ph.D. Dissertation, Purdue University, West

Lafayette, Indiana, 1982.
11. Pontyagin, L . S . , V.G. Boltyanski, R.V. Gamkrelidze and E.F.
Mischenko, "The Mathematical Theory of Optimal
Processes," Interscience, Ν.Y., 1962.
12. Kompala, D.S. M.S. Dissertation, Purdue University, West
Lafayette, Indiana, 1982.

8
Strategies for Optimizing Microbial Growth

and P r o d u c t Formation
CHARLES L. COONEY
Massachusetts Institute of Technology, Department of Nutrition and Food Science,
Biochemical Engineering Laboratory, Cambridge, MA 02139
An essential element for fermentation processes, wheth

er for cell mass o
the organism. Optimizin
requires balancing the economic feasibility of maintain
ing a suitable chemical and physical environment, while
meeting the physiological needs of the organism. Using
several examples of bacteria, yeast and mold fermenta
tions, strategies for optimizing growth are examined
and compared. In the first case, the objective of the
fermentation is cell mass production; two examples are
Baker's yeast (Saccharomyces cerevisiae) and single
-cell protein (Hansenula polymorpha) production. In
seeking to optimize productivity and conversion yields,
carbon source and oxygen become key operating vari
ables. The second case focuses on production of en
zymes; in particular, the production of heparinase by
Flavobacterium heparinum and α-glucosidase (maltase)
by Saccharomyces italicus. The objective functions are
maximum total activity and maximum productivity. Im
portant variables are growth rate and composition and
concentration of nutrients in the environment. The
level of these enzymes is subject to metabolic regulation
in response to the environment and the productivity de
pends also on microbial growth. Lastly, the third case
to be examined is metabolite production; the production
of penicillin by Penicillium chrysogenum is discussed.
Optimization of growth must consider not only rapid
accumulation of cell mass, but also conditioning of the
cell to maximize the rate and extent of product forma
tion. Maintenance of adequate nutrients in the environ
ment, with high levels of some and low levels of others,
is essential. An analysis of strategies to meet the meta
bolic demands provides insight into ways to improve the
organism through genetic engineering, as well as the
process through bioreactor design.
0097-6156/83/0207-0179$06.00/0

T h e common theme i n the development of optimum fermenta-

tion strategies i s "environmental management". T h e examples de-
s c r i b e d here cover a spectrum of fermentations i n c l u d i n g cell
mass, enzyme a n d secondary metabolite p r o d u c t i o n , y e t i t will be-
come apparent that there i s a common need for c a r e f u l e n v i r o n -
mental management with particular emphasis on carbon source
management throughout the fermentation.
T h e objective functions important to fermentation process
optimization a r e : volumetric p r o d u c t i v i t y , p r o d u c t concentration,
c o n v e r s i o n y i e l d a n d p r o c e s s r e p r o d u c i b i l i t y . Maintaining a h i g h
p r o d u c t i v i t y will allow one to maximize r e t u r n on capital i n v e s t -
ment. T h i s i s p a r t i c u l a r l y important f o r the development o f new
p r o c e s s e s , as a consequence of the capital i n t e n s i v i t y o f fermen-
tation processes. T h e need to achieve a n d maintain h i g h p r o d -
uct concentrations reflect th importanc f i th
of p r o d u c t i o n . Achievemen
s a r y to minimize raw material , hig
v e r s i o n y i e l d from the carbon source will minimize the demand for
o x y g e n a n d the need f o r heat removal, thus minimizing these op-
e r a t i n g costs ( 1 ) . L a s t l y , r e p r o d u c i b i l i t y i s essential to minimize
both capital and o p e r a t i n g c o s t s , as well as meet p r o d u c t i o n goals.
T h e s e objective functions are not equally important, however, i n
all fermentations as i l l u s t r a t e d i n the following b r i e f case studies
which a r e compiled here to illustrate the importance of e n v i r o n -
mental management i n d e v e l o p i n g strategies for optimizing micro-
bial growth a n d p r o d u c t formation.
Production o f C e l l Mass
T h e manufacturing costs for the p r o d u c t i o n of microbial cell

mass are dominated b y : (i) the raw materials cost, especially the
c a r b o n source; (ii) capital investment, i n the case o f a new manu-
f a c t u r i n g facility; a n d , (iii) e n e r g y c o s t s , with most of the ener-
gy b e i n g used for o x y g e n t r a n s f e r a n d heat removal (1, 2). A s
a consequence, it i s essential to achieve h i g h c o n v e r s i o n y i e l d s
while maintaining h i g h p r o d u c t i v i t i e s from the b i o r e a c t o r . T w o
approaches to these goals a r e i l l u s t r a t e d b y the following:
Baker's Yeast P r o d u c t i o n . One of the oldest fermentation

processes for the p r o d u c t i o n o f cell mass i s Baker's yeast p r o d u c -
t i o n . T h e actual p r o d u c t of the fermentation i s cell mass a n d i t s
" b a k i n g power", o r ability to generate carbon dioxide u n d e r bak-
i n g conditions. T h i s i n d u s t r y , which was modernized b y Pasteur
i n the 1800's, i s c h a r a c t e r i z e d b y i t s h i g h s e n s i t i v i t y to raw ma-
terial costs v a r i a b l e s o r seasonal demand a n d need to maintain a

8. COONEY Strategies for Optimizing Growth 181
p r o d u c t acceptable to the baker. T h e objective i n process devel-

opment i s to achieve h i g h c o n v e r s i o n y i e l d s of yeast from t h e
c a r b o n source at a h i g h p r o d u c t i v i t y while maintaining the best
b a k i n g power. T h e solution to meet these objectives has been
the evolution of the fed-batch fermentation p r o c e s s .
It has often been suggested that Baker's yeast could be most
economically p r o d u c e d b y a continuous p r o c e s s , however, there
is little i n c e n t i v e to c o n v e r t e x i s t i n g (and fully depreciated c a p i -
tal investment) from fed-batch to continuous operation. T h e fed-
b a t c h fermentation allows a plant c o n t a i n i n g many fermentors to
operate with a variable p r o d u c t demand. F u r t h e r m o r e , the food
i n d u s t r y p r o d u c i n g Baker's yeast i s not i n t e r e s t e d i n a process
change which may alter their p r o d u c t i d e n t i t y a n d hence d e s i r -
ability b y the baker. F o r these reasons, fed-batch operation
o f f e r s maximum plant operatio f l e x i b i l i t y It i i n t e r e s t i n t
note, however, i n c o n s i d e r i n
be more i n c e n t i v e to g proces
r e d u c i n g the e n t r y costs associated with this capital i n t e n s i v e i n -
dustry.
T h e s t r a t e g y u n d e r l y i n g the operation of the fed-batch ferm-
entation i s b a s e d on matching the s u p p l y of c a r b o n source with
the demand b y the microorganism. T h e rationale f o r this i s i l l u s -
t r a t e d i n F i g u r e 1 ( 3 ) . In o r d e r to maximize the c o n v e r s i o n
y i e l d , there i s a narrow r a n g e of growth rates between 0.075 a n d
1
0.25 h " that c a n be u s e d . It i s important to stay above 0.075
h"* to p r e v e n t excessive maintenance metabolism ( 4 ) . In a d d i -
1
t i o n , the growth rate must be below 0.25 h " to p r e v e n t ethanol
p r o d u c t i o n which o c c u r s i n response to the C r a b tree effect. In
addition, it i s imperative that sufficient o x y g e n be available to
p r e v e n t anaerobic p r o d u c t i o n o f ethanol.
Several i n v e s t i g a t o r s (3, 5) have p r o p o s e d the application o f
computer process control to this p r o c e s s . U s i n g on-line measure-
ments o f a i r flow rate, o x y g e n consumption a n d carbon dioxide
p r o d u c t i o n i n combination with a set of material balance equations
d e s c r i b e d b y Cooney et a l . ( 6 ) , it i s possible to calculate on-line
the cell concentration ancT growth r a t e a n d a p p l y a modified feed-
f o r w a r d control s t r a t e g y to increase (or decrease) the s u g a r a d -
dition rate so that t h e specific growth rate does not exceed a
c r i t i c a l value while at the same time maximizing the volumetric
p r o d u c t i v i t y (3. 7). R e s u l t s of a t y p i c a l fermentation are shown
i n F i g u r e 2 a n d a summary o f r e s u l t s from a v a r i e t y o f fermenta-
tions u n d e r v a r i o u s o p e r a t i n g conditions are shown i n T a b l e I.
From an a n a l y s i s o f these fermentations, it i s clear that the
k e y to a s u c c e s s f u l fermentation i s c a r e f u l management o f the
c a r b o n source; this i s achieved b y continuous assessment of the

00
Ν)
0.1 02 0.3 0.4 05 0.1 j 0.2 0.3 0.4

CELLULAR YIELD (g/g) j SUGAR CONC. ( g/1.)
0.2 0.3 0.4

SUGAR CONC. ( g / I.)
Figure 1. Relationships in baker's yeast fermentation. Left, specific growth rate and cell yield;

upper right, sugar concentration and specific growth rate; and lower right, sugar concentration and
ethanol production rate. Reproduced, with permission, from Ref. 3. Copyright 1979, John Wiley
& Sons, Inc.
Figure 2. Kinetics of baker's yeast fermentation under feedback-modified feed-

forward computer control. Reproduced, with permission, from Ref. 3. Copyright
1979, John Wiley & Sons, Inc.

Table I
COMPARISON O F C O M P U T E R - C O N T R O L L E D Y E A S T F E R M E N T A T I O N S WITH DIFFERENT

INITIAL C E L L C O N C E N T R A T I O N S
Overall
Initial Final Fermentation cell y i e l d Volumetric
cell cone cell cone time (g cells/ productivity
( g/liter) (g/liter) (hr) g sugar) (g/liter-hr)
3.8 59.1 17.0 0.49 3.3

4.9 52.0 16.0 0.49 3.1
5.8 54.6 12.8 0.50 4.0
8.9 54.1 14.0 0.51 3.4
9.0 49.3 11.7 0.49 3.7
14.4 63.2 10.2 0.50 5.2
Source: Ref. 3.

demand for the c a r b o n source a n d a feedback modified feedfor-

w a r d c o n t r o l s t r a t e g y to match s u p p l y a n d demand.
S i n g l e - C e l l P r o t e i n . T h e concept of single-cell p r o t e i n ,
while not new i n that it was utilized b y both the A z t e c s a n d v a r -
ious c u l t u r e s i n c e n t r a l A f r i c a , has only r e c e n t l y (since 1960)
been p r o p o s e d as a means for p r o d u c i n g l a r g e - s c a l e , h i g h quality
protein to supplement traditional p r o t e i n resources ( 8 ) . T h e man-
u f a c t u r i n g cost for S C P i s dominated b y the raw material cost, as
well as capital investment ( 1 ) . A s a consequence, the objective
i n p r o c e s s development i s to maximize the c o n v e r s i o n y i e l d a n d
the volumetric p r o d u c t i v i t y . In this way, the o p e r a t i n g a n d cap-
ital investment cost c a n be minimized. Continuous c u l t u r e o f f e r s
a u s e f u l a p p r o a c h i n which cells c a n be grown u n d e r c a r b o n
source to p r o t e i n u n d e
ity.
Two strategies fo optimizatio o productio are sugges
ted i n F i g u r e 3. F i g u r e 3A shows a traditional continuous c u l t u r e
isotherm. T h e maximum p r o d u c t i v i t y o c c u r s at a value D x ( 9 ) . m a
However, this analysis does not lead to a n economic optimum op-

e r a t i n g condition. It assumes that the inlet feed concentration,
S , i s a constant u n d e r all o p e r a t i n g conditions, a n d furthermore
0
does not take into account the need to operate the fermentor at
i t s maximum p r o d u c t i v i t y , the value o f which i s determined b y the
o x y g e n t r a n s f e r rate (or i n the case o f v e r y large r e a c t o r s , the
heat t r a n s f e r r a t e ) . T h i s i s i l l u s t r a t e d i n Equation 1:
m a x
W m = DX m = Y n R (1)
m m 0 2 0 2
where W m i s the maximum p r o d u c t i v i t y

D i s the dilution rate
max
X_ i s the maximum cell concentration allowed b y
m C>2
a x
Rj? i s the maximum o x y g e n t r a n s f e r rate
u
2
Plotting the maximum cell density permitted at a given o x y g e n

t r a n s f e r rate ( a n d hence constant volumetric p r o d u c t i v i t y ) , gives
the r e s u l t s shown i n F i g u r e 3B. In this a n a l y s i s , S , the feedQ
concentration o f c a r b o n source, i s not constant. C o n s i d e r i n g the

s t r a t e g y for operation, one will choose a dilution rate t h a t : (1)
will minimize the r e s i d u a l limited n u t r i e n t concentration a n d t h u s
achieve highest substrate utilization a n d minimum waste treatment
load, (2) will maximize the conversion y i e l d o f the limiting n u t r i -
ent, the c a r b o n s o u r c e , a n d (3) will maximize the cell concentra-
tion to minimize r e c o v e r y c o s t s . T h e d e s i r e d dilution rate i s i n -
dicated b y the arrow on F i g u r e 3B. A g a i n , the s t r a t e g y relates
to effective management o f the c a r b o n s o u r c e . In this r e g a r d ,

Dilution Rate
Figure 3. Strategies for optimization of continuous culture for production of

single-cell protein. Top, under continuous culture isotherm with a fixed value for
carbon source feed concentration. D is the dilution rate of maximum productivity.
m
Bottom, comparison of isotherm for fixed substrate feed concentration, S , with0
that for fixed oxygen transfer rate, OTR (curves show the maximum cell concen-
tration for a given OTR and cell yield).

the use of continuous c u l t u r e i s an important tool f o r S C P p r o d u c -

tion.
It i s important, however, to go one step f u r t h e r i s this anal-
y s i s . One o f the major c a r b o n sources of interest f o r single cell
protein p r o d u c t i o n i s methanol. T h e reasons f o r this relate prim-
a r i l y to i t s low cost a n d availability. It has been shown that h i g h
methanol concentrations are toxic to cell growth a n d more import-
antly lead to decreased cell y i e l d (10). T h e r e f o r e , a f u r t h e r ob-
jective i s to maintain the r e s i d u a l methanol concentration low at all
times. T h e importance o f this i s i l l u s t r a t e d i n F i g u r e 4 ( 1 1 ) ,
which shows the response of Hansenula polymorpha to a p e r t u r b a -
tion i n methanol c o n c e n t r a t i o n . T h e p e r t u r b a t i o n was initiated b y
a p e r i o d of o x y g e n s t a r v a t i o n (achieved b y r e d u c i n g the agitation
r a t e ) . Because methanol metabolism i s obligately aerobic, u n d e r
o x y g e n limiting conditions methanol r a p i d l y accumulates When
the o x y g e n limitation i
methanol i s r a p i d l y catabolize
acid accumulates. B o t h these compounds are extremely toxic a n d
lead to instability i n c u l t u r e operation. T o overcome this problem,
an on-line computer control s t r a t e g y that would c o n t i n u o u s l y
assess r e s i d u a l methanol concentration a n d , should methanol ac-
cumulate, take c o r r e c t i v e action to p r e v e n t c u l t u r e i n s t a b i l i t y was
developed (12).
T h e s t r a t e g y f o r optimizing single-cell p r o t e i n p r o d u c t i o n i s
based not only on a dilution rate that will give a low r e s i d u a l sub-
strate concentration and h i g h c o n v e r s i o n y i e l d , b u t also that will
operate with the maximum cell d e n s i t y permitted b y the o x y g e n
t r a n s f e r rate. Simultaneously, it i s important to p r e v e n t accumu-
lation of r e s i d u a l methanol to achieve both h i g h y i e l d s a n d process
stability.
Enzyme Production
T h e economics of enzyme p r o d u c t i o n are dominated i n most

cases b y r e c o v e r y c o s t s . F o r this reason, the objective functions
for optimization a r e to maximize the specific activity (units o f en-
zyme a c t i v i t y p e r weight of cell mass) a n d volumetric p r o d u c t i v i t y
(units p e r l i t e r - h ) . Maximization o f specific a c t i v i t y e n s u r e s both
efficient c o n v e r s i o n o f raw materials to the d e s i r e d p r o d u c t as
well as facilitating enzyme r e c o v e r y ; maximization of total volume-
t r i c p r o d u c t i v i t y helps to minimize r e c o v e r y costs a n d maximize
economic r e t u r n on capital investment. T o achieve these objec-
tives i n process development, one utilizes both genetic manipula-
tions a n d environmental management to achieve a h i g h enzyme spe-
cific activity a n d a h i g h cell d e n s i t y .
Two case studies a r e examined here to illustrate the import-
ant elements i n developing a s t r a t e g y f o r optimal enzyme p r o d u c -
tion.

Figure 4. Response of a steady state culture of H . polymorpha to an interruption

in agitation causing oxygen limitation. Reproduced, with permission, from Ref. 11.
Copyright 1981, American Society for Microbiology.

Maltase P r o d u c t i o n
T h e intracellular enzyme maltase (α-glucosidase E . C .

3.2.1.20) i s an important enzyme f o r analysis of blood amylase.
T h e enzyme i s intracellular a n d i s subject to c a r b o n catabolite r e
p r e s s i o n b y glucose as well as i n d u c t i o n b y maltose. A s t u d y was
initiated with the objective of d e v e l o p i n g a lower cost p r o c e s s f o r
maltase manufacture; the goals were to minimize the fermentation
medium cost while maximizing the total a c t i v i t y a n d p r o d u c t i v i t y
of maltase formation. T h e s t a r t i n g point was a fermentation with
Saccharomyces italicus grown i n complex medium c o n t a i n i n g maltose
as the sole carbon source. R e s u l t s from t h i s fermentation are
shown i n F i g u r e 5. T h e problems with this fermentation are h i g h
medium cost a n d the point of h a r v e s t o f maximum maltase activity
was difficult to assess maltos disappeared th
cific activity of the enzym
Medium optimization was achieved b y d e v e l o p i n g a defined
medium without maltose. Saccharomyces italicus does not use s u
crose because it l a c k s i n v e r t a s e , however, maltase will h y d r o l y z e
sucrose a n d allow it to be u s e d for growth. A mutation a n d se
lection program was initiated to select f o r mutants o f S. italicus
that would grow on sucrose as the sole c a r b o n s o u r c e . It was
h y p o t h e s i z e d that s u c h mutants would have a h i g h concentration
of maltase. T h e r e s u l t s are shown i n Table II, i n which mutant
1-4 i s compared with the wild type (13). A s expected, the mu
tant, when grown on s u c r o s e , has h i g h maltase a c t i v i t y . When
grown i n d e f i n e d medium with sucrose, the volumetric a n d spe
cific activity of maltase are greatly e n h a n c e d .
Table II
MALTASE P R O D U C T I O N ON VARIOUS C A R B O N SOURCES
Maltase (units/g-cell)
Carbon Source Wild T y p e Mutant 1-4
Sucrose no growth
Maltose 870 770
Glycerol 10 1100
Acetate 10 770
Fructose 10 380
Glucose 10 320
A comparison of alternative methods f o r p r o d u c i n g this en

zyme i n b a t c h a n d continuous c u l t u r e i s shown i n Table III. T h e
r e s u l t s a r e somewhat s u r p r i s i n g . It was anticipated that i n con
tinuous c u l t u r e u n d e r carbon-limited conditions the specific activ
i t y of maltase would be h i g h e r than o b s e r v e d i n b a t c h c u l t u r e .
However, f o r reasons that are not c l e a r , the specific a c t i v i t y i s
substantially h i g h e r i n b a t c h c u l t u r e . T h e s e r e s u l t s illustrate

TIME (HOURS)
Figure 5. Batch fermentation for maltase production by Saccharomyces italicus.

Reproduced, with permission, from Ref. 13. Copyright 1982, American Society
for Microbiology.

again the importance of u s i n g good carbon source management a n d

d e v e l o p i n g an optimal s t r a t e g y f o r enzyme p r o d u c t i o n . It i s also
i n t e r e s t i n g to note that b y going from a complex to a defined
medium one not only r e d u c e d the medium cost b u t also stabilized
the enzyme to subsequent degradation.
Table III
MALTASE P R O D U C T I O N BY M U T A N T AND WILD T Y P E
Specific
Activity Productivity
(units/ ( unit s7
Process g-cell) g-cell-hr) (units/1-hr)
Batch
(1-4)
Continuous 2 9 Q 3 4 0 0
(1-4)
Batch
890 70 470
(wild type)
Heparinase Production
T h e enzyme heparinase i s o f interest as a means f o r d e g r a d -

i n g h e p a r i n i n blood a n d f o r d e g r a d i n g l a r g e molecular weight
h e p a r i n into smaller fragments h a v i n g potential as novel anticoag-
ulants (14). T h e p r o d u c t i o n of heparinase b y Flavobacterium
heparinum i n a complex medium with h e p a r i n as an i n d u c e r i s
shown i n F i g u r e 6. T h e requirement f o r h e p a r i n makes the med-
ium expensive a n d it i s difficult to p i c k the best h a r v e s t time be-
cause the enzyme a c t i v i t y r a p i d l y decays late i n the fermentation.
The first step i n process optimization was to develop a d e f i n e d
medium; r e s u l t s are shown i n F i g u r e 7 (JL5). A l t h o u g h h e p a r i n
i s still r e q u i r e d as an i n d u c e r , this lowers medium cost a n d i n -
creases the stability o f heparinase. It was expected that the p r o -
duction o f this enzyme, i n addition to b e i n g u n d e r i n d u c t i o n b y
h e p a r i n , would be u n d e r control b y c a r b o n catabolite r e p r e s s i o n .
T h e r e s u l t s showed that i n c r e a s i n g the initial concentrations of
glucose leads to both decreased growth a n d heparinase p r o d u c t i o n
(15). F o r this reason, we examined a fed-batch c u l t u r e designed
to keep the glucose low throughout the fermentation. T h e r e s u l t s
showed that it i s possible to achieve h i g h enzyme activity which
is r e l a t i v e l y stable at the e n d of the fermentation. A g a i n , the
s t r a t e g y of c a r b o n source management has p r o v e d to be v e r y ef-
fective i n optimizing this fermentation. C u r r e n t l y we are i n the
p r o c e s s of looking f o r mutants which no longer r e q u i r e h e p a r i n
as an i n d u c e r . We a r e also e x p l o r i n g the use of continuous c u l -

CE
Lu
I
O 10 20 30
HOURS
Figure 6. Batch fermentation for the production of heparinase by Flavobacterium

heparinum on complex medium. Key: heparinase specific activity; Δ , dry cell
weight; O , heparin concentration. Reproduced, with permission, from Ref. 15.
Copyright 1981, American Society for Microbiology.
Figure 7. Batch fermentation for the production of heparinase by F . heparinum

in defined medium. Key: O , dry cell weight, (D) glucose; Δ , heparin; and V ,
heparinase activity. Reproduced, with permission, from Ref. 15. Copyright 1981,
American Society for Microbiology.

t u r e as a means f o r maintaining low glucose concentrations with

the hope of maximizing the specific activity of heparinase.
Metabolite Production
T h e p r i m a r y objective f u n c t i o n s f o r p r o d u c t i o n o f metabolites
are to maximize the concentration (to minimize r e c o v e r y costs)
while maintaining h i g h c o n v e r s i o n y i e l d s o f costly raw materials to
the p r o d u c t , u n d e r conditions o f h i g h volumetric p r o d u c t i v i t y (to
r e d u c e capital c o s t ) . T h e u s u a l a p p r o a c h i s to r a p i d l y p r o d u c e a
h i g h cell concentration u n d e r conditions that maximize the c o n v e r -
sion rate o f raw materials to the d e s i r e d p r o d u c t .
The example c o n s i d e r e d here i s penicillin p r o d u c t i o n b y Péni-
cillium c h r y s o g e n u m . Penicillin i s u n d e r control of c a r b o n catab-
olite r e p r e s s i o n a n d is t y p i c a l l formed secondar product
after p r i m a r y growth i
oped i n i n d u s t r y f o r thi generally
fed-batch fermentation which r e s t r i c t s the flow o f c a r b o n to the
c u l t u r e to slow down growth, t h u s p r e v e n t i n g c a r b o n catabolite
r e p r e s s i o n . Mou a n d Cooney (16) initiated a p r o g r a m to examine
the application of on-line computer control as a means f o r achiev-
i n g improved c a r b o n source management f o r penicillin p r o d u c t i o n .
The rationale was to utilize on-line monitoring o f growth to assess
the demand o f the c u l t u r e for the c a r b o n source a n d i n t h i s way
develop a means to control the specific growth rate i n the fermen-
tation. It was reasoned that on-line computer c o n t r o l would p r o v e
to be more flexible i n allowing one to balance s u p p l y a n d demand
of the c a r b o n source d u r i n g this fermentation.
A series o f studies were conducted i n which a computer con-
t r o l system was utilized to control both growth phase a n d p r o d u c -
tion phase growth rates a n d to a s k the question, "what i s the ef-
fect o f changes i n these growth rates on the specific p r o d u c t i v i t y
of p e n i c i l l i n ? " . In F i g u r e 8 are shown r e s u l t s i n which the p r o -
1
duction phase growth rate was manipulated from 0 to 0.015 h "
while the initial growth rate was manipulated constant at i t s maxi-
mum value o f 0.107. T h e specific rates of penicillin p r o d u c t i o n
are shown i n F i g u r e 9. It i s clear that control o f the p r o d u c t i o n
phase growth rate i s important to maintaining the ability of the
cells to make p e n i c i l l i n . While it i s not clear from these r e s u l t s
what the optimum value i s , this a p p r o a c h does allow one to b e g i n
to design a set of experiments to answer that q u e s t i o n . In a
second set o f experiments, two growth phase growth rates were
compared. In the first case, the growth was controlled at 0.11
1
h " a n d i n the second case at 40% o f this value. T h e r e s u l t s ,
shown i n F i g u r e 10, indicate that cells grown more slowly d u r i n g
the initial growth phase have a h i g h e r specific rate of penicillin
p r o d u c t i o n . A g a i n , it i s not clear what the optimum values f o r
growth phase growth rate are; i t i s clear, however, that c a r b o n
source management d u r i n g both the growth a n d p r o d u c t i o n phase
is important to maximize the specific rate of penicillin p r o d u c t i o n .

600Γ
HOURS
Figure 8. Growth kinetics of P. chrysogenum grown in computer controlled fed-

batch culture fsee text for details).

6Γ
HOURS
Figure 9. Specific penicillin production rate for the fermentation shown in

Figure 8.

HOURS
Figure 10. Growth and penicillin production by P. chrysogenum grown at two

different growth phase growth rates.

T h e s e r e s u l t s suggest that f u r t h e r refinement o f this important

fermentation c a n be achieved t h r o u g h the use o f on-line monitor-
ing and control.
Conclusions
F o r the p r o d u c t i o n of cell mass, enzymes a n d metabolites, it

is clear from the above examples that c a r e f u l c a r b o n source man-
agement i s important to achieve h i g h c e l l concentrations with h i g h
y i e l d , maximum specific enzyme activities a n d the efficient c o n v e r -
sion o f raw materials to p r o d u c t s . One approach to achieve good
environmental management i s t h r o u g h the use of on-line p r o c e s s
c o n t r o l . In this way, it i s possible to match the s u p p l y with the
demand f o r c a r b o n source i n a wide v a r i e t y o f fermentations.
A n o t h e r approach i s to use both traditional genetics as well as
genetic e n g i n e e r i n g to
source utilization to achiev
Genetics c a n be used to eliminate c a r b o n catabolite r e p r e s s i o n ,
side reactions to u n d e s i r e d p r o d u c t s a n d the need for i n d u c e r s
of d e s i r e d enzymes. In addition, the use o f recombinant D N A to
increase gene dosage c a n be important to i n c r e a s i n g the specific
activity o f d e s i r e d enzymes; these may b e enzymes that a r e de-
s i r e d as p r o d u c t s o r that a r e rate limiting enzymes i n metabolic
pathways. Simultaneously with optimizing the genetic constitution
and the application o f p r o c e s s control s t r a t e g i e s , it i s essential i n
bioreactor d e s i g n a n d scale-up to take into account the organism's
s e n s i t i v i t y to carbon source concentrations. In situations s u c h as
methanol fermentations where the organism may b e v e r y sensitive
to small variations i n c a r b o n source c o n c e n t r a t i o n s , mixing time i s
a k e y parameter a n d n o v e l e n g i n e e r i n g d e s i g n s must often be con-
s i d e r e d to avoid this problem. Despite what appears s u p e r f i c i a l l y
to be d i v e r s i t y among fermentation p r o c e s s e s , there are s e v e r a l
common factors important i n the d e s i g n o f optimum process strate-
gies. C a r b o n source management i s k e y among these.
Literature Cited
1. Cooney, C . L . Microb. Growth on C . Compounds, 1975, pp.

1
183-197.
2. Cooney, C.L.; Rha, C . K . ; Tannenbaum, S.R. Adv. in
Food Res., 1980, 26, 1-52.
3. Wang, H . Y . ; Cooney, C . L . ; Wang, D.I.C. Biotech. Bio-
engr., 1979, 21, 975-995.
4. Pirt, S . J . "Principles of Microbe and Cell Cultivation",
1975, Blackwell Scientific Publications, London.
5. Aiba, S.; Nagai, S.; Nishizawa, Y. Biotechnol. Bioengr.,
1976, 19, 1001.
6. Cooney, C . L . ; Wang, H . ; Wang, D.I.C. Biotech. Bioengr.,
1977, 19, 55-66.

7. Wang, H . Y . ; Cooney, C . L . ; Wang, D.I.C. Biotech. Bio-

engr., 1978, 19, 67-86.
8. Cooney, C.L.; Rha, C . K . ; Tannenbaum, S.R. Adv. Food
Res., 1981, 26, 1-47.
9. Herbert, D . ; Elsworth, R . ; Telling, R . C . J . Gen. Micro-
biol., 1956, 14, 601-622.
10. Books, J . D . ; Meers, J . L . J . Gen. Microbiol., 1973, 77,
513.
11. Swartz, J . R . ; Cooney, C.L. Appl. Env. Microbiol., 1981,
41, 1206.
12. Swartz, J . R . Ph.D. Thesis, M . I . T . , Cambridge, MA, 1979.
13. Schaefer, E.J.; Cooney, C . L . Appl. Environ. Microbiol.,
1982, 43(1), 75-80.
14. Langer, R . ; Linhardt, R.J.; Hoffberg, S.; Larsen, A.K.;
Cooney, C . L . Tapper D.; Klein M Science 1982
in press.
15. Galliher, P . M . ; Cooney, C.L.; Langer, R . ; Linhardt, R . J .
Appl. Environ. Microbiol., 1981, 41(2), 360-5.
16. Mou, D - G . ; Cooney, C . L . Biotech. Bioeng., 1982, sub-
mitted.

9
I n t e r a c t i o n s of Microbial Populations in Mixed

Culture Situations
A. G. FREDRICKSON
University of Minnesota, Department of Chemical Engineering and
Materials Science, Minneapolis, MN 55455
This paper reviews the classification and dynam-

ics of interactio
lations inhabitin
of interaction between three or more populations are
considered, also. The nature of the scheme of
classification of interaction is described and its
utility as well as its weaknesses are mentioned.
Several interactions, competition for resources and
feeding of one population upon another in particular,
have been studied in some detail and reasonably
reliable mathematical models of some simple types of
these interactions are available. Other interactions
have not received so much attention, however, and in
some cases nothing but qualitative statements about
an interaction can be made. The review is concluded
with a listing of areas where research is needed to
provide and improve knowledge of interspecific micro-
bial interactions.
Probably the main reason f o r the predominance of pure

c u l t u r e techniques i n the fermentation i n d u s t r y i s that i n most
cases the product i s a complicated, v a l u a b l e organic molecule
which can be made by a s i n g l e m i c r o b i a l p o p u l a t i o n , and when we
have i s o l a t e d and improved such a p o p u l a t i o n we do not want to
undo the l a b o r s of the m i c r o b i o l o g i s t s and g e n e t i c i s t s by l e t t i n g
i n t o our propagation apparatus other microorganisms which might
compete with o r be a n t a g o n i s t i c to our producer p o p u l a t i o n , o r
which might make substances which would have to be separated from
the d e s i r e d product, and so on. The reasons why most fermentation
technology i s pure c u l t u r e technology a r e t h e r e f o r e s i m i l a r to the
reasons why our a g r i c u l t u r e tends to be monoculture of p l a n t s .
Nevertheless, there a r e reasons f o r studying mixed c u l t u r e s ,
and I w i l l l i s t f o u r . F i r s t , c e r t a i n i n d u s t r i a l o p e r a t i o n s ,
notably waste d i s p o s a l s , do u t i l i z e mixed c u l t u r e s . Second,
i n v a s i o n by contaminants or formation of mutants turn pure
0097-6156/83/0207-0201 $ 0 7 . 7 5 / 0

c u l t u r e s i n t o mixed c u l t u r e s . T h i r d , mixed c u l t u r e s o f f e r some

p o t e n t i a l advantages, such as ( i ) a b i l i t y to perform sequences of
chemical transformations which no pure c u l t u r e can do, ( i i )
a b i l i t y to grow on simpler and so cheaper media, ( i i i ) a b i l i t y to
continue f u n c t i o n i n g over wider ranges of environmental c o n d i t i o n s ,
and ( i v ) a b i l i t y to r e s i s t i n v a s i o n by contaminants. F i n a l l y , a
f o u r t h reason f o r studying mixed c u l t u r e s i s that n a t u r a l systems
always i n v o l v e the a c t i v i t i e s of mixed c u l t u r e s .
C l a s s i f i c a t i o n of population interactions
When s e v e r a l populations of microorganisms i n h a b i t a common

a b i o t i c environment they w i l l almost i n v a r i a b l y i n t e r a c t with one
another. Attempts to construct t h e o r i e s of the dynamics of such
s y s t e m s — t h a t i s , to construc t h e o r i e which w i l l p r e d i c ho th
systems evolve i n time—mus
what i n t e r a c t i o n s occur
Hence, the r e s t of what I have to say w i l l focus on what i s known
about i n t e r a c t i o n s i n simple mixed c u l t u r e systems.
Economy of d i s c u s s i o n makes i t necessary to devise some scheme
f o r naming m i c r o b i a l i n t e r a c t i o n s . In a d d i t i o n , development and
use of such a scheme w i l l help organize our t h i n k i n g on the subject
and w i l l even suggest research that should be done.
A l l schemes of naming i n t e r a c t i o n s that I have seen are based
on naming i n t e r a c t i o n s between p a i r s of populations. This i s
p e r f e c t l y understandable, s i n c e a p a i r of populations i s the sim
p l e s t u n i t of b i o l o g i c a l o r g a n i z a t i o n that can e x h i b i t i n t e r a c t i o n s
other then i n t r a s p e c i f i c ones. However, the use of the b a s i s named
leads to some d i f f i c u l t i e s when we consider systems with three or
more populations, as w i l l be explained s h o r t l y .
The scheme of naming i n t e r a c t i o n s between a p a i r of popula
t i o n s , say A and B, u s u a l l y adopted by e c o l o g i s t s i s based on the
q u a l i t a t i v e e f f e c t s that the presence of A has on Β as w e l l as on
the e f f e c t s that Β has on A. I f the presence of A stimulates the
growth of Β somehow, then A i s s a i d to have a p o s i t i v e e f f e c t on
Β whereas A i s s a i d to have a negative e f f e c t on Β i f the presence
of A represses or slows the growth of B. Nothing i s s a i d about the
p r e c i s e means by which one population a f f e c t s the other, and thus,
by t h i s scheme, q u i t e d i f f e r e n t mechanisms of i n t e r a c t i o n w i l l be
c l a s s i f i e d i n the same way; t h i s i s not altogether undesirable, of
course. A t y p i c a l scheme of such c l a s s i f i c a t i o n i s given by
Odum (1).

9. FREDRICKSON Interactions of Microbial Populations 203
I am going to r e t a i n the foregoing scheme, but i n a d d i t i o n ,

as I have suggested elsewhere ( 2 ) , the scheme w i l l be combined
with another that names i n t e r a c t i o n s as d i r e c t or i n d i r e c t .
D i r e c t i n t e r a c t i o n s are those f o r which p h y s i c a l contact of
i n d i v i d u a l organisms from the two d i f f e r e n t populations i s a
necessary part of the i n t e r a c t i o n . I n d i r e c t i n t e r a c t i o n s r e q u i r e
no such contact, but r a t h e r are those i n t e r a c t i o n s which occur
when changes i n the a b i o t i c environment produced by the a c t i v i t i e s
of one p o p u l a t i o n a f f e c t the growth r a t e of the other, and v i c e
v e r s a . D i r e c t i n t e r a c t i o n s are thus p h y s i c a l i n nature, whereas
i n d i r e c t i n t e r a c t i o n s are chemical i n nature.
The scheme of naming b i n a r y i n t e r a c t i o n s based on these two
s e t s of c r i t e r i a i s shown i n F i g u r e 1. T h i s f i g u r e i s l a r g e l y
s e l f explanatory and most of the names used are f a m i l i a r , although
not every e c o l o g i s t woul
of them that the f i g u r e
antagonism to r e f e r to any i n t e r a c t i o n that has a negative e f f e c t
but i n the f i g u r e , antagonism means that each member of the p a i r
exerts a negative e f f e c t on the other.
In two cases, however, I have used words which are new or
d e v i a t e from common usage. The i n t e r a c t i o n which r e s u l t s when
population Β grows on products of l y s i s of c e l l s of p o p u l a t i o n A,
the l y s i s being caused by exoenzymes released by p o p u l a t i o n B, i s
c a l l e d e c c r i n o l y s i s . T h i s i s a word which I got from d i s c u s s i o n s
with C. Takoudis, S. Pavlou, and R. A r i s . E c c r i n o l y s i s i s t y p i f i e d
by the i n t e r a c t i o n of myxobacteria with c e r t a i n other b a c t e r i a ;
see, e. g., K a i s e r ejt a l . (3). The word which d e v i a t e s from common
usage i s feeding, which i s here used i n preference to prédation.
Prédation has connotations of hunting and one-on-one a c t i o n which
do not c h a r a c t e r i z e a l l of the i n t e r a c t i o n s that I c a l l f e e d i n g .
I agree that feeding i s too general a word to be used as F i g u r e 1
uses i t , and I hope that someone can suggest a b e t t e r word to
replace i t .
Very o f t e n the i n t e r a c t i o n between a p a i r of populations
w i l l be more complicated than any one of the i n t e r a c t i o n s named
and described i n F i g u r e 1. When we are confronted with such
s i t u a t i o n s , i t seems best not to t r y to invent new words to des-
c r i b e them but r a t h e r to s t a t e what combination of the i n t e r -
a c t i o n s of F i g u r e 1 are i n v o l v e d . For example, consider a
s i t u a t i o n where a by-product of the metabolism of one population
acts as a growth f a c t o r f o r a second p o p u l a t i o n , and where the
two populations consume a common s u b s t r a t e to supply t h e i r needs
f o r carbon and a v a i l a b l e energy. The i n t e r a c t i o n between the
populations i s n e i t h e r commensalism nor competition, but we do not
invent a new word to d e s c r i b e i t ; i n s t e a d , we say that i t i s
commensalism p l u s competition. When the i n t e r a c t i o n between two
populations i s f u l l y described by j u s t one of the items l i s t e d i n
F i g u r e 1, i t i s convenient to emphasize that f a c t by saying that
the i n t e r a c t i o n i s pure.

Effect Effect
of presence of presence
of Β on of A on
growth rate growth rate Name of
of A of Β Qualifying remarks interaction
-
—
—
—
Ο
—
Negative effects caused by
removal of resources
COMPETITION
ANTAGONISM
— Ο production of toxins or inhibitors AMENSALISM
- + production of lytic agents;
positive effects caused by
solubilization of biomass
ECCRINOLYSIS
a stimulus for growth of A

ο (commensal) or by removal COMMENSALISM
by Β of an inhibitor
for growth of A
See remarks for Commensalism.
Also presence of both PROTO -
+ • populations not necessary COOPERATION
for growth of both
See remarks for Commensalism.
Also presence of both
• + populations is necessary for MUTUALISM
growth of either
FEEDING -
- + Β feeds on A
INCLUDES
PREDATION AND
SUSPENSION-
FEEDING
The parasite (B) penetrates
- +
the body of its host (A) and
therein converts the host's
biomaterial or activities
PARASITISM
into its own

• A and Β are in physical contact;
+ SYMBIOSIS
(or perhaps Ο ) interaction highly specific
- - Competition for space CROWDING
Figure 1. Scheme of classification of binary population interactions. The roles

of A and Β may be reversed. Top, indirect interactions; bottom, direct interactions.

9. FREDRiCKSON Interactions of Microbial Populations 205
In a system i n h a b i t e d by ρ d i f f e r e n t populations, there i s a

p o s s i b i l i t y f o r p!/2!(p-2)! b i n a r y i n t e r a c t i o n s to occur. A
q u a n t i t a t i v e understanding of the dynamics of each of these
i n t e r a c t i o n s i s necessary but not s u f f i c i e n t f o r a q u a n t i t a t i v e
understanding of the dynamics of the whole system. Insufficiency
may be demonstrated by c o n s i d e r i n g as an example the feeding of a
s i n g l e protozoan population on two b a c t e r i a l populations which
compete f o r a common resource. C l e a r l y , study of the two i n d i
v i d u a l food chains i n v o l v e d here w i l l not allow us to p r e d i c t the
behavior of the three-population system, because u n t i l the p r o t o
zoans are a c t u a l l y presented with the choice of the two kinds of
b a c t e r i a , we w i l l not know whether and to what extent the p r o t o
zoans w i l l e x e r c i s e preference i n t h e i r uptake of food.
D i s c u s s i o n of the i n t e r a c t i o n
I w i l l now d i s c u s s these v a r i o u s i n t e r a c t i o n s i n turn,

beginning with the i n d i r e c t or chemical ones; the p r i n c i p a l
o b j e c t s of the d i s c u s s i o n w i l l be to summarize the s t a t e of know
ledge about each and to p o i n t out research that needs to be done
on each.
Competition. Probably the m a j o r i t y of s t u d i e s of competition

that have been published have d e a l t with what has been c a l l e d pure
and simple competition ^4). In t h i s type of competition, there i s
only one resource whose a v a i l a b i l i t y or c o n c e n t r a t i o n a f f e c t s the
growth r a t e of a competitor p o p u l a t i o n , t h i s resource i s the same
f o r both competitors i n the i n t e r a c t i o n , and the growth r a t e s are
not a f f e c t e d by changes i n the concentrations of other substances
present i n the common environment.
Pure and simple competition has a strong tendency to r e s u l t
i n the e x c l u s i o n of one of the competitors, and v a r i o u s attempts
to formulate a competitive e x c l u s i o n p r i n c i p l e have appeared i n
the l i t e r a t u r e . One such f o r m u l a t i o n , which i s supported by many
experiments as w e l l as by the p r e d i c t i o n s of mathematical models,
i s that pure and simple competitors w i l l not c o e x i s t i n d e f i n i t e l y
i n a system that i s s p a t i a l l y homogeneous and that i s s u b j e c t to
t i m e - i n v a r i a n t e x t e r n a l i n f l u e n c e s . For example, the p r e d i c t i o n
i s that pure and simple competitors w i l l not c o e x i s t i n a w e l l -
mixed chemostat having a v a n i s h i n g l y s m a l l surface-to-volume r a t i o
(so that the s p a t i a l heterogeneity due to the presence of the
chemostat w a l l s i s n e g l i g i b l e ) i f the d i l u t i o n r a t e and temperature
of the chemostat, the composition of feed to the chemostat, e t c . ,
are a l l independent of time. Moreover, the p r e d i c t i o n i s that not
only w i l l the competitors not c o e x i s t i n a steady s t a t e i n such a
system but that they w i l l not even c o e x i s t i n a p e r p e t u a l l y t r a n
s i e n t s t a t e , such as sustained o s c i l l a t i o n s of t h e i r population
d e n s i t i e s ; see F r e d r i c k s o n and Stephanopoulos (4) f o r a d i s c u s s i o n
of the l i t e r a t u r e on these p o i n t s .

F u r t h e r a n a l y s i s of pure and simple competition suggests that

populations which so compete can c o e x i s t i f two s e t s of c o n d i t i o n s
are s a t i s f i e d . F i r s t , the dependence of the growth r a t e s of the
populations on the c o n c e n t r a t i o n of the l i m i t i n g resource must be
such that there i s a range (or set of ranges) of concentration
which causes the f i r s t population to grow f a s t e r than the second,
and another range (or set of ranges) of concentration which
causes the second population to grow f a s t e r than the f i r s t . If
one population grows f a s t e r than the other at a l l concentrations
of the l i m i t i n g resource, coexistence seems never to be p o s s i b l e .
Second, the environment must be e i t h e r s p a t i a l l y or temporally
heterogeneous, and heterogeneous i n such a way that i t favors the
growth of one population here (now) and of the other population
there (then). S p a t i a l heterogeneity can be imposed on a system,
as f o r example, by usin tw chemostat with i n t e r s t a g flow (5)
and temporal heterogeneit
p e r i o d i c v a r i a t i o n of d i l u t i o , composition, ,
studied by many workers (6-11). Analyses of the competition
equations used by the foregoing workers shows that so long as
s p a t i a l homogeneity i s imposed on systems, as by mixing i n a
chemostat, temporal heterogeneity i s not generated by the a c t i v i -
t i e s of pure and simple competitors, so that temporal heterogeneity
must be imposed from outside the system (12,13).
Now the competition equations j u s t r e f e r r e d to are based on
the hypothesis that we are d e a l i n g with a resource that i s not
self-renewing; that i s , which i s e i t h e r a n o n - l i v i n g substance or
a b i o l o g i c a l resource which f o r some reason or other i s not grow-
ing and reproducing. When t h i s hypothesis i s changed, and one
assumes that the resource competed f o r i s capable of s e l f - r e n e w a l ,
i t appears that the competitive e x c l u s i o n p r i n c i p l e s t a t e d above
i s no longer true.
Some years ago A. L. Koch (14) published computer simulations
of s i t u a t i o n s i n which two predator populations competed purely
and simply f o r one prey p o p u l a t i o n . Koch's simulations showed
these three populations c o e x i s t i n g i n what appeared to be l i m i t
c y c l e s , a c l e a r v i o l a t i o n of the competitive e x c l u s i o n p r i n c i p l e
stated above, but s i n c e h i s s i m u l a t i o n s were based on Lotka-
V o l t e r r a type equations whch I consider to be q u i t e i n a p p r o p r i a t e
for m i c r o b i a l p o p u l a t i o n s , I disregarded h i s r e s u l t s and d i d not
see t h e i r s i g n i f i c a n c e . S i m i l a r r e s u l t s published by Hsu et a l .
(15,16) were disregarded f o r the same reason. However, P.
Waltman of the U n i v e r s i t y of Iowa r e c e n t l y pointed out to me i n a
personal communication that even when L o t k a - V o l t e r r a concepts are
discarded e n t i r e l y and Monod's model i s used f o r a l l growth r a t e s ,
the r e s u l t i n g competition equations f o r two predators and one prey
seem to have l i m i t c y c l e s o l u t i o n s f o r c e r t a i n c o n d i t i o n s of
o p e r a t i o n . Mr. B a s i l B a l t z i s has found that use of a s o - c a l l e d
m u l t i p l e s a t u r a t i o n model f o r the predators, which seems to be
more appropriate than Monod's model f o r protozoans at any r a t e
(17), and of Monod's model f o r the prey, a l s o leads to p r e d i c t i o n

of coexistence of the three p o p u l s t i o n s i n what appear to be l i m i t

c y c l e s . Hence, i t i s not the p a r t i c u l a r growth model that makes
the d i f f e r e n c e here; r a t h e r , i t i s the presence of the a d d i t i o n a l
t r o p h i c l e v e l that allows competing predators to c o e x i s t .
I t seems i n t u i t i v e l y evident that a necessary c o n d i t i o n f o r
coexistence ( i n a l i m i t c y c l e ) of two feeding populations that
compete purely and simply f o r a growing and reproducing food popu-
l a t i o n i s that there must be a range (or s e t of ranges) of food
c o n c e n t r a t i o n f o r which the f i r s t feeder grows f a s t e r than the
second and another range (or s e t of ranges) of food c o n c e n t r a t i o n
f o r which the second feeder grows f a s t e r than the f i r s t . I f this
i s so, then what appears t o be happening i n these three-population
systems i s that the a c t i v i t i e s of the populations c r e a t e temporal
heterogeneity o f the environment even when none i s imposed from
without, and when t h i s heterogeneit f a v o r i successio h
of f i r s t one feeder an
population density w i l l possible
The p r e d i c t i o n s of these recent modeling s t u d i e s should be
tested by experimental research. T h i s could be done by observing,
say, the competition of two protozoan populations, which do not
feed on each other, f o r a s i n g l e , growing b a c t e r i a l p o p u l a t i o n i n
a chemostat. In a d d i t i o n , modeling and mathematical analyses of
other s i t u a t i o n s where competition ends a food chain might be
rewarding. An example might be a system where two populations
compete f o r a growth f a c t o r which i s r e l e a s e d i n t o the environment
by the a c t i v i t i e s of a host p o p u l a t i o n . T h i s would not be
expected to produce coexistence of the competitors i f the i n t e r -
a c t i o n of each with the host was pure commensalism, but i f the
competitors a l s o d i d something that i n f l u e n c e d the host p o p u l a t i o n ,
coexistence i n l i m i t c y c l e s might be p o s s i b l e . F i n a l l y , these
recent r e s u l t s suggest that f u r t h e r examination o f pure and simple
competition f o r a resource that does not renew i t s e l f might be
profitable. I t should be remembered i n t h i s connection that
experimental devices l i k e the chemostat impose s p a t i a l homogeneity
on competition systems. I f we d i d not impose homogeneity by
mixing, i s i t p o s s i b l e that the a c t i v i t i e s of the populations could
create s p a t i a l heterogeneity even where none i s imposed? I f t h i s
i s p o s s i b l e , could i t allow pure and simple competitors to c o e x i s t ?
I think that P r o f . Lauffenberger has been working on questions l i k e
t h i s , and I hope that he w i l l provide us with some answers today.
There are s e v e r a l other p o i n t s about competition that I would
l i k e t o make b e f o r e going on to other kinds of i n t e r a c t i o n . Com-
p e t i t i o n between populations i n c e r t a i n environments might be
p u r e — 1 . e., the only i n t e r a c t i o n between the p o p u l a t i o n s — b u t i t
might not be simple because the concentrations of two o r more
n u t r i e n t s competed f o r may a f f e c t the growth r a t e s of the popu-
lations. " N u t r i e n t s " as used here means chemicals not produced by
the competing populations o r by others present, so there i s no
question here of competition f o r self-renewing resources. In
n u t r i e n t - p o o r environments, the concentrations of s e v e r a l

substances which are complementary resources ( f u l f i l l d i f f e r e n t

needs i n the c e l l u l a r economies) may become r a t e - l i m i t i n g whereas
i n complex, n u t r i e n t - r i c h environments, the concentrations of
s e v e r a l substances which are s u b s t i t u t a b l e resources ( f u l f i l l the
same need i n the c e l l u l a r economies) may become r a t e - l i m i t i n g .
Many mathematical models of s i t u a t i o n s l i k e the foregoing
have been published; see (6, 18-25). Analyses of these models
suggests that pure but not simple competition should o f t e n
r e s u l t i n coexistence of competitors, even i n systems i n which
s p a t i a l homogeneity of the environment i s imposed and f o r which
a l l e x t e r n a l i n f l u e n c e s are t i m e - i n v a r i a n t . Experimental data of
Yoon e£ a l . (22) on competition of B a c i l l u s cereus and Candida
t r o p i c a l i s f o r the s u b s t i t u t a b l e resources glucose and f r u c t o s e
show that coexistence i n a chemostat i s indeed p o s s i b l e here.
Experimental t e s t i n g of model p r e d i c t i o n i s i t u a t i o n f elemen
tary but not simple competitio
models used are n e c e s s a r i l y multipl
of growth, and a l l such models have low c r e d i b i l i t y , i n my o p i n i o n .
Another form of elementary but not simple competition i s what
has been c a l l e d (4) p a r t i a l competition. In t h i s , two populations
compete f o r a resource whose a v a i l a b i l i t y a f f e c t s both t h e i r
growth r a t e s , but i n a d d i t i o n , the growth r a t e of one of the popu-
l a t i o n s , at l e a s t , i s also a f f e c t e d by the c o n c e n t r a t i o n of a
substance which i s exempt from competition, e i t h e r because i t i s a
resource which only one of the populations can use, or because i t
i s a substance that i s not used by e i t h e r population but never-
t h e l e s s exerts an e f f e c t , say of a u t o i n h i b i t i o n , on the growth
r a t e of one of them. G o t t s c h a l ejt a l . (26) have provided an
elegant experimental example of the former s i t u a t i o n with t h e i r
mixotroph-obligate heterotroph system fed on a combination of
acetate and t h i o s u l f a t e ; coexistence of these populations i n a
chemostat occurred even though the populations competed f o r
acetate. A s i m i l a r example i s given by Laanbroek et_ a l . (27).
De F r e i t a s and F r e d r i c k s o n (28) Tiave analyzed mathematical models
of s i t u a t i o n s of the l a t t e r type, and these show that the pro-
duction of a u t o i n h i b i t o r s can allow c o m p e t i t o r s — p a r t i a l com-
p e t i t o r s — t o c o e x i s t . F i n a l l y , Miura et a l . (29) have analyzed a
mathematical model of a s i t u a t i o n where p a r t i a l competition f o r a
resource i s coupled with commensalism; again, coexistence i s pre-
d i c t e d to be p o s s i b l e . Broad as w e l l as deep knowledge of micro-
b i a l n u t r i t i o n and physiology are probably n e c e s s i t i e s f o r
c r e a t i n g s u c c e s s f u l experimental systems of p a r t i a l competition,
and one hopes that more poeple having such knowledge w i l l attempt
to apply i t i n the d i r e c t i o n noted.
The l a s t thing that I want to say about competition i s that
i t might be rewarding to consider models of i t that take i n t o
account some of the n o n - i d e a l f a c t o r s that f r e q u e n t l y complicate
growth of microorganisms. By n o n - i d e a l f a c t o r s I mean such things
as the occurrence of maintenance, v a r i a b i l i t y of biomass y i e l d ,
time l a g of metabolic process r a t e s i n response to changes i n

environmental c o n d i t i o n s , and so on. These things are u s u a l l y

ignored i n mathematical models of competition, but i t seems
p o s s i b l e that t h e i r occurrence could have strong e f f e c t s on the
r e s u l t s of competition. F o r example, Alexander (30) mentions that
c a p a c i t y of a population t o s y n t h e s i z e and s t o r e reserve foods
when e x t e r n a l food i s abundant and then to use the s t o r e d food
when the e x t e r n a l supply dwindles i s l i k e l y to be an important
f
aspect of a p o p u l a t i o n s s competitive a b i l i t y . The experiments of
van Gemerden (31) on competition of purple s u l f u r b a c t e r i a grown
i n a chemostat subjected to a regimen of a l t e r n a t i n g l i g h t and
dark seem to v e r i f y Alexander's suggestion. Wilder et a l . (32)
have considered r e c e n t l y a model of competition i n which time lags
of metabolic response were accounted f o r . But a s i d e from these
examples, I do not know of other papers which consider the e f f e c t s
that n o n - i d e a l phenomen might hav competitiv situations
Mathematical models tha
t e d l y would not have hig c r e d i b i l i t y y migh
i n t e r e s t i n g p r e d i c t i o n s which could then be tested experimentally.
Amensalism and antagonism. A computerized l i t e r a t u r e search

on these i n t e r a c t i o n s w i l l produce a l a r g e number of r e f e r e n c e s , a
f a c t which i n d i c a t e s that the importance of these i n t e r a c t i o n s i s
recognized widely. However, examination of the references pro-
duced w i l l show that very l i t t l e mathematical modeling work or
experimental work that i s h e l p f u l i n c o n s t r u c t i n g such models has
been done. The most important reason f o r t h i s i s probably that
models o f amensalism and antagonism have to be based on models f o r
the k i n e t i c s of production and a c t i o n of i n h i b i t o r s and t o x i n s ,
and current models f o r the k i n e t i c s of these processes are not of
high c r e d i b i l i t y . B e t t e r models could, no doubt, be constructed
from data from experiments on systems e x h i b i t i n g amensal o r
a n t a g o n i s t i c i n t e r a c t i o n s , but the best remedy f o r the d i f f i c u l t y
noted i s the performance of a u t e c o l o g i c a l — m e a n i n g u s u a l l y pure
c u l t u r e — w o r k on organisms that produce toxins o r i n h i b i t o r s and
a l s o on organisms that are a f f e c t e d by such substances.
Use of simple models which are p l a u s i b l e but which have low
c r e d i b i l i t y because they have not had extensive experimental
t e s t i n g and refinement suggests that a p a i r of populations which
i n t e r a c t by amensalism o r antagonism only, or by a combination of
amensalism o r antagonism w i t h competition f o r a s i n g l e resource,
cannot c o e x i s t i n d e f i n i t e l y i n a common, homogeneous environment
(28). An experimental system which involved competition and
amensalism was devised by Adams et a l . (33). They found that the
two populations, both of which were s t r a i n s of E s c h e r i c h i a c o l i ,
d i d not c o e x i s t i n a chemostat and that the i d e n t i t y of the popu-
l a t i o n which was excluded depended on the d e n s i t i e s of the popu-
l a t i o n s at the beginning of the experiment. Both of these r e s u l t s
are p r e d i c t e d by simple models (28), and thus, the n o t i o n that
populations which i n t e r a c t by amensalism or antagonism cannot
c o e x i s t gains some p l a u s i b i l i t y .

Amensalism i s probably a means by which some organisms or

groups of organisms can defend t h e i r h a b i t a t against invaders who
would compete w i t h them f o r the resources of the h a b i t a t . I f an
a s s o c i a t i o n of organisms occurs i n a h a b i t a t , then the c o n j e c t u r a l
p r i n c i p l e s t a t e d above r e q u i r e s that these organisms s h a l l not
i n t e r a c t by amensalism; r a t h e r , amensalism comes i n t o play when an
invader appears. A r e l a t i v e l y simple example of t h i s s o r t of thing
i s shown i n F i g u r e 2. In t h i s f i g u r e , A and Β are the organisms of
the a s s o c i a t i o n . They compete f o r the resources, S, of t h e i r
h a b i t a t , but Β i s commensally dependent on A through the by-product,
P, of the growth of A; Ρ i s r e q u i r e d by Β but cannot by synthesized
by that organism from a v a i l a b l e precursors. T h i s a s s o c i a t i o n leads
to s t a b l e coexistence steady s t a t e s , i n s p i t e of the competition;
see the d i s c u s s i o n of commensalism given below. In a d d i t i o n , the
commensal population produce substance T which i t
i n h i b i t o r y to i t s e l f or
many p o t e n t i a l invaders o
appears, amensalism comes i n t o p l a y , and i f the amensalism i s
strong enough, the invader w i l l be destroyed. I t would be very
i n t e r e s t i n g to f i n d an experimental r e a l i z a t i o n of the scheme
shown i n F i g u r e 2, and to see what i t s dynamics were.
Commensalism. One of the d i f f i c u l t i e s with the scheme of

c l a s s i f i c a t i o n used here i s that i t does not recognize d i f f e r e n c e s
i n mechanisms of i n t e r a c t i o n s . T h i s may a l s o be one of i t s
s t r e n g t h s , though I am not so sure about t h a t . The d i f f i c u l t y i s
w e l l i l l u s t r a t e d by the i n t e r a c t i o n of commensalism; a l l of the
f o l l o w i n g s i t u a t i o n s are commensalism, and a l l i n v o l v e q u i t e d i f
f e r e n t mechanisms. ( i ) Population Η r e l e a s e s a by-product of
growth, P, which i s required by another p o p u l a t i o n , C, f o r i t s
growth; ( i i ) p o p u l a t i o n Η produces a set of exoenzymes, E, which
1
a t t a c k i n s o l u b l e m a t e r i a l s , S , to produce s o l u b l e s u b s t r a t e s , S,
which are then used by another p o p u l a t i o n , C; ( i i i ) p o p u l a t i o n Η
consumes a substance, I , which i n h i b i t s the growth of another
p o p u l a t i o n , C; and ( i v ) p o p u l a t i o n Η produces an exoenzyme, E,
which destroys a substance, I, that i s t o x i c to another p o p u l a t i o n ,
H. In a l l of these s i t u a t i o n s , and i n
others that one can imagine, the host population (H) performs a
f u n c t i o n which changes the chemical environment i n a way that
favors the growth of the commensal population (C), but the a c t i v i
t i e s of the commensal are such that the favor i s not r e c i p r o c a t e d .
Mechanisms ( i ) - ( i v ) above are d i f f e r e n t forms of commensalism,
and we may t h i n k of them as elementary or simple forms of t h i s
i n t e r a c t i o n . C l e a r l y , these forms may occur i n combination with
one another, and thus, even a s i t u a t i o n of pure commensalism might
be complex i n the sense that s e v e r a l d i f f e r e n t mechanisms are
involved i n i t . The same t h i n g can be s a i d about most of the
other i n t e r a c t i o n s named i n F i g u r e 1.
The nature of the commensal i n t e r a c t i o n s j u s t described i s
such that many of those that we might f i n d i n nature or construct

FREDRiCKSON Interactions of Microbial Populations
A
Β I Figure 2. An association of two popu-
lations that interact by commensalism
Î / and competition, and that are protected

from invasion by the production of a
toxin.

i n the l a b o r a t o r y would not be pure but would be complicated by

competition of host and commensal f o r a s u b s t r a t e . Meers (34)
pointed t h i s out f o r mechanism ( i ) above and i t i s c l e a r that i t
i s l i k e l y to be true of the other mechanisms as w e l l . The
occurrence of commensalism of mechanism ( i ) can prevent competitive
e x c l u s i o n from happening, and that the a s s o c i a t i o n of host and
commensal i s s t a b l e even though they compete has been demonstrated
c o n v i n c i n g l y both by experiment and by mathematical models; see
Megee et a l . (35), G o t t s c h a l et a l . (26), Miura et_ a l . (29,36).
I would think that the other kinds of commensalism l i s t e d above
would a l s o permit competing host and commensal populations to
coexist.
R e i l l y (37) pointed out another s e t of complications that
o f t e n a f f e c t commensal systems; t h i s was suggested by some e x p e r i -
ments performed by Chao
R e i l l y mentioned a r i s e fro
by the commensal population that have e f f e c t s , e i t h e r s t i m u l a t o r y
or i n h i b i t o r y , on the host p o p u l a t i o n . The production of such
substances changes the i n t e r a c t i o n of pure commensalism i n t o an
i n t e r a c t i o n of pure mutualism or protocooperation, or i n t o a com-
b i n a t i o n of commensalism and amensalism. R e i l l y presented com-
puter s o l u t i o n s of the d i f f e r e n t i a l equations f o r v a r i o u s model
systems of t h i s type, and more r e c e n t l y Sheintuch (39) made a
d e t a i l e d a n a l y s i s of commensal systems complicated by the produc-
t i o n of substances having i n h i b i t o r y e f f e c t s .
In s p i t e of the l i k e l i h o o d that the foregoing complications
w i l l occur, there are probably a f a i r number of s i t u a t i o n s where
commensalism i s pure or r e l a t i v e l y pure; that i s , not much com-
p l i c a t e d by competition or by the production of substances by the
commensal which a f f e c t the host. As an example, I c i t e the
s i t u a t i o n s t u d i e d by Lee et a l . (40). The organisms used,
L a c t o b a c i l l u s plantarum and Propionibacterium shermanii, are
a s s o c i a t e d together i n the manufacture of Swiss cheese. The
p o t e n t i a l f o r competition of these organisms i s present, f o r both
can use, say, glucose as the source of carbon and a v a i l a b l e energy.
However, P. shermanii can s u b s t i t u t e l a c t i c acid f o r glucose, and
i n f a c t , i t was shown (41) that t h i s organism takes up no glucose
when s u f f i c i e n t amounts of l a c t i c a c i d are present. Hence, the
l a c t i c acid produced by L. plantarum i s used by shermanii, and
by i t s preference f o r l a c t i c a c i d over glucose, P. shermanii
avoids competition with L. plantarum, although i t does so at the
expense of becoming commensally dependent on the l a t t e r organism.
One would expect that many s i t u a t i o n s where a p o p u l a t i o n having
the c a p a c i t i e s to use s e v e r a l to many s u b s t i t u t a b l e resources and
to e x h i b i t s u b s t r a t e p r e f e r e n c e — a g e n e r a l i s t p o p u l a t i o n — f o r m s an
a s s o c i a t i o n w i t h a population l a c k i n g these features but having
the a b i l i t y to grow r a t h e r r a p i d l y i n c e r t a i n r a t h e r s p e c i a l i z e d
e n v i r o n m e n t s — a s p e c i a l i s t p o p u l a t i o n — o c c u r i n nature.

Mutualism and Protocooperation. I f the a c t i v i t i e s of two

populations are such that each produces and excretes i n t o the
common environment a substance o r s e t of substances which serves
as r e q u i r e d s u b s t r a t e or growth f a c t o r f o r the other, and i f such
substances are not s u p p l i e d t o the environment from e x t e r n a l
sources, then the i n t e r a c t i o n between the populations w i l l i n v o l v e
mutualism. Other mechanisms g i v i n g r i s e to mutualism might be
imagined a l s o , but the one d e s c r i b e d , which i s sometimes c a l l e d
syntrophism and sometimes c r o s s - f e e d i n g , i s l i k e l y to be the form
of mutualism most o f t e n encountered. Experimental examples of i t
have been provided by Nurmikko ( 4 2 ) Wolfe and Pfennig (43),
9
S l a t e r (44), and Lamb and Garver (45), as w e l l as by others.

Three remarks about t h i s kind of mutualism seem p e r t i n e n t .
F i r s t , the f a c t that two organisms w i l l grow together i n a batch
of medium i n which they
be an i n d i c a t i o n that th
many cases where t h i s observatio
c o r r e c t . But even i f i t i s v a l i d , i t does not f o l l o w that the
syntrophism can be put to use i n , say, a chemostat type o f appara-
tus. I have shown elsewhere (2) that i n order f o r m u t u a l i s t i c
steady s t a t e s to be p o s s i b l e i n a chemostat i t i s necessary that
the production and consumption of the substances which produce the
i n t e r a c t i o n must be such that more o f them i s produced than i s
consumed. I f that i s the case, the system i s s u p e r c r i t i c a l , and
mutualism can produce a steady s t a t e of coexistence i n a chemostat.
But i f the system i s only c r i t i c a l or s u b c r i t i c a l , no such steady
s t a t e i s p o s s i b l e . Hence, i f we wish to e x p l o i t a s i t u a t i o n of
syntrophism i n a chemostat, we have t o be sure that the s i t u a t i o n
at hand i s s u p e r c r i t i c a l or the attempt at e x p l o i t a t i o n w i l l f a i l .
The second p o i n t i s that even f o r syntrophic p a i r s of
organisms which are s u p e r c r i t i c a l , the steady s t a t e of washout from
a chemostat i s always s t a b l e with respect to s m a l l p e r t u r b a t i o n s
(46) . Therefore, the steady s t a t e o f coexistence of the popula-
t i o n s cannot be s t a b l e with respect to a l l l a r g e p e r t u r b a t i o n s , so
coexistence of the partners i s always menaced by l a r g e p e r t u r b a -
t i o n s of t h e i r system.
The t h i r d p o i n t i s that there w i l l o f t e n (always?) be two
coexistence steady s t a t e s f o r a p a i r of s y n t r o p h i c organisms i n a
chemostat. In one of these, the only i n t e r a c t i o n between the
populations i s mutualism but i n the other, some other i n t e r a c t i o n ,
such as competition f o r a n u t r i e n t s u p p l i e d from o u t s i d e the s y s -
tem, occurs a l s o . I t turns out that the steady s t a t e of pure
mutualism i s unstable always (46), so that i f we wanted to study
i t we would have t o t r y t o s t a b i l i z e i t by adding some c o n t r o l s to
the chemostat.
Protocooperation d i f f e r s from mutualism i n that i n mutualism
n e i t h e r population can s u r v i v e without the other whereas i n proto-
cooperation one o r both populations can s u r v i v e without the other.
One could d e f i n e two sub-cases of protocooperation, depending upon
whether the presence of the second population i s necessary f o r

s u r v i v a l of one population or n e i t h e r p o p u l a t i o n . The example

provided by Wilkinson et_ a l (47) seems to be of the former kind of
protocooperation. Here, a Pseudomonas species produces methanol
from methane, and i s s t r o n g l y a u t o - i n h i b i t e d by the a l c o h o l ; a
Hyphomicrobium species consumes the a l c o h o l and thus r e l e a s e s the
Pseudomonas from a u t o - i n h i b i t i o n . The i n t e r a c t i o n may be somewhat
more complex, f o r Wilkinson et a l . were unable to grow t h e i r
Pseudomonas i n pure c u l t u r e , and two other populations, an
Acinetobacter species and a Flavobacter s p e c i e s , were present a l s o
i n t h e i r mixed c u l t u r e system and may have played some r o l e i n i t .
I t appears that removal of i n h i b i t o r y substances plays a l a r g e
r o l e i n many protocooperative i n t e r a c t i o n s that we can imagine, and
when we attempt to construct mathematical models of these i n t e r -
a c t i o n s we are faced with the d i f f i c u l t y that we do not have models
f o r the k i n e t i c s of i n h i b i t o r a c t i o i which plac lot
of confidence. Advancemen
therefore would seem to depen autecologica
knowledge of the k i n e t i c s of i n h i b i t o r a c t i o n .
E c c r i n o l y s i s . C o n s t r u c t i o n of t h e o r i e s of t h i s i n t e r a c t i o n i s
faced with d i f f i c u l t i e s s i m i l a r to those f a c i n g c o n s t r u c t i o n of
t h e o r i e s of protocooperation, but the d i f f i c u l t i e s are even more
severe. In order to construct such a theory, we would need know-
ledge of the k i n e t i c s of formation of exoenzymes by the one popu-
l a t i o n and of the k i n e t i c s of a c t i o n of those enzymes on the other
population of the p a i r . The k i n e t i c s of both processes are complex,
i f we may judge from some recent attempts at modeling exoenzyme
production and a c t i o n presented by Van Dedem and Moo Young (48),
and thus, i t i s not s u r p r i s i n g that attempts to make models of sys-
tems e x h i b i t i n g e c c r i n o l y s i s seem not to have been made. The
i n t e r a c t i o n i s probably of importance i n some n a t u r a l systems, f o r
i t must be involved i n the c y c l i n g of minerals. Therefore, attempts
to study i t q u a n t i t a t i v e l y should be made.
We turn now to d i r e c t i n t e r a c t i o n s between p a i r s of m i c r o b i a l
populations.
Feeding. The f i r s t thing to be s a i d about t h i s subject at the

present time i s that i n s u f f i c i e n t a t t e n t i o n has been paid to the
d i f f e r e n c e s i n the modes of feeding e x h i b i t e d by phagotrophic
microorganisms. Such d i f f e r e n c e s may be i l l u s t r a t e d by c o n s i d e r i n g
the feeding of Didinium nasutum, D i c t y o s t e l i u m discoideum, and
Tetryhymena p y r i f o r m i s , a l l organisms which have been used i n
l a b o r a t o r y s t u d i e s of what i s u s u a l l y c a l l e d m i c r o b i a l prédation.
Didinum nasutum i s a c i l i a t e d protozoan that was used by Gause

(49) i n h i s seminal s t u d i e s of m i c r o b i a l i n t e r a c t i o n s , and many
subsequent s t u d i e s using t h i s organism have been made; f o r a resume
of l i t e r a t u r e , see Berger (50). Didinium feeds by a t t a c k i n g and
i n g e s t i n g other protozoa, normally Paramecium, which i t encounters
during i t s swimming a c t i v i t y . Attacks are on one Paramecium c e l l

at a time, and chemotaxis seems to play a r o l e i n these (51,52) .

Feeding i s s e l e c t i v e both i n regard to d i f f e r e n c e s i n the f r e -
quency of attacks on p r e f e r r e d and non-preferred food as w e l l as
i n regard t o d i f f e r e n c e s i n the percentages of u n s u c c e s s f u l attacks
on d i f f e r e n t organisms (50,52). I t seems q u i t e appropriate to
apply the term prédation to t h i s kind o f feeding behavior. I t
should be noted that Didinium i s not what Slobodkin (53) has c a l l e d
a "prudent predator," f o r i t commonly makes the p o t e n t i a l l y l e t h a l
mistake o f consuming a l l of i t s prey when i t i s i n o c u l a t e d i n t o a
batch of them (49,54,55).
D i c t y o s t e l i u m discoideum i s an a c e l l u l a r slime mold that has

been used much by b i o l o g i s t s studying such processes as chemotaxis,
c e l l aggregation, and morphogenesis. Feeding forms of t h i s
organism are amoebae, and i n a t u r a l s i t u a t i o n s thes g l i d
s o l i d surfaces and engul
encounter them. Tsuchiy (56) gre organis
i n l i q u i d c u l t u r e i n chemostats, and they observed the o s c i l l a t i o n s
of population d e n s i t i e s that t h e o r e t i c i a n s had been p r e d i c t i n g f o r
so long. Dr. Bazin has been working with t h i s organism i n recent
y e a r s , and I hope he w i l l t e l l us more about i t .
Tetrahymena p y r i f o r m i s i s a c i l i a t e d protozoan whose feeding

has been s t u d i e d by many workers; a recent l i t e r a t u r e survey i s
given by Swift et a l . (57). A c e l l of T\ p y r i f o r m i s has a b u c c a l
c a v i t y which has a l a r g e undulating membrane on one s i d e and three
s m a l l e r , moving membranes on the other. The beating of these mem-
branes d i r e c t s water i n t o the b u c c a l c a c i t y , and p a r t i c l e s ,
e s p e c i a l l y b a c t e r i a , suspended i n t h i s water are c o l l e c t e d by the
organism i f they are of appropriate s i z e , n e i t h e r too l a r g e nor too
s m a l l . I t would seem that t h i s k i n d of f o o d - c o l l e c t i n g apparatus
should have no a b i l i t y t o s e l e c t p a r t i c l e s except on the b a s i s of
p r o p e r t i e s that are hydromechanically s i g n i f i c a n t , such as s i z e ,
shape, and d e n s i t y . I t i s not appropriate to apply the name
prédation to t h i s kind o f feeding, and the term suspension-feeding
advocated by J^rgensen (58) w i l l be used i n s t e a d .
The remainder of my remarks on feeding w i l l be about suspen-
s i o n - f e e d i n g . This i s not because I consider t h i s to be the most
important kind of m i c r o b i a l feeding but r a t h e r because i t i s the
kind with which I have f i r s t hand experience.
An i n t e r e s t i n g f a c t which emerges from many l a b o r a t o r y s t u d i e s
of suspension-feeding of Tetrahymena and s i m i l a r bacterivorous
protozoans i s that these organisms have not been observed t o con-
sume a l l of the b a c t e r i a i n t h e i r h a b i t a t . Hence, they are examples
1
of S l o b o d k i n s "prudent predators," although I w i l l change h i s term
to prudent feeders.
The f e a t u r e s of a suspension-feeder's behavior which make i t
"prudent" are not the same f o r a l l such organisms. F o r example,
Bader ejt a l . (59) studied feeding on the blue-green a l g a Anacystis
nidulans by the c i l i a t e d protozoan Colpoda s t e i n i i . [The i d e n t i -

f i c a t i o n of t h i s protozoan was challenged by Frenchel (60) on the

grounds that "the r e a l Colpoda s t e i n i i i s about 10 times smaller
than that recorded i n the reference (to a new handbook of suspen-
s i o n - f e e d i n g by J ^ r g e n s e n ) I have not yet seen J^rgensen's work.
A c t u a l l y , Bader ej: a l . made no statement about the s i z e of t h e i r
c i l i a t e , but they used the same c u l t u r e used by Drake and
Tuschiya (61) , and the c e l l volume data reported by these workers
i s w i t h i n , comfortably so, the range of c e l l s i z e s commonly s t a t e d
f o r Colpoda s t e i n i i , as by Kudo (62), f o r example]. When c e r t a i n
conditions a r i s e i n c u l t u r e s of Colpoda s t e i n i i , i t s m o t i l e , feed-
ing c e l l s undergo morphological change and become non-motile, non-
feeding c y s t s . Conditions which cause encystment are by no means
f u l l y understood, but they seem to i n c l u d e a low density of food,
a high d e n s i t y of feeding c e l l s , or, at intermediate d e n s i t i e s of
the two populations, some combination of the d e n s i t i e s Many sus
pension-feeders are know
a l l having t h i s capacit
P r o t o z o o l o g i s t s have not been able to confirm a few reports
that Tetrahymena p y r i f o r m i s encysts ( C o r l i s s (63)) and so t h i s
organism must be regarded to be non-encysting. Nevertheless, i t
appears to be "prudent" because when i t i s i n o c u l a t e d i n t o a batch
of v i a b l e but non-growing b a c t e r i a i t does not consume a l l of them
but instead only reduces the d e n s i t y of v i a b l e b a c t e r i a to the
5 7 1
order of 10 - 10 mlT . This observation was made by Habte and
Alexander (64) and confirmed by Watson et a l . (65). Experiments
reported i n these papers prove that f a i l u r e to consume a l l of the
b a c t e r i a i s not due to death of the protozoa, to a u t o i n h i b i t i o n of
the protozoa, to exhaustion of some e s s e n t i a l m a t e r i a l which the
protozoa get from the l i q u i d medium r a t h e r than from feeding on the
b a c t e r i a , or to l o s s of the protozoan's a b i l i t y to feed on the bac-
t e r i a . One could t r y to e x p l a i n the r e s u l t s mentioned by saying
that the b a c t e r i a present i n i t i a l l y have a d i s t r i b u t i o n of s t a t e s
and that b a c t e r i a f a l l i n g i n t o some domain or domains of t h i s d i s -
t r i b u t i o n cannot be consumed by the protozoa. For example,
Fenchel (60,66) has shown that the a b i l i t y of a suspension-feeder
to c o l l e c t l a t e x beads i s confined to beads of a c e r t a i n s i z e range,
and s i n c e the same r e s u l t a p p l i e s without much doubt to c o l l e c t i o n
of b a c t e r i a , the argument may be made that the b a c t e r i a which com-
p r i s e the residuum l e f t a f t e r v i a b l e b a c t e r i a l d e n s i t y i n a batch
ceases to f a l l are those that are too s m a l l , or too l a r g e , or too
small and too l a r g e , to be c o l l e c t e d by the protozoans. One cannot
t e s t t h i s hypothesis d i r e c t l y by measuring the changes produced i n
the s i z e d i s t r i b u t i o n of v i a b l e b a c t e r i a by the feeding because the
d e n s i t y of v i a b l e b a c t e r i a present toward the middle and end of the
experiment i s always much l e s s than the density of d e t r i t a l par-
t i c l e s having s i z e s comparable to the b a c t e r i a and which are always
produced by feeding.
The foregoing hypothesis, w h i l e p l a u s i b l e , i s c o n t r a d i c t e d by
some a d d i t i o n a l experiments done by Habte and Alexander (64) . They
found that T. p y r i f o r m i s f a i l e d to s t a r t c o l l e c t i n g b a c t e r i a when

i n o c u l a t e d a t high d e n s i t y i n t o a c u l t u r e of non-growing b a c t e r i a ,
even though i n o c u l a t i o n of T_. p y r i f o r m i s at low d e n s i t y i n t o a
c u l t u r e of non-growing b a c t e r i a d i d r e s u l t i n c o l l e c t i o n (the
i n i t i a l b a c t e r i a l d e n s i t i e s were the same i n both experiments). I
do not think that the observed f a i l u r e to s t a r t feeding i n the high
d e n s i t y case i s an a r t i f a c t to be a t t r i b u t e d to the procedures used
to prepare the protozoan inoculum, although that does need to be
checked out.
An a l t e r n a t e working hypothesis which i s suggested by the
foregoing d i s c u s s i o n i s that T. p y r i f o r m i s c e l l s possess sensing
and c o n t r o l mechanisms which lead to c e s s a t i o n of t h e i r feeding
a c t i v i t y under c e r t a i n c o n d i t i o n s of environmental s t a t e . A
c o r o l l a r y of t h i s hypothesis i s that T. p y r i f o r m i s c e l l s which do
not eat b a c t e r i a l c e l l s under one set of c o n d i t i o n s can eat these
c e l l s — t h e same o n e s — u n d e different t f conditions Testin
of these d i f f e r e n t hypothese
at the present time.
A d d i t i o n a l adaptations which make m i c r o b i a l feeders "prudent"
or which allow m i c r o b i a l "predators" to c o e x i s t with t h e i r "prey"
are discussed i n a recent review by Alexander (67).
C e s s a t i o n of feeding, whether i t be caused by encystment o r
by some process that i s not accompanied by a morphological t r a n s -
formation of the feeders, implies that there i s some threshold
d e n s i t y of food. The threshold d e n s i t y i s such that feeding w i l l
stop (or f a i l to s t a r t ) i f the density f a l l s (or i s ) below the
threshold d e n s i t y . One would expect that the threshold density of
food would depend on the i d e n t i t i e s of the feeding and fed-upon
populations as w e l l as on such things as the composition of the
medium. However, there i s some evidence that i n a d d i t i o n to these
f a c t o r s the threshold d e n s i t y of food changes with the density of
the feeders themselves. F o r example, i n the experiment of Habte
and Alexander c i t e d above (64) , Tetrahymena c e l l s i n o c u l a t e d at
high d e n s i t y f a i l e d to s t a r t feeding whereas Tetrahymena c e l l s
i n o c u l a t e d at a low density d i d s t a r t , and t h i s i s evidence that
the threshold density of b a c t e r i a i s r a i s e d by increase of the
protozoan d e n s i t y . Bader £t a l . (59) reached s i m i l a r conclusions
about thresholds f o r encystment of Colpoda s t e i n i i , and a d d i t i o n a l
evidence from the l i t e r a t u r e could be c i t e d .
Results such as these suggest that tht threshold r e l a t i o n
between the two d e n s i t i e s might be as shown i n F i g u r e 3. Herein,
feeding w i l l occur i f the combination of d e n s i t i e s l i e s above and
to the l e f t of the curve but feeding w i l l not occur i f the combi-
n a t i o n l i e s below and to the r i g h t of the curve.
The curve shown i s drawn with a h o r i z o n t a l asymptote because,
i f the d e n s i t y of the feeding p o p u l a t i o n i s s u f f i c i e n t l y low, there
can be no e f f e c t s o f crowding i n t h i s population. Under such con-
d i t i o n s , the threshold d e n s i t y of the fed-upon p o p u l a t i o n — i f there
i s such a threshold d e n s i t y — m u s t be independent of the density of
the feeding population. The curve i s drawn with a v e r t i c a l
asymptote, a l s o . This i s because one expects t h a t , under very

° c
> ο
W CO
I! Feeding
ο,ο.
ο
tr α No Feeding
Figure 3. Conjectural relation between

densities of feeding and fed-on popula
tion where cessation of feeding by a Logarithm of the Density
"prudent" feeder occurs. of the Feeding Population

crowded c o n d i t i o n s , the best t h i n g f o r a feeding population to do

would be to stop e a t i n g and so prevent f u r t h e r increase of crowd
i n g . Under such c o n d i t i o n s , there i s no threshold d e n s i t y of the
fed-upon population, of course.
F i g u r e 3 must be regarded almost e n t i r e l y as s p e c u l a t i o n ,
s i n c e , so f a r as I know, a complete f i g u r e l i k e i t has never been
e s t a b l i s h e d f o r any p a i r of microorganisms. S a l t (68) gave data
f o r the predator-prey system Woodruffia metabolica-Paramecium
a u r e l i a , but these are of l i m i t e d extent and do not show the
asymptotes of F i g u r e 3. Bader ejt a l . ( 5 9 ) gave a f i g u r e f o r con
d i t i o n s of population d e n s i t i e s which lead to encystment of the
suspension-feeder Colpoda s t e i n i i when i t feeds on Anacystis
nidulans. T h e i r data do suggest that asymptotes e x i s t , but the
data are i n s u f f i c i e n t to prove that they do, and i n a d d i t i o n , are
badly s c a t t e r e d . Threshold d intraspecifi crowdin phenomen
are of fundamental b i o l o g i c a
work to c l a r i f y the p i c t u r
pursued with v i g o r .
I f there i s indeed a threshold density of b a c t e r i a below which
protozoans l i k e Tetrahymena do not feed, then i t follows that
growth and feeding of such organisms cannot be described by Monod's
model of growth (69). A model that suggests i t s e l f f o r s i t u a t i o n s
l i k e this i s
b < b t
Φα) = (1)
φ (b - b.)
m t b > b_
L + b
where φ ^ ) i s the feeding r a t e per protozoan c e l l when the bac

t e r i a l d e n s i t y i s b, b i s the threshold density of b a c t e r i a , and
t
φ and L are model parameters, the former being the maximum, or

π1
s a t u r a t i o n v a l u e , of φ ^ ) . Models l i k e t h i s are inconvenient from

the mathematical and computational point of view, however. The
m u l t i p l e s a t u r a t i o n model of J o s t et a l . (17) i s
* ( b )
" (L, Λ(L 2 + b) ( 2 )
and i t avoids the mathematical and computational d i f f i c u l t i e s

inherent i n use of an e x p l i c i t threshold model, l i k e Equation (1).
Equation (2) does not have a threshold value of b, of course, but
i t does p r e d i c t that
(3)

which i s c h a r a c t e r i s t i c of a t r u e threshold model but not of

Monod's model. I t has been shown that the m u l t i p l e s a t u r a t i o n
model does a much b e t t e r job of c o r r e l a t i n g data f o r feeding of
Tetrahymena on b a c t e r i a than does Monod's model (17 ,70 ,71 ,72) .
As pointed out above, Tetrahymena p y r i f o r m i s c o n s i s t e n t l y
f a i l s to c l e a r batches of water of b a c t e r i a , and the explanation
f o r t h i s may be that some of the b a c t e r i a are too small or too
l a r g e to be captured by the protozoans. I f t h i s i s the case, then
there i s no t h r e s h o l d d e n s i t y of the b a c t e r i a , of course, so there
would be l i t t l e j u s t i f i c a t i o n f o r using models l i k e Equations (1)
or ( 2 ) . In t h i s circumstance, what one needs to do i s to d i v i d e
the b a c t e r i a i n t o sub-populations, one which i s eaten by the p r o t o -
zoans and one or more which i s (are) not eaten by them. Monod's
model might be assumed f o r feeding of the protozoans on the sub-
population of b a c t e r i a that they can eat and models f o r t r a n s f e r
of b a c t e r i a from one sub-populatio
also.
A c t u a l l y use of models l i k e those that we have been d i s c u s s i n g
can probably never y i e l d b e t t e r than order-of-magnitude p r e d i c t i o n s
of p o p u l a t i o n d e n s i t i e s i n dynamic, t r a n s i e n t s i t u a t i o n s . Recent
s t u d i e s of the responses of Tetrahymena p y r i f o r m i s to sudden
changes i n the b a c t e r i a l d e n s i t y of i t s surroundings r e v e a l phe-
nomena (57 ,65) which seem to r e q u i r e p a r t i a l d i f f e r e n t i a l equations
r a t h e r than o r d i n a r y d i f f e r e n t i a l equations f o r t h e i r accurate
description.
F i n a l l y , i t should be mentioned that feeding by protozoans
would be expected to exert strong r e g u l a t o r y e f f e c t s on the bac-
t e r i a l populations on which they feed, e s p e c i a l l y i f these l a t t e r
populations compete with one another f o r n u t r i e n t s . J o s t et a l .
(17), f o r example, found that f e e d i n g of Tetrahymena p y r i f o r m i s on
E s c h e r i c h i a c o l i and Azotobacter v i n e l a n d i i i n a chemostat seemed
to lead to coexistence of the three populations i n a p e r p e t u a l l y
t r a n s i e n t s t a t e ; i n the absence of the protozoans, however, Azoto-
b a c t e r was excluded by E. c o l i .
C l e a r l y , an important f a c t o r to be considered i n s i t u a t i o n s
l i k e the foregoing i s the p o s s i b i l i t y that the protozoans may
e x h i b i t preference f o r one b a c t e r i a l species over the other. As
mentioned p r e v i o u s l y , i t seems l i k e l y that food preference by
suspension-feeding microorganisms must be based e n t i r e l y , or almost
e n t i r e l y , on d i f f e r e n c e s i n hydromechanically s i g n i f i c a n t proper-
t i e s l i k e s i z e , shape, and d e n s i t y , of the food organisms.
Experiments i n which protozoans are presented which choice of food,
the food organisms d i f f e r i n g i n the p r o p e r t i e s noted, need to be
done. We are c u r r e n t l y p r e s e n t i n g Tetrahymena p y r i f o r m i s with E.
c o l i and A. v i n e l a n d i i , these being b a c t e r i a whose s i z e s are q u i t e
d i f f e r e n t . But experiments i n which a suspension-feeder i s pre-
sented with b a c t e r i a having the same s i z e and shape, but d i f f e r i n g
i n some p r o p e r t i e s that are not hydromechanically s i g n i f i c a n t , need
to be done, too. One expects no preference i n such cases, and the
one experiment of the kind that I know of showed no preference

(73), but more data of the same kind are needed before one can
conclude that food preference by suspension-feeders i s based
s o l e l y on d i f f e r e n c e s of hydromechanically s i g n i f i c a n t p r o p e r t i e s
of food organisms.
P a r a s i t i s m . In t h i s d i r e c t i n t e r a c t i o n a p a r a s i t e c e l l or
p a r t i c l e attaches i t s e l f to a host c e l l and makes a p a r t i a l or
t o t a l p e n e t r a t i o n i n t o i t , where i t then uses the host's biomass
or metabolic a c t i v i t i e s to grow and reproduce i t s e l f . Examples
are provided by the p a r a s i t i s m of v i r u s e s on b a c t e r i a and other
microorganisms, by the p a r a s i t i s m of the very small bacterium
B d e l l o v i b r i o on other b a c t e r i a , and by the p a r a s i t i s m of b a c t e r i a
on protozoa (30,74_,75) . P a r a s i t i s m i s c h a r a c t e r i z e d by a v a r i a b l e
but always high degree of host s p e c i f i c i t y . That i s , a given
p a r a s i t e i s able to i n f e c t onl l i m i t e d numbe f hosts d i
some cases, the number ma
A number of mathematica host-parasit
have been published. For references to l i t e r a t u r e appearing
before 1977, see F r e d r i c k s o n (2). A more recent model has been
given by L e v i n ^ t a l . (76).
P a r a s i t i s m can serve to r e g u l a t e competition between d i f f e r e n t
host populations. An example i s provided by the work of L e v i n
et a l . (76). They found that p a r a s i t i s m by the v i r u l e n t b a c t e r i o
phage T2 on two s t r a i n s of E s c h e r i c h i a c o l i s t a b i l i z e d the compe
t i t i o n of the s t r a i n s f o r sugar, and allowed the competitors to
c o e x i s t i n a chemostat. One of the s t r a i n s was s u s c e p t i b l e to
i n f e c t i o n by T2 but the other was not.
A most i n t e r e s t i n g aspect of the v i r u s - b a c t e r i a h o s t - p a r a s i t e
i n t e r a c t i o n i s the tendency f o r genetic changes of the populations
to keep a l t e r i n g the dynamics of the i n t e r a c t i o n . T h i s i s w e l l
i l l u s t r a t e d by some a d d i t i o n a l work of Chao et a l . (77). A host
bacterium B Q (a s t r a i n of E. c o l i ) and a bacteriophage T were Q
introduced i n t o a chemostat; B was s u s c e p t i b l e to i n f e c t i o n by

Q
T .
0 Mutation of B produced a new s t r a i n of b a c t e r i a , B^, which
Q
was not s u s c e p t i b l e to i n f e c t i o n by T . Q However, mutation of the

phage produced a new v i r u s , T^, which was capable of i n f e c t i n g
both B and B^.
Q A second mutuation of the b a c t e r i a produced a
t h i r d s t r a i n , Β£, which was immune to i n f e c t i o n by T and T-^. q In
these experiments, the v a r i o u s s t r a i n s of b a c t e r i a competed with
one another f o r sugar, but the presence of the p a r a s i t e s prevented
competitive e x c l u s i o n s from o c c u r r i n g . When a s t r a i n of b a c t e r i a
that was not s u s c e p t i b l e to i n f e c t i o n by any of the p a r a s i t e s was
present, sugar c o n c e n t r a t i o n was low and b a c t e r i a l and phage den
s i t i e s were high. However, when a l l s t r a i n s of b a c t e r i a present
were s u s c e p t i b l e to i n f e c t i o n by the phages, sugar c o n c e n t r a t i o n
was high and b a c t e r i a l and phage d e n s i t i e s were low. These obser
v a t i o n s of Chao et a l . (77) suggest that the h o s t - p a r a s i t e
r e l a t i o n i s a kind of genetic race between the two groups of
organisms. Undoubtedly, analagous s i t u a t i o n s occur with other
i n t e r m i c r o b i a l i n t e r a c t i o n s , and e f f o r t s to detect and analyze
such s i t u a t i o n s would very l i k e l y prove to be most f r u i t f u l .

Symbiosis. T h i s i s a d i r e c t i n t e r a c t i o n between two micro-

b i a l populations which i s c h a r a c t e r i z e d not only by the mutual
(even i f i m p e r f e c t l y understood) b e n e f i t s which i t confers upon
the partners i n the a s s o c i a t i o n but a l s o , l i k e p a r a s i t i s m , by i t s
high degree of s p e c i f i c i t y . That i s , the two partners are more or
l e s s uniquely adapted to l i v e together, and i t i s impossible or
very d i f f i c u l t to r e p l a c e one of the partners i n the a s s o c i a t i o n
by another organism of s i m i l a r k i n d . Undoubtedly, syntrophism i s
involved i n many m i c r o b i a l symbioses, and perhaps models l i k e
those f o r t h i s m u t u a l i s t i c i n t e r a c t i o n would be a p p l i c a b l e to
symbiosis. Q u a n t i t a t i v e models f o r symbiosis seem to be non-
e x i s t e n t at the present time, however.
Crowding. T h i s i n t e r a c t i o n i s important when population

d e n s i t i e s become so l a r g that th a v a i l a b i l i t f become
a l i m i t i n g f a c t o r i n th
s p e c i f i c i n t e r a c t i o n of g probably importanc
i n s i t u a t i o n s where the i n t e r a c t i n g populations are suspended i n
a l i q u i d medium, because d e n s i t i e s t h e r e i n are not l i k e l y to
become so high that a v a i l a b i l i t y of space becomes a r a t e - l i m i t i n g
f a c t o r f o r both populations. I n t r a s p e c i f i c crowding can become
important i n l i q u i d c u l t u r e s , i t appears; the existence of the
v e r t i c a l asymptote i n the graph of F i g u r e 3 i s an example of an
e f f e c t due to i n t r a s p e c i f i c crowding.
I n t e r s p e c i f i c crowding may be of great importance when we are
d e a l i n g with systems i n which much s u r f a c e area, upon which the
organisms attach themselves, i s present. A good d e a l of e f f o r t
has been expended i n recent years to understand the mechanisms
involved i n attachment of organisms to s u r f a c e s . The mechanisms
are complex, even when only a s i n g l e population i s i n v o l v e d , f o r
the d e n s i t y of attached c e l l s i s found to depend on the i d e n t i t y
and p h y s i o l o g i c a l s t a t e of the organism, the i d e n t i t y of the
m a t e r i a l of which the surface i s made as w e l l as the p r i o r t r e a t -
ment of the s u r f a c e , the composition of the l i q u i d medium adjacent
to the s o l i d s u r f a c e , the d e n s i t y of the organisms i n the l i q u i d
medium, and the time of contact between the suspension of organisms
and the s u r f a c e (78). F a m i l i a r concepts that apply to adsorption
of molecules on s u r f a c e s , l i k e that of the a c t i v e s i t e , seem not
to apply, and t h i s means that the Langmuir-Hinshelwood model of
adsorption, or one of i t s g e n e r a l i z a t i o n s , i s not l i k e l y to apply,
either. Instead, i t seems that we need to construct dynamic
s t o c h a s t i c models which w i l l make the p r o b a b i l i t y of attachment of
one c e l l to a given area of s u r f a c e i n a short i n t e r v a l of time
dependent upon the number of c e l l s already attached to that s u r -
face as w e l l as to the d e n s i t y of c e l l s present i n the bulk
l i q u i d , e t c . Since I am not aware that models l i k e t h i s have been
worked out even f o r s i n g l e populations, I cannot say anything
meaningful about models f o r competition of two populations f o r

space on a s o l i d substratum. We are t r y i n g to study t h i s i n t e r -

a c t i o n i n our l a b o r a t o r y a t the present time, but i t i s not an
easy t h i n g to do.
I t was pointed out above that "crowding" as I have used the
term r e f e r s t o an i n t e r s p e c i f i c i n t e r a c t i o n but that b i o l o g i s t s
o f t e n use the term f o r an i n t r a s p e c i f i c i n t e r a c t i o n . The context
w i l l make i t c l e a r i n most cases which kind of i n t e r a c t i o n i s
being r e f e r r e d to, so there should be no d i f f i c u l t y i n using the
same word f o r two d i f f e r e n t kinds of s i t u a t i o n s .
Summary and d i s c u s s i o n
The foregoing d i s c u s s i o n shows that improved understanding

of i n t e r a c t i o n s between m i c r o b i a l populations w i l l r e q u i r e
research i n a number of r e l a t e d A partial list i
follows.
Much work remains autecology
populations; that i s , on the responses of i n d i v i d u a l populations
to changes i n t h e i r environment and on the changes produced i n the
a b i o t i c environment by the a c t i v i t i e s of such p o p u l a t i o n s . Of
p a r t i c u l a r importance i n s o f a r as osmotrophic microorganisms a r e
concerned i s research aimed at p r o v i d i n g good models f o r the
e f f e c t s of m u l t i p l e n u t r i e n t l i m i t a t i o n on growth r a t e s , f o r the
k i n e t i c s of s u b c e l l u l a r processes i n general, f o r the k i n e t i c s of
formation of e x t r a c e l l u l a r chemicals and enzymes, f o r the k i n e t i c s
of a c t i o n of i n h i b i t o r s , t o x i n s , and l y t i c enzymes, e t c . More
work i s needed on the autecology of phagotrophic microorganisms,
a l s o . The mechanisms that make such organisms "prudent" feeders
need t o be e s t a b l i s h e d and compared and the question of the extent
to which they s e l e c t t h e i r food needs to be examined f u r t h e r .
Almost a l l attempts t o c o n s t r u c t mathematical models of popu-
l a t i o n i n t e r a c t i o n s assume that the populations present are
e n t i t i e s of f i x e d genetic c o n s t i t u t i o n . I f they allow f o r the
occurrence of mutuation, i t i s almost always i n the sense of
assuming that a mutant of given p r o p e r t i e s has formed, and then
t r y i n g to see how the system of mutant, w i l d organism, and any
other organisms present, w i l l evolve i n time. Models which are
b u i l t on some of the e x c i t i n g new information that m i c r o b i a l
g e n e t i c i s t s have provided need t o be developed and a p p l i e d to
systems of i n t e r a c t i n g populations. High p r i o r i t y should be given
to producing models which take i n t o account mechanisms of mutation
and genetic recombination.
Several b i n a r y i n t e r a c t i o n s have been neglected by people who
take a q u a n t i t a t i v e , mathematical approach t o such processes.
Amensalism, antagonism, and e c c r i n o l y s i s are i n d i r e c t i n t e r a c t i o n s
which f a l l i n t o t h i s category, and there i s room and motivation
for much work on them. In a d d i t i o n , there are many elementary
mechanisms of commensalism, mutualism, and protocooperation that
have been neglected. Among the d i r e c t i n t e r a c t i o n s , nothing
q u a n t i t a t i v e i s known about crowding and symbiosis, and the former
i n t e r a c t i o n , at l e a s t , should be made the object o f study q u i t e
soon.

New experimental techniques and apparatus f o r studying popu-

l a t i o n i n t e r a c t i o n s need to be developed. A l t e r n a t e and improved
methods of o b t a i n i n g census data on systems c o n t a i n i n g two or more
populations are c r i t i c a l needs, and improved, automated means of
making chemical analyses of a b i o t i c media would be most h e l p f u l ,
too.
New mathematical techniques f o r d e a l i n g with the systems of
n o n l i n e a r d i f f e r e n t i a l equations that r e s u l t from models of popu-
l a t i o n i n t e r a c t i o n s need to be found. D i s c u s s i o n s with mathema-
t i c i a n s make i t c l e a r that techniques which would seem to be
u s e f u l have not been much developed, so r a p i d progress i s not to
be expected i n t h i s d i f f i c u l t l i n e of endeavor. However, some
progress has been made. For example, Stephanopoulos (79,80) has
shown how the theory of the Poincaré index can be a p p l i e d to the
equations of b i n a r y p o p u l a t i o i n t e r a c t i o n s Th proble with
t h i s technique i s that i
reduced to two o r d i n a r y equations y
of b i n a r y i n t e r a c t i o n f a l l i n t o t h i s category (79), but many do
not and of course ternary, quaternary, e t c . i n t e r a c t i o n s do not,
either.
Another l i n e of research, e n t i r e l y d i f f e r e n t from what has
j u s t been mentioned, concerns the a p p l i c a t i o n s of mixed c u l t u r e s
and of knowledge about i n t e r a c t i o n s i n mixed c u l t u r e s . I t seems
to me that i t would now be worthwhile to t r y to think of i n d u s t r i a l
operations where mixed c u l t u r e s could be used to advantage.
Development of g e n e t i c engineering techniques f o r making micro-
organisms capable of doing s p e c i f i c , assigned tasks would seem to
have increased the l i k e l i h o o d that s u c c e s s f u l mixed c u l t u r e pro-
cesses can be developed, and work on developing them might now
prove to be rewarding. There i s a l s o the a d d i t i o n a l f i e l d of
a p p l i c a t i o n of models and t h e o r i e s of p o p u l a t i o n i n t e r a c t i o n s to
problems of understanding the dynamics of n a t u r a l ecosystems.
M i t i g a t i o n of p o l l u t i o n of lakes and streams, cleanup of o i l and
chemical s p i l l s , prevention of acid mine drainage, and so on, are
a l l r e a l and l a r g e problems, and i t i s reasonable to expect that
a p p l i c a t i o n of the models and t h e o r i e s noted can be of help i n
s o l v i n g such problems.
Acknowledgement
The support of the N a t i o n a l Science Foundation, through

grants ENG77-21632 and CPE-8020783, i s acknowledged with thanks.

Literature Cited
1. Odum, P. "Fundamentals of Ecology", 3rd. ed., Saunders:
Philadelphia, 1971, pp. 211-213.
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10
The Role of S p e c i a l i s t s a n d G e n e r a l i s t s
in Microbial Population Interactions
J. G. KUENEN
Delft University of Technology, Laboratory of Microbiology,
Julianalaan 67a, 2628 BC Delft, The Netherlands
In many environments highly specialized bacteria co-

exist with generalists
bolize a large diversit
understand the mechanisms of interaction between
these types of bacteria, model experiments in con-
tinuous culture have been carried out with two
typical specialists and one generalist. It could be
shown that the generalist can compete successfully
with specialists for growth limiting substrates
when mixtures of substrates are available. Under
these conditions the generalist can utilize these
mixtures simultaneously. Another advantage of gene-
ralists might lie in their capability to continue
to grow when the supply of different substrates a l -
ternate. In that case specialists would alternati-
vely grow and starve. Model-competition experiments
indicate that, in general, the success of specia-
lists was favoured by increased length of growth
and starvation periods.
The breakdown of organic and i n o r g a n i c compounds i n nature i s

c a r r i e d out by an enormous d i v e r s i t y of b a c t e r i a which are adapted
to a v a r i e t y of p h y s i c a l and chemical environmental parameters. In
a given environment, many microorganisms may c o e x i s t which o f t e n
have completely d i f f e r e n t metabolic c a p a b i l i t i e s , but sometimes
a l s o have overlapping p r o p e r t i e s . T y p i c a l l y , one can f i n d h i g h l y
s p e c i a l i z e d organisms, able to metabolize only one or a few com-
pounds, c o e x i s t i n g with very v e r s a t i l e organisms, termed genera-
l i s t s . The l a t t e r are able to metabolize a great d i v e r s i t y of o r -
ganic compounds.
From the e c o l o g i c a l p o i n t of view one may ask how to e x p l a i n
t h i s coexistence of microorganisms and how t h e i r r e s p e c t i v e phy-
s i o l o g i c a l p r o p e r t i e s may give them an advantage or disadvantage
under a given growth c o n d i t i o n . S i m i l a r l y , from the p o i n t of view
of management of sewage treatment p l a n t s , one may ask how the
0097-6156/83/0207-0229$06.75/0

regime of a p l a n t may s e l e c t c e r t a i n metabolic types and how t h i s

may have a bearing on p r o p e r t i e s of the p l a n t such as s u s c e p t i b i -
l i t y to pulse charges and loading c a p a c i t y .
In order to understand the complicated r e a c t i o n s occuring be-
tween s p e c i a l i s t s and g e n e r a l i s t s w i t h i n a complex mixture of
other organisms, a thorough i n s i g h t i n t o t h e i r b a s i c metabolic
p r o p e r t i e s , that i s t h e i r p h y s i o l o g i c a l behaviour, i s needed. Un-
f o r t u n a t e l y the physiology of most microorganisms i s impossible
to study i n the very complicated mixtures i n which they n a t u r a l l y
occur. Therefore, as a i n i t i a l approach, i t has been necessary to
study s p e c i a l i s t s and g e n e r a l i s t s i n pure c u l t u r e s , and i n a r t i f i -
c i a l composite-mixtures of organisms i n order to obtain i n s i g h t
i n t o t h e i r e c o l o g i c a l niches.
When grown on t h e i r s p e c i f i c substrate i n the laboratory, the
s p e c i a l i s t s are c h a r a c t e r i z e d b high s p e c i f i growth rate
(ymax), whereas the g e n e r a l i s t
low maximum s p e c i f i c growt y
l i z e . Even at very low concentrations of a s u b s t r a t e , as they
g e n e r a l l y occur i n the environment, the v e r s a t i l e organisms grow
r e l a t i v e l y slowly.
F i g u r e 1 shows a g e n e r a l i z e d p i c t u r e of the r e l a t i o n s h i p be-
tween the growth l i m i t i n g substrate and the s p e c i f i c growth r a t e
of a s p e c i a l i s t and a g e n e r a l i s t .
I t may be asked whether the g e n e r a l i s t s might be able to grow
f a s t e r than s p e c i a l i s t s i n mixtures of s u b s t r a t e s . However, under
such c o n d i t i o n s the g e n e r a l i s t s o f t e n show s e q u e n t i a l substrate
u t i l i z a t i o n , known as d i a u x i e . A w e l l known example i s the growth
of the bacterium Escherichia coli on mixtures of glucose and l a c -
tose. F i r s t the glucose i s u t i l i z e d , and only when t h i s compound
i s completely metabolized, the enzymes needed f o r l a c t o s e u t i l i z a -
t i o n w i l l be induced, allowing growth on l a c t o s e . Thus at high
concentrations of mixtures of s u b s t r a t e s , simultaneous u t i l i z a -
t i o n i n t h i s case i s not p o s s i b l e .
One should r e a l i z e that i n many n a t u r a l and seminatural e n v i -
ronments concentrations of substrates are g e n e r a l l y low, u s u a l l y
below the mM and even o f t e n below the pM range. Under such condi-
t i o n s , the growth r a t e of microorganisms w i l l be l i m i t e d by the
concentration of t h e i r s u b s t r a t e s , and mixed substrate u t i l i z a -
t i o n might be p o s s i b l e . In the l a b o r a t o r y , growth under dual sub-
s t r a t e l i m i t a t i o n can be conveniently created i n a f l o w - c o n t r o l l e d
chemostat, or continuous c u l t u r e . In the growth medium supplied
to the c u l t u r e , a l l i n g r e d i e n t s necessary f o r growth are i n ex-
cess except f o r the two substrates i n question. I t has been shown
for E. coli by S i l v e r and Mateles (1) that under such c o n d i t i o n s
simultaneous u t i l i z a t i o n of glucose and l a c t o s e was p o s s i b l e .
Analogous r e s u l t s have been obtained f o r other b a c t e r i a , such as
Pseudomonas oxalaticus growing on mixtures of formate and acetate
(2) and Thiobacillus s t r a i n A2 growing on t h i o s u l f a t e and acetate
(J3). Our own work on the g e n e r a l i s t Thiobacillus A2 may serve as

Microbial Specialists and Generalists
Specialist
Generalist Figure 1. The relationship between the

concentration (s) of the growth-limiting
substrate and the specific growth rate (μ)
of a typical specialist and a typical gen
eralist bacterium growing on the same
-> s substrate.

an example, T. A2 i s a very v e r s a t i l e organism able to grow on at

l e a s t 25-30 d i f f e r e n t organic s u b s t r a t e s , and, i n a d d i t i o n , a l s o
capable of a u t o t r o p h i c growth on i n o r g a n i c reduced s u l f u r com
pounds. (See Table I)· Under the l a t t e r c o n d i t i o n s i t o x i d i z e s ,
f o r example, t h i o s u l f a t e or s u l f i d e as a source of energy while
using carbon d i o x i d e as the only carbon source f o r growth. When
grown i n batch c u l t u r e i n the presence of high concentrations of
acetate and t h i o s u l f a t e Τ. A2 c l e a r l y shows a b i p h a s i c u t i l i z a t i o n
of the two compounds (Figure 2). However when grown i n the chemos
tat under growth l i m i t a t i o n by t h i s mixture, simultaneous u t i l i
z a t i o n of acetate and t h i o s u l f a t e i s p o s s i b l e i r r e s p e c t i v e of the
a v a i l a b l e r a t i o of t h i o s u l f a t e and acetate (Figure 3). Under such
c o n d i t i o n s , acetate and t h i o s u l f a t e concentrations i n the c u l t u r e
are below the d e t e c t i o n l e v e l . The metabolic machinery of T. A2
adapts to the r e q u i r e d turnover rates of the r e s p e c t i v e sub
s t r a t e s . For example, th
sent at maximum c a p a c i t
c u l t u r e , whereas no t h i o s u l f a t e r e s p i r a t i o n c a p a c i t y i s present
when only acetate i s a v a i l a b l e . I n t e r e s t i n g l y , the a b i l i t y of the
c e l l s to a s s i m i l a t e C O 2 f o r c e l l carbon i s e f f i c i e n t l y adapted to
the a v a i l a b l e organic carbon i n the c u l t u r e . This implies that
when the r a t i o of t h i o s u l f a t e to acetate i s r e l a t i v e l y h i g h , ace
tate i s used p r i m a r i l y as a carbon source to "save" energy f o r
C O 2 f i x a t i o n . F u r t h e r work i n our laboratory by others (Ji and j )
has shown that simultaneous u t i l i z a t i o n of mixtures of other
substrates i s e q u a l l y p o s s i b l e .
The aim of our research was then focussed on the question of
whether t h i s g e n e r a l i s t would be able to compete s u c c e s s f u l l y f o r
g r o w t h - l i m i t i n g substrates with s p e c i a l i s t s during mixotrophic
growth under n u t r i e n t l i m i t a t i o n . For our purpose we used the
three model organisms shown i n Table I I . These included a t y p i c a l
g e n e r a l i s t , namely the v e r s a t i l e Thiobacillus A2, and two s p e c i a
l i s t s . The f i r s t s p e c i a l i s t was the chemolithoautotroph, Thioba
cillus neap olitonus
9 which can grow very f a s t i n m i n e r a l s - t h i o
s u l f a t e medium using C O 2 as i t s carbon source, and the second was
a chemoorganoheterotroph Spirillum G7, which can grow very r a p i d l y
on m i n e r a l s - a c e t a t e medium using acetate as carbon and energy
source. (See a l s o Table I ) .
A s e r i e s of experiments was c a r r i e d out i n continuous c u l t u r e
to study the competition between sets of two and three organisms.
In the first experiment, Thiobacillus A2 and Thiobacillus neapoli-
tanus were each grown s e p a r a t e l y i n a t h i o s u l f a t e - l i m i t e d chemos
t a t . Once steady s t a t e s had been e s t a b l i s h e d at a f i x e d d i l u t i o n
r a t e , the c u l t u r e s were mixed one to one (v/v) and the change i n
the percentages of the two organisms was followed u n t i l no f u r t h e r
change could be observed f o r one or two volume changes. F i g u r e 4
shows that i n m i n e r a l s - t h i o s u l f a t e medium, Thiobacillus A2 was
out-competed by the s p e c i a l i s t . T. A2 was not completely e l i m i n a
ted s i n c e i t has been shown that the s p e c i a l i s t excretes g l y c o l -
l a t e (6) which can be consumed by T. A2.

10. KUENEN Microbial Specialists and Generalists 233
Table I
11
Types of metabolism of "model b a c t e r i a used f o r the study of
competition between " s p e c i a l i s t s " and " g e n e r a l i s t s " .
GENERALIST:
Facultative chemolitho(auto)troph ("mixotroph") (Thiobacillus A2)
= °2
S 0 2 3 (or S ) > S0 4
Energy source: and/or Λ
U
2
(many) organic compound(s) > CO^
CO2 — * cellmaterial
Carbon source: and/or
(many) organic compound(s) — ^ c e l l m a t e r i a l
SPECIALIST I
O b l i g a t e chemolitho(auto)troph (Thiobacillus neapolitanus)
0 o
2
Energy source: S20 3
5
«
?
u
(or S ) =-> SO,
Carbon source: CO^—^ c e l l m a t e r i a l
SPECIALIST I I
Chemo(organo)heterotroph (Spirillum G7)
°
2
Energy source: (few) organic compounds > CO^
Carbon source: (few) organic compounds—^ c e l l m a t e r i a l

0 2 4 6 8 10 12 % 16
time (h)
Figure 2. Maximum substrate oxidation capacities ( Q o ^ ' j and substrate concen

tration during growth of T. A2 on a mixture of acetate and thiosulfate in batch
culture. The inoculum consisted of cells from an acetate-grown culture. Key: · ,
q ™*-thiosulfate;
0 A , Qog^'-acetate; O , thiosulfate concentration; Δ , acetate
concentration. Reproduced, with permission, from Ref. 3. Copyright 1980, Springer-
Verlag.

0 2 4 6 8 10 12 14 16 18 20
acetate (mM)
40 36 32 28 24 20 16 12 8 4 0
thiosulfate (mM)
Figure 3. Maximum substrate oxidation potentials and carbon dioxide fixation
potential of whole cells of T . A2 as a function of different acetate and thiosulfate
concentrations in the reservoir medium of the chemostat cultures. Key: · , Qo™*-
thiosulfate; A , QoJ™ *-acetate;
1
M, C0 -fixation potential. Reproduced, with per-
2
mission, from Ref. 3. Copyright 1980, Springer-Verlag.

Data were obtained with cells from thiosulfate- and/or acetate-limited chemostat cultures in
1
steady state at a dilution rate of 0.05 h~ . Acetate and thiosulfate concentrations in the cultures
were below the detection level. Cellular carbon in the cultures ranged from 110 mg C/L (40
mM thiosulfate) to 200 mg C/L (20 mM acetate).

When acetate was s u p p l i e d to the i n f l o w i n g medium, the number

of T. A 2 c e l l s increased s i n c e T. neapolitanus cannot u t i l i z e
acetate. F i g u r e 5 shows that with i n c r e a s i n g acetate c o n c e n t r a t i o n
the percentage of T. A 2 g r a d u a l l y increased and coexistence of the
two organisms became p o s s i b l e . Above a c o n c e n t r a t i o n of about 10
mM acetate, the s p e c i a l i s t was e l i m i n a t e d from the c u l t u r e . The
explanation f o r t h i s l i e s i n the a b i l i t y of T. A 2 to u t i l i z e
acetate and t h i o s u l f a t e simultaneously under these growth c o n d i -
t i o n s . With i n c r e a s i n g acetate c o n c e n t r a t i o n , the absolute concen-
t r a t i o n of the T. A 2 c e l l s increased and allowed these organisms
to c l a i m an i n c r e a s i n g l y l a r g e r share of t h i o s u l f a t e . E v e n t u a l l y ,
the r a t i o of acetate to t h i o s u l f a t e became such that T. A 2 was
able to reduce the t h i o s u l f a t e c o n c e n t r a t i o n to below the l e v e l at
which T. neapolitanus could maintain the r e q u i r e d growth r a t e , r e -
s u l t i n g i n the wash out of the s p e c i a l i s t Very s i m i l a r r e s u l t s
were obtained with mixture
t h i o s u l f a t e and glucose
Competition experiments between the s p e c i a l i s t Spirillum G7
and T. A 2 gave analogous r e s u l t s . In acetate medium Spirillum G7
out-competed T. A 2 completely. Thiobacillus A 2 out-competed the
s p e c i a l i s t when more than 10 mM t h i o s u l f a t e was present i n the
acetate-minerals medium. Obviously, under " n a t u r a l " conditions i n
the environment, or i n sewage treatment p l a n t s , the g e n e r a l i s t
would have to compete with both s p e c i a l i s t s at the same time.
Therefore, three-membered c u l t u r e s were a l s o s t u d i e d . The r e s u l t s ,
as summarized i n F i g u r e 6, show that coexistence of the three
organisms was p o s s i b l e over a large range of c o n c e n t r a t i o n r a t i o ' s
with T. A 2 dominating the c u l t u r e . The coexistence of three orga-
nisms does not agree with t h e o r e t i c a l p r e d i c t i o n s which w i l l be
discussed below. In any case the outcome of these experiments
c l e a r l y i n d i c a t e d that when mixed substrates are s u p p l i e d , genera-
l i s t s have metabolic advantages which allow them to c l a i m a
"niche", that i s , a r i g h t of existence i n the n a t u r a l environment.
The v a l i d i t y of t h i s g e n e r a l i s a t i o n was confirmed by the r e s u l t s
of enrichment c u l t u r e s c a r r i e d out i n the chemostat i n o c u l a t e d
with n a t u r a l samples. Table I I I shows the outcome of such e n r i c h -
ment c u l t u r e s performed with d i f f e r e n t mixtures of t h i o s u l f a t e and
a c e t a t e . In a l l cases when f r e s h water i n o c u l a were used, a domi-
nant c u l t u r e of a g e n e r a l i s t Thiobacillus was obtained. I n t e r e s -
t i n g l y , i n 4 out of 5 cases the g e n e r a l i s t was a f a c u l t a t i v e
chemolithotroph able to grow a u t o t r o p h i c a l l y . However, i n one
case when r e l a t i v e l y high amounts of acetate were presented to the
c u l t u r e , a bacterium able to o b t a i n energy from t h i o s u l f a t e but
not able to f i x C O 2 came to the f o r e . This i s not s u r p r i s i n g ,
s i n c e at t h i s r a t i o C O 2 f i x a t i o n i s not r e q u i r e d (compare F i g u r e 3
f o r T. A2).
As mentioned b e f o r e , a somewhat p u z z l i n g r e s u l t of the compe-

t i t i o n experiments with the three-membered c u l t u r e was that
coexistence of three organisms seemed p o s s i b l e . In s e v e r a l theore-

Table I I
Maximum s p e c i f i c growth rates ν^οχ (h ^) of the specialized,

o b l i g a t e l y c h e m o l i t h o ( a u t o ) t r o p h i c Thvobacillus neapolitanus,
the v e r s a t i l e , f a c u l t a t i v e l y chemolithοtrophic Thiobacillus A2 (a
g e n e r a l i s t ) , and a s p e c i a l i z e d h e t e r o t r o p h i c Spirillum G7, during
growth i n minerals medium supplemented with t h i o s u l f a t e ( t ) ,
acetate (a) or mixtures of both substrates ( t + a ) .
T. neapolitanus and Spirillum G7 cannot grow on acetate or t h i o
sulfate respectively (10).
Maximum s p e c i f i c growth r a t e P m a x (h ^)
t a t +a
T. neapolitanus 0.3
Thiobacillus A2 0.10 0.22 0.22
Spirillum G7 - 0.43 0.43
3 A 5 6 7 8
Volume changes
Figure 4. Competition in continuous culture between T. neapolitanus (the special
ist) and T. A2 (the generalist) for thiosulfate as the only growth-limiting substrate.
Key: Φ, relative cell number of Τ. A2; Ο , T. neapolitanus. Reproduced, with per
mission, from Ref. 4. Copyright 1979, Springer-Verlag.
1
The chemostat was run at a dilution rate of 0.05 h' with a 40 mM thiosulfate concentration in
the reservoir medium. Organisms had been pregrown separately in continuous culture at
1
Ό = 0.05 h' and at zero time mixed in a 1:1 rate.

Figure 5A. Effect of different concentrations of organic substrate on the outcome

of the competition between T . A 2 and T. neapolitanus for thiosulfate. Protein
concentration and organic content in the cultures are shown, limited by thiosulfate
plus acetate or glycollate. Reproduced, with permission, from Ref. 4. Copyright
1979, Springer-Verlag.
1
The chemostat was run at a dilution rate of 0.07 h' . The inflowing medium contained thiosulfate
(40 mM) together with either acetate or glycollate at concentrations ranging from 0-7 mM.
Relative cell numbers, protein content, and organic cell carbon in the culture were determined
after steady states had been established.

(• glycollate)
J. neapolitanus
(•glycollate)
Oh -L -L JL
3 4 5 6 7 8 9 10
GLYCOLLATE OR ACETATE (mM)
Figure 5B. Effect of different concentrations of organic substrate on the outcome

of the competition between T . A2 and T. neapolitanus for thiosulfate. Conditions
as in Figure 2A. The percentage of T . A2 cells and of T . neapolitanus cells in
cultures are shown, with thiosulfate plus acetate or glycollate in the reservoir
medium. Reproduced, with permission, from Ref. 4. Copyright 1979, Springer-
Verlag.

mM THIOSULFATE
Figure 6. Results of competition between the generalist T . A2, and two specialists,
T. neapolitanus and Spirillum G7 for thiosulfate and acetate as growth-limiting
1
substrates in the chemostat at a dilution rate of 0.07 h' . Concentrations in the
inflowing medium ranged from 0-20 mM for acetate and from 0-40 mM for thio-
sulfate. After a steady state had been established relative cell numbers were
determined. Solid line, experimental data; dashed line, outcome of the competition
as predicted from mathematical modeling.

Table I I I
Results of enrichment c u l t u r e s (I - V) from f r e s h water samples

a f t e r 15-20 volume changes i n the chemostat under dual substrate l i m i t a t i o n
1
of t h i o s u l f a t e ( t ) and a c t e t a t e (a) at a d i l u t i o n r a t e of 0.05 h " , using
d i f f e r e n t mixtures of t + a. Adapted from G o t t s c h a l and Kuenen, ( Π ) .
substrate Dominant population

concentration
Number sample i n medium (mM) % of t o t a l metabolic type
I canal 30 t + 5a 82 facultative chemolithotroph

II canal 10 t + 15 a 75 tt It
III canal 30 t + 5a 85 tt ft
IV ditch 20 t + 10 a 50 It II
V ditch 10 t + 15 a 86 chemolithoheterotroph

t i c a l treatments of the growth of m i c r o b i a l populations on mix

tures of substrates i t has been p r e d i c t e d that the maximum number
of organisms which can c o e x i s t can never be more than the number
of g r o w t h - l i m i t i n g substrates supplied to the c u l t u r e . This holds
i f no i n t e r a c t i o n other than mere competition takes place i n the
c u l t u r e . Based on previous p u b l i c a t i o n s of other workers G o t t s c h a l
and Things tad (_7) have developed a mathematical model f o r growth
of the three organisms i n the p a r t i c u l a r case.
The model i s based on simple Monod k i n e t i c s f o r growth. The
s p e c i f i c growth r a t e of the s p e c i a l i s t autotroph (A) on t h i o s u l
fate (t) i s
max
y S
U tA (8 ) = t A · t
i n which s i s the c o n c e n t r a t i o n of the t h i o s u l f a t e and K ^ the

t t
substrate s a t u r a t i o n constant of the autotroph f o r t h i o s u l f a t e .

S i m i l a r l y , f o r growth of the heterotroph (H) on acetate (a)
one obtains
max
M aH <·.) - y - s
»
+ s
aH a
The growth r a t e f o r the mixotrophic g e n e r a l i s t (M) has been taken

as the sum of the two separate growth r a t e :
max max
P S y S
\L, ( s . s . ) = aM * a tM ' t
+
Κ + s Κ + s,
aM a tM t
As pointed out by G o t t s c h a l and Thingstad (J) t h i s i s an approxi

9
mation which can only be v a l i d at low growth r a t e s where the r e

l a t i o n s h i p between μ and s i n nearly l i n e a r . This model does not
take i n t o account that the g e n e r a l i s t adapts i t s substrate o x i d i
zing c a p a c i t y to the r a t i o between the two substrates a v a i l a b l e i n
the mixture. This would probably lead to an over-estimation of
max max
μ ~ and μ ^ . I t could be shown, however, that i n a two-membered
c u l t u r e the decrease of μ ^ would have a constant value s i n c e the
p h y s i o l o g i c a l s t a t e of the g e n e r a l i s t (M) would be constant.
Although the model allowed accurate p r e d i c t i o n of the compe
t i t i o n between two organisms (Figure 7 a,b), i t could indeed not
account f o r the anomaly observed i n the experiments using three
organisms. The dotted l i n e s of F i g u r e 6 d e s c r i b e the p r e d i c t e d
steady s t a t e v a l u e s . The parameters used i n t h i s p r e d i c t i o n had
been derived from the outcome of the r e s u l t s obtained from the
two-membered c u l t u r e s .
Several p o s s i b i l i t i e s e x p l a i n i n g t h i s anomaly have been con-

0 2 4 6 8 10
acetate (mM)
Figure 7A. Cell density of the generalist (mixotroph T. A2, O) and the autotroph
(specialist T . neapolitanus, Φ) in mixed chemostat culture at steady state, growing
1
at a dilution rate of 0.075 h' . Growth was simultaneously limited by thiosulfate
(reservoir medium concentration, S° —40 mM) and by increasing concentrations
of acetate in the reservoir medium (S ° = 0-10 mM).
a

2 4 6 8
thiosulfate (mM)
Figure 7B. Cell density of the mixotroph (O) and the heterotroph (specialist
Spirillum G7, %) in mixed chemostat-culture at steady state, growing at a dilution
1
rate of 0.075 h . Growth was simultaneously limited by acetate (S ° = 20 mM)
a
and by increasing concentrations of thiosulfate in the inflowing medium (0-10 mM).

s i d e r e d . F i r s t of a l l , the assumption that no i n t e r a c t i o n s other

than competition f o r the substrates occur may be i n c o r r e c t . A l
though t h i s p o s s i b i l i t y cannot be r u l e d out, i t i s u n l i k e l y since
the spent medium from each of the pure c u l t u r e s d i d not i n f l u e n c e
the growth of the o t h e r ( s ) . T. neapolitanus d i d however excrete
g l y c o l l a t e , which can be u t i l i z e d by T. A2 and by Spirillum G7.
However, the e f f e c t of g l y c o l l a t e should be g r e a t e s t when the con
c e n t r a t i o n of T. neapolitanus i s h i g h e s t , and t h i s i s apparently
not the case.
A more complicated explanation may l i e i n the f a c t , i n d i c a t e d
above, that i n the t h e o r e t i c a l treatment, i t has been assumed that
the growth of T. A2 i s the sum of the separate s p e c i f i c growth
rates on the i n d i v i d u a l s u b s t r a t e s . As long as only two organisms
are present i n the c u l t u r e , T. A2 may indeed be i n a constant
metabolic s t a t e . However th p h y s i o l o g i c a l s t a t f T. A2 i th
presence of T. neapolitanus
i n the presence of Spirillum y
politanus i s removed from the c u l t u r e the metabolic s t a t e of T. A2
w i l l change to that found i n pure c u l t u r e during mixed substrate
consumption (Figure 3). As a r e s u l t , the assumed K and μ g a x
values f o r T. A2 w i l l a l t e r and i n f a c t i t can be shown that t h i s

w i l l make T. A2 l e s s competitive. As pointed out by G o t t s c h a l and
Thingstad Ç7), i t may therefore be that the r a t e a t which the
s h i f t i n p o p u l a t i o n occurs i n the three-membered c u l t u r e i s much
smaller than that i n the two-membered c u l t u r e s . Mathematical simu-
l a t i o n experiments show that i f a s h i f t i n parameters happened,
i t might very w e l l take 100 or more volume changes before a r e a l
steady s t a t e would be a t t a i n e d . I f t h i s were the case, changes
might have taken place so slowly that they were not n o t i c e d i n
the a c t u a l experiments, even though, i n one experiment, a measure-
ment of r a t i o s was made a f t e r 30 volume changes.
In s p i t e of the discrepancy between the t h e o r e t i c a l and prac-
t i c a l models, i t should be s t r e s s e d that both models c l e a r l y i n d i -
cate the e c o l o g i c a l advantages of a g e n e r a l i s t type of physiology.
Recent work by Laanbroek, Smit, Klein-Nulend and Veldkamp (8)
shows that t h i s phenomenom may a l s o e x p l a i n the coexistence of
v e r s a t i l e and s p e c i a l i s t Clostridium s p e c i e s . Furthermore, r e s u l t s
obtained by Harder and co-workers (W. Harder, U n i v e r s i t y of
Groningen, unpublished) i n d i c a t e d that the same p r i n c i p l e may
apply to g e n e r a l i s t s and s p e c i a l i s t s among the methanol u t i l i z i n g
bacteria.
Mixotrophic growth of the g e n e r a l i s t s i s apparently only one
p o s s i b l e advantage of a v e r s a t i l e metabolism. Another p o s s i b l e
b e n e f i t might l i e i n the c a p a b i l i t y of v e r s a t i l e organisms to grow
continuously with a l t e r n a t i n g s u p p l i e s of two s u b s t r a t e s . This has
a l s o been studied i n d e t a i l f o r the same s e t of three organisms
shown i n Table I I . (9). F i g u r e 8 gives an example of one s e r i e s
of experiments designed to show that the g e n e r a l i s t can grow un-
i n t e r r u p t e d l y during an a l t e r n a t i n g supply of 4h t h i o s u l f a t e ,

-433
150
ι
125| protein
ο 100
ε 50
ο 25|
• I > I •I ' I ' I

0
Ô ^ C02-f'xotion
CM
ο
4-+
* -1000k
<u -c I
•o J 80θ|
QQ£ - thiosulfate
S Ϊ X
Φ £ 600|
i -S 4001
QQ -9 acetate
1 | 200h — 0
(A)
1 _L
1
X I I
-2 k 6 8 10
t i m e (h)
a c e t a t e — ι — t h i o s u l f a t e —|!— a c e t a t e —!—-thiosulfate
Figure 8. T. A 2 grown under alternate limitation of acetate and thiosulfate in

1
continuous culture at a dilution rate of 0.05 h . Thiosulfate (40 mM) or acetate
(10 mM) was supplied to the culture, each for 4 h. The dashed lines indicate the
minimum activities needed for uninterrupted growth. Reproduced, with permission,
from Ref. 9. Copyright 1981, Society for General Microbiology.

4h acetate, i n a chemostat run a t a d i l u t i o n r a t e of 0.05 h

Under these c o n d i t i o n s , the overcapacity of T. A2 t o o x i d i z e
e i t h e r of the substrates was marginal. During the acetate p e r i o d ,
the a b i l i t y to o x i d i z e t h i o s u l f a t e was repressed more than the
acetate o x i d a t i o n p o t e n t i a l was during the t h i o s u l f a t e p e r i o d .
When the acetate p e r i o d was extended r e l a t i v e to the t h i o s u l f a t e
p e r i o d , T. A2 no longer maintained a s u f f i c i e n t l y high t h i o s u l -
f a t e o x i d a t i o n p o t e n t i a l to permit continued growth a t the same
r a t e . As a r e s u l t , p o p u l a t i o n d e n s i t i e s s t a r t e d to f l u c t u a t e and
t h i o s u l f a t e t r a n s i e n t l y accumulated.
I n t e r e s t i n g l y , the two s p e c i a l i s t organisms grew w e l l i n pure
c u l t u r e on t h e i r s p e c i a l i s t substrate under a regime of 4h sub-
s t r a t e ( t h i o s u l f a t e or acetate) and 4h s t a r v a t i o n . Of course,
during the s t a r v a t i o n p e r i o d a p r o p o r t i o n of the s p e c i a l i s t s was
washed out of the chemostat
a very high overcapacit
to grow instantaneously at a high r a t e a f t e r the s t a r v a t i o n p e r i o d .
When a competition experiment was c a r r i e d out between T. A2
and T. neapolitanus under a l t e r n a t i n g substrate c o n d i t i o n s , the
two organisms appeared to c o e x i s t i n equal numbers. This suggested
that T. neapolitanus would u t i l i z e p r a c t i c a l l y a l l of the t h i o s u l -
f a t e , whereas T. A2 was growing on acetate only.
The explanation of t h i s r e s u l t l i e s i n the v e r s a t i l i t y of
T. A2. During the acetate p e r i o d T. A2 reduced i t s t h i o s u l f a t e
o x i d i z i n g c a p a c i t y w h i l s t T. neapolitanus was washed out but r e -
tained i t s high t h i o s u l f a t e o x i d a t i o n p o t e n t i a l . As a r e s u l t , at
the beginning of the t h i o s u l f a t e p e r i o d , the T. neapolitanus popu-
l a t i o n could o x i d i z e the t h i o s u l f a t e a t a much higher r a t e than
T. A2. D i r e c t measurement of the substrate l e v e l s i n the chemostat
showed that under these c o n d i t i o n s T. neapolitanus could maintain
the c o n c e n t r a t i o n of the substrate 10 f o l d lower than T. A2 was
able to (Figure 9 ) . Thus, during the t h i o s u l f a t e p e r i o d i n the
mixed c u l t u r e T. neapolitanus could grow much f a s t e r than T . A2.
Furthermore, the very low c o n c e n t r a t i o n of t h i o s u l f a t e imposed by
T. neapolitanus obviously l e d to a f u r t h e r r e d u c t i o n of the t h i o -
s u l f a t e o x i d i z i n g p o t e n t i a l of T. A2. In other words, the genera-
l i s t was f o r c e d by the s p e c i a l i s t to grow as a heterogroph on ace-
tate only.
T. A2 i s , however, not a s p e c i a l i s t heterotroph and the a d d i -
t i o n of the s p e c i a l i s t a c e t a t e - u t i l i z i n g Spirillum G7 to the two-
membered c u l t u r e l e d to wash out of T. A2, r e s u l t i n g i n a c o e x i s -
t i n g p o p u l a t i o n of the two s p e c i a l i s t s (Figure 10a).
Further experiments showed that an a l t e r n a t i n g supply of two
d i f f e r e n t mixtures of t h i o s u l f a t e and acetate l e d to coexsistence
of a l l three organisms (Figure 10 b , c ) . Obviously, under an a l t e r -
n a t i n g supply of s p e c i a l i s t s u b s t r a t e s , g e n e r a l i s t s s i m i l a r to
T. A2 c l e a r l y are a t a disadvantage. However, i t could be shown
that nature habours organisms akin to T. A2 which are b e t t e r
American Chemical
Society Library
In Foundations 1155 16th St.,
of Biochemical N.W. Blanch, H., et al.;
Engineering;
Washington,
ACS Symposium Series; U.C. 20036
60,
Figure 9. The accumulation of sulfide under alternate supply of 4 h acetate, 4 h

sulfide to a continuous culture of T. A2. O, sulfide concentration in the culture at
pH 8.0; and · , at pH 7.5. Competition experiments all had been carried out at pH
7.5. Sulfide is a substrate which, in these experiments, is entirely equivalent to thio
sulfate, but has the advantage of being detectable down to a concentration of less
than 1 μΜ. In cultures of T . neapolitanus grown at pH 7.5 the sulfide concen
tration never increased to above 4 μΜ. Reproduced, with permission, from Ref. 12.
Copyright 1982, H. Veenman en ZonenBV.

°h . ι . • . • • • • ι . ι • Π
0 4 8 12 16 20 24 28
volume changes
Figure 10. Competition in continuous culture between T . A2, T . neapolitanus and

Spirillum G7 for thiosulfate and acetate as growth-limiting substrates. The dilution
1
rate was 0.05 h' with intermittent feeding of two media containing thiosulfate, or
acetate, or both. These media were supplied alternately to the culture, each during
4 h. Key: • , T. A2; Spirillum G7; and Ο , T. neapolitanus as percentage of the
total cell number present in the culture. A: One medium contained 10 mM acetate,
the other 40 mM thiosulfate. B : One medium contained 2.2 mM acetate plus 34.4
mM thiosulfate, the other 10 mM thiosulfate plus 6.4 mM acetate. C: One medium
contained 5.7 mM acetate plus 31.0 mM thiosulfate, the other 6 mM thiosulfate plus
11.7 mM acetate. Reproduced, with permission, from Ref. 9. Copyright 1981,
Society for General Microbiology.

adapted to the regime described i n F i g u r e 10a. When a continuous

c u l t u r e under t h i s regime was i n o c u l a t e d w i t h f r e s h water mud, a
dominant p o p u l a t i o n of a s p i r i l l u m - s h a p e d f a c u l t a t i v e chemolitho-
troph developed (9). The success of t h i s organism was probably due
to i t s a b i l i t y to " s t o r e " acetate as a polymer (poly-f$-hydroxy-
b u t y r a t e ) i n the c e l l s . This polymer was used d u r i n g the t h i o s u l -
f a t e p e r i o d as a source of carbon (J.-G. Kuenen and A . Spijkerman,
unpublished r e s u l t s ) . In t h i s way, the t h i o s u l f a t e u t i l i z i n g s p i -
r i l l u m could save energy f o r CO2 f i x a t i o n d u r i n g the t h i o s u l f a t e
period.
The experiments described i n t h i s paper c l e a r l y show the p o s -
s i b l e advantages and disadvantages of the s p e c i a l i s t versus géné-
r a l e s t s t r a t e g i e s f o r s u r v i v a l . Our present hypothesis i s that
s p e c i a l i s t organisms w i l l be s u c c e s s f u l under c o n d i t i o n s where
the turnover r a t e of t h e i r s p e c i a l i s t s u b s t r a t e i s h i g h r e l a t i v e
to that of other s u b s t r a t e s
ganisms might have an advantage when the r e l a t i v e turnover r a t e s
of s e v e r a l of t h e i r growth s u b s t r a t e s are i n the same order of
magnitude. Obviously when the s u p p l i e s of d i f f e r e n t substrates
s t r o n g l y f l u c t u a t e or even a l t e r n a t e , the outcome of competition
between s p e c i a l i s t s and g e n e r a l i s t s f o r g r o w t h - l i m i t i n g s u b s t r a t e s
w i l l be determined by the length of the supply p e r i o d s . As yet i t
has not been p o s s i b l e to confirm these p r e d i c t i o n s w i t h d i r e c t
measurements of g e n e r a l i s t s and s p e c i a l i s t s i n n a t u r a l mixed c u l -
t u r e s . A t present we are developing methods f o r the measurement
of the c o n t r i b u t i o n s of the d i f f e r e n t p o p u l a t i o n i n s u i t a b l e
systems, namely sewage treatment p l a n t s , which r e c e i v e s u p p l i e s
of, f o r example, s u l f i d e and organic s u b s t r a t e s .
A b e t t e r understanding of the s e l e c t i o n of d i f f e r e n t p o p u l a -
t i o n s i n the mixed c u l t u r e s of sewage treatment p l a n t s may f a c i -
l i t a t e c o n t r o l and management of these p l a n t s . An example may
help to ads t r u c t t h i s p o i n t . The presence of s p e c i a l i s t p o p u l a -
t i o n s w i l l improve the " r e s i l i e n c e " of the p l a n t to strong f l u c -
t u a t i o n s , s i n c e the organisms u s u a l l y possesss a h i g h overcapa-
c i t y . I n c o n t r a s t i f , d u r i n g supply of s u l f i d e and organic com-
pounds, chemolithotrophic heterotrophs are s e l e c t e d , the p o p u l a -
t i o n w i l l become extremely s e n s i t i v e to strong f l u c t u a t i o n s of
s u l f i d e , not only because t h e i r maximum r e s p i r a t o r y c a p a c i t y i s
low, but a l s o because such organisms tend to produce t o x i c p r o -
ducts i n h i b i t o r y f o r t h e i r own metabolism of s u l f i d e . In such a
case, a sudden pulse of s u l f i d e would lead to accumulation of
s u l f i d e , subsequent formation of t o x i c p r o d u c t s , and f u r t h e r i n -
h i b i t i o n of the s u l f i d e o x i d a t i o n , l e a d i n g to even f u r t h e r accumu-
l a t i o n of t h i s compound. With p r i o r knowledge of t h i s s e l e c t i o n ,
one might be able to absorb t h i s peak l o a d i n g , thus a v o i d i n g u n -
s a t i s f a c t o r y performance of the sewage treatment p l a n t .

Acknowledgements
Many thanks are due to my two former co-workers Dr. J . C . Gott-
schal and Dr. R.F. Beudeker who did most of the experimental work
described i n this paper. I thank Miss L . A . Robertson for correc-
ting the English and Mrs. C.M. Paanakker-Kuipers and Mrs. I . Hag-
man-v.d. Bulk for typing the manuscript.
Literature Cited
1. Silver, R.S.; Mateles, R . I . J. Bacteriol. 1969, 97, 535-543.

2. Dijkhuizen, L.; Harder, W. Arch. Microbiol. 1979, 123,
47-53.
3. Gottschal, J.C.; Kuenen J.G Arch Microbiol 1980a 126
33-42.
4. Gottschal, J.C.;
b i o l . 1979, 121, 241-249.
5. Smith, A . L . ; Kelly, D.P. J. Gen. Microbiol. 1979, 115,
377-384.
6. Cohen, Y . ; De Jonge, I . ; Kuenen, J.G. Arch. Microbiol. 1979,
122, 189-194.
7. Gottschal, J.C.; Thingstad, T.F. Biotechnology and Bioengi-
neering 1982, in press.
8. Laanbroek, H . J . ; Smit, A.J.; Klein-Nulend, G.; Veldkamp, H.
Arch. Microbiol. 1979, 120, 61-67.
9. Gottschal, J.C.; Nanninga, H.; Kuenen, J.G. J. Gen. Micro-
b i o l . 1981, 126, 85-96.
10. Gottschal, J.C.; Kuenen, J.G. "Microbial Growth on C Com- 1
pounds"; Dalton, H., Ed.; Heyden: London, Philadelphia,

Rheine, 1981, 91-104.
11. Gottschal, J.C.; Kuenen, J.G. FEMS Microbiol. Lett. 1980b,
7, 241-247.
12. Beudeker, R.F.; Kuenen, J.G. Antonie van Leeuwenhoek 1982,
in press.

11
Microbial Predation Dynamics
1 1
M. J. BAZIN, C. CURDS , A. DAUPPE , Β. Α. OWEN, and
P. T. SAUNDERS
Queen Elizabeth College, Department of Microbiology, London, England W87AH
The specific growth rate of a microbial predator

(λ) was found t
the prey (H) to
rather than the prey density alone. Predatory
amoebae of the cellular slime mould Dictyostelium
discoideum were grown in single-stage and two
-stage chemostat cultures with the gut bacterium,
Escherichia coli as their source of food. Predator
growth in the cultures was compared to each of
three functions which have been proposed for λ .
In addition to showing that λ changed with respect
to H/P, the results indicated also that it depended
explictly on time, so that the differential
equations describing the predator-prey system are
probably non-autonomous.
Quite c l e a r l y , the growth of a predator population i s i n

some way dependent upon the abundance of i t s prey. The most
f r e q u e n t l y c i t e d model of predator-prey dynamics i s the set o f
l i n k e d , non-linear d i f f e r e n t i a l equations known as the Lotka-
V o l t e r r a equations ( 1 ) . This model assumes that i n the absence
of predator, the prey grows e x p o n e n t i a l l y , while i n the absence
of prey the predator d i e s e x p o n e n t i a l l y , and that the predator
growth r a t e i s d i r e c t l y p r o p o r t i o n a l to the product o f the prey
(H) and predator (P) population d e n s i t i e s . The equations a r e :
H = k x Η - k PH
2 (1)
Ρ = -k Ρ 3 + k PH
4 (2)
where k^ s are constants. The s p e c i f i c growth r a t e of the
1
Current address: British Museum (Natural History), Cromwell Road,
London SW7, England
0097-6156/83 /0207-0253$06.00/0

predator p o p u l a t i o n (λ) according to the model i s t h e r e f o r e

d i r e c t l y p r o p o r t i o n a l to the prey d e n s i t y :
λ - k 4 Η (3)
In microbiology the r e l a t i o n s h i p between the s p e c i f i c growth

r a t e of a m i c r o b i a l predator and i t s prey i s o f t e n expressed i n
terms of the h y p e r b o l i c f u n c t i o n suggested by Monod (2) f o r the
growth of b a c t e r i a on a l i m i t i n g d i s s o l v e d n u t r i e n t . Applied to
prédation the f u n c t i o n i s :
λ = λ H/(L + Η) (4)
m
where λ i s the maximum s p e c i f i c growth r a t e and L i s the
c o n c e n t r a t i o n of prey s u f f i c i e n t f o growth t s p e c i f i rat
2
V ·
Both the L o t k a - V o l t e r r a and the Monod functions f o r predator
s p e c i f i c growth r a t e are dependent upon a s i n g l e v a r i a b l e , the
c o n c e n t r a t i o n of prey organisms. A t h i r d f u n c t i o n , proposed by
Contois (3) f o r b a c t e r i a l growth as an a l t e r n a t i v e to that of
Monod, when a p p l i e d to predator growth takes the form:
λ = λ Η/(LP + Η) (5)
m
In t h i s case the s p e c i f i c growth rate i s a f u n c t i o n of both prey
and predator d e n s i t y .
Using the c i l i a t e protozoan Tetrahymena p y r i f o r m i s as a
predator and K l e b s i e l l a aerogenes as prey, Curds and Cockburn
(4) found that of the three f u n c t i o n a l forms suggested f o r
predator s p e c i f i c growth r a t e represented by equations (3) - ( 5 ) ,
the Contois equation gave the best f i t to t h e i r data. D i v i d i n g
the numerator and denomenator of equation (5) by Ρ gives
λ = λ ((H/P) / (L + H/P)) (5a)

m
showing that predator s p e c i f i c growth r a t e according to the
Contois equation can be considered to be a f u n c t i o n of the r a t i o
of prey to predator p o p u l a t i o n d e n s i t i e s . Using, catastrophe
theory, a n a l y s i s of r e s u l t s from experiments i n which slime
mould amoebae f e d on E. c o l i i n d i c a t e d that i t was t h i s r a t i o
that was the c r i t i c a l v a r i a b l e i n the system 05).
The goal of the research we report here was to determine
whether the s p e c i f i c growth r a t e of predatory amoebae of the
c e l l u l a r slime mould D i c t y o s t e l i u m discoideum feeding on the gut
b a c t e r i a , E s c h e r i c h i a c o l i was dependent upon prey d e n s i t y alone
or upon the r a t i o of prey to predator. Two experimental systems
were employed, both based on the chemostat type of continuous
c u l t u r e . A chemostat i s a continuously s t i r r e d tank r e a c t o r i n
which microorganisms grow i n a homogeneous environment and are
supplied with n u t r i e n t s o l u t i o n a t the same volumetric r a t e at

11. BAZIN ET A L . Microbial Prédation Dynamics 255
which the c u l t u r e i s harvested. The advantages of such a system

for studying m i c r o b i a l predator-prey dynamics have been described
p r e v i o u s l y (6), but e s s e n t i a l l y , chemostat c u l t u r e extends the
period of time over which an experiment can be performed,
reduces the e f f e c t of s t a t i s t i c a l f l u c t u a t i o n s and sampling
e r r o r s and has w e l l - d e f i n e d parameters which can be c o n t r o l l e d
by the experimenter while other aspects of the e x t e r n a l e n v i r o n -
ment can be kept constant.
Our f i r s t experimental system c o n s i s t e d of a s i n g l e - s t a g e
chemostat to which n u t r i e n t s o l u t i o n f o r the b a c t e r i a l prey was
fed a t a constant r a t e and i n which the i n t e r a c t i o n between the
prey and the predator took p l a c e . The equation of balance f o r
the two populations can be w r i t t e n as:
Ρ - ( λ - D) Ρ (6)
Η = ( u - D ) H (7)
W
where D = d i l u t i o n r a t e (the r a t e of flow through the c u l t u r e
v e s s e l d i v i d e d by i t s volume), u i s the s p e c i f i c growth r a t e of
the prey and W i s the y i e l d of predator per u n i t of prey consumed,
assumed to be constant.
When slime mould amoebae and b a c t e r i a l prey are grown
together i n a chemostat, the p o p u l a t i o n d e n s i t i e s of both
organisms f l u c t u a t e s i n u s o i d a l l y f o r s e v e r a l days (7, 8 ) .
We estimated the s p e c i f i c growth r a t e of the predator p o p u l a t i o n
i n such c u l t u r e s from the slope of the curve generated by
p l o t t i n g the logarithm of the predator d e n s i t y a g a i n s t time.
T h i s slope i s
Ρ/Ρ = λ - D (8)
from which r e l a t i o n s h i p λ could be simply c a l c u l a t e d . Prey

d e n s i t y was a l s o measured so that the change i n λ as a f u n c t i o n
of Η and H/P could be determined.
Our second experimental system c o n s i s t e d of two chemostats
l i n k e d i n s e r i e s . In the f i r s t v e s s e l the b a c t e r i a l prey was
allowed to come to steady s t a t e and then fed i n t o the second
stage v e s s e l which contained the amoebae. As the l i m i t i n g
n u t r i e n t source f o r the b a c t e r i a i s v i r t u a l l y exhausted under
steady s t a t e c o n d i t i o n s , i t was assumed that no f u r t h e r growth
of prey occurred i n the second v e s s e l . In the second v e s s e l
the equation of balance f o r the predator i s :
Ρ = (λ - D ) Ρ (9)
so that at steady s t a t e
λ = D (10)

By feeding b a c t e r i a to the predator p o p u l a t i o n at d i f f e r e n t

d i l u t i o n r a t e s and measuring the steady state prey d e n s i t y i n the
second v e s s e l i t i s therefore p o s s i b l e to r e l a t e λ and Η to each
other.
Methods
E s c h e r i c h i a c o l i B/r and D i c t y o s t e l i u m discoideum NC4 were
maintained r o u t i n e l y and grown i n s i n g l e - s t a t e chemostat c u l t u r e
as described p r e v i o u s l y ( 8 ) . The outflow of chemostat c u l t u r e s
was passed along the same l i n e as e x p e l l e d a i r and by t h i s means
was forced i n t o tubes r e s t i n g i n a r e f r i g e r a t e d f r a c t i o n
c o l l e c t o r . F r a c t i o n s were c o l l e c t e d at hourly i n t e r v a l s .
For two-stage continuous c u l t u r e , the f i r s t stage v e s s e l had
a maximum c a p a c i t y of 1 L while the v e s s e l used f o r the second
stage had a maximum c a p a c i t 1 500
upon the volume appropriat
v e s s e l s were f i t t e d wit , flange g
access p o r t s . Both v e s s e l s were mixed by means of magnetic
s t i r r e r s and immersed i n water maintained at 25°. F i l t e r -
s t e r i l i s e d a i r was supplied to both stages and the flow of n u t r i e n t
to the f i r s t v e s s e l and the flow from the f i r s t to the second
v e s s e l was regulated by p e r i s t a l t i c pumps. Two-stage chemostat
experiments were performed by i n o c u l a t i n g the f i r s t v e s s e l with
I>» c o l i and i n c u b a t i n g with the flow on u n t i l the system came to
steady s t a t e . This was considered to have occurred when no
s i g n i f i c a n t change i n the t u r b i d i t y of the e f f l u e n t c e l l suspension
could be detected. At t h i s time the second v e s s e l was f i l l e d with
c u l t u r e from the f i r s t and i n o c u l a t e d with a suspension of
D. discoideum spores. The c u l t u r e i n the second v e s s e l was
incubated under batch c o n d i t i o n s f o r about 48 h during which time
the spores germinated to form amoebae. Flow from the f i r s t
v e s s e l to the second was then i n i t i a t e d and the amoebae c u l t u r e d
on a continuous b a s i s . Samples were taken d i r e c t l y from the
culture vessels for analysis.
The c e l l number d e n s i t i e s of the b a c t e r i a and the amoebae i n
both systems was measured on a C o u l t e r Counter (Coulter
E l e c t r o n i c s L t d . , Harpenden, England). Mean c e l l volumes were
estimated using a C o u l t e r C1000 Channelyzer. A 30 um diameter
aperture was used f o r the b a c t e r i a and a 50 um aperture f o r the
amoebae. Samples were suspended e i t h e r i n c u l t u r e medium or
"Isoton" (Coulter E l e c t r o n i c s Ltd) immediately p r i o r to c o u n t i n g .
Biomass was estimated e i t h e r i n terms of biovolume, the product of
the mean c e l l volume and the number of c e l l s present, or, f o r
b a c t e r i a l biomass where appropriate, as t u r b i d i t y at 560 nm.
Within the range of readings made there i s a l i n e a r r e l a t i o n s h i p
between c e l l volume d e n s i t y and t u r b i d i t y at 560 nm f o r E. c o l i .
Sing-estage continuous c u l t u r e data was smoothed by the method of
cubic s p l i n e s using Nottingham Algorithms Routine E02AAF. A l l
computations were performed on a CDC 6600 d i g i t a l computer.

11. BAZIN ET AL. Microbial Prédation Dynamics 257
Results
Single-stage chemostat c u l t u r e . The s i n g l e stage chemostat

system represents a food chain i n which glucose i s converted to
b a c t e r i a l biomass which, i n turn, i s converted to amoebal biomass.
As glucose i s measured on a weight per u n i t volume b a s i s i t i s
appropriate that the other dependent v a r i a b l e s of the system, H and
P, be measured i n the same terms, i . e . biomass per u n i t volume.
The number d e n s i t i e s of the prey and predator populations would be
appropriate u n i t s provided that the average mass per b a c t e r i a l or
amoebal c e l l remained constant. Results of a t y p i c a l s i n g l e -
stage experiment are shown i n Figure 1. The change i n the mean
c e l l volume (MCV) of each species i n d i c a t e s q u i t e c l e a r l y that
c e l l mass does not remain constant. Therefore, we have used the
biovolume d e n s i t y , the product f th numbe d e n s i t d MCV
estimates of H and Ρ i
considerable d i f f e r e n c e syste
numbers and biovolumes i s shown by i n s p e c t i n g phase p l a n t p l o t s
of our r e s u l t s . There were constfucted using smoothed data to
p l o t predator d e n s i t y against prey d e n s i t y . Figure 2 shows the
r e s u l t s obtained when number d e n s i t i e s were used. An i n t e r
dependence between the two v a r i a b l e s i s not r e a d i l y apparent. On
the otherhand, as shown i n F i g u r e 3, when biovolume d e n s i t i e s were
used a more r e a d i l y interprétable r e l a t i o n s h i p emerges. I t i s
c l e a r from the t r a j e c t o r y i n phase space that the system damps
slowly at f i r s t and then moves r a p i d l y towards an e q u i l i b r i u m
value. Figure 4 shows the change i n P/P as a f u n c t i o n o f t i m e , β
c a l c u l a t e d from smoothed data, and Figure 5 i s a p l o t of P/P

against Η constructed from the data i n Figure 4.
Two-stage chemostat c u l t u r e . In the two-stage continuous

c u l t u r e system, steady s t a t e i n the second v e s s e l which contained
the amoebal p o p u l a t i o n took more than two weeks to achieve.
Steady s t a t e was accompanied by considerable clumping of the c e l l s
i n d i c a t i n g , p o s s i b l y , that the slime mould amoebae were
aggregating. T h i s c o n d i t i o n was r e l i e v e d only s l i g h t l y by
i n c r e a s i n g the concentration of EDTA i n the medium from 0.65 mM
to 0.8 mM. Both the amount of time r e q u i r e d to reach steady s t a t e
and the tendency of the amoebae to form clumps of c e l l s once
steady s t a t e has been reached made c o l l e c t i n g s u f f i c i e n t data to
c h a r a c t e r i s e the system with respect to the amoebal s p e c i f i c
growth r a t e d i f f i c u l t . Therefore, steady-state data was augmented
with estimates made near to steady s t a t e and r e s u l t s recorded i n
terms of the s p e c i f i c r a t e or prédation, Φ = -fi/P + D ^ - I ^ ) /Ρ,
which i s d i r e c t l y p r o p o r t i o n a l to predator s p e c i f i c growth r a t e
i f the y i e l d , W, i s constant. The s p e c i f i c r a t e of prédation was
estimated by applying the f o l l o w i n g c a l c u l a t i o n :
Φ = D (Η χ - H )/P
2 (11)

7
io E
Ε ο
ο
w 10- Mo» ë
Φ
I
Φ °% .·/·· β
ο°;°ο·-
β % ν <- 1
1C
10-ï 8
; E
.2
*c
S
& 10 7 E
<
M00
co
E ÎaooE
a.
— ο·4Η
> >
o o
100.2
φ
O
E
<
100 200 300
TIME (h)
Figure L Change in mean cell volume and number density of D . discoideum

amoebae and Ε . coli grown together in single-stage chemostat culture.

11. BAZIN E T A L . Microbial Prédation Dynamics 259
1
Smoothed prey number density ml"
Figure 2. Phase plane plot of predator and prey number densities. Data from a
single stage chemostat culture of D . discoideum and E . coli were smoothed by the
method of cubic splines, and the smoothed data were used to construct this figure.
The arrows indicate the direction of increasing time.

1
Smoothed prey biovolume density (μηι ml')
Figure 3. Phase plane plot of predator and prey densities constructed as for Figure
2, using biovolume density as the coordinates. The arrows indicate the direction
of increasing time.

11. BAZIN ET A L . Microbial Prédation Dynamics 261
008
i\ A
ooH
s Λ Λ
o • · · · / \ m
• · · · · \ S
o ooH f :
φ
\ - \/ v
w
υ
Φ
α
y
^ -004H
0
1
l
I
50 100 150 200 250
Time ( h )
Figure 4. Change in the specific rate of change of D. discoideum in a single-stage
chemostat as a function of time. Values were obtained from smoothed data. The
specific growth rate of the amoebae is obtained by adding the dilution rate of the
1
culture (0.065 h' ) to the values on the ordinate.
0.08 h
ο 0.04
-0.04
σ
-D
6.0 7.0 8.0

Log prey population density
Figure 5. Specific rate of change of predator in a single-stage chemostat culture

as a function of prey density. The specific growth rate of the predator is the sum
1
of the specific rate of change and the dilution rate (0.065 h' ).

where H and H^ are the p o p u l a t i o n d e n s i t i e s o f the b a c t e r i a l

prey i n the f i r s t and second stages of the continuous c u l t u r e
system r e s p e c t i v e l y and D i s the d i l u t i o n r a t e of the second
stage v e s s e l . For the purposes o f t h i s c a l c u l a t i o n , b a c t e r i a l
d e n s i t y was estimated i n terms of t u r b i d i t y at 560 nm and i t was
assumed that the c o n t r i b u t i o n by the amoebal p o p u l a t i o n i n the
second v e s s e l at t h i s wavelength was n e g l i g i b l e . The change i n
Φ as a f u n c t i o n of steady s t a t e prey d e n s i t y i s shown i n
Figure 6.
Discussion
The three models we tested our experimental r e s u l t s against

are given i n equations (3), (4) and ( 5 ) . The L o t k a l V o l t e r r a
equations p r e d i c t a l i n e a r e l a t i o n s h i betwee predato specifi
growth r a t e (or the s p e c i f i
when λ i s p l o t t e d agains g ,
f a m i l i a r rectangular hyperbola i s generated. A h y p e r b o l i c
r e l a t i o n s h i p between λ and H/P i s p r e d i c t e d by the Contois
expression. Figures 5 and 6 show the way i n which λ changes as
a f u n c t i o n of prey d e n s i t y taken from s i n g l e - and double-stage
chemostat c u l t u r e s r e s p e c t i v e l y . In n e i t h e r case do the
r e l a t i o n s h i p s i n d i c a t e d i n equations (3) and ( 4 ) , a s t r a i g h t l i n e
and a rectangular hyperbola, become apparent! Figure 7 shows
λ p l o t t e d against the r a t i o of prey to predator, H/P, c a l c u l a t e d
from data from the two-stage experiment. Quite c l e a r l y , t h i s
r e l a t i o n s h i p can be i n t e r p r e t e d i n terms of equation ( 5 ) .
Figure 8 shows a s i m i l a r p l o t using data from s i n g l e - s t a g e
experiments. Here i t seems that a family of rectangular
hyperbola are represented with H/P as the independent v a r i a b l e
and with λ and L of equation (5) decreasing with time. I t
appears, t h e r e f o r e , that the s p e c i f i c growth r a t e of the amoebal
predator i s dependent not j u s t on H, but upon the r a t i o of prey to
predator present. The mechanism with which the amoebae are able
to c o n t r o l t h e i r growth r a t e i n t h i s way i s , of course, not known.
We have suggested (5) that f o l i c a c i d , which i s secreted by the
b a c t e r i a and i s , t h e r e f o r e , a f u n c t i o n of the prey population
d e n s i t y , might be i n a c t i v a t e d by the amoebae so that i t s
concentration depends a l s o on the d e n s i t y of predator present.
This indeed appears to be the case as reported by Pan and
Wurster (9). I t i s conceivable, t h e r e f o r e , that the concentration
of f o l i c a c i d i n the media might serve to regulate the rate of
growth of the predator population. Furthermore, the r e s u l t s
i n d i c a t e that the s p e c i f i c growth rate might depend e x p l i c i t l y on
time. I f such i s the case then the d i f f e r e n t i a l equations
d e s c r i b i n g the dynamics of the system are non-autonomous with
time appearing on the r i g h t hand side of the equals s i g n and not
autonomous as are the L o t k a - V o l t e r r a equations and the vast
majority of equations that have been suggested f o r d e s c r i b i n g
m i c r o b i a l predator-prey i n t e r a c t i o n s .

11. BAZIN E T A L . Microbial Prédation Dynamics 263
0.1 0.2 0.3

Prey density
Figure 6. Data from a two-stage chemostat system showing the change in the
specific feeding rate of D . discoideum in the second stage as a function of E . coli
density. The specific feeding rate is directly proportional to specific growth rate
providing that the yield of predator produced per unit of prey consumed is constant.
Prey density is measured in terms of absorbance at 560 nm in the second-stage vessel. Specific
feeding rate was calculated as the product of the difference in turbidity between the cultures in
the two vessels and the dilution rate, divided by the prey biovolume density. The units on the
1 3
ordinate are therefore absorbance at 560 nm/mL culture h' μm~ .
6h
-6 4
Q.
10 20 30
Prey/predator ratio
Figure 7. Specific feeding rate plotted against the ratio of prey to predator in the
second vessel, estimated in terms of absorbance at 560 nm divided by the predator
ζ 1
biovolume density (μ/η mL ). Other conditions as in Figure 6.

0.08 h
• · · ·*··.
#
.·.·*. »
0.04 h
• ··
0 h
£ -0.04
ν..·.
LÎ!_
0.8 0.9 1.0 l.l 1.2
Ratio of prey t o predator population densities
Figure 8. Specific rate of change of the predator population in a single-stage

chemostat culture, plotted as a fraction of the ratio of prey to predator biovolume
1
densities. The dilution rate of the culture was 0.065 h' .
Acknowledgments
T h i s r e s e a r c h was supported by the U.K. N a t u r a l Environmental

Research C o u n c i l and an SRC CASE studentship.
Literature Cited
1. Curds, C.R.; Bazin, M.J. "Advances in Aquatic Microbiology";

Droop, M.R.; Jannasch, H.W., Ed.; Academic: London, 1977;
Vol. 1, p. 115.
2. Monod, J . Ann. Inst. Pasteur 1950, 79, 390.
3. Contois, D.E. J . Gen. Microbiol. 1959, 21, 40.
4. Curds, C.R.; Cockburn, A. J . Gen. Microbiol. 1968, 54, 343.
5. Bazin, M.J.; Saunders, P.T. Nature 1978, 275, 52-54.
6. Bazin, M.J.; Rapa, V.; Saunders, P.T. "Ecological Stability";
Usher, M.B.; Williamson, M.E., Ed; Chapman and Hall: London,
1974; p. 159.
7. Tsuchiya, H.M.; Drake, J . F . ; Jost, J . F . ; Fredrickson, A.G.
J . Bacteriol. 1972, 100, 1147-1153.
8. Dent, V . E . ; Bazin, M.J.; Saunders, P.T. Arch. Microbiol.
1976, 109, 187-194.
9. Pan, P.; Wurster, B. J . Bacteriol. 1978, 955-959.

12
Effects of Cell Motility Properties on Cell

P o p u l a t i o n s in E c o s y s t e m s
DOUGLAS A. LAUFFENBURGER
University of Pennsylvania, Department of Chemical Engineering,
Philadelphia, PA 19104
Because most natural ecosystems

cannot r e a l l
conclusions regardin
population growth and i n t e r a c t i o n s drawn
from well-mixed, chemostat studies may
not necessarily be v a l i d . Many microbial
species, in f a c t , possess sophisticated
movement behavioral properties by which
d i s t r i b u t i o n of a population in space i s
influenced by concentrations and
gradients of chemicals commonly present
in their environment. These chemotactic
and chemokinetic properties can require
s i g n i f i c a n t devotion of genetic information
and sometimes s i g n i f i c a n t energy expenditure,
yet are of little apparent use i n artificial
well-mixed systems. However, in non-
-mixed environments, the e f f e c t s of chemosensory
movement properties may be extremely
important in determining the a b i l i t y of
a species to grow, or in deciding the
outcome of competitive i n t e r a c t i o n s .
This paper summarizes the a v a i l a b l e
experimental evidence that t h i s i s indeed
the case, that movement properties can
have c r u c i a l e f f e c t s in microbial ecosystems.
We then present some mathematical models
that help explain and predict these
effects.
0097-6156/83/0207-0265 $08.00/0

S t u d i e s o f m i c r o b i a l p o p u l a t i o n dynamics have
focused p r i m a r i l y on well-mixed, macroscopically
homogeneous s y s t e m s . T h i s emphasis i s e a s i l y
d i s c e r n i b l e from t h e c o n t e n t s o f t h i s symposium
volume. However, t h e s i t u a t i o n i n most n a t u r a l l y
o c c u r i n g m i c r o b i a l systems i s f a r from i d e a l
l a b o r a t o r y c o n d i t i o n s , so that understanding gained
a b o u t w e l l - m i x e d s y s t e m s may n o t p r o v i d e t h e
appropriate insight into ecological situations.
Environments which a r e not well-mixed can allow
formation o f s p a t i a l gradients o f chemical concentra-
t i o n s and c e l l d e n s i t i e s . Also, chemical d i f f u s i o n
and c e l l m o t i l i t y ( i . e . , s e l f - p r o p e l l e d movement
r e q u i r i n g energy expenditure) can replace convection
a s t h e d o m i n a n t mod
common c a t e g o r i z a t i o n
c o n t a i n a t l e a s t o n e m o t i l e s p e c i e s (1), including
many o f t h e commonly o c c u r i n g s p e c i e s . F u r t h e r , most
m o t i l e b a c t e r i a e x h i b i t c h e m o t a x i s , w h i c h i s most
s i m p l y d e f i n e d a s c e l l movement t o w a r d o r away f r o m
chemicals ( 2 ) , o r a s p r e f e r e n t i a l c e l l movement t o w a r d
h i g h e r o r lower c o n c e n t r a t i o n s o f a c h e m i c a l stimulus
{3). A c t u a l l y , t h e r e a r e a number o f d i f f e r e n t types
o f movement r e s p o n s e s w h i c h l e a d t o b e h a v i o r o f t h i s
g e n e r a l d e s c r i p t i o n ( 4 ) . For peritrichously flagellated bacteria,
w h i c h a p p e a r t o be t h e m o s t c o m m o n l y e n c o u n t e r e d group
(and o n w h i c h t h i s p a p e r w i l l a c c o r d i n g l y f o c u s ) ,
k l i n o k i n e s i s ( i n which the t u r n i n g frequency o f
swimming b a c t e r i a i s m o d u l a t e d b y s t i m u l u s c o n c e n t r a -
t i o n ) a p p e a r s t o be c l o s e s t t o o b s e r v e d b e h a v i o r ( 5 ) .
T h i s i s i l l u s t r a t e d i n F i g u r e 1. In the absence o f a
chemical stimulus gradient, these b a c t e r i a swim i n
roughly s t r a i g h t l i n e steps c a l l e d "runs" f o r a short
time (about 1 second) and t h e n s t o p and change
d i r e c t i o n , o r " t u m b l e " , f o r a b o u t 1/10 s e c o n d ( 6 ) .
The d i r e c t i o n c h a n g e i s p u r e l y r a n d o m , b u t t h e
p r o b a b i l i t y o f tumbling i s constant d u r i n g a r u n ,so
t h a t t h e r u n time d i s t r i b u t i o n i s P o i s s o n i a n ( 6 ) .
T h i s movement b e h a v i o r i s t e r m e d r a n d o m m o t i l i t y . In
the presence o f a g r a d i e n t , t h e d i r e c t i o n change
r e m a i n s random b u t t h e t u m b l i n g p r o b a b i l i t y decreases
f o r a c e l l swimming t o w a r d h i g h e r a t t r a c t a n t c o n c e n t r a -
t i o n s o r lower r e p e l l e n t c o n c e n t r a t i o n s (6), l e a d i n g
to n e t m i g r a t i o n i n t h e d i r e c t i o n o f t h e g r a d i e n t .
T h i s mechanism p r o v i d e s v e r y e f f i c i e n t response (]_) ;
i n an o p t i m a l g r a d i e n t t h e n e t m i g r a t i o n v e l o c i t y i s
r o u g h l y h a l f t h e l i n e a r c e l l swimming s p e e d ( 8 , 9 ) .

12. LAUFFENBURGER Cell Motility 267
RANDOM MOTILITY CHEMOTAXIS
Figure 1. Illustration of typical movement of peritrichously flagellated bacteria.

The left-handfigureshows movement of a cell in the absence of a chemical stimulus
concentration gradient. The right-handfigureshows that the run length is increased
when the cell moves in the direction of increasing attractant concentration, toward
the top of the figure. The angles between respective runs in the two figures are
identical. The increase in run length results in a greater drift in the direction of
increasing attractant concentration for chemotactic movement than for random
movement.

S t i m u l u s c o n c e n t r a t i o n s a r e m e a s u r e d by o c c u p a n c y
o f c e l l membrane r e c e p t o r s w h i c h c a n r e v e r s i b l y b i n d
stimulus molecules according to Michaelis-Menten
enzyme k i n e t i c s w i t h d i s s o c i a t i o n c o n s t a n t .
G r a d i e n t s a r e d e t e c t e d by a t e m p o r a l s e n s i n g m e c h a n i s m
by w h i c h r e c e p t o r o c c u p a n c y o v e r t h e e n t i r e c e l l i s
m o n i t o r e d d u r i n g a r u n (1Λ) . A spatial sensing
m e c h a n i s m by w h i c h d i f f e r e n c e s i n r e c e p t o r o c c u p a n c y
a c r o s s the c e l l l e n g t h i s measured i s i m p r a c t i c a l f o r
s u c h r a p i d l y swimming c e l l s , b e c a u s e o f f a l s e
a p p a r e n t g r a d i e n t s due t o c e l l m o t i o n i t s e l f (12).
A l a r g e v a r i e t y o f c h e m i c a l s c a n s e r v e as
chemotactic s t i m u l i
r e c e p t o r s a n d 10 r e p e l l e n
i d e n t i f i e d f o r E s c h e r i c h i a c o l i and Salmonella
t y p h i m u r i u m , t h e two m o s t w i d e l y - s t u d i e d s p e c i e s ( Γ 3 ) .
T h e s e r e c e p t o r s a r e s p e c i f i c f o r one o r two chemicals
at h i g h a f f i n i t i e s (low K ^ ) , but w i l l a l s o b i n d a
range o f r e l a t e d m o l e c u l e s w i t h lower a f f i n i t y (2).
Table 1 l i s t s a number o f t h e w e l l - k n o w n s t i m u l i f o r
a v a r i e t y of species, although t h i s i s c e r t a i n l y
i n c o m p l e t e a s new r e s p o n s e s a r e b e i n g d i s c o v e r e d
almost d a i l y . I n g e n e r a l , s u b s t a n c e s b e n e f i c i a l t o an
o r g a n i s m s u c h as n u t r i e n t s and o x y g e n ( i f a e r o b i c )
s e r v e as a t t r a c t a n t s w h i l e t o x i c compounds o r compounds
c a u s i n g pH e x t r e m e s a c t a s r e p e l l e n t s ( 2 ) . Some a m i n o
a c i d s a r e a t t r a c t a n t s and o t h e r s r e p e l l e n t s , d e p e n d i n g
upon t h e s p e c i e s . A l t h o u g h t h e r e i s n o t an e x a c t
correspondence between m e t a b o l i z a b l e compounds and
a t t r a c t a n t s n o r b e t w e e n u n f a v o r a b l e c o m p o u n d s and
r e p e l l e n t s (L4), the observed responses can g e n e r a l l y
be r a t i o n a l i z e d i n t e r m s o f p a r t i c u l a r s p e c i e s
b i o c h e m i c a l p a t h w a y s and by r e c o g n i t i o n o f some
p u z z l i n g s t i m u l i as a n a l o g u e s o f o t h e r s t i m u l i (2).
Speculation regarding a possible survival
advantage o f chemotaxis i s thus not s u r p r i s i n g .
Approximately 40 g e n e s a r e d e v o t e d s p e c i f i c a l l y t o t h e
c h e m o t a c t i c r e s p o n s e i n E ^ c o l i and S^ t y p h i m u r i u m (15) y
and a s i m i l a r number o f g e n e s may be d e v o t e d t o t h e

m o t i l i t y apparatus. S u c h an i n v e s t m e n t m u s t p r o v i d e
some b e n e f i t i n t h e c o m p e t i t i v e w o r l d o f m i c r o b i a l
ecology. However, f u n d a m e n t a l u n d e r s t a n d i n g o f the
c i r c u m s t a n c e s i n w h i c h a s i g n i f i c a n t a d v a n t a g e due t o
m o t i l i t y a n d c h e m o t a x i s w i l l a c t u a l l y be p r e s e n t i s
l a c k i n g , a s a r e e s t i m a t e s o f t h e m a g n i t u d e o f s u c h an
advantage. This understanding i s l a c k i n g even f o r a
s i n g l e s t i m u l u s , and t h e p r o b l e m b e c o m e s e v e n more
complex i n any n a t u r a l e n v i r o n m e n t i n w h i c h m u l t i p l e

Table 1. Classification of Weil-Known Bacterial

Chemotactic Responses
Genus Classes o f Classes o f

Attractants Repellents
Escherichia sugars pH extremes

amino acids a l i p h a t i c alcohols
C>
Pseudomonas sugars inorganic ions

amino acids pH extremes
nucleotides amino acids
Bacillus sugars inorganic ions

metabolic poisons
°2
Salmonella sugars a l i p h a t i c alcohols
amino acids
0„
Vibrio amino acids
Spirillum sugars inorganic ions

Rhodospirilium nucleotides pH extremes

s u l f h y d r y l compounds poisons
Clostridium
Bdellovibrio amino acids
Proteus sugars inorganic acids
°2
Erwinia sugars inorganic ions
pH extremes
Sarcina inorganic ions
pH extremes
Serratia sugars inorganic ions
0„
Bordetella
Pasteurella
Marine b a c t e r i a algal culture f i l t r a t e s heavy metals
t o x i c hydrocarbons
Source: Refs. 36-38.

s t i m u l i , p r o b a b l y b o t h a t t r a c t a n t s and r e p e l l e n t s , a r e
present. M u l t i p l e s i g n a l s a r e a d d i t i v e i n some s e n s e
( 1 6 ) , and p r e s u m a b l y t h e b a c t e r i a a t t e m p t t o o p t i m i z e
t h e i r growth through p r e f e r e n t i a l m i g r a t i o n . F r o m an
e n g i n e e r i n g p e r s p e c t i v e , t h e r e i s no q u a n t i t a t i v e
b a s i s f o r p r e d i c t i o n o f the e f f e c t s o f c e l l m o t i l i t y
and c h e m o t a x i s o n m i c r o b i a l p o p u l a t i o n d y n a m i c s i n a n y
given s i t u a t i o n at present. T h i s paper i s devoted to
r e v i e w o f the s m a l l body o f i n f o r m a t i o n a v a i l a b l e i n
t h i s area at t h i s time.
Experimental Observations
T h e r e e x i s t s o n l y a s m a l l number o f p u b l i s h e d
experiments p e r t a i n i n t th effect f cell motilit
on p o p u l a t i o n g r o w t h
g i v e s one example w i t h o u
c o m p e t i t i o n b e t w e e n an a e r o t a c t i c ( i . e . , c h e m o t a c -
t i c a l l y a t t r a c t e d by o x y g e n ) P s e u d o m o n a s s p e c i e s a n d
an i m m o t i l e A c i n e t o b a c t e r s p e c i e s , f o r o x y g e n ( 1 7 ) .
When t h e g r o w t h medium i s w e l l - a e r a t e d t h e
A c i n e t o b a c t e r predominate, thus showing s u p e r i o r
g r o w t h k i n e t i c s on t h e r a t e - l i m i t i n g s u b s t r a t e
(presumably oxygen) s i n c e c e l l m o t i l i t y i s a p p a r e n t l y
irrelevant. But i n a non-mixed c u l t u r e the
Pseudomonas p r e d o m i n a t e . The c h e m o t a c t i c a b i l i t y of
the Pseudomonas s p e c i e s p r o v i d e s , i n t h i s s i t u a t i o n ,
enough o f a b e n e f i t t o overcome i t s growth k i n e t i c
inferiority.
The f i r s t l i t e r a t u r e r e p o r t i n t h i s a r e a was by
S m i t h a n d D o e t s c h (]J*) , who s t u d i e d c o m p e t i t i o n
b e t w e e n a e r o t a c t i c P s e u d o m o n a s f l u o r e s c e n s and an
i m m o t i l e m u t a n t s t r a i n o f t h e same s p e c i e s , f o r o x y g e n
(see Figure 2 ). In a e r a t e d mixed c u l t u r e b o t h s t r a i n s
g r e w t o a r o u g h l y 1:1 r a t i o o v e r a 2 4 - h o u r p e r i o d
i n d i c a t i n g t h a t t h e i r g r o w t h k i n e t i c p r o p e r t i e s were
i d e n t i c a l as e x p e c t e d . In n o n - a e r a t e d media, the
a e r o t a c t i c s t r a i n outgrew the mutant t o a f i n a l ratio
o f o v e r 10:1 a f t e r 24 h o u r s . U n f o r t u n a t e l y , the
a u t h o r s c r e d i t e d m o t i l i t y per se f o r t h i s a d v a n t a g e ,
e v e n t h o u g h i t i s not c l e a r whether random m o t i l i t y
without chemotaxis i s n e c e s s a r i l y always b e n e f i c i a l .
In the c o u r s e o f s t u d y i n g t h e r o l e o f f i m b r i a e i n
b a c t e r i a l g r o w t h , O l d and D u g u i d l o o k e d a t c o m p e t i t i o n
f o r o x y g e n b e t w e e n two n o n f i m b r i a t e s t r a i n s o f
Salmonella typhimurium: one a e r o t a c t i c and one
immotile (_19) (see Table 2 ) . In a e r o b i c shaken b r o t h ,
t h e a e r o t a c t i c s t r a i n m u l t i p l i e d by a f a c t o r o f 46
w i t h i n 48 h o u r s , w h i l e t h e i m m o t i l e s t r a i n m u l t i p l i e d
by a f a c t o r o f 52. Again the growth k i n e t i c

Time (hr) Time (hr)
Figure 2. Multiplication of aerotactic (O) and immotile (Φ) strains of Pseudo

monas fluorescens in two different experiments, each in aerated mixed culture
(left) and nonaerated mixed culture (right). Reproduced, with permission, from
Ref. 18. Copyright 1969, Society for General Microbiology.

Table 2. Growth i n Mixed C u l t u r e s of P a i r s of Variant Strains of Salmonella typhimurium -J
to
COMPETING STRAINS Viable Count (10 bacteria)/ml o f
Time o f Amt
Challenged Challenging Conditions o f Growth Challenged Challenging
Incuba- of
(rha-) (rha+) bacteria bacteria
tion Growth
hr
+ +
S6352, fim f l a S6358, f i m f l a Aerobic s t a t i c broth 0 0.16 130 0.00033
6 0.37 530 0.0053
24 0.71 380 165
48 1.84 1,030 880
Aeiobic shaken broth 0 0.16 130 0.00033

6 3.27 4,200 0.0012
24 3.48 4,600 0.0029
48 3.61 4,700 0.0093
S6351, fim f l a S6355, fim f l a Aerobic s t a t i c broth 0 0.19 65 0.00017

6 0.35 270 0.00080
24 0.46 195 4.8
48 0.48 86 74
72 0.66 105 185
96 1.41 190 450
S6351, fim f l a S6353, fim f l a Aerobic s t a t i c broth 0 0.15 94 0.00024

6 0.38 380 0.00080
24 0.55 500 0.0016
48 0.69 540 4.5
Aerobic shaken broth 0 0.15

94 0.00024
6 2.91 6,900 0.016
9
24 3.00 3,800 0.011 W

48 2.94 4,900 0.011
Reproduced w i t h p e r m i s s i o n f r o m R e f e r e n c e 19. Copyright 1970, A m e r i c a n Society f o r Microbiology.

p r o p e r t i e s appeared n e a r l y the same. In a e r o b i c

s t a t i c b r o t h , however, the a e r o t a c t i c s t r a i n
m u l t i p l i e d by a f a c t o r o f 18,000 i n 48 hours w h i l e the
immotile s t r a i n m u l t i p l i e d by a f a c t o r o f 6. Inter-
p r e t a t i o n o f t h i s c a s e i s not s t r a i g h t f o r w a r d , t h o u g h ,
because p e l l i c l e f o r m a t i o n was noted a f t e r 24 h o u r s ,
c o m p l i c a t i n g the s i t u a t i o n .
More r e c e n t l y , P i l g r a m and W i l l i a m s s t u d i e d a
s l i g h t l y d i f f e r e n t c a s e — c o m p e t i t i o n between
c h e m o t a c t i c P r o t e u s m i r a b i l i s and a nonchemotactic but
randomly m o t i l e mutant o f the s p e c i e s (20) (see
F i g u r e s 3 and 4 ) . In both pure and mixed c u l t u r e s o f
p e r i o d i c a l l y a g i t a t e d amino a c i d b r o t h , the two
s t r a i n s grew t o a 1:1 r a t i o a f t e r 14 h o u r s . On the
o t h e r hand, i n both
s o l i d agar the r a t i
s t r a i n s was g r e a t e r than 5:1 a f t e r 14 h o u r s .
The f i n a l e x p e r i m e n t a l r e p o r t s were by F r é t e r et_
al. (21, 22, 2 3 ) , who s t u d i e d growth o f V i b r i o
c h o l e r a e i n mouse and r a b b i t l a r g e i n t e s t i n e (see
Figure 5 and Table 3. Here t h r e e s t r a i n s were compared:
the c h e m o t a c t i c w i l d t y p e , an immotile mutant s t r a i n ,
and a nonchemotactic but randomly m o t i l e mutant
strain. In w e l l - s t i r r e d c o n t i n u o u s flow c u l t u r e , a l l
t h r e e s t r a i n s grew i n p r o p o r t i o n . In the i n t e s t i n a l
l o o p s , the nonchemotactic s t r a i n was r a p i d l y d i s p l a c e d
by the c h e m o t a c t i c w i l d t y p e . Most i n t e r e s t i n g l y , i n
another experiment the randomly m o t i l e s t r a i n was a l s o
r a p i d l y d i s p l a c e d by the immotile s t r a i n . A p p a r e n t l y ,
i n t h i s s i t u a t i o n at l e a s t , m o t i l i t y w i t h o u t chemo-
t a x i s was a l i a b i l i t y f o r the c e l l s .
It i s e v i d e n t t h a t t h e o r e t i c a l a n a l y s i s o f
m o t i l i t y and chemotaxis i s n e c e s s a r y i n o r d e r t o
p r o v i d e q u a n t i t a t i v e i n t e r p r e t a t i o n o f these r e s u l t s ,
and even q u a l i t a t i v e e x p l a n a t i o n o f the l a s t , perhaps
c o u n t e r - i n t u i t i v e , o b s e r v a t i o n by F r é t e r et al_. This
w i l l be the c o n c e r n o f the next s e c t i o n o f t h i s p a p e r .
But i s i m p o r t a n t to emphasize at t h i s p o i n t t h a t the
e x p e r i m e n t a l r e s u l t s c i t e d here demonstrate t h a t the
e f f e c t s o f c e l l movement p r o p e r t i e s can c l e a r l y be
s i g n i f i c a n t , and even dominant, i n d e t e r m i n i n g the
c o m p e t i t i v e a b i l i t i e s o f b a c t e r i a l p o p u l a t i o n s i n non-
mixed e n v i r o n m e n t s .
Theoretical Analyses
E a r l y attempts at t h e o r e t i c a l a n a l y s i s o f the
e f f e c t s o f c e l l m o t i l i t y on p o p u l a t i o n growth c e n t e r e d
on uptake o f n u t r i e n t by a s i n g l e c e l l i n a medium o f
i n f i n i t e e x t e n t (12, 24, 25, 2 6 ) . These a n a l y s e s have

J- J
6 8 10 12 H
Hours
6 8 10 12 H 2L
Hours
Figure 3. Growth of pure cultures of chemotactic (O) and nonchemotactic ( O

strains of Proteus mirabilis in periodically shaken amino acid broth (top) and
soft-agar amino acid medium (bottom). Reproduced, with permission, from Ref. 20.
Copyright 1976, National Research Council of Canada.

12. LAUFFENBURGER Cell Motility
I 1 ι ι ι ι ι IK ι
0 2 4 6 8 10 12 Η 24
Hours
Figure 4. Growth of mixed cultures of chemotactic (O) and nonchemotactic ( Q )

strains of Proteus mirabilis in periodically shaken amino acid broth (top) and
soft-agar amino acid medium (bottom). Reproduced, with permission, from Ref. 20.
Copyright 1976, National Research Council of Canada.

c 100 η
I 90
5 80
Ο
^ 70
° ~ 60
5 2 50 -
6 30-
ζ ζ
!: 20-1 < LU
< <
ο 10 CL
LU
° 0
<
DAY AFTER INFECTION
Figure 5. Time course of growth of chemotactic parent and randomly motile

mutant strains of Vibrio cholerae in mouse large intestine. Reproduced, with per
mission, from Ref. 21. Copyright 1979, American Society for Clinical Nutrition.

T a b l e 3. D i s p l a c e m e n t o f Randoml Motil Mutant Strai f

V i b r i o c h o l e r a e by I m m o t i l
Day a f t e r Percentage o f non-motile No. o f

monoassociation v i b r i o s i n cecum (+ S.D.) mice cultured
0 (inoculum) 0.0
-
1 44 (+ 27) 6
2 50 (+ 35) 11
3 39 <+ 36) 3
5 96 1
10 o r more 93 (+ 7.9) 11
R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f e r e n c e 21. C o p y r i g h t 1979,
American S o c i e t y f o r C l i n i c a l N u t r i t i o n .

i n v a r i a b l y found that s u b s t r a t e uptake i s o s t e n s i b l y

i n c r e a s e d by movement, a l t h o u g h t h e i n c r e a s e w i l l be
n e g l i g i b l e f o r t y p i c a l c e l l s i z e s , swimming
s p e e d s , and n u t r i e n t d i f f u s i v i t i e s . However, the
r e s u l t f o r a p o p u l a t i o n may be q u i t e d i f f e r e n t b e c a u s e
t h e n u t r i e n t i s s u b j e c t t o d e p l e t i o n by a l l t h e cells,
and i f t h e e n v i r o n m e n t i s n o t w e l l - m i x e d t h e e f f e c t o f
movement w i l l v a r y d e p e n d i n g o n t h e d i r e c t i o n o f
movement. S i n c e n u t r i e n t u p t a k e r e l i e s e s s e n t i a l l y on
d i f f u s i o n t o t h e c e l l s u r f a c e (22), c e l l growth
depends on t h e l o c a l n u t r i e n t c o n c e n t r a t i o n i n w h i c h
the c e l l i s s i t u a t e d . Thus the c h i e f e f f e c t o f
m o t i l i t y c o m e s n o t f r o m t h e a c t o f movement i t s e l f ,
but from the f a c t t h a t a c e l l can e x p e r i e n c e a new
l o c a l n u t r i e n t c o n c e n t r a t i o n when i t c h a n g e s p o s i t i o n
We h a v e a c c o r d i n g l
for the e f f e c t s o f c e l
l e v e l ( 3 , 28, 29, 3 0 ) . F i g u r e 6 i l l u s t r a t e s the
physical situation considered: "confined growth" of
b a c t e r i a l populations i n a one-dimensional s p a t i a l l y
f i n i t e non-mixed r e g i o n , w i t h the d i f f u s i b l e r a t e -
l i m i t i n g n u t r i e n t e n t e r i n g the r e g i o n from a boundary.
The e q u a t i o n s g o v e r n i n g a s i n g l e b a c t e r i a l p o p u l a t i o n
g r o w i n g on a s i n g l e n u t r i e n t a r e
3 J
3b _ b
+ G
3t 3x "b L
3s !^s
at ax s
over 0<x<L, w h e r e t h e n o t a t i o n i s a s f o l l o w s :
b (x,t) = v i a b l e c e l l d e n s i t y
s (x,t) = n u t r i e n t c o n c e n t r a t i o n
Jfc = viable cell flux
Js = nutrient flux
Gfc = net c e l l growth r a t e
Gs = n u t r i e n t consumption rate
The b o u n d a r y c o n d i t i o n s a r e
Jb = 0 s = s at χ = L
0
Jfc = 0 J s = 0 at χ = 0
i f the n u t r i e n t c o n c e n t r a t i o n at the source boundary
remains constant.
F o r t h e n e t c e l l g r o w t h r a t e , we u s e Monod's
m o d e l f o r g r o w t h and a f i r s t o r d e r r a t e o f l o s s o f
viability:
ks .

w h e r e k i s t h e g r o w t h r a t e c o n s t a n t , Κ i s t h e Monod
c o n s t a n t , and k i s the n o n - v i a b i l i t y r a t e constant.
e
The n u t r i e n t c o n s u m p t i o n r a t e i s
1
r _ ks t_
vas
*S -~ Ϋ77 K+S
where Y i s the y i e l d c o e f f i c i e n t .
In order to permit a n a l y t i c a l s o l u t i o n o f the
m o d e l e q u a t i o n s , we r e p l a c e t h e s e e x p r e s s i o n s by s t e p -
function approximations:
i(k - k ) e b
G
b= - k P b s <
γ kb s > s c
where s c is a threshold nutrient concentration

required to support net growth:
k e
S c = (
kH^) Κ
T h i s a p p r o x i m a t i o n a l l o w s d i r e c t a n a l y s i s o f the model
p r e d i c t i o n s , and n u m e r i c a l c o m p u t a t i o n s show g o o d
q u a n t i t a t i v e agreement w i t h the o r i g i n a l e x p r e s s i o n s
(31) .
For the nutrient flux we can use Fick's law:
s
3x
where D i s t h e d i f f u s i v i t y . F o r t h e c e l l f l u x we use
an e x p r e s s i o n o r i g i n a l l y f o r m u l a t e d by K e l l e r and
Segel (32):
b y X
"3x âx
w h e r e μ i s t h e r a n d o m m o t i l i t y c o e f f i c i e n t and χ
i s the chemotaxis c o e f f i c i e n t . T h e r e a r e some
e s t i m a t e s o f μ and χ a v a i l a b l e ( 3 3 , 3±, 35)t although
a s a t i s f a c t o r y a n a l y s i s of a convenient assay for
t h e s e p a r a m e t e r s n e e d s t o be developed.

Defining dimensionless variables and p a r a m e t e r s
. Ys D
u = s_ v = b_ ξ = χ τ . Dt b = - ^
s 0
b
o L
L kL
k L* 1 u>u
= ML.
S
X 0
λ = K θ = -4- β = -^p F(u) =
D D
0 u<u c
we o b t a i n d i m e n s i o n l e s s model equations
If • λ
w ~ δ λ
i l (
2
3u _ 3 u
F ( U ) V
3T " ~
over 0 < ξ < 1, w i t h boundary c o n d i t i o n s
f f - 6 v f ! = 0 u « 1 at ζ - 1
|f - 0 - 0 at ς - 0
Using these equations, the steady-state
population
Β = Ifo£ ν
with 2
kL
V = / vdÇ 0
h a s b e e n a n a l y z e d f o r t h r e e c a s e s s o f a r : a) a s i n g l e
r a n d o m l y m o t i l e p o p u l a t i o n {3, 28), b) a s i n g l e
c h e m o t a c t i c p o p u l a t i o n (3, 2j)) , a n d c ) two r a n d o m l y
m o t i l e p o p u l a t i o n s g r o w i n g i n c o m p e t i t i o n {30, 31). A
s t e a d y s t a t e i s s e t up b y t h e b a l a n c e b e t w e e n c e l l
g r o w t h i n a n u t r i e n t - r i c h zone and l o s s o f v i a b i l i t y
i n a n u t r i e n t - p o o r zone, m e d i a t e d by c e l l f l u x from
t h e n u t r i e n t - r i c h zone t o t h e n u t r i e n t - p o o r zone ( s e e
F i g u r e 7 ) . The d i m e n s i o n l e s s p o s i t i o n , ω , o f t h e
d i v i s i o n b e t w e e n t h e s e two z o n e s i s t h e p o i n t a t w h i c h
u = u = s /s .
c ω i s g i v e n by t h e l a r g e s t r o o t , l e s s
c 0
than u n i t y , o f the equation
α tanh αω = β t a n β(1-ω)
1 / 2 1 / 2
where α = (θ/λ) and β = ([κ - θ])/λ) .

12. LAUFFENBURGER Cell Motility
Ambient
Substrate
Concentration
So
-Bacteria Density b (x)
Substrate
diffusion
Substrate Concentration s(x)
x= 0
Figure 6. Illustration of confined-growth model system.
Figure 7. Typical steady state profiles of dimensionless bacterial density, v, and

dimensionless substrate concentration, u. In the growth zone, ω < ξ < 1, u > u , c
and bacterial growth can be supported. In the depleted zone, 0 < £ < ω, u = u , c
and bacterial growth cannot be supported.

T h u s , ω i n c r e a s e s a s κ i n c r e a s e s and λ d e c r e a s e s .
Although i n n a t u r a l systems such a steady s t a t e may
n o t u s u a l l y be a t t a i n e d , i t i s a g o o d i n d i c a t i o n o f
t h e s i t u a t i o n w h i c h w i l l be a p p r o a c h e d i f e n v i r o n
mental c o n d i t i o n s are not changed.
In the f i r s t c a s e , s i n g l e p o p u l a t i o n growth o f a
n o n c h e m o t a c t i c s p e c i e s (6 = 0 ) , t h e s t e a d y s t a t e
p o p u l a t i o n s i z e Β d e p e n d s u p o n two q u a n t i t i e s : λ/κ
and κ/θ (see Figure 8 ) . The l a t t e r q u a n t i t y i s t h e
r a t i o o f growth to n o n v i a b i l i t y r a t e c o n s t a n t s ,
r e p r e s e n t i n g t h e g r o w t h p o t e n t i a l a t any g i v e n n u t r i e n t
concentration. F i g u r e 11 shows t h a t f o r any v a l u e o f
λ/κ Β i n c r e a s e s a s κ/θ i n c r e a s e s . The f o r m e r q u a n t i t y
2
is equal to μ / k L , and t h u s i s t h e r a t i o o f t h e r a t e
o f c e l l movement a c r o s th regio t th growth r a t
constant. Figure 1
κ / θ , Β i r c r e a s e s a s λ/κ d e c r e a s e s . T h i s i s because
r a n d o m m o t i l i t y l e a d s t o d i s p e r s a l o f c e l l s away f r o m
the n u t r i e n t - r i c h zone. F r o m t h i s r e s u l t we e x p e c t
t h a t an i m m o t i l e s t r a i n c a n o u t g r o w a r a n d o m l y m o t i l e
s t r a i n , a l l o t h e r f a c t o r s b e i n g e q u a l , a s was observed
by F r é t e r e t a l . ( Z I ) . One c a n a l s o s e e f r o m F i g u r e
11 t h a t a s p e c i e s w i t h s m a l l e r g r o w t h r a t e c o n s t a n t
can outgrow a s p e c i e s with l a r g e r growth r a t e
c o n s t a n t , i f t h e random m o t i l i t y c o e f f i c i e n t o f t h e
former s p e c i e s i s s m a l l enough r e l a t i v e t o t h a t o f the
latter species. F i g u r e 11 a l s o s h o w s t h a t f o r λ/κ
much g r e a t e r t h a n 1, Β i s i n d e p e n d e n t o f t h e r a n d o m
m o t i l i t y c o e f f i c i e n t , because the system behaves
e s s e n t i a l l y l i k e a well-mixed system.
In the second c a s e , s i n g l e p o p u l a t i o n growth o f a
chemotactic s p e c i e s , Β a d d i t i o n a l l y d e p e n d s u p o n 6,
t h e r a t i o o f t h e c h e m o t a x i s c o e f f i c i e n t t o t h e random
m o t i l i t y c o e f f i c i e n t (see Figure 9 ) . Β i n c r e a s e s as 6
i n c r e a s e s , b e c a u s e c h e m o t a x i s c o u n t e r a c t s t h e random
d i s p e r s a l o f c e l l s away f r o m t h e n u t r i e n t - r i c h z o n e
(see F i g u r e 10 )· The e f f e c t o f c h e m o t a x i s b e c o m e s
s i g n i f i c a n t o n l y when δ b e c o m e s g r e a t e r t h a n a b o u t
1
10" . T h e s e c o m p u t a t i o n s were done u s i n g a p e r t u r b a
t i o n m e t h o d , s o t h a t r e s u l t s f o r 6> 1 a r e m e r e l y
extrapolations. N u m e r i c a l c o m p u t a t i o n s h a v e shown t h e
p e r t u r b a t i o n r e s u l t s t o be a c c u r a t e up t o a t l e a s t 6 =
1.1, however ( 3 1 ) .
An i n t e r e s t i n g i n f e r e n c e w h i c h c a n be d r a w n f r o m
F i g u r e 12 i s t h a t t h e r e i s a minimum v a l u e o f 6 that
m u s t be e x c e e d e d i n o r d e r f o r m o t i l i t y t o c o n f e r an
advantage i n t h i s c o n f i n e d growth s i t u a t i o n . I f λ χ
r e p r e s e n t s t h e B r o w n i a n m o t i o n c o e f f i c i e n t f o r an

LAUFFENBURGER Cell Motility 283

Figure 9. Plot of dimensionless total steady state bacterial density vs. 8 for single
chemotactic populations. Extrapolations of perturbation computations beyond 8
= 1 shown ( ). Asymptotic values for δ = 0 shown (- · - · -).

Figure 10. Typical steady state profiles of dimensionless bacterial density, v.

i m m o t i l e s p e c i e s (_33) , t h e n a c h e m o t a c t i c s p e c i e s m u s t
have a l a r g e r v a l u e , say λ > λι.
2 By i t s e l f t h i s
w o u l d y i e l d a s m a l l e r v a l u e o f B. Increasing 6 from
0 i n c r e a s e s B, and t h e r e w i l l be a c r i t i c a l v a l u e , 6*,
a t w h i c h Β f o r λ2 and 6 b e c o m e s e q u a l t o Β f o r λ 1 #
O n l y f o r 6>6* w i l l t h e c h e m o t a c t i c s t r a i n o u t g r o w t h e
immotile s t r a i n . Thus, s p e c u l a t i o n t h a t a m o t i l e
c h e m o t a c t i c s t r a i n s h o u l d a l w a y s be s u p e r i o r t o an
immotile s t r a i n i s not n e c e s s a r i l y t r u e .
T h e s e s i n g l e p o p u l a t i o n r e s u l t s s u g g e s t some
i m p o r t a n t i m p l i c a t i o n s f o r c o m p e t i t i o n b e t w e e n two o r
more p o p u l a t i o n s g r o w i n g t o g e t h e r o n a s i n g l e r a t e -
limiting nutrient. I f one s p e c i e s h a s s u p e r i o r g r o w t h
k i n e t i c p r o p e r t i e s but t h e o t h e r has s u p e r i o r m o t i l i t y
p r o p e r t i e s , we m i g h
A n a l y s i s o f the t h i r
r a n d o m l y m o t i l e p o p u l a t i o n s , shows t h a t t h i s i s i n d e e d
possible. T h e r e a r e now a c t u a l l y t h r e e p e r m i s s i b l e
steady s t a t e s : 1) c o e x i s t e n c e , 2) s p e c i e s 1 o n l y , a n d
3) s p e c i e s 2 o n l y . For sake o f c l a r i t y , l e t the two
s p e c i e s have i d e n t i c a l p r o p e r t i e s e x c e p t t h a t
ki > k2« Then s p e c i e s 1 w i l l have a g r e a t e r growth
rate at a l l n u t r i e n t concentrations. In a d d i t i o n , the
t h r e s h o l d c o n c e n t r a t i o n f o r net growth o f s p e c i e s 2
u > u
must be g r e a t e r t h a n t h a t o f s p e c i e s 1; i . e . , c 2c i *
We c a n i m m e d i a t e l y see t h a t a n e c e s s a r y c o n d i t i o n f o r
> ω
coexistence i s that ω 2 * · The v a l u e s o f ω f o r
e a c h s p e c i e s a r e d e t e r m i n e d by t h e same e q u a t i o n a s i n
t h e s i n g l e p o p u l a t i o n c a s e , u n a f f e c t e d by t h e p r e s e n c e
o f the other s p e c i e s . We c a n , t h e r e f o r e , move
d i r e c t l y to a g r a p h i c a l d e s c r i p t i o n o f the steady-
s t a t e behavior f o r our c o m p e t i t i o n model. F i g u r e 11
shows i s o c l i n e s o f ω i n t h e p l a n e o f (κ,λ) v a l u e s . If
we s p e c i f y v a l u e s λ χ and κι f o r p o p u l a t i o n 1, t h i s
y i e l d s a value for ωι. We c a n t h e n immediately
d i s c o v e r t h e p e r m i s s i b l e s t e a d y - s t a t e s f o r any s p e c i e s
2 with parameter values λ 2 and κ 2 (see Figure 1 2 ) . If
κ 2 < K l r o n l y s p e c i e s 1 can s u r v i v e , u n l e s s λ2 i s such
t h a t o)2 > ω χ — which would a l l o w c o e x i s t e n c e . If
κ 2 > Κ χ , o n l y s p e c i e s 2 can s u r v i v e , u n l e s s λ 2 is
s u c h t h a t α)2 < ω ι , w h i c h a g a i n a l l o w s c o e x i s t e n c e .
S o , t h e c o m p e t i t i o n o u t c o m e c a n be p r e d i c t e d f r o m
t h e s i n g l e p o p u l a t i o n r e s u l t s , w i t h one m i n o r
modification. Remember t h a t t h e ω c r i t e r i o n i s o n l y a
necessary condition for coexistence. I t turns out
t h a t a s e c o n d , s l i g h t l y more r e s t r i c t i v e c o n d i t i o n i s
a l s o r e q u i r e d , t o e n s u r e t h a t t h e c e l l d e n s i t i e s and
n u t r i e n t c o n c e n t r a t i o n r e m a i n p o s i t i v e e v e r y w h e r e (3JL) .
The d i f f e r e n c e b e t w e e n t h e two c o n d i t i o n s i s s m a l l

LAUFFENBURGER Cell Motility
Figure 11. Sample plot of curves of constant ω in plane of (Χ, λ).

Ω
1
10 e ! !
10*
I COEXISTENCE j
ΙΟ 2
SPECIES 1 !
ONLY ! /
/
ι ι ι •
8 ! 32
λ, =10"'
•*• II
Χ Ι
• I
ΙΟ" - 2
/
/ ι SPECIES 2
/ s ONLY
/ ι
-/ I
4
/ ι
,ο -ι Ι
! COEXISTENCE j
ίο j 6
J
I 1
K 1 = 12
Figure 12. Illustration of predicted results for competition between two randomly
motile populations with identical properties except for Κ and λ.

for t y p i c a l parameter v a l u e s , however, so t h a t g i v e n

the a p p r o x i m a t i o n i n v o l v e d i n the model i t s e l f , we
n e e d n o t be t o o c o n c e r n e d w i t h i t .
When t h e c e l l d e n s i t i e s a r e c o m p u t e d f o r a
c o e x i s t e n c e s t a t e , t h e s p e c i e s w i t h s m a l l e r k c a n grow
to a g r e a t e r p o p u l a t i o n s i z e than the s p e c i e s w i t h
l a r g e r k, i f i t s m o t i l i t y p r o p e r t i e s a r e s u f f i c i e n t l y
superior. T h i s g i v e s an e x p l a n a t i o n t o t h e r e s u l t
c i t e d by S t a n i e r e t a l (1_7) .
S i m i l a r r e s u l t s a r e e x p e c t e d when c h e m o t a x i s i s
present i n competing p o p u l a t i o n s , although the
a n a l y s i s has not y e t been c a r r i e d o u t . Chapman (5)
f o r m u l a t e d a m o d e l f o r c o m p e t i t i o n o f two chemotactic
s p e c i e s i n a t r a v e l i n g band which p r e d i c t e d that
superior chemotaxi
i n f e r i o r growth r a t
species. T h i s r e s u l t i s c o n s i s t e n t w i t h our
e x p e c t a t i o n s ; the model, however, i s r a t h e r e m p i r i c a l .
Conclusions
R e v i e w o f t h e l i t e r a t u r e r e v e a l s a s m a l l number
o f e x p e r i m e n t s t h a t show t h a t c e l l m o t i l i t y p r o p e r t i e s
c a n h a v e d r a m a t i c e f f e c t s o n p o p u l a t i o n g r o w t h and
c o m p e t i t i o n i n non-mixed systems. Simple mathematical
models have been d e v e l o p e d w h i c h p r o v i d e q u a l i t a t i v e
e x p l a n a t i o n f o r a l l t h e o b s e r v e d p h e n o m e n a , and yield
q u a n t i t a t i v e p r e d i c t i o n of the magnitude of e f f e c t s
w h i c h m i g h t be e x p e c t e d i n a v a r i e t y o f s i t u a t i o n s .
Ac k nowledgme n t s
T h i s work h a s b e e n p a r t i a l l y s u p p o r t e d by
NSF C h e m i c a l and B i o c h e m i c a l P r o c e s s e s Program
G r a n t CPE80-06701. D.A.L. w o u l d a l s o l i k e t o t h a n k
P a t Thompson f o r t y p i n g t h i s m a n u s c r i p t and R e n a t e
S c h u l t z f o r many o f t h e f i g u r e s .
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"Bergey's Manual of Determinative Bacteriology";
Williams and w i l k i n s : Baltimore, 7th e d i t i o n ,
1957.
2. Koshland, Jr., D. E. "Bacterial Chemotaxis as a

Model Behavioral System"; Raven Press: New York,
1980.

3. Lauffenburger, D. A. "Effects of M o t i l i t y and

Chemotaxis in C e l l Population Dynamical Systems";
PhD Thesis, University of Minnesota, 1979.
4· Fraenkel, G. S.; Gunn, D. L . "The Orientation of

Animals"; Dover: New York, 1961.
5. Chapman, P. A. "Chemotaxis of Bacteria"; PhD Thesis,

University of Minnesota, 1973.
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500-504.
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Development", v o l . 2; R. Goldberger, e d . ;
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Acad. S c i . USA 1972, 69, 2509-2512.
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193-219.
13. Hazelbauer, G. L.; Parkinson, J. S. In "Receptors
and Recognition: Microbial Interactions"; J.
R e i s s i g , e d . ; Chapman and Wall: London, 1977;
pp. 60-80.
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D. E . J. B a c t e r i a l 1979, 139, 107-114.
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Science 1973, 181, 60-63.
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Cliffs, New Jersey, 1976.
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19. O l d , D. C., Duguid, J. P. J. B a c t e r i o l . 1970,

103, 447-456.
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M i c r o b i o l . 1976, 22, 1771-1773.
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Halstead, S. A. In "Proceedings of the 13th
Joint Conference on Cholera", U.S. - Japan
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Govt. Printing O f f i c e , Washington, D . C . ;
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22. F r e t e r , R.; O'Brien, P. C. M . ; Halstead, S. A.

Adv. Exp. Med
23. F r e t e r , R.; O'Brien, P. C. M . ; Macsai, M. S.
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Bishop, ed.; AAAS Publication No. 72:
Washington, D. C., 1962.
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147-217.
26. Brunn, P. O. J. Biomech. Eng. 1981, 103, 32-37.
27. P u r c e l l , Ε. M. Am. J. Physics 1977, 45, 3-11.
28. Lauffenburger, D. Α . ; A r i s , R.; K e l l e r , Κ. H.

Microb. E c o l . 1981, 7, 207-227.
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Biophys. J . (submitted for p u b l i c a t i o n ) .
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and Competition of Motile B a c t e r i a l Popula
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30, 225-234.
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M o t i l . 1973, 2, 25-34.
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1976, 30, 221-239.

13
Macroscopic Thermodynamics and the
Description of Growth and Product Formation
in Microorganisms
J. A. ROELS
Delft University of Technology, Netherlands Central Organization for Applied
Scientific Research, T.N.O., P.O. Box 108, 3700 A C ZEIST, The Netherlands
From the point o

living organisms are energy transducers c o n v e r t i n g a
source of energy,e.g. chemical substances or
photons, i n t o other forms o f energy. As such they
are subject to the c o n s t r a i n t s posed by the first
and second laws o f thermodynamics. As micro
organisms are open systems and as such e x i s t i n
a s t a t e o u t s i d e e q u i l i b r i u m , n o n - e q u i l i b r i u m thermo
dynamics provide the p e r f e c t v e h i c l e f o r a first
approach to the d e s c r i p t i o n of t h e i r behaviour.
The concept o f the thermodynamic e f f i c i e n c y
of growth i s developed and it is shown t h a t ,
as a r u l e of thumb, the maximum observed e f f i c i e n c i e s
are about 0.65 i r r e s p e c t i v e the nature of the
energy supplying process. A number of notable
exceptions are shown to be most probably caused
by l i m i t a t i o n s other than a v a i l a b l e energy.
The nature o f growth and product formation
i s discussed i n terms o f the c o u p l i n g o f the
transformation of a given amount of substrate
energy i n t o biomass energy to the energy obtained
from a flow of e l e c t r o n s to a l e v e l of high
to a l e v e l of low energy. The treatment i s shown
to r e s u l t i n a r e l i a b l e r u l e of thumb f o r a
first estimate o f the order of magnitude of
the growth y i e l d o f an organism feeding on a
given energy supplying t r a n s f o r m a t i o n process.
The b i o s p h e r e o n e a r t h i s , t h e r m o d y n a m i c a l l y s p e a k i n g , i n a
c e r t a i n s e n s e an o p e n s y s t e m . I t r e c e i v e s e n e r g y f r o m t h e s u n
i n t h e f o r m o f r a d i a t i o n . The e n e r g y o f t h e p h o t o n s r e a c h i n g e a r t h
i s , i n p a r t , converted t o chemical energy i n a process c a l l e d
0097-6156/83/0207Ό295$08.00/0

p h o t o s y n t h e s i s . F o r the l a r g e r p a r t t h i s energy takes the form

o f c a r b o h y d r a t e s , e.g. s u g a r s , s t a r c h e s and c e l l u l o s i c s . The
components o f t h e b i o m a s s o f t h e p r i m a r y p r o d u c e r s a r e t h e
s t a r t i n g p o i n t o f a wide v a r i e t y o f t r a n s f o r m a t i o n s , which takes
p l a c e under c a t a l y t i c a c t i o n o f l i v i n g organisms.
As a f i n a l r e s u l t t h e s o l a r e n e r g y i s c o n v e r t e d t o l e s s
energy r i c h forms o f r a d i a t i o n w h i c h t r a n s p o r t energy t o o u t e r
space o r i s used t o d e c r e a s e t h e e n t r o p y o f t h e b i o s p h e r e .
The r e a s o n i n g d e v e l o p e d above shows t h a t t h e b i o s p h e r e i s
a s y s t e m , w h i c h i s s u b j e c t t o a f l o w o f e n e r g y . The e n e r g y e n t e r s
t h e s y s t e m a t a l o w e n t r o p y l e v e l and l e a v e s i t a t a
s u b s t a n t i a l l y h i g h e r e n t r o p y l e v e l . As s u c h t h e b i o s p h e r e c a n
m a i n t a i n a s t a t e w i t h a n e n t r o p y l o w e r t h a n t h e maximum c o r r e s
p o n d i n g t o t h e r m o d y n a m i c e q u i l i b r i u m and p r o c e s s e s known as
" l i f e " r e s u l t (J_).
A s i n g l e organism o
supplying process e x i s t positio
b i o s p h e r e as a w h o l e . I t i s an open s y s t e m t h r o u g h w h i c h e n e r g y
f l o w s f r o m a low e n t r o p y s t a t e , e.g. c h e m i c a l e n e r g y s t o r e d i n
compounds more r e d u c e d t h a n C 0 , t o a h i g h e n t r o p y s t a t e , e.g.
2
h e a t a t a l o w t e m p e r a t u r e l e v e l . As a r e s u l t t h e o r g a n i s m s c a n
m a i n t a i n a s t a t e o u t s i d e t h e r m o d y n a m i c e q u i l i b r i u m and c a n
continue performing the processes c h a r a c t e r i s t i c f o r t h e i r " l i f e " .
As o r g a n i s m s a r e s y s t e m s w h i c h e x i s t o u t s i d e t h e r m o d y n a m i c
e q u i l i b r i u m and i r r e v e r s i b l e p r o c e s s e s a r e t a k i n g p l a c e , t h e
f o r m a l i s m o f thermodynamics of i r r e v e r s i b l e p r o c e s s e s c o n s t i t u t e s
the l o g i c a l v e h i c l e t o t r e a t t h e i r b e h a v i o u r . I n the p r e s e n t
a r t i c l e t h e f o r m a l i s m w i l l be b r i e f l y s u m m a r i z e d f o r t h e p u r p o s e
o f i t s a p p l i c a t i o n t o m i c r o o r g a n i s m s engaged i n g r o w t h and
p r o d u c t f o r m a t i o n . F o r a more t h o r o u g h t r e a t m e n t o f t h e b a s i c
f o r m a l i s m t h e r e a d e r i s r e f e r r e d t o t h e s t a n d a r d t e x t s ( 2 - 4 ) and
e a r l i e r w o r k o f t h e p r e s e n t a u t h o r ( 5 , 6^).
Macroscopic t h e r m o d y n a m i c s and p r o c e s s e s i n open s y s t e m s . 05, 6)
For t h e purpose o f the present a n a l y s i s o f m i c r o b i a l metabolism,

a g i v e n amount o f m i c r o o r g a n i s m s i s c o n s i d e r e d t o be an e n e r g y
t r a n s d u c e r . I t i s s c h e m a t i c a l l y r e p r e s e n t e d i n f i g . 1. The s y s t e m
e x c h a n g e s c h e m i c a l e n e r g y and h e a t w i t h t h e e n v i r o n m e n t . F o r
s i m p l i c i t y ' s s a k e t h e c a s e o f p r o c e s s e s i n v o l v i n g radiâtional
e n e r g y i s e x c l u d e d . The b a s i c f o r m a l i s m , h o w e v e r , c a n be e a s i l y
extended t o i n c l u d e these s i t u a t i o n s .
The s t a t e o f t h e s y s t e m c a n be c h a r a c t e r i z e d by a number
o f e x t e n s i v e q u a n t i t i e s ; t h e s e s p e c i f y t h e amount o f t h e v a r i o u s
c h e m i c a l s u b s t a n c e s and t h e amount o f e n e r g y p r e s e n t i n t h e
s y s t e m . F o r e a c h e x t e n s i v e q u a n t i t y , w h i c h c a n be a t t r i b u t e d
t o t h e s y s t e m , a b a l a n c e e q u a t i o n c a n be f o r m u l a t e d ; i t e x p r e s s e s
t h e a c c u m u l a t i o n o f t h e q u a n t i t y i n s i d e t h e s y s t e m as t h e sum
o f t h e c h a n g e s o f i t s amount due t o t r a n s f o r m a t i o n and t r a n s p o r t
p r o c e s s e s r e s p e c t i v e l y . M a t h e m a t i c a l l y t h i s c a n be e x p r e s s e d a s :

13. ROELS Macroscopic Thermodynamics and Growth 297
\
HEAT
Figure 1. An open system for macroscopic analysis. It exchanges chemical sub

stances and heat with the environment. The flow of chemical substances, Φι, is
characterized by the elemental composition and the partial enthalpy of the chemical
substances.

(1)
V V s
I n t h e f o r m u l a t i o n o f eqn. (1) i t i s assumed t h a t t h e volume and
t h e s u r f a c e a r e a o f t h e s y s t e m do n o t change. I n t h e p r e s e n t
a r t i c l e t h e d i s c u s s i o n w i l l be r e s t r i c t e d t o s y s t e m s i n a
s t a t i o n a r y s t a t e , i . e . systems f o r which the time d e r i v a t i v e
a p p e a r i n g a t t h e l e f t hand s i d e o f eqn. (1) has become z e r o . I n
s u c h a c a s e eqn. (1) c a n be s i m p l i f i e d t o :
(2)
V
W i t h respect to th
n o n - e q u i l i b r i u m system the e x t e n s i v e q u a n t i t i e s c h a r a c t e r i z i n g
t h e s y s t e m can be d i s t i n g u i s h e d i n t o two g r o u p s : c o n s e r v e d and
non-conserved q u a n t i t i e s . S o - c a l l e d conserved q u a n t i t i e s cannot
be p r o d u c e d o r consumed i n t h e t r a n s f o r m a t i o n p r o c e s s e s o p e n t o
a g i v e n s y s t e m . T h e r e f o r e , t h e f i r s t t e r m a t t h e r i g h t hand s i d e
of eqn. (1) i s n e c e s s a r i l y z e r o and eqn. (2) c a n be s i m p l y
w r i t t e n as :
(3)
E q u a t i o n (2) e x p r e s s e s t h e f a c t t h a t f o r e a c h c o n s e r v e d
q u a n t i t y t h e t r a n s p o r t t o a s y s t e m i n s t a t i o n a r y s t a t e must
e x a c t l y match t r a n s p o r t from t h a t system. For a non-conserved
q u a n t i t y such s i m p l i f i c a t i o n i s not p o s s i b l e .
The a p p l i c a t i o n o f t h e f o r m a l m a c r o s c o p i c t h e o r y t o t r a n s
f o r m a t i o n p r o c e s s e s i n o p e n s y s t e m s i s b a s e d on t h e f o r m u l a t i o n
o f b a l a n c e e q u a t i o n s f o r a number o f c o n s e r v e d q u a n t i t i e s and
an a d d i t i o n a l t h e r m o d y n a m i c c o n s t r a i n t a l l o w i n g t h e f o r m u l a t i o n
of a u s e f u l e f f i c i e n c y measure.
The e l e m e n t a l b a l a n c e e q u a t i o n s . In m i c r o b i a l conversion
p r o c e s s e s t h e amounts o f t h e v a r i o u s a t o m i c s p e c i e s a r e c o n s e r v e d .
T h i s o b s e r v a t i o n r e s u l t s i n the f o r m u l a t i o n of e l e m e n t a l balance
e q u a t i o n s . U s i n g eqn. (3) t h e e l e m e n t a l b a l a n c e e q u a t i o n f o r
a t o m i c s p e c i e s j c a n , a g a i n a s s u m i n g a s t a t i o n a r y s t a t e , be
e x p r e s s e d as ( 5 , 6 ) :
η
Σ Φ.«*. .= 0 (4)
1 1 J
i-i

I n e q n . ( 4 ) Φ. s t a n d s f o r t h e n e t m o l a r f l o w o f compound
i t o t h e s y s t e m , e. . s t a n d s f o r t h e number o f m o l e s o f a t o m i c
s p e c i e s j i n one m J l e o f compound i . E q u a t i o n s o f t h e t y p e o f
eqn. ( 4 ) p r o v i d e one c o n s t r a i n t t o t h e n e t exchange f l o w s f o r e a c h
atomic species considered.
The thermodynamic c o n s t r a i n t s . The a p p l i c a t i o n o f n o n - e q u i

l i b r i u m thermodynamics t o t r a n s f o r m a t i o n p r o c e s s e s i s based on
t h e f o r m u l a t i o n o f two b a s i c b a l a n c e e q u a t i o n s . The f i r s t o n e ,
a b a l a n c e e q u a t i o n f o r e n e r g y , c a n , by v i r t u e o f t h e f a c t t h a t
the f i r s t l a w o f t h e r m o d y n a m i c s a s s u r e s e n e r g y t o be a c o n s e r v e d
q u a n t i t y i n any s y s t e m , f o r a s y s t e m i n s t a t i o n a r y s t a t e be
expressed as:
Φ Ε = 0 (5
i n w h i c h Φ^ i s t h e n e t f l o w o f e n e r g y t o w a r d s t h e s y s t e m .
Thermodynamics show t h a t , f o r a n open s y s t e m o n w h i c h no work
i s p e r f o r m e d by e x t e r n a l f o r c e f i e l d s , t h e e n e r g y f l o w t o w a r d s
t h a t s y s t e m c a n be e x p r e s s e d as f o l l o w s :
Φ
Ε - Η Φ +
Σ Φ
Α ( 6 )
I n t h i s e q u a t i o n Φ^ i s t h e s o - c a l l e d h e a t f l o w o f P r i g o g i n e ( 2 )
and t h e h. a r e t h e p a r t i a l m o l a r e n t h a l p i e s o f t h e compounds
exchanged w i t h t h e environment.
I f eqns. ( 5 ) and ( 6 ) a r e combined t h e f a m i l i a r b a l a n c e
equation f o r enthalpy i s obtained. I t allows the c a l c u l a t i o n of
the heat exchanged w i t h t h e environment from t h e f o l l o w i n g
equation:
Φ Η = - Σ Φ h. (7)
1
Equation ( 7 ) i n t r o d u c e s one new unknown f l o w , t h e h e a t f l o w Φ^,

and one a d d i t i o n a l c o n s t r a i n t , hence t h e t o t a l number o f unknown
f l o w s does n o t change by t h e a p p l i c a t i o n o f t h e f i r s t l a w o f
thermodynamics.
A second r e s t r i c t i v e e q u a t i o n r e s u l t s from t h e b a l a n c e
e q u a t i o n f o r e n t r o p y . F o r a system i n s t a t i o n a r y s t a t e the b a l a n c e
equation f o r entropy r e s u l t s i n the f o l l o w i n g expression:
i n w h i c h Tig i s t h e t o t a l e n t r o p y p r o d u c t i o n i n t h e s y s t e m , Φ<,
i s the f l o w o f entropy t o the system.
The s e c o n d l a w o f t h e r m o d y n a m i c s p o s e s an i m p o r t a n t
r e s t r i c t i o n t o Tig » w h i c h must n e c e s s a r i l y e x c e e d z e r o f o r any
possible process:

Π δ > 0 (9)
Furthermore, t h e r m o d y n a m i c s show t h a t t h e e n t r o p y f l o w c a n
be w r i t t e n as :
<& = Φ„/Τ + Σ Φ.s.

Q (10)
1
Combining eqns. (8) - (10) the f o l l o w i n g c o n s t r a i n t i s seen

to d e r i v e from t h e second law o f thermodynamics :
Φ / Τ + ΣΦ.β. < 0
υ (11)
η . 1 1
1
Equation (11) shows t h a t t h e e n t r o p y f l o w t o w a r d s a s y s t e m

in a stationary state outsid
two c o n t r i b u t i o n s : on
environment, the other g exchang
s u b s t a n c e s . As c a n be s e e n eqn. ( 1 1 ) does n o t i m p l y t h a t h e a t
should necessarily be p r o d u c e d ( i . e . Φ < 0) i n any p r o c e s s
a l l o w e d by t h e s e c o n d l a w , a s t a t e m e n t , w h i c h c a n be shown (6)
t o be t h e r a t i o n a l e b e h i n d t h e M i n k e v i c h and E r o s h i n ( 7 )
e f f i c i e n c y , η.
Thermodynamics do n o t e x c l u d e p r o c e s s e s i n w h i c h h e a t i s
t a k e n up ( i n f a c t e n d o t h e r m i c p r o c e s s e s a r e w e l l known i n c h e m i c a l
e n g i n e e r i n g ) as l o n g as i t i s c o m p e n s a t e d b y a s u f f i c i e n t l y l a r g e
e n t r o p y f l o w a s s o c i a t e d w i t h t h e exchange o f c h e m i c a l s u b s t a n c e s .
The c o m b i n a t i o n o f e q n s . (11) and ( 7 ) a l l o w s t h e f o r m u l a t i o n
of an a l t e r n a t i v e e x p r e s s i o n o f t h e second law, i t i n t r o d u c e s
t h e p a r t i a l m o l a r f r e e e n t h a l p y , g., ( a l s o termed G i b b s f r e e
energy):
Σ Φ 8· > 0 ί (12)
i
The p a r t i a l m o l a r f r e e e n t h a l p y i s d e f i n e d by:
g = h T s ( 1 3 )
i i " i
E q u a t i o n (12) w i l l be shown t o a l l o w t h e f o r m u l a t i o n o f an
e f f i c i e n c y m e a s u r e , w h i c h c a n be used t o a n a l y s e g r o w t h and
p r o d u c t f o r m a t i o n i n m i c r o o r g a n i s m s , i t s d e v e l o p m e n t w i l l be
undertaken i n the next s e c t i o n .
The t h e r m o d y n a m i c efficiency.
The e n e r g y and e n t r o p y c o n t e n t o f c h e m i c a l s u b s t a n c e s .
The t h e r m o d y n a m i c t h e o r y o u t l i n e d above c a n , i n p r i n c i p l e , be
s t r a i g h t f o r w a r d l y a p p l i e d t o t h e d e s c r i p t i o n o f m i c r o b i a l growth
and p r o d u c t f o r m a t i o n . I n o r d e r t o p e r f o r m s u c h an a n a l y s i s ,
t h e r m o d y n a m i c d a t a a r e needed r e g a r d i n g t h e compounds w h i c h a r e
exchanged w i t h t h e environment, i . e . t h e p a r t i a l molar e n t h a l p i e s

and e n t r o p i e s o f t h e compounds i n v o l v e d i n t h e p r o c e s s e s i n t h e
s y s t e m need t o be known. I n p r i n c i p l e t h e p a r t i a l m o l a r
q u a n t i t i e s , e n t h a l p y a s w e l l as e n t r o p y , a r e f u n c t i o n s o f t h e
c o n c e n t r a t i o n s o f e a c h and e v e r y c h e m i c a l compound p r e s e n t , i . e .
d e f i n i t e v a l u e s c a n n o t be a t t r i b u t e d t o a s i n g l e compound. I n
the approximation t o the e n e r g e t i c s o f m i c r o b i a l growth presented
h e r e , i d e a l i t y o f t h e m i x t u r e o f compounds i n v o l v e d w i l l be
assumed. T h i s i m p l i e s t h e p a r t i a l m o l a r t h e r m o d y n a m i c q u a n t i t i e s
to be e q u a l t o t h e s p e c i f i c m o l a r q u a n t i t i e s , t h e s e l a t t e r
q u a n t i t i e s depend o n i n t e n s i v e v a r i a b l e s l i k e t e m p e r a t u r e and
p r e s s u r e and t h e c o n c e n t r a t i o n o f t h e compound c o n s i d e r e d o n l y .
To a good d e g r e e o f a p p r o x i m a t i o n , e n t h a l p y c a n , a t a g i v e n
t e m p e r a t u r e and p r e s s u r e , be assumed i n d e p e n d e n t o f t h e c o n c e n t r a
t i o n o f t h e compound u n d e r c o n s i d e r a t i o n . The f r e e e n t h a l p y i s ,
h o w e v e r , by v i r t u e o f t h e n t r o p c o n t r i b u t i o t that quantit
(eqn. 1 3 ) , d e f i n i t e l y c o n c e n t r a t i o
relationship holds:
g L = g? + R T l n C i (14)
i n w h i c h g? i s t h e f r e e e n t h a l p y a t a g i v e n t e m p e r a t u r e and
p r e s s u r e and u n i t c o n c e n t r a t i o n o f t h e compound, w h i c h i s c a l l e d
standard free enthalpy. C. i s t h e c o n c e n t r a t i o n o f compound i .
As a f i r s t a p p r o a c h s t a n d a r d q u a n t i t i e s , i . e . a s s u m i n g u n i t
9
c o n c e n t r a t i o n s , w i l l be u s e d i n t h e p r e s e n t e v a l u a t i o n o f g r o w t h
and product formation.
One f u r t h e r c o n v e n i e n t c o n v e n t i o n needs t o be d i s c u s s e d .
E n e r g y , and hence a l s o d e r i v e d q u a n t i t i e s l i k e e n t h a l p y and f r e e
e n t h a l p y , c a n n o t be a t t r i b u t e d a d e f i n i t e v a l u e ; i t s m a g n i t u d e
c a n o n l y be d e f i n e d w i t h r e s p e c t t o a g i v e n r e f e r e n c e s t a t e w h i c h
i s a t t r i b u t e d a zero energy l e v e l . A convenient r e f e r e n c e s t a t e
f o r t h e e v a l u a t i o n o f g r o w t h and p r o d u c t f o r m a t i o n i n m i c r o
o r g a n i s m s i s o b t a i n e d i f C 0 , H 0 , 0 and N a r e a s s i g n e d a z e r o
2 2 2 2
e n e r g y l e v e l . The e n e r g y o f a compound t h u s becomes e q u a l t o i t s

e n e r g y o f c o m b u s t i o n t o C 0 , H 0 and N . T h i s c o n v e n t i o n c a n be
2 2 2
m o t i v a t e d by t h e f a c t t h a t m i c r o o r g a n i s m s c a n u n d e r no c i r c u m
stances d e r i v e u s e f u l energy from processes i n which o n l y C 0 , 2
H 0 , 0 and N a r e i n v o l v e d .
2 2 2
The m o l a r f r e e e n t h a l p i e s and e n t h a l p i e s _ o f c o m b u s t i o n a t
0
s t a n d a r d c o n d i t i o n s w i l l be termed Ag°. and A h , r e s p e c t i v e l y . ^
C 1
From e q n . (7) i t i s c l e a r t h a t t h e e n f n a l p y o f c o m b u s t i o n , A h ° ^ ,
equals t h e heat o f combustion.
0
The f r e e e n t h a l p y o f c o m b u s t i o n , A g . , i s m a r k e d l y d e p e n d e n t
on t h e c o n c e n t r a t i o n s o f t h e r e a c t a n t s i n v o l v e d ( e q n . ( 1 4 ) ) and
most b i o l o g i c a l p r o c e s s e s t a k e p l a c e i n aquous s o l u t i o n s a t a
h y d r o g e n i o n c o n c e n t r a t i o n c o r r e s p o n d i n g t o a pH o f 7 r a t h e r t h a n
a t u n i t c o n c e n t r a t i o n o f t h e H - i o n , c o r r e s p o n d i n g t o a pH o f
z e r o . The f r e e e n t h a l p i e s o f c o m b u s t i o n t o l i q u i d w a t e r , t h e HC0 3
i o n (the p r e d o m i n a n t f o r m i n w h i c h C 0 e x i s t s a t a pH o f 7) and
2
N a t a pH o f 7 c a n t h u s be c o n s i d e r e d more r e l e v a n t t o b i o l o g i c a l
2

t r a n s f o r m a t i o n s . The f r e e e n t h a l p y o f c o m b u s t i o n u n d e r t h e s e
0
c o n d i t i o n s w i l l be i n d i c a t e d A g ! . The e n t h a l p y o f c o m b u s t i o n
i s h a r d l y a f f e c t e d by t h e pH. Tê f r e e e n t h a l p i e s and e n t h a l p i e s
o f c o m b u s t i o n a r e known t o obey r e g u l a r i t i e s ( 6 , 8 ) . T h e s e c a n
be t r e a t e d u s i n g t h e c o n c e p t o f t h e d e g r e e o f r e d u c t i o n a s i n t r o
d u c e d by M i n k e v i c h and E r o s h i n (_7) and e x t e n d e d and g e n e r a l i z e d
by t h e p r e s e n t a u t h o r ( 5 , 6 ) . The g e n e r a l i z e d d e g r e e o f r e d u c t i o n ,
γ., o f a compound w i t h r e s p e c t t o m o l e c u l a r n i t r o g e n i s d e f i n e d
by:
Ύ = 4 + a 2 b ( 1 5 )
ί i" i
I n w h i c h a. and b. a r e t h e number o f m o l e s o f Η and 0 p r e s e n t
i n one C-mole ( b e i n g t h e amount c o n t a i n i n g 12 grams o f c a r b o n )
o f coumpound i . F o r a component i t h e n t h a l p i e d fre
e n t h a l p i e s o f combustio
Ag°. r e s p e c t i v e l y , a r e approximation uniqu
f u n c t i o n o f t h e d e g r e e o f r e d u c t i o n , γ., as i n t r o d u c e d i n e q n .
( 1 5 ) . A s t a t i s t i c a l a n a l y s i s o f d a t a f o r some 60 o r g a n i c
compounds o f b i o l o g i c a l s i g n i f i c a n c e revealed the existence of
the f o l l o w i n g r e g u l a r i t i e s :
0
Ah . = 115γ. (16)
CL ' 1
Ag°. = 94.4γ. + 86.6 (17)
The r e s i d u a l e r r o r o f t h e e s t i m a t e i s 18 k J f o r b o t h e q n s . ( 1 6 )
and ( 1 7 ) . E q u a t i o n (16) s t a t e s t h a t t h e h e a t o f c o m b u s t i o n p e r
C-mole i s more o r l e s s d i r e c t l y p r o p o r t i o n a l t o t h e d e g r e e o f
r e d u c t i o n . As i s a p p a r e n t f r o m eqn. (17) s u c h a s i m p l e p r o p o r
t i o n a l i t y r e l a t i o n does n o t a p p l y t o f r e e e n t h a l p i e s o f
combustion.
E q u a t i o n s (16) and (17) show t h a t s y s t e m a t i c d e v i a t i o n s
b e t w e e n f r e e e n t h a l p i e s and h e a t s o f c o m b u s t i o n must e x i s t . F o r
s u b s t r a t e s o f a low degree o f r e d u c t i o n the f r e e e n t h a l p i e s o f
combustion exceed t h e heats o f combustion, f o r s u b s t r a t e s of a
h i g h d e g r e e o f r e d u c t i o n t h e r e v e r s e a p p l i e s . T h i s phenomenon
c a n be i l l u s t r a t e d i f t h e e n t r o p y c o n t r i b u t i o n t o t h e f r e e
0
enthalpy o f combustion, T A s . , i s c a l c u l a t e d . I t i s obtained from
the equation:
0 0
Ag°. = Ah . - TAs . (18)
6
C1 CI CI
I n f i g . 2 t h e r e l a t i o n i s shown b e t w e e n t h e s a i d e n t r o p y c o n t r i
b u t i o n and t h e d e g r e e o f r e d u c t i o n u s i n g d a t a f o r a v a r i e t y o f
o r g a n i c compounds. A d e f i n i t e t r e n d c a n i n d e e d be shown t o e x i s t
( a p a r t f r o m an i n c i d e n t a l o u t l y e r ) : The e n t r o p y c o n t r i b u t i o n
i n c r e a s e s w i t h i n c r e a s i n g degree o f r e d u c t i o n . I n v i e w o f the
o b s e r v a t i o n t h a t f r e e e n t h a l p y changes a t a pH o f 7 may w e l l be
more r e l e v a n t i n m i c r o b i o l o g i c a l p r o c e s s e s , t h e e n t r o p y c o n t r i b u -

501 ι ι ί
ο 2.5 5 7.5
Γ
Figure 2. The entropy contribution, T A s ° (kJ/C-mole), to the free enthalpy of

c
combustion at standard conditions, as a function of the degree of reduction, y, of

the compounds considered, for acids (%), carbohydrates (A), alkanes (Ο), ethene
and ethyne (O), alcohols (%), acetone (|Λ aldehydes (A), and amino acids (*).

0 f
t i o n t o t h e f r e e e n t h a l p y o f c o m b u s t i o n a t a pH o f 7, T A s , was
a l s o c a l c u l a t e d . T h e s e v a l u e s a r e p l o t t e d as a f u n c t i o n o?
t h e d e g r e e o f r e d u c t i o n i n f i g . 3. The f e a t u r e s o f f i g s . 2 and
3 a r e s e e n t o be v e r y much a l i k e , e x c e p t f o r t h e a c i d s , w h i c h
0 f
have a m a r k e d l y h i g h e r T A s a t a pH o f 7, i . e . t h e i r f r e e
e n t h a l p y o f c o m b u s t i o n i s îower a t t h a t pH. T h i s d i f f e r e n c e
b e t w e e n s t a n d a r d c o n d i t i o n s and a s i t u a t i o n i n w h i c h t h e c o n c e n
t r a t i o n s d i f f e r f r o m u n i t y , e.g. a t a pH o f 7, i s c h a r a c t e r i s t i c
f o r a l i m i t a t i o n o f t h e use o f s t a n d a r d f r e e e n t h a l p y changes
i n t h e a n a l y s i s o f p r o c e s s e s , where t h e c o n c e n t r a t i o n s a t t h e
l o c a l e o f t h e p r o c e s s may d i f f e r f r o m u n i t y : t h e Ag° v a l u e s c a n
o n l y be a p p l i e d t o an a p p r o x i m a t e a n a l y s i s . F o r d e t a i l e d
c o n s i d e r a t i o n s the A g v a l u e s at the c o n c e n t r a t i o n s at the l o c a l e
Q
o f e n e r g y g e n e r a t i o n a r e n e e d e d . S t i l l , as w i l l be shown, t h e
approximate analyses g r e a t l c o n t r i b u t t th understandin
of m i c r o b i a l energetics
T h e r e a l s o e x i s t compounds o f w h i c h t h e e n e r g y r e l e a s e d on
t h e i r c o m b u s t i o n does d e f i n i t e l y n o t f o l l o w t h e t r e n d s i n d i c a t e d
by e q n s . (16) and ( 1 7 ) . E x a m p l e s a r e o x y g e n and n i t r i c a c i d w h i c h
have a " h e a t o f c o m b u s t i o n " w h i c h i s c l o s e t o z e r o and a d e g r e e o f
r e d u c t i o n o f -4 and -5 r e s p e c t i v e l y . By v i r t u e o f t h i s f e a t u r e
t h e s e compounds can s e r v e as v e r y e f f i c i e n t " e l e c t r o n a c c e p t o r s " .
The t h e r m o d y n a m i c e f f i c i e n c y . The t h e r m o d y n a m i c t h e o r y
d e v e l o p e d e a r l i e r was shown t o r e s u l t i n eqn. ( 1 2 ) , a r e s t r i c t i v e
e q u a t i o n r e g a r d i n g the f l o w s o f m a t t e r exchanged w i t h the e n v i r o n
ment by an open s y s t e m . On eqn. (12) a d e f i n i t i o n o f t h e t h e r m o
d y n a m i c e f f i c i e n c y c a n be b a s e d i f t h e d i s s i p a t i o n , ΤΠ , i s
compared t o t h e t o t a l o f t h e f l o w s o f f r e e e n t h a l p y e n t e r i n g t h e
s y s t e m . However, a p r o b l e m w h i c h was a l r e a d y i n d i c a t e d above
has t o be t a c k l e d . The amount o f e n e r g y c a n n o t be s p e c i f i e d i n
a u n i q u e way and i t can o n l y be d e f i n e d w i t h r e f e r e n c e t o a b a s e
l e v e l , w h i c h i s a r b i t r a r i l y a t t r i b u t e d z e r o e n e r g y c o n t e n t . As
s o o n as s u c h a r e f e r e n c e s t a t e has been c h o s e n , t h e t h e r m o d y n a m i c
e f f i c i e n c y i s e a s i l y c a l c u l a t e d . The p r o c e d u r e i s i l l u s t r a t e d
i n f i g . 4. The t h e r m o d y n a m i c e f f i c i e n c y , fL,» i s d e f i n e d e q u a l
t o t h e r a t i o o f t h e f r e e e n t h a l p y g a i n e d i f t h e compounds l e a v i n g
t h e s y s t e m were t r a n s f o r m e d t o t h e r e f e r e n c e s t a t e , t o t h a t ,
w h i c h w o u l d be o b t a i n e d i f t h i s p r o c e d u r e were a p p l i e d t o t h e
compounds e n t e r i n g t h e s y s t e m . I t i s e a s i l y u n d e r s t o o d t h a t
η , i s c o n s t r a i n e d between z e r o , i f a l l t h e f r e e e n t h a l p y e n t e r i n g
trie s y s t e m i s d i s s i p a t e d and u n i t y i f Ag!j e q u a l s Ag^ , i . e . i f t h e
d i s s i p a t i o n equals zero. I t i s important to r e a l i z e t h a t the
f o r m e r c o n s t r a i n t s t r o n g l y depends on t h e c o r r e c t c h o i c e o f t h e
reference state.
A p p l i c a t i o n s of the theory
A e r o b i c g r o w t h w i t h o u t p r o d u c t f o r m a t i o n . The a p p l i c a t i o n
of the theory to a e r o b i c growth without f o r m a t i o n of products

k J/C-mole
100
50
Π
Ο
Δ*
* *
*
**
* *
-50
2.5 7.5
Υ
Figure 3. The entropy contribution, T A S C (kJ/C-mole), to the free enthalpy of

0 /
combustion at a pH of 7, as a function of the degree of reduction, y, of the com

pounds considered; symbols as in Figure 2.

w i l l now be shown. F i g u r e 5 shows t h e s y s t e m and t h e exchange

f l o w s f o r t h i s c a s e . The e l e m e n t a r y c o m p o s i t i o n s and t h e h e a t s
o f c o m b u s t i o n o f t h e compounds exchanged w i t h t h e e n v i r o n m e n t
a r e i n d i c a t e d . A e r o b i c g r o w t h i s f o r t h e p r e s e n t a n a l y s i s assumed
t o i n v o l v e u p t a k e o f a n i t r o g e n s o u r c e , a c a r b o n s o u r c e and o x y g e n
and t r a n s p o r t t o t h e e n v i r o n m e n t o f c a r b o n d i o x i d e , w a t e r and
new b i o m a s s . The b i o m a s s i s assumed t o be one compound, w h i c h
c a n be c h a r a c t e r i z e d f u l l y by i t s e l e m e n t a l c o m p o s i t i o n . As t h e
e n t h a l p y as w e l l as t h e f r e e e n t h a l p y o f c o m b u s t i o n , i . e . t h e
e n t h a l p y and f r e e e n t h a l p y c o n t e n t w i t h r e s p e c t t o t h e r e f e r e n c e
s t a t e adopted i n t h e p r e s e n t a n a l y s i s , i s zero f o r oxygen, carbon
d i o x i d e and w a t e r , i t i s e a s i l y u n d e r s t o o d t h a t t h e thermodynamic
e f f i c i e n c y c a n be d e f i n e d a s f o l l o w s :
Φ Ag°
n'th
. u - — ^ — ^ (19
cN
i n which Φ , and Φ^ a r e t h e f l o w s o f b i o m a s s ( C - m o l e / h r ) ,
s u b s t r a t e ^ C - m o l e / h r ) and n i t r o g e n s o u r c e ( m o l e / h r ) t o o r f r o m
t h e s y s t e m ( a s i n d i c a t e d i n f i g u r e 5 ) . Ag° and A g ° a r e t h e
g
f r e e e n t h a l p i e s _ o f c o m b u s t i o n o f a C-mole o i b i o m a s s and s u b s t r a t e
r e s p e c t i v e l y . A g ^ i s t h e f r e e e n t h a l p y o f combustion o f a mole
of t h e n i t r o g e n source.
E q u a t i o n (19) c a n a l s o be w r i t t e n a s :
Ag°
η,. — (20)
A o / r -
g s + ( /c )Ai»
C ) 4 N
In the formulation o f t h i s equation a balance f o r atomic n i t r o g e n

f f
i s u s e d t o r e l a t e t h e f l o w s Φ , and Φ . Y % i sa yield factor
• Ν χ sx ·
f o r b i o m a s s o n s u b s t r a t e o n a p e r C-mole b a s e ; C-moles o f b i o m a s s
p r o d u c e d p e r C-mole o f s u b s t r a t e consumed, t h u s i t i s d e f i n e d
by:
γ " = φ /φ (21)
SX X s
A n o t h e r u s e f u l e f f i c i e n c y measure i s t h e s o - c a l l e d e n t h a l p y
e f f i c i e n c y o f g r o w t h , η^, i t i s by a n a l o g y o b t a i n e d i f t h e Ag°.
0
i n eqn. (20) a r e r e p l a c e d by t h e r e s p e c t i v e A h . . As was alreaày
i n d i c a t e d t h e e n t h a l p y e f f i c i e n c y does n o t have t h e f u n d a m e n t a l
p r o p e r t i e s o f t h e thermodynamic e f f i c i e n c y a s t h e r e s t r i c t i o n
f o l l o w i n g from t h e a p p l i c a t i o n o f t h e second law o f thermodynamics
( s e e e q n . ( 1 1 ) ) d o e s n o t pose an u p p e r l i m i t t o Φ^. H e n c e , p r o c e s s e s
f o r w h i c h η„ e x c e e d s u n i t y c a n by no means be e x c l u d e d . I t c a n
e a s i l y b e shown t h a t t h e e f f i c i e n c y m e a s u r e s d e v e l o p e d a b o v e ,
i . e . η^, and η^, a l l o w t h e f o r m u l a t i o n o f e x p r e s s i o n s f o r t h e
d i s s i p a t i o n ΤΠ and t h e h e a t p r o d u c t i o n (-Φ„). The f o l l o w i n g
S n

FREE ENTHALPY
ENTERING SYSTEM
FREE ENTHALPY
LEAVING SYSTEM
FREE ENTHALPY OF REFERENCE STATE
Figure 4. The thermodynamic efficiency, ηα, of a process. A g / and A g / are the

amounts of free enthalpy gained when the compounds entering and leaving the
system, respectively, are transformed to the reference state.
_\h°-0
c
co 2
_H 0
2
cs
W W CHa b
i° i i Nc
Figure 5. System and flows for thermodynamic analysis of aerobic growth without
formation of products.

equations hold:
(22)
(23)
I n eqn. (22) D e q u a l s t h e d i s s i p a t i o n ΤΠ .
For a e r o b i c growth w i t h o u t product f o r m a t i o n the a b s o l u t e
m a g n i t u d e second t e r m a p p e a r i n g a t t h e l e f t hand s i d e o f eqn.
(11) c a n be shown t o be s m a l l as compared t o Φ^/Τ, i . e . t h e
exchange o f h e a t l a r g e l y e x c e e d s t h e exchange o f " c h e m i c a l
e n t r o p y " ( 5 , 6^. T h i s i m p l i e s t h a t D and a r e , t o a good a p p r o x
a n a
i m a t i o n e q u a l and t h i s , o f c o u r s e , a l s o a p p l i e s t o η * Π^·
I n t h i s c a s e an a n a l y s i s b a s e d on an e n t h a l p y e f f i c i e n c y o f
g r o w t h i s more o r l e s s v a l i
of t h e s e c o n d law as t
As has been shown e a r l i e r (6) t h e v e r y f a c t t h a t eqn. (16)
i s more o r l e s s v a l i d i m p l i e s t h a t h e a t p r o d u c t i o n and oxygen
consumption, Φ , are p r o p o r t i o n a l a c c o r d i n g to the f o l l o w i n g
equation :
Φ„ = 460 Φ (24)
ο
The a p p r o x i m a t e v a l i d i t y o f t h i s e q u a t i o n i s e a s i l y u n d e r s t o o d
as f o l l o w s . The d e g r e e o f r e d u c t i o n γ. i n d i c a t e s t h e number o f
moles o f e l e c t r o n s a v a i l a b l e f o r t r a n s f e r t o o x y g e n on c o m p l e t e
c o m b u s t i o n o f a C-mole o f a compound t o C0 , H 0 and N . On
2 2 2
t r a n s f e r t o oxygen t h e e n e r g y o f t h e e l e c t r o n s i s d i s s i p a t e d ,
r e s u l t i n g i n a h e a t p r o d u c t i o n o f 115 k J p e r mole o f e l e c t r o n s .
On a e r o b i c g r o w t h p a r t o f t h e e n e r g y c o n t e n t o f t h e s u b s t r a t e
and the n i t r o g e n source i s conserved i n the form o f newly
s y n t h e s i z e d biomass, the e l e c t r o n s c o r r e s p o n d i n g to the remainder
a r e t r a n s f e r r e d t o o x y g e n and p r o v i d e d i s s i p a t i o n . As f o u r moles
o f e l e c t r o n s a r e a c c e p t e d by one m o l e o f o x y g e n eqn. (16) shows
460 k J t o be g e n e r a t e d f o r e a c h m o l e o f oxygen consumed.
The v a l i d i t y o f eqn. (24) shows t h a t a t r e a t m e n t can a l s o
be b a s e d on o x y g e n e f f i c i e n c y o f g r o w t h C5"\7) ·
A s u b s t a n t i a l body o f d a t a on a e r o b i c g r o w t h w i t h o u t p r o d u c t
f o r m a t i o n s u p p o r t e d by ammonia as a n i t r o g e n s o u r c e has r e c e n t l y
been r e v i e w e d ( 9 ) . From t h e s e d a t a t h e v a l u e s o f η (- η ) and
t h e d i s s i p a t i o n p e r u n i t b i o m a s s p r o d u c e d (D/Φ , k J / C - m o l e ) were
c a l c u l a t e d . The r e s u l t s were a v e r a g e d f o r e a c h o f t h e c a r b o n
s o u r c e s c o n s i d e r e d and a r e shown i n f i g s . 6 and 7.
A l t h o u g h , not u n e x p e c t e d l y , i t i s c l e a r t h a t c o n s i d e r a b l e
s c a t t e r i s e x i s t e n t i n b o t h f i g s . 6 and 7,some g l o b a l r e g u l a r i t i e s
seem t o be p r e s e n t , w h i c h a r e i n d i c a t e d i n t h e f i g u r e s . F o r s u b
s t r a t e s w i t h a d e g r e e o f r e d u c t i o n l o w e r t h a n about 5, t h e thermo
dynamic e f f i c i e n c y a v e r a g e s 0.58 ( t h e o n l y s i g n i f i c a n t o u t l y e r
b e i n g t h e v e r y low e f f i c i e n c y o b s e r v e d f o r g r o w t h s u p p o r t e d by

0.25
Figure 6. Thermodynamic efficiency, η , of aerobic growth with NH

ιη S as the
nitrogen source, plotted as a function of the degree of reduction, γ, of the substrate.
Theoretical limits due to the second law and C-limitation. Shown are averages of
experimental data for methane (%), n-alkanes (A), methanol (J^), ethanol (*ψ),
glycerol (®), mannitol (O), acetic acid (Δλ lactic acid glucose (*), formalde
hyde (VA gluconic acid (S), succinic acid (@), citric acid (®), malic acid (®),
formic acid (®), oxalic acid (A)-

A Î 1270 1240 I·
Figure 7. Dissipation of aerobic growth (kJ/C-mole of biomass produced) for

aerobic growth with ΝΗ Άas a nitrogen source as a function of the degree of
reduction of substrate. Theoretical limits due to second law and C-limitation.
Average of experimental data for various substrates, symbols as in Figure 6.

o x a l i c a c i d ) , t h i s corresponds to a d i s s i p a t i o n of roughly
400 k J / m o l e o f b i o m a s s p r o d u c e d . F o r s u b s t r a t e s w i t h a d e g r e e
o f r e d u c t i o n e x c e e d i n g 5 t h e thermodynamic e f f i c i e n c y
p r o g r e s s i v e l y d e c r e a s e s and t h e d i s s i p a t i o n i n c r e a s e s up t o 1400
kJ/C-mole f o r t h e c a s e o f g r o w t h s u p p o r t e d by methane.
I n f i g s . 6 and 7 t h e r e s t r i c t i o n s o f u n i t e f f i c i e n c y and
z e r o d i s s i p a t i o n r e s p e c t i v e l y , a s imposed by t h e s e c o n d law a r e
i n d i c a t e d as w e l l as a l i m i t imposed by a l i m i t a t i o n o f a
d i f f e r e n t n a t u r e , i . e . c a r b o n l i m i t a t i o n . The l a t t e r l i m i t m e r i t s
a more t h o r o u g h d i s c u s s i o n . The d e g r e e o f r e d u c t i o n , γ , o f b i o
mass f r o m a v a r i e t y o f s o u r c e s a v e r a g e s 4.8 (5,6,10) and hence
by v i r t u e o f eqn. (16) i t s e n e r g y c o n t e n t i s a b o u t 550 k J / C - m o l e .
I f g r o w t h i s s u p p o r t e d by a s u b s t r a t e o f a d e g r e e o f r e d u c t i o n
e x c e e d i n g 4.8 i t s e n e r g y c o n t e n t w i l l e x c e e d t h e s a i d 550
k J / C - m o l e and h e n c e , eve i f a l lsubstrat carbo fixed
i n b i o m a s s , a thermodynami
be o b t a i n e d . A c o m p l e t e x p l o i t a t i o energy p r e s e n
t h e s u b s t r a t e w o u l d r e q u i r e f i x a t i o n o f a d d i t i o n a l low e n e r g y
c a r b o n e.g. f r o m C0 . T h i s i s , however, e x c l u d e d , as o n l y one
2
c a r b o n s o u r c e i s assumed t o be s u p p l i e d .
On o b s e r v a t i o n o f f i g . 6 i t becomes c l e a r t h a t t h e t r e n d s
o b s e r v e d i n t h e e x p e r i m e n t a l v a l u e s o f t h e thermodynamic
e f f i c i e n c y mimic t h e shape o f t h e t h e o r e t i c a l r e s t r i c t i o n s a t
a l e v e l o f a b o u t 60%. The d e v i a t i o n between t h e t h e o r e t i c a l l i m i t
and t h e v a l u e s a c t u a l l y o b s e r v e d i s q u i t e e a s i l y u n d e r s t o o d i n
g e n e r a l terms i n t h e r e g i o n o f low d e g r e e s o f r e d u c t i o n , where
the energy a v a i l a b l e i n the s u b s t r a t e r a t h e r than i t s carbon
M
c o n t e n t l i m i t s the v a l u e s o f Y . The t h e o r e t i c a l l i m i t o f u n i t y
sχ ·
c a n n e v e r be r e a c h e d as any p r o c e s s needs a n o n - z e r o d i s s i p a t i o n
t o proceed at a non-zero r a t e (2-4). In f a c t the r a t e at which
a p r o c e s s p r o c e e d s , e.g. t h e r a t e o f g r o w t h o f t h e amount o f
biomass, i s to a c e r t a i n extent i n c r e a s i n g w i t h i n c r e a s i n g
d i s s i p a t i o n . V a r i o u s o p t i m a l i t y p r i n c i p l e s ( 1 1 ) , may d i c t a t e
an o p t i m a l thermodynamic e f f i c i e n c y o f r o u g h l y t h e m a g n i t u d e
o b s e r v e d h e r e ( 1 1 , 1 2 ) . The b e h a v i o u r a t h i g h d e g r e e s o f r e d u c t i o n
i s l e s s e a s i l y u n d e r s t o o d on f u n d a m e n t a l g r o u n d s . The t h e r m o
dynamic e f f i c i e n c y c o u l d w e l l a p p r o a c h t h e t h e o r e t i c a l l i m i t c l o s e r
with a s u f f i c i e n t l y l a r g e d i s s i p a t i o n . A p p a r e n t l y o t h e r phenomena
t o w h i c h t h e f o r m a l m a c r o s c o p i c t r e a t m e n t p r o v i d e s no c l u e s ,
l i m i t t h e maximum c o n s e r v a t i o n o f s u b s t r a t e c a r b o n i n b i o m a s s
t o a b o u t 2/3 o f t h e maximum. O b v i o u s l y one has t o r e s o r t t o b i o
c h e m i c a l t h e o r y t o o b t a i n c l u e s t o the n a t u r e o f t h e mechanisms
u n d e r l y i n g t h e phenomenon.
T h e r e e x i s t s s t i l l a n o t h e r u s e f u l q u a n t i t y , w h i c h has been
used t o s y s t e m a t i z e t h e e x p e r i m e n t a l d a t a on t h e e f f i c i e n c y
1
o f g r o w t h s u p p o r t e d by v a r i o u s c a r b o n s o u r c e s ; i t i s P a y n e s
y i e l d on a v a i l a b l e e l e c t r o n s , Υ , ( 1 3 ) . I t i s d e f i n e d as t h e
amount o f b i o m a s s p r o d u c e d p e r mole o f e l e c t r o n s a v a i l a b l e f o r
t r a n s f e r t o o x y g e n on c o m p l e t e c o m b u s t i o n . N u m e r i c a l l y t h e number
o f m o l e s o f e l e c t r o n s a v a i l a b l e f o r t r a n s f e r t o o x y g e n on

c o m b u s t i o n o f a C-mole o f a g i v e n s u b s t r a t e i s e q u a l t o t h e
d e g r e e o f r e d u c t i o n , γ., as d e f i n e d by eqn. ( 1 5 ) . As a f i r s t
a p p r o x i m a t i o n , t a k i n g i n t o account the f a c t t h a t biomass from
a«-·• vVW
a.
rAi.^
eWtWy^ νo fΛ. I
mUX
i.V
c.rLVUo b iJLaj.
a l sIo7uWUr1c.eU^t7
s can
COU beVJ t.w eW lI— lJ. J- r e p r e s e n t e d by t h e
composition formula C H O . 5 N 0 . 2 » l e 8 0
Y
/ C c a n be c o n s i d e r e d
p r o p o r t i o n a l t o X]^ ( o r r a t h e r n ) (§, 6 ) H
e
Υ , = 5.85 η., (25)

av/e th
F i g u r e 8 shows t h e d a t a d e p i c t e d i n f i g s . 6 and 7 i n a p l o t o f
Y . v e r s u s t h e d e g r e e o f r e d u c t i o n γ. The t r e n d s and t h e o r e t i c a l
l i m i t s a r e a l s o shown.
Growth w i t h e l e c t r o
preceeding s e c t i o n a cas
e l e c t r o n a c c e p t i n g m o i e t y . Oxygen i s , h o w e v e r , by no means t h e
o n l y compound, w h i c h c a n p e r f o r m s u c h f u n c t i o n . O t h e r examples
a r e n i t r a t e and s u l p h a t e . The c h a r a c t e r i s t i c f e a t u r e o f an
e l e c t r o n a c c e p t o r i s t h a t f r e e e n t h a l p y i s gained i n the o v e r a l l
p r o c e s s o f t r a n s f e r o f an e l e c t r o n f r o m a g i v e n s o u r c e (a
s u b s t r a t e ) to the acceptor. T h i s f r e e enthalpy i s p a r t l y
d i s s i p a t e d t o p r o v i d e t h e n e c e s s a r y i r r e v e r s i b i l i t y and i t c a n
be u s e d t o t r a n s f e r c a r b o n ( - d i o x i d e ) f r o m a low e n e r g y l e v e l
t o a h i g h e n e r g y one. I n t a b l e I a summary i s p r o v i d e d o f t h e
e n e r g y b e c o m i n g a v a i l a b l e on t r a n s f e r o f one m o l e o f e l e c t r o n s
f r o m g l u c o s e t o a g i v e n e l e c t r o n a c c e p t o r engaged i n a g i v e n
reduction process.
Table I. F r e e e n t h a l p y g a i n e d a t pH = 7 and s t a n d a r d
c o n d i t i o n s on t r a n s f e r o f one m o l e o f e l e c t r o n s
f r o m g l u c o s e t o a g i v e n e l e c t r o n a c c e p t o r (14)
0 1
e l e c t r o n acceptor moles of e l e c t r o n s Ag ^ ^ ^Sai/^
+ * +
accepted kJ kjfmole
N 2 (NH ) 4 6 79.1 13.2
S (HS~) 2 27.7 13.9
2-
SO, (HS ) 8 150.7 18.8
4 9
S 0
(HS~) 6 171.9 28.7
3
N0 ~ 2 (NH ) 4
+
6 435.5 72.6
N0 " 3 (NH ) 4
+
8 598.4 74.8
N0 " 3 (N )
2
5 559.5 111.9
(H 0)
2
4 473.9 118.5
°2
* I n b r a c k e t s reduced form of e l e c t r o n a c c e p t o r

ROELS Macroscopic Thermodynamics and Growth 313
'av/e
g DM/mole
6h SECOND
4H
2h
1h
Figure 8. Yield on electrons available for transfer to oxygen, Yav/e, (g DM/mole

av/e) for aerobic growth with NH as a nitrogen source. Theoretical limits due to
3
the second law and C-limitation. Average of experimental data for various sub-
strates, symbols as in Figure 6.

The v a l u e s o f t h e f r e e e n t h a l p y g a i n e d p e r m o l e o f e l e c t r o n s
t r a n s f e r r e d a r e a r r a n g e d i n o r d e r o f i n c r e a s i n g m a g n i t u d e . The
" e f f i c i e n c y " o f an e l e c t r o n a c c e p t o r t h u s i n c r e a s e s f r o m t h e t o p
to t h e b o t t o m o f t h e t a b l e and f a i t h i n l i f e ' s u n i v e r s a l s t r i v i n g
f o r more o p t i m a l g r o w t h w o u l d assume p r e f e r e n c e f o r t h e e l e c t r o n
a c c e p t o r s a t t h e b o t t o m o f t h e t a b l e i f two o r more e l e c t r o n
acceptors are simultaneously a v a i l a b l e .
Growth w i t h o u t e x t e r n a l l y s u p p l i e d e l e c t r o n a c c e p t o r s . I n
a number o f c a s e s g r o w t h i s o b s e r v e d t o t a k e p l a c e w i t h o u t an
i d e n t i f i a b l e seperate e l e c t r o n a c c e p t o r b e i n g p r e s e n t . I n such
case t h e s u b s t r a t e o r a s u b s t r a t e d e r i v e d moiety i s both donor
and a c c e p t o r o f e l e c t r o n s . F o r s i m p l i c i t y ' s s a k e o n l y t h e c a s e
o f a n a e r o b i c g r o w t h on a s i n g l e c a r b o n s o u r c e w i t h f o r m a t i o n o f
a s i n g l e p r o d u c t and NH^ as t h e n i t r o g e n s o u r c e w i l l be t r e a t e d
I t i s e a s i l y understoo
be b a s e d o n a b a l a n c e o
eqn. ( 1 6 ) . T a b l e I I shows t h e f l o w s o f t h e v a r i o u s compounds and
t h e i r c o n t e n t o f e l e c t r o n s a v a i l a b l e f o r t r a n s f e r t o oxygen on
f o r m a t i o n o f Φ C-moles of b i o m a s s .
χ
Table I I . A degree o f r e d u c t i o n balance f o r anaerobic
growth w i t h o u t e x t e r n a l e l e c t r o n a c c e p t o r s
molecular degree of flow contribution

species reduction to degree o f
reduction
balance
substrate Y
Φ /Υ"
χ
γ Φ /Y"
s X
SX 's X SX
N-source (NH^) 3 C φ 3ο.Φ
1 χ 1 χ
B i o m a s s (CH ,0, N -) 4 Φ - γ Φ
a1 b1 c1 Y
x χ χ
χ
Product φ - γ φ
Y
P Ρ Ρ Ρ
co 2
0 Φ /Υ" --Φ -φ 0
χ sx χ ρ
H 02 0 Φ 0
W
By v i r t u e o f t h e f a c t t h a t no e x t e r n a l e l e c t r o n a c c e p t o r s
are present the c o n t r i b u t i o n s t o the degree o f r e d u c t i o n balance
as shown i n t h e l a s t c o l u m n o f t a b l e 2 must add up t o z e r o , i t
follows :
Φ (26)
<w v SX ( Y
+ 3 C
I
Φ = Φ {(1 γ /γ ) / Υ " - 1 3c,/γ • γ /γ } (27)
c χ ρ χ ρ
s 'ρ SX

I t becomes c l e a r t h a t on t h e a n a e r o b i c g r o w t h p r o c e s s an
amount o f p r o d u c t Φ , g i v e n by eqn. ( 2 6 ) , i s formed. I t
c o r r e s p o n d s t o t h e E r a n s f e r o f (γ /Y" + 3c, - γ )Φ moles o f
s sχ . 1 χ χ
e l e c t r o n s from the s u b s t r a t e to r e s u l t i n g i n the f o r m a t i o n
of the p r o d u c t . C l e a r l y , the d r i v i n g f o r c e of the r e a c t i o n , w h i c h
m u s t , amongst o t h e r s , p r o v i d e t h e n e c e s s a r y d i s s i p a t i o n , must
r e s t i n th e f a c t t h a t t h e e l e c t r o n s i n t h e s u b s t r a t e a r e a t a
h i g h e r f r e e e n t h a l p y l e v e l t h a n t h o s e i n t h e p r o d u c t . The
mechanism o f t h i s p r o c e s s becomes c l e a r e r i f eqns. (16) and (17)
a r e o b s e r v e d c l o s e r . The e n t h a l p y o f c o m b u s t i o n i s , f o r compounds
o f d i f f e r e n t d e g r e e o f r e d u c t i o n ^ a p p a r e n t l y t o a v e r y good d e g r e e
o f a p p r o x i m a t i o n e q u a l p e r mole o f e l e c t r o n s a v a i l a b l e f o r
t r a n s f e r t o o x y g e n . H e n c e , i f e n t h a l p y w e r e , as i n t h e c a s e o f
a e r o b i c g r o w t h , t o be t h e s o u r c e p r o v i d i n g dissipât i o n , a n a e r o b i c
growth would to a degre approximatio involv negligibl
d i s s i p a t i o n and be i m p o s s i b l
view. E q u a t i o n ( 1 7 ) , however, enthalpy
o f a s u b s t r a t e p e r mole o f a v a i l a b l e e l e c t r o n s d e c r e a s e s , as a
g e n e r a l t e n d e n c y , w i t h i n c r e a s i n g d e g r e e o f r e d u c t i o n . Hence,
t h e d i s s i p a t i o n n e c e s s a r y f o r e f f i c i e n t g r o w t h c a n be g a i n e d i f
a product of a h i g h e r degree of r e d u c t i o n i s , w i t h f o r m a t i o n of
CO^, formed f r o m a s u b s t r a t e w i t h a low d e g r e e o f r e d u c t i o n 0 6 ) .
T h i s c a n be shown t o be t h e g e n e r a l t e n d e n c y u n d e r l y i n g a n a e r o b i c
g r o w t h o f t h e t y p e t r e a t e d h e r e . The g e n e r a l t e n d e n c y u n d e r l y i n g
a n a e r o b i c growth i s t h a t , c o n t r a r y t o the s i t u a t i o n i n a e r o b i c
g r o w t h , t h a t i s d r i v e n by t h e e n t h a l p y r a t h e r t h a n the " c h e m i c a l
entropy" c o n t r i b u t i o n to the d i s s i p a t i o n , i t i n v o l v e s a l a r g e
c o n t r i b u t i o n of "chemical entropy" d i s s i p a t i o n at a g e n e r a l l y
m i n o r c o n t r i b u t i o n due t o h e a t p r o d u c t i o n . F o r p r a c t i c a l p u r p o s e s ,
t h i s means t h a t on t h e s e a n a e r o b i c p r o c e s s e s c l o s e t o 100% o f
the energy of combustion o f the s u b s t r a t e s u p p l i e d i s conserved
i n t h e p r o d u c t and b i o m a s s f o r m e d , t h e i r r e v e r s i b i l i t y b e i n g
0
p r o v i d e d by t h e c o n t r i b u t i o n , T A s .
The t e n d e n c y s k e t c h e d above o n l y a c c o u n t s f o r t h e m a i n
f e a t u r e s , another important aspect b e i n g t h a t anaerobic growth
p r o c e e d s t o compounds, w h i c h have a low e n t h a l p y a t a g i v e n d e g r e e
o f r e d u c t i o n , i . e . a n a e r o b i c g r o w t h i s a l s o d r i v e n by t h e
d e v i a t i o n s from e q n s . (16) and (17) r a t h e r t h a n by t h e g e n e r a l
tendencies these equations d e s c r i b e .
The r e a s o n t h a t t h e l i m i t e d a c c u r a c y o f eqns. (16) and (17)
becomes i m p o r t a n t h e r e , w h i l s t b e i n g o f v i r t u a l l y no i m p o r t a n c e
i n a e r o b i c growth, r e s t s i n the f a c t t h a t l a r g e f l o w s of s u b s t r a t e
and p r o d u c t a r e i n v o l v e d i n a n a e r o b i c g r o w t h . T h u s , d i s s i p a t i o n
becomes t h e d i f f e r e n c e o f two l a r g e numbers and m i n o r e r r o r s i n
t h e s e c o n t r i b u t i o n s become q u i t e s i g n i f i c a n t .
As a t y p i c a l example o f t h e f e a t u r e s e x h i b i t e d by a n a e r o b i c
g r o w t h the f o l l o w i n g q u a n t i t i e s a r e w o r t h w h i l e . On a n a e r o b i c
g r o w t h o f a y e a s t on g l u c o s e w i t h f o r m a t i o n o f e t h a n o l t y p i c a l l y
a b o u t 90 k J o f h e a t a r e g e n e r a t e d p e r C-mole o f b i o m a s s p r o d u c e d ;
t h i s v a l u e i s s u b s t a n t i a l l y l o w e r t h a n t h e h e a t p r o d u c t i o n on

a e r o b i c g r o w t h o f t h a t same y e a s t , w h i c h t y p i c a l l y amounts t o
300 - 350 k J / C - m o l e . The f r e e e n t h a l p y d i s s i p a t i o n on a n a e r o b i c
g r o w t h i s , h o w e v e r , c l o s e t o 300 k J / C - m o l e p r o d u c e d , i . e . s l i g h t l y
l o w e r t h a n o r e q u a l t o t h a t on a e r o b i c g r o w t h .
An e a r l i e r s t u d y p e r f o r m e d by t h e p r e s e n t a u t h o r (5_, 6^)
showed t h a t , i f a r e a l i s t i c r e f e r e n c e s t a t e f o r e n e r g y c o n t e n t
i s c h o s e n , a n a e r o b i c g r o w t h p r o c e e d s as a g e n e r a l t e n d e n c y w i t h
a t h e r m o d y n a m i c e f f i c i e n c y o f t h e same o r d e r o f m a g n i t u d e as t h a t
o b s e r v e d on a e r o b i c g r o w t h . T h i s p r o v i d e s t h e n u c l e u s t o a
p o s t u l a t e t h a t w i l l be somewhat more c l o s e l y i n v e s t i g a t e d i n t h e
l a s t s e c t i o n o f t h i s p a p e r : Growth y i e l d s can as a r u l e o f thumb
be e s t i m a t e d f r o m t h e a s s u m p t i o n o f a c o n s t a n t t h e r m o d y n a m i c
e f f i c i e n c y o r , e q u i v a l e n t l y , a constant d i s s i p a t i o n per u n i t
biomass produced.
The constant e f f i c i e n c
F o r g r o w t h s u p p o r t e d by d i f f e r e n t s o u r c e s and s i n k s o f
e l e c t r o n s , e.g. a e r o b i c g r o w t h , g r o w t h s u p p o r t e d by e l e c t r o n
a c c e p t o r s o t h e r t h a n o x y g e n o r g r o w t h p r o c e s s e s i n w h i c h no
e x t e r n a l e l e c t r o n a c c e p t o r s are p r e s e n t , w i d e l y v a r y i n g s u b s t r a t e
y i e l d s , e.g. e x p r e s s e d as Υ" , a r e o b s e r v e d . As was a l r e a d y
i n d i c a t e d w i t h r e f e r e n c e t o a e r o b i c g r o w t h , m a t t e r s can be
g e n e r a l i z e d to a c e r t a i n extent i f y i e l d s are expressed i n
terms o f Y / > t h e y i e l d e x p r e s s e d p e r mole o f s u b s t r a t e
a v e
e l e c t r o n s a v a i l a b l e f o r t r a n s f e r t o o x y g e n . However, as was
p o i n t e d o u t i n d i s c u s s i n g t a b l e I , l a r g e l y d i f f e r e n t amounts
o f e n e r g y may r e s u l t from t h e t r a n s f e r o f one mole o f e l e c t r o n s
i n dependence on t h e d o n o r / a c c e p t o r c o m b i n a t i o n . T a b l e I I I
p r o v i d e s a summary o f l i t e r a t u r e d a t a . F o r a number o f g r o w t h
p r o c e s s e s t h e y i e l d on a v a i l a b l e e l e c t r o n s , Y , , as w e l l
ο
1
av / e
as t h e Ag , , t h e f r e e e n t h a l p y o b t a i n e d on t r a n s f e r o f one
m o l e o f eîecfrons f r o m t h e s u b s t r a t e t o t h e e l e c t r o n a c c e p t o r
( s t a n d a r d c o n d i t i o n s and a pH = 7 ) , a r e g i v e n .
Even a s u p e r f i c i a l s t u d y o f t a b l e I I I c l e a r l y shows t h e
e x i s t e n c e o f a r e l a t i o n s h i p between Y . and A g ° ,. T h i s
1
r e l a t i o n s h i p can be s y s t e m a t i z e d as f o l l o w s . I f t K e t h e r m o d y n a m i c
e f f i c i e n c y , η , , i s known eqn. (22) a l l o w s t h e c a l c u l a t i o n
of the d i s s i p a t i o n per u n i t biomass produced. Furthermore i t
has t o be r e c o g n i z e d t h a t t h e d i s s i p a t i o n p e r mole o f b i o m a s s
p r o d u c e d can be c a l c u l a t e d f r o m :
0
D/Φ = A g ' , (25.8/Y - 4.8) + 65.8 (28)
χ °av/e av/e
I n w h i c h t h e c o n v e r s i o n f a c t o r 25.8 a c c o u n t s f o r t h e
c o n v e r s i o n o f g.DM. t o C-moles, 4.8 i s a c o r r e c t i o n a c c o u n t i n g
f o r t h e number o f m o l e s o f a v a i l a b l e e l e c t r o n s c o n s e r v e d i n
one C-mole o f b i o m a s s f o r m e d , and 65.3 a c c o u n t s f o r t h e f r e e
e n t h a l p y o f c o m b u s t i o n o f t h e ammonia u s e d i n the p r o c e s s o f

Table I I I . Y i e l d s on a v a i l a b l e e l e c t r o n s and e n e r g y
a v a i l a b l e p e r mole o f e l e c t r o n s t r a n s f e r r e d
for various processes
substrate electron acceptor Y . Ag° , S o u r c e

av/e °av/e
g.D.M./
mole
Average
Glycerol 3. 65 1 16.4
°2 from(9)
Mannitoi 3. 26 117.5
°2
Acetic acid 2. 63 105.5
°2
Lactic acid 3. 26 110.1
°2
Glucose
°2
Formaldehyde 2. 98 124.0
°2
Gluconic acid 3. 53 117.9
°2
Succinic acid 2. 90 107.1
°2
Citric acid 3. 09 1 11 .0
°2
Malic acid 3. 18 112.1
°2
Formic acid 3. 17 118.0
°2
Oxalic acid 1. 78 119.0
°2 _
Succinic a c i d N0 3 2. 28 100.6 (15)
Succinic a c i d N0 ~ 2 2. 71 120.9 (15)
Succinic acid 2. 75 107.1 (15)
°2
Gluconic acid 3. 26 117.9 (15)
°2
Gluconic a c i d N0 3 3. 44 111.4 (15)
Glucose G1 uco s e->e t hano 1 0. 88 9.5 from (6)
Glucose Glucose+lactic acid 0. 81-1 .5 8.3
Glucose Glucose+propionic/acetic a c i d 1 .48 12.9
Glucose Glucose-hue t h a n e / a c e t i c acid 1. 63 14.4
Acetic acid Acetic acid-miethane 0. 36 3.9
Methanol Me t hano l->me t hane 0. 91 1 3.0
Formate Formate-*me t h a n e 0. 27 16.4
Propionic P r o p i o n i c acid->methane 0. 41 4.0
acid

g r o w t h . The b i o m a s s was assumed t o have t h e s t a n d a r d composition

C H N a n c t 0 c o n t a n
1
8°0 5 0 2 * ^ ^% a s h .
I r e q n s . (28) and (22) a r e combined t h e f o l l o w i n g expression
can be d e r i v e d :
0 t
25.8Ag , &
av/e
Y
a v / e ( 2 9 )
536(1 - n t h )/n t h + 4.8Ag- / e - 65.8
From eqn. (29) t h e s o l i d l i n e d e p i c t e d i n f i g . 9, w h i c h

c o r r e s p o n d s t o an η ^ o f 0.65, was c a l c u l a t e d . P a r t o f t h e e x p e r i
mental data depicteS i n t a b l e I I I are a l s o i n d i c a t e d i n the
f i g u r e . As c a n be s e e n t h e g e n e r a l f e a t u r e s o f t h e e x p e r i m e n t a l
m a t e r i a l f o l l o w the r e g u l a r i t y of a constant e f f i c i e n c y of
a b o u t 0.65. Of c o u r s e , some d e v i a t i o n s and o u t l y e r s a r e p r e s e n t .
T h i s may be due t o l i m i t a t i o n
as w e l l as o f t h e a p p r o x i m a t
as t h e f r e e e n t h a l p y changes a r e b a s e d on s t a n d a r d c o n d i t i o n s
f o r compounds a p a r t f r o m t h e H i o n and w a t e r .
As a r e p r e s e n t a t i v e example t h e f o l l o w i n g a n a l y s i s may
p r o v e i l l u s t r a t i v e . A e r o b i c g r o w t h o f the y e a s t S.cerevisiae
proceeds w i t h a y i e l d , Υ , , o f about 3.75 (g DM/mole a v / e ) .
a
E q u a t i o n (29) a l l o w s r\^ ¥br t h i s c a s e t o be e s t i m a t e d a t 0.63.
0 t
(Ag , e q u a l s 1 1 8 . 5 ) . F o r a n a e r o b i c g r o w t h on g l u c o s e w i t h
0
f o r m a t i o n of e t h a n o l A g ' , e q u a l s 9.5 and i f η , w o u l d r e m a i n
c o n s t a n t a t 0.63 a Y * o f 0.83 w o u l d be e x p r e c t e d . T h i s
e
c o r r e s p o n d s w e l l t o t K e e x p e r i m e n t a 1 v a l u e o f 0.88.
The a n a l y s i s p r e s e n t e d above may i n some c a s e s p r o v i d e a
u s e f u l a l t e r n a t i v e to the treatment o f the e f f i c i e n c y of growth
jnax
i n terms o f a Y «m-n o r γ value (16, 1 7 ) , w h i c h i n v o l v e s t h e
A1 κ AT Ρ ' _ '
n e c e s s i t y of knowledge of the d e t a i l e d b i o c h e m i s t r y of the
pathways used i n t h e g e n e r a t i o n o f m e t a b o l i c e n e r g y . The t r e a t m e n t
d e v e l o p e d above does n o t need s u c h i n f o r m a t i o n as i t i s b a s e d
on an a n a l y s i s o f t h e e n e r g y f l o w s t o and f r o m a s y s t e m , w i t h o u t
c o n s i d e r a t i o n o f t h e d e t a i l e d m e t a b o l i c mechanism c a u s i n g c o u p l i n g
o f e n e r g y g e n e r a t i o n t o b i o m a s s f o r m a t i o n and d i s s i p a t i o n
processes i n f u n c t i o n i n g l i v i n g organisms.
Of c o u r s e e x c e p t i o n s t o t h e g e n e r a l r u l e s d e v e l o p e d above
e x i s t as i s a l r e a d y a p p a r e n t f r o m t h e f a c t t h a t t h e low
e f f i c i e n c i e s o b s e r v e d on a e r o b i c g r o w t h on h i g h l y r e d u c e d
compounds, where f a c t o r s o t h e r t h a n a v a i l a b l e e n e r g y are
l i m i t i n g , were, f o r obvious r e a s o n s , not i n c l u d e d i n the a n a l y s i s
p e r f o r m e d i n the p r e s e n t s e c t i o n .

4h
av/e
g D.MV
/note av/e
r/ th = 0 65
10 50 100
9 [kJ molej
^ av e
Figure 9. Relationship between yield on available electrons, Y / (g DM/mole)

av C
and the energy gained per mole electrons transferred to the electron acceptor,
A g a , / r ' (kJ/mole).
0
Expected relation if the thermodynamic efficiency were
constant at 0.65 (—). Experimental data are shown for aerobic growth on glucose
( ), succinate (®), and gluconate (®). Data are shown for growth with Ν Of
as an electron acceptor on succinate ( @ ), and on gluconate ( @ ). A naerobic growth
on glucose is shown with formation of ethanol (*), lactate (®), propionic/acetic
acids ( Δ J, and methane/acetic acid (O). Anaerobic growth is shown with formation
of methane from acetic acid (\7), formic acid (V), propionic acid (A), and
methanol (•,).


As°. p a r t i a l e n t r o p y o f c o m b u s t i o n o f a C-mole o f ( k J / C - m o l e K)
compound i a t s t a n d a r d c o n d i t i o n s
As°. p a r t i a l e n t r o p y o f c o m b u s t i o n o f a C-mole o f ( k J / C - m o l e K)
compound i a t a pH = 7
Τ absolute temperature Κ
3
ι
V vo 1 urne m
Y
sx yi^d f° r
b i o m a s s on s u b s t r a t e (Omole/C-mole)
1
Y , J
y i e l d on t h e s u b s t r a t e s available electrons (g.D.M./mole)
&
av/e
d e g r e e o f r e d u c t i o n o f compound i (-)
f
η M i n k e v i c h and E r o s h i n s e f f i c i e n c y (-)
η enthalpy e f f i c i e n c
Η
η ^ thermodynamic e f f i c i e n c y (-)
TTg entropy production r a t e (kJ/Ks)
r a t e o f f l o w o f compound i t o t h e s y s t e m (mole/s)
Φ_ r a t e o f f l o w o f energy to the system (kJ/s)
heat f l o w t o the system (kJ/s)
Subscripts :
c carbon d i o x i d e
Ν nitrogen source
ο oxygen
ρ product
s substrate
χ biomass
Literature Cited
1. Morowitz, H.J. Energy Flow i n Biology 1968, Academic Press:

New York
2. P r i g o g i n e I. Thermodynamics of I r r e v e r s i b l e Processes T h i r d
E d i t i o n . Wiley: New York, 1967
3. G l a n s d o r f f , P., I. Prigogine Thermodynamic Theory of
Structure S t a b i l i t y and F l u c t u a t i o n s ; Wiley: New York, 1971
4. Haase, R. Thermodynamics of I r r e v e r s i b l e Processes; Addison-
Wesley. Reading (MA) 1969
5. Roels, J.A. B i o t e c h n o l . Bioeng. 1980, 22, 2457
6. Roels,J.A. Ann. New York Acad. S c i . , 1981, 369, 113

7. Minkevich, I.G., E r o s h i n , V.K. F o l i a Microbiol., 1973, 18, 376

8. Kharasch, M.S. Bur. Stand. J . Res., 1929, 2, 359
9. Heijnen, J . J . ; Roels, J.A. B i o t e c h n o l . Bioeng., 1981, 23, 739
10. Roels, J.A. Bioengineering E n e r g e t i c s and K i n e t i c s , E l s e v i e r
North Holland (In prep.)
11. S t u c k i , J.W. Eur. J . Biochem.; 1980, 109, 269
12. Payne, W.J. Annu. Rev. M i c r o b i o l . ; 1970, 24, 17
13. Caplan S.R. Curr. Top. Bioenerg.; 1971, 4, 1
14. Thauer, R.H.; Jungerman, K.; Decker, K. B a c t e r i o l . Rev.; 1977,
41, 100
15. Van Verseveld, H.W. Influence of Environmental Factors on the
E f f i c i e n c y of Energy Conservation i n Paracoccus d e n i t r i f i c a n s
Ph. D. T h e s i s , Free U n i v e r s i t y of Amsterdam; Meppel Krips
Repro, 1979
16. Bauchop. T.; Elsden
17. Stouthamer, A.H. M i c r o b i a
R E C E I V E D June 29, 1982

14
Ion Pumps as Energy Transducers
ERICH HEINZ
Cornell University Medical College, New York, NY 10021
Ionmotive forces, i.e. the electrochemical potential differences

produced by ion pump
by exergonic reaction
synthesis of energy-rich compounds o r uphill transport of
organic solutes. A typical example is the "chemiosmotic
coupling" by a proton pump in oxidative and photosynthetic
phosphorylation. The effectiveness of this coupling depends on
its (energetic) efficiency as well as on its (kinetic) expeditious-
ness. The efficiency is mainly limited by inner and outer
leakages to the transported ion species, the expeditiousness
mainly by the delay in generating the protonmotive force. Where-
as little is known about the various leakages to assess the
efficiency, some preliminary information concerning the
expeditiousness may be considered here. The generation of an
ionmotive force, to the extent that it depends on ion gradients is
a rather slow process. Electrogenic pumps, however, can more
rapidly raise the electrical potential, p r i o r to appreciable ion
translocation by loading the static membrane capacity. Such
rapid electric effects may significantly increase the expeditious-
ness of the coupling but only under the condition that the pump
works at constant driving force. On the other hand, i f the pump
works at constant rate the r i s e of the protonmotive force is
largely determined by the formation of ion gradients which r i s e
more slowly and are less sensitive towards variations in energy
input. Using a recently developed optical method of rapidly
monitoring the P D , it could be shown that the light-driven
proton pump of halobacterium halobium has the high expeditious-
ness predicted for an electrogenic pump working at constant
driving force.
0097-6156/83/0207-0323$06.00/0

In o r d e r that e n e r g y be t r a n s f e r r e d b e t w e e n two d i f f e r e n t pro

cesses these have to b e coupled. Homogenous c o u p l i n g , i . e. between
two c h e m i c a l r e a c t i o n s w i t h i n the s a m e c o m p a r t m e n t i s usually
b a s e d on the p r i n c i p a l of the " c o m m o n i n t e r m e d i a t e " a s i s i l l u s t r a t e d
b y the " c l a s s i c a l " c o u p l i n g b e t w e e n the d e h y d r o g e n a t i o n of triose-
phosphate a n d the f o r m a t i o n of a d e n o n i s i n e t r i p h o s p h a t e , for which
diphosphoglycerate was identified as the " c o m m o n i n t e r m e d i a t e " that
l i n k s t h e s e two reactions. A n analagous, though s o m e w h a t d i f f e r e n t
p r i n c i p l e u n d e r l i e s the c o u p l i n g b e t w e e n two o s m o t i c processes,
e. g. in (secondary active) ion-linked cotransport, namely through a
ternary complex supposedly formed b y the two solutes with a carrier
or a mobile gate. More c o m p l i c a t e d and l e s s well understood is
the c o r r e s p o n d i n g p r i n c i p l e of h e t e r o g e n o u s coupling,between a
c h e m i c a l and an o s m o t i
has been discussed in mor
mainly been considered a means of t r a n s f e r r i n g c h e m i c a l e n e r g y to
drive an endergonic osmotic process, until M i t c h e l l ( 2 ) postulated
the r e v e r s e for m i t o c h o n d r i a l and b a c t e r i a l " o x i d a t i v e phosphory
l a t i o n " w h e r e the s y n t h e s i s of e n e r g y r i c h phosphate should be
e n e r g i z e d b y the d o w n h i l l m o v e m e n t o f Η - i o n s . In the e v o l v i n g
" c h e m i o s m o t i c h y p o t h e s i s " o f o x i d a t i v e p h o s p h o r y l a t i o n two s t e p s of
heterogenous coupling o c c u r in s e r i e s : the first one l i n k s a n o x i
d a t i o n - r e d u c t i o n r e a c t i o n to the a c t i v e e x t r u s i o n o f p r o t o n s , thereby
generating an e l e c t r o c h e m i c a l potential difference of H-ions
("protonmotive force"); the s e c o n d one l i n k s the p a s s i v e r e e n t r y of
protons, d r i v e n by this force, to the s y n t h e s i s of energy-rich
phosphate b y means o f the " p r o t o n t r a n s l o c a t i n g A T P a s e " (Fig. 1).
There is good e v i d e n c e that the s a m e s e q u e n c e of s i m i l a r c o u p l i n g
s t e p s o c c u r s i n the o x i d a t i v e p h o s p h o r y l a t i o n o f a e r o b i c bacteria (3)
a s w e l l a s i n the l i g h t - d r i v e n p h o s p h o r y l a t i o n o f c h l o r o p l a s t s (4, 5 ),
and h a l o p h i l i c b a c t e r i a ( 6 ) . Various plausible models of such
s y s t e m s have s i n c e been d e v i s e d and c r i t i c a l l y d i s c u s s e d (7, 8, 9 ).
T h o u g h the p o s t u l a t e d " c h e m i o s m o t i c " c o u p l i n g h a s not b e e n proven
y e t b e y o n d doubt, it is so far well enough supported b y experimental
evidence to j u s t i f y the wide b e l i e f i n i t s v a l i d i t y . It i s n o n e t h e l e s s
s u r p r i s i n g that t h i s c o u p l i n g , w h i c h u l t i m a t e l y l i n k s two t r u l y c h e m i
cal r e a c t i o n s , d o e s i n c o n t r a s t to the c l a s s i c a l m o d e l s not i n v o l v e a
common chemical intermediate, b u t r a t h e r two h e t e r o g e n o u s coupling
steps i n s e r i e s . In o t h e r w o r d s , energy is transferred from one
chemical r e a c t i o n to a n o t h e r one i n o s m o t i c f o r m , with the h e l p of
two " c h e m i o s m o t i c e n e r g y transductions". Whether this p e c u l i a r
k i n d of c o u p l i n g i s a mere vestige o r whether it has c e r t a i n
advantages as c o m p a r e d to o t h e r k i n d s of c o u p l i n g , for instance, by
b e i n g m o r e e f f e c t i v e , is h a r d to t e l l . L e t u s s e e how a n d u n d e r w h i c h
conditions a g r e a t e r effectiveness m i g h t c o m e about.

14. HEINZ Ion Pumps 325
Figure 1. Chemiosmotic coupling in

oxidative phosphorylation. The model is
drawn according to Mitchell's hypothesis
for mitochondria, but might also apply
to other systems of phosphorylation.
A hydrogen ion pump transports the H-ions
from left to right, energized by an oxidation-
reduction reaction, a light-driven photochemical
mechanism, or any other appropriate energy
source. This pumping is tantamount to pumping
Ο H-ions in the opposite direction. A membrane-
bound enzyme, the proton translocating AT-
Pase catalyzes the interconversion of A DP and
Pi and ATP by a heterophasic (or vectorial)
reaction, i.e., a reaction in which the Ο H-ions,
released by the synthesis or incorporated by the
hydrolysis of ATP, can interact with the en
zyme only from the phase contralateral to that
from which the other reactants interact. In this
way, the direction of the chemical reaction de
pends on the spatial direction of the H- (or
OH-) gradient.

T h e o v e r a l l effectiveness of a n y c o u p l i n g m e c h a n i s m m a y b e
a s s e s s e d i n t e r m s o f i t s ( e n e r g e t i c ) e f f i c i e n c y a s w e l l a s i n t e r m s of i t s
(kinetic) e x p e d i t i o u s n e s s . T h e e n e r g e t i c e f f i c i e n c y i s l i m i t e d b y the
e x t e n t of u n a v o i d a b l e l e a k a g e s , w h i c h a r e p a r t l y due to p a s s i v e permea-
b i l i t y of the m e m b r a n e b a r r i e r to the t r a n s p o r t e d s o l u t e (outer l e a k a g e )
a n d p a r t l y r e s i d e i n the t r a n s p o r t m e c h a n i s m i t s e l f ( i n n e r l e a k a g e ) i n
that " s l i p p i n g " a n d " b a c k c y c l i n g " m a y p e r m i t the e s c a p e of u n u t i l i z e d
i n p u t e n e r g y (1 ). T o o l i t t l e i s k n o w n about the e x t e n t of l e a k a g e s w i t h -
i n the c h e m i o s m o t i c m e c h a n i s m o f p h o s p h o r y l a t i o n to c o m p a r e i t s
e n e r g e t i c e f f i c i e n c y w i t h that o f i t s c h e m i c a l c o u n t e r p a r t .
N o t m u c h m o r e i s known about the e x p e d i t i o u s n e s s w h i c h , i n
t h i s c o n t e x t , m a y be d e f i n e d a s the r a t e at w h i c h the p r o t o n m o t i v e
f o r c e r i s e s a f t e r the o n s e t of the p u m p , but s o m e g e n e r a l p r e d i c t i o n s
c a n be m a d e . The expeditiousnes
r a t e of p r o t o n s i s r e l a t i v
tend to shunt the e l e c t r o g e n i c p u m p effect, i n o t h e r w o r d s , to m a i n t a i n
electroneutrality. W e s e e that the two c o m p o n e n t s of the p r o t o n m o t i v e
force, the e l e c t r i c a l a n d the c h e m i c a l P D , a r e c o n t r o l l e d b y d i f f e r e n t
f a c t o r s a n d m a y t h e r e f o r e d e v e l o p at d i f f e r e n t r a t e s , a s i s i l l u s t r a t e d
b y the f o l l o w i n g two e x t r e m e c o n d i t i o n s :
If the p a s s i v e c o n d u c t a n c e i s e x t r e m e l y l o w , the e l e c t r i c a l
p o t e n t i a l w i l l b e g e n e r a t e d p u r e l y e l e c t r o g e n i c a l l y , i . e. without
a p p r e c i a b l e net m o v e m e n t of p a s s i v e i o n s . A s the e l e c t r i c c a p a c i t y of
the m e m b r a n e i s r a t h e r l o w , ^F/cm , 2
e l e c t r o g e n i c e x p u l s i o n of 3 0 0 -
400 p r o t o n s p e r c e l l , p a s s i v e s h u n t i n g effect b e i n g n e g l i g i b l e at t h i s
stage, s h o u l d s u f f i c e to r a i s e the e l e c t r i c a l P D b y 1 m v o l t .
O n the o t h e r h a n d , i f the p a s s i v e c o n d u c t a n c e i s v e r y h i g h , i t
w i l l k e e p e l e c t r i c a l e f f e c t s s m a l l but a l l o w i n s t e a d a n e q u i v a l e n t r i s e
o f the c h e m i c a l P D of p r o t o n s . T h i s r i s e w i l l d e p e n d o n the b u f f e r
c a p a c i t y o f the m e d i u m , w h i c h i s n o r m a l l y m u c h h i g h e r than the
e l e c t r i c a l capacity. F r o m i t s v a l u e i n the m i t o c h o n d r i a we w o u l d e s t i -
m a t e that about a m i l l i o n p r o t o n s h a v e to be e x p e l l e d p e r 1 m v o l t r i s e
of the c h e m i c a l P D , a n d h e n c e o f the p r o t o n m o t i v e f o r c e . Obviously,
the r i s e i n c h e m i c a l P D i n t h i s c a s e i s t h o u s a n d o r m o r e t i m e s s l o w e r
than the e l e c t r o g e n i c one i n the f o r m e r c a s e . A t the r a t e a s s u m e d for
the m i t o c h o n d r i a l p r o t o n p u m p , we would p r e d i c t that the c r i t i c a l
protonmotive force, i . e . about 200 m v o l t s , c o u l d be r e a c h e d w i t h i n a
few h u n d r e d m i l l i s e c o n d s i n the f i r s t c a s e , but would take a few
h u n d r e d s e c o n d s i n the l a s t c a s e . It f o l l o w s that the e x p e d i t i o u s n e s s of
the c o u p l i n g , i . e. the s p e e d at w h i c h t h i s c r i t i c a l p r o t o n m o t i v e f o r c e
i s r e a c h e d a f t e r the i n i t i a t i o n of the p u m p d e p e n d s on the r e l a t i v e c o n -
t r i b u t i o n of the i n i t i a l p u m p i n g r a t e r e l a t i v e to the s h u n t i n g p a t h w a y s
for passive i o n s .

T o a p p r o a c h t h i s p r o b l e m q u a n t i t a t i v e l y , we m a y f o r s i m p l i c i t y
r e a s o n s f o l l o w the c o n v e n t i o n a l p r o c e d u r e u s e d b y e l e c t r o p h y s i o l o g i s t s
i n d e a l i n g with e l e c t r o g e n i c p u m p s in t h e i r b i o l o g i c a l s y s t e m s , e t c . In
the e q u i v a l e n t c i r c u i t s u s e d b y t h e m to v i s u a l i z e the e l e c t r i c p h e n o m e n a
of b i o l o g i c a l s y s t e m s , an electrogenic ion p u m p i s u s u a l l y represented
e i t h e r as a constant voltage s o u r c e o r as a constant c u r r e n t s o u r c e ,
b a s e d o n the s i m p l i f y i n g a s s u m p t i o n that d u r i n g the e x p e r i m e n t a l
o b s e r v a t i o n e i t h e r the d r i v i n g f o r c e o f the p u m p o r the p u m p i n g r a t e
remains fairly constant. T h e d i f f e r e n c e b e t w e e n t h e s e two
a l t e r n a t i v e s c o r r e s p o n d s w i t h s o m e c r u d e a p p r o x i m a t i o n to the a b o v e
d i s c u s s e d d i f f e r e n c e b e t w e e n two k i n d s o f c o u p l i n g m o d e l s ; the c o n s t a n t
v o l t a g e s o u r c e n e t w o r k m o s t c l o s e l y w o u l d a c c o u n t f o r the e x p e d i t i o u s -
n e s s , but l e s s s t a b l e c o u p l i n g m o d e l , w h e r e a s the c o n s t a n t c u r r e n t
s o u r c e w o u l d d o s o f o r th
most cases, it i s difficul
f o r e i t h e r one s o m e p l a u s i b l e e x p l a n a t i o n c a n b e g i v e n . As a conse-
q u e n c e , the t r e a t m e n t b y e l e c t r o p h y s i o l o g i s t s i n t U s r e s p e c t i s n o t
u n i f o r m , b u t a s l o n g a s the s t e a d y s t a t e i s c o n c e r n e d , e i t h e r one m a y
give reasonable a n s w e r s . T h e d i f f e r e n c e b e t w e e n the two m o d e l s
becomes, however, c r u c i a l i n transient states, for instance, i m m e d i -
a t e l y a f t e r the p u m p h a s b e e n t u r n e d o n o r off.
M i t c h e l l ( 8 ) was p r o b a b l y the f i r s t t o i n v e s t i g a t e t h e s e questions»
a n d b y r i g o r o u s c a l c u l a t i o n s , b a s e d on the a s s u m p t i o n o f c o n s t a n t
p u m p i n g r a t e , h e p r e d i c t e d that the e l e c t r i c a l P D r i s e s v e r y f a s t but
i n s i g n i f i c a n t l y h i g h w h i l e the f u l l d e v e l o p m e n t o f the p r o t o n m o t i v e f o r c e
h a s to a w a i t the g e n e r a t i o n o f a s u f f i c i e n t l y h i g h c o n c e n t r a t i o n g r a d i e n t
w h i c h w o u l d t a k e m o r e t h a n 30 s e c o n d s . The same approach, except
f o r the a s s u m p t i o n that the d r i v i n g f o r c e , r a t h e r t h a n the r a t e o f the
p u m p r e m a i n s c o n s t a n t , g a v e a g r e a t l y d i f f e r e n t r e s u l t (10, 11 ).
D e p e n d i n g o n the p u m p i n g r a t e r e l a t i v e to the p a s s i v e i o n m o b i l i t i e s ,
t h e i n i t i a l s u r g e o f the e l e c t r i c a l p o t e n t i a l m a y i n d e e d b e h i g h e n o u g h
to a n t i c i p a t e m o s t o f the final p r o t o n m o t i v e f o r c e a t s t a t i c h e a d
( F i g . 2). O b v i o u s l y , a c o n s t a n t p u m p i n g r a t e i m p l i e s that the d r i v i n g
f o r c e i s i n i t i a l l y l o w but r i s e s a c c o r d i n g l y a s the o p p o s i n g p r o t o n m o t i v e
force grows. B y contrast, at constant d r i v i n g force, the p u m p i n g r a t e
s h o u l d i n i t i a l l y b e v e r y h i g h a n d l a t e r d e c l i n e a c c o r d i n g l y a s the
opposing protonmotive force r i s e s . A p r i o r i , both models a r e i n s o m e
w a y p l a u s i b l e a n d o n l y the e x p e r i m e n t c a n d e c i d e w h i c h one i s the v a l i d
one.
So f a r , e x p e r i m e n t a l t e s t s a r e s c a r c e , p a r t l y due t o the
d i f f i c u l t i e s to m e a s u r e r a p i d l y e n o u g h c h a n g e s i n e l e c t r i c a l m e m b r a n e
p o t e n t i a l (or p r o t o n m o t i v e f o r c e ) w i t h s m a l l c o m p a r t m e n t s s u c h a s
mitochondria and b a c t e r i a . T h e usually applied chemical probes for
p o t e n t i a l c h a n g e s a r e e i t h e r too s l o w o r too n o i s y . Recently, however,

,,ιι
H + -pump M II
on
,,1
Η -pump o f f
II
Figure 2. The calculated response of the protonmotive force, and its electrical and
chemical components, to the sudden initiation or stopping of an electrogenic proton
pump.
Ordinate: protonmotive force (X )
H in relative units. Abscissa: time. Solid line, electric PD;
dashed line, chemical PD of H*; dotted line, X . H Top: constant rate of pumping (constant
current source). Bottom: constant driving force of the pump (constant voltage source). It is
seen that only under the condition of constant driving force is the initial rise of the electrical
PD, and hence of the total protonmotive force, high enough to make pumping power available
almost instantaneously after the start of the pump. It also is seen that the pump is more stable
under constant pumping rate, because after the pump is turned off, the PMF drops almost
instantaneously in the lower curve whereas it declines gradually in the upper curve. Reproduced,
with permission, from Ref. 11. Copyright 1982, Academic Press, Inc.

a m e t h o d that a l l o w s r a p i d m o n i t o r i n g o f s u c h c h a n g e s b a s e d o n a c h a n g e
i n l i g h t a b s o r p t i o n o f the n a t u r a l l y p r e s e n t s u b s t a n c e , bacteriorhodopsm,
h a s b e e n d e v i s e d f o r the l i g h t - d r i v e n p r o t o n p u m p s i n h a l o b i u m (12 ).
B y this m e t h o d , i t c o u l d be shown with envelope v e s i c l e s of this b a c
t e r i u m , that a f t e r i n i t i a t i n g the p u m p b y i l l u m i n a t i o n , the e l e c t r i c a l
p o t e n t i a l (or the p r o t o n m o t i v e f o r c e ) r i s e s w i t h a h a l f t i m e of a b o u t
2 0 m s e c to a v a l u e c l o s e to 2 0 0 m v e v e n t h o u g h the b u f f e r c a p a c i t i e s o f
a l l p e r m e a n t i o n s i n s i d e a n d o u t s i d e was k e p t h i g h e n o u g h to p r e c l u d e
a p p r e c i a b l e c h a n g e s i n i o n d i s t r i b u t i o n d u r i n g that t i m e (13 ). This
r e s u l t c l o s e l y a g r e e s w i t h what h a s b e e n p r e d i c t e d o n the b a s i s of the
c o n s t a n t d r i v i n g f o r c e m o d e l , a n d t h u s c o n f i r m s the h y p o t h e s i s o f the
c r u c i a l r o l e o f the e l e c t r i c a l p o t e n t i a l f o r the e x p e d i t i o u s n e s s o f the
coupling system.
This result also confirm
d i n g to the f o l l o w i n g e q u a t i o n d e r i v e d i n t e r m s o f n o n - e q u i l i b r i u m
thermodynamics (14):
a r e leakage coefficients of H and Κ , respectively. and

a r e s t o i c h i o m e t r i c c o e f f i c i e n t s of the p u m p f o r the i o n s i n d i c a t e d . It i s
s e e n that a t c o n s t a n t c h e m i c a l p o t e n t i a l s o f the i o n s c o n c e r n e d the
p a r t i a l d i f f e r e n t i a l i s d i f f e r e n t f r o m z e r o o n l y i n the p r e s e n c e o f a n
e l e c t r o g e n i c p u m p , i . e. i f Φ Though buffer c a p a c i t i e s of
a l l p e r m e a n t i o n s c o n c e r n e d w e r e k e p t h i g h e n o u g h to p r e v e n t the f o r -
m a t i o n o f a n y s i g n i f i c a n t i o n g r a d i e n t d u r i n g the t i m e o f o b s e r v a t i o n
a d i s t i n c t e l e c t r i c r e s p o n s e was r e g i s t e r e d u p o n i l l u m i n a t i o n a f t e r a
few m i l l i s e c o n d s a s w o u l d b e i n c o n s i s t e n t w i t h a n e l e c t r i c a l l y s i l e n t
H /K pump
+ +
( i T R = JT ).
K
W h e t h e r the'higfr e x p e d i t i o u s n e s s r e s u l t i n g f r o m the e l e c t r o g e n i c
P D r i s e w o u l d b e a n a d v a n t a g e f o r a n y s y s t e m i s h a r d to p r e d i c t . Owing
to the l o w e l e c t r i c m e m b r a n e c a p a c i t y the i o n m o t i v e f o r c e at t h i s s t a g e ,
being predominantly electrogenic i s v e r y sensitive towards changing the
driving force. B y contrast a m o r e slowly developing ionmotive force
g e n e r a t e d b y i o n t r a n s l o c a t i o n i s c e r t a i n l y m o r e s t a b l e a s the energy
s t o r e d i n i t w o u l d s t i l l b e a v a i l a b l e f o r s o m e t i m e a f t e r the p u m p h a s
stopped. If the s y s t e m w e r e t o s u p p l y e n e r g y - r i c h c o m p o u n d s a t a
s t e a d y r a t e i n s p i t e of h i g h l y f l u c t u a t i n g e n e r g y i n p u t , o b v i o u s l y the
s l o w e r but m o r e stable a r r a n g e m e n t would be m o r e suitable. If, o n the
o t h e r h a n d , the s y s t e m w e r e to a d j u s t i t s a c t i v i t y to a r a p i d l y c h a n g i n g
r e q u i r e m e n t at a r a t h e r steady input of e n e r g y , the m o r e expeditious
but l e s s stable a r r a n g e m e n t would be m o r e suitable.

O n the o t h e r h a n d , i t i s v e r y l i k e l y that the e l e c t r o g e n i c P D of a

h i g h l y e x p e d i t i o u s c o u p l i n g s y s t e m a p p e a r s o n l y a s a wave of r a t h e r
s h o r t d u r a t i o n , b e i n g r e p l a c e d l a t e r b y the c h e m i c a l P D , which m a y
p r e d o m i n a t e a f t e r the s y s t e m h a s r e a c h e d a s t a t i o n a r y state ( F i g . 2).
S u c h i s to be e x p e c t e d i f the " b u f f e r c a p a c i t i e s " of the p a s s i v e i o n s ,
c h i e f l y K - i o n s , a r e b i g e n o u g h to p r e v e n t the f o r m a t i o n of a m o r e
stable diffusion potential. H e n c e the p r o t o n m o t i v e f o r c e g e n e r a t e d b y
s u c h a s y s t e m m a y h a v e a d i f f e r e n t c o m p o s i t i o n i n the s t a t i o n a r y state
f r o m that d u r i n g the i n i t i a l e l e c t r i c a l p o t e n t i a l r i s e . In t h i s way, the
a d v a n t a g e of a h i g h e x p e d i t i o u s n e s s at the b e g i n n i n g m a y be c o m b i n e d
with that of a h i g h e r s t a b i l i t y o b t a i n e d a f t e r s o m e t i m e of c o n t i n u o u s
pumping activity. F i g 2. a l s o s h o w s that the d e g r e e of s t a b i l i t y at a n y
s t a g e s h o u l d b e c o m e a p p a r e n t i f the p u m p i s s u d d e n l y s t o p p e d . A f t e r the
p u m p h a s r e a c h e d i t s (stable
that s t a g e would decay only s l o w l y and would t h e r e f o r e be measurable
i m m e d i a t e l y a f t e r the p u m p h a s s t o p p e d . In the i n i t i a l s t a g e , however,
when the e l e c t r o g e n i c c o m p o n e n t of the P M F d o m i n a t e s , the P M F
s h o u l d i m m e d i a t e l y d i s a p p e a r a f t e r s t o p p i n g the p u m p , a n d thus e s c a p e
detection. T h i s m a y b e the r e a s o n why the p r o t o n m o t i v e f o r c e h a s often
b e e n found i n a d e q u a t e t o s u p p o r t the c h e m i o s m o t i c h y p o t h e s i s o f
o x i d a t i v e p h o s p h o r y l a t i o n , a s the e l e c t r o g e n i c c o m p o n e n t i s too l a b i l e to
s u r v i v e the d i s t u r b a n c e a s s o c i a t e d with the u s u a l m e a s u r e m e n t s of
electrical potential.
Acknowledgment
T h e s e studies were supported by a U S P H S - N I H grant No. R O I G M
26554-01.
Literature Cited
1. Heinz, Ε., "Mechanics and Energetics of Biological Transport";

Springer V e r l a g : Heidelberg, New York, London, 1978
2. M i t c h e l l , P., Nature, 1961, 191, 144-8
3. Maloney, P.C., Wilson, T.H., J. Membrane B i o l , 1975, 25, 285
4. Junge, W., Ber. Dtsch, Bot. Ges., 1975, 88, 283-301
5. Witt, Η. Τ., Biochim. Biophys. Acta, 1979, 505, 355-427
6. Stoeckenius, W., Lozier, R.H., Bogomolni, R.A., Biochim.
Biophys. Acta, 1979, 505, 215
7. Mitchell, P., "Chemiosmotic Coupling i n Oxidative and Photo-
synthetic Phosphorylation"; Glynn Research Ltd. : Bodmin, 1966
8. Mitchell, P., "Chemiosmotic Coupling and Energy Transduction";
Glynn R e s e a r c h Ltd. : Bodmin, 1968
9. Skulachev, V. P., "Membrane Energetics"; Lee, C. P., Schatz, G.,
Ernster, L., Eds; Addison-Wesley; p. 373

10. Heinz, E., "Electrogenic and E l e c t r i c a l l y Silent Proton Pumps in

Hydrogen Ion Transport i n Epithelia"; Shultz, I., Sachs, G.,
Forte, J.G., U l l r i c h , K.J., Eds; E l s e v i e r / N o r t h Holland
Biomedical, 1980, p. 41
11. Heinz, E., "Current Topics i n Membranes and Transport";
Kleinzeller, Α . , Bronner, F., Eds; Academic, 1982; V o l 16, p.249
12. Dancshazy, Ζ., Helgerson, S. L., Stoeckenius, W., i n preparation
13. Helgerson, S. L., Dancshazy, Z., Stoeckenius, W., Heinz, E.;
(Abstract); Biophys. J . , 1982, 37, 266a
14. Heinz, Ε . , " E l e c t r i c a l Potentials i n Biological Membrane T r a n s
port"; (Monograph); Springer, 1981

15
Kinetics and Transport Phenomena in Biological

Reactor Design
M . MOO-YOUNG
University of Waterloo, Department of Chemical Engineering,
Waterloo, Ont. N2L 3G1 Canada
H . W. B L A N C H
University of California, Department f Chemical Engineering Berkeley CA 94720
The application of c h e m i c a l e n g i n e e r i n g principles is

u s e f u l i n an a n a l y s i s of the d e s i g n and o p e r a t i o n of bioreactors.
However, classical approaches to the a n a l y s i s a r e limited by the
following special constraints:
1. The reactant mixture i s r e l a t i v e l y complex. Microbial

biomass increases with the biochemical transformation and
c a t a l y s t i s synthesized as the r e a c t i o n s proceed.
2. The bulk d e n s i t i e s of suspended m i c r o b i a l c e l l s and sub-

s t r a t e p a r t i c l e s g e n e r a l l y approach those of t h e i r l i q u i d
environment so that r e l a t i v e flow between the dispersed
and continuous phases is normall low. T h i s s i t u a t i o n may be
conetrasted to the r e l a t i v e l y heavy m e t a l l i c c a t a l y s t
p a r t i c l e s g e n e r a l l y used i n chemical r e a c t o r s .
3. The s i z e s of s i n g l e m i c r o b i a l c e l l s are very small ( i n the

range of a few microns) compared to chemical c a t a l y s t par-
ticles: coupled with the above c o n s t r a i n t s it i s
generally difficult to promote high v e l o c i t i e s and
turbulent-flows conditions
4. Polymeric substrates or metabolites and m y c e l i a l growth

often produce very viscous r e a c t i o n mixtures which are
g e n e r a l l y pseudoplastic non-Newtonian; a g a i n , these
conditions tend to l i m i t d e s i r a b l y high flow dynamics
in bioreactors.
5. Many m u l t i c e l l u l a r m i c r o b i a l growths, e s p e c i a l l y fungal

ones g e n e r a l l y form r e l a t i v e l y large cell aggregates such
as clumps or p e l l e t s : compared to c a t a l y s t particles,
0097-6156/83/0207-0335$06.00/0

i n t r a p a r t i c l e d i f f u s i o n a l resistances are often serious

i n these systems* e . g . , l e a d i n g to a n a e r o b i o e i e .
6. Bioreactors frequently require c r i t i c a l l y close control of

s o l u t e c o n c e n t r a t i o n s * pH* t e m p e r a t u r e * and l o c a l
p r e s s u r e s i n o r d e r t o a v o i d damage o r d e s t r u c t i o n o f l i v e
o r l a b i l e components w h i c h a r e e s s e n t i a l t o t h e p r o c e s s .
7. V e r y low c o n c e n t r a t i o n s o f r e a c t a n t s a n d / o r p r o d u c t s i n
a c q u e o u s m e d i a a r e n o r m a l l y i n v o l v e d i n b i o r e a c t o r s so
c o n c e n t r a t i o n d r i v i n g f o r c e s f o r mass t r a n s f e r a r e o f t e n
severely limited.
8. M i c r o b i a l growth r a t e s a r e s u b s t a n t i a l l y lower t h a n
c h e m i c a l r e a c t i o n r a t e s so t h a t r e l a t i v e l y l a r g e r e a c t o r
v o l u m e s and r e s i d e n c
A s a n i l l u s t r a t i o n o f some o f t h e p r o b l e m s i m p o s e d by t h e
a b o v e c o n s t r a i n t s * we n o t e t h a t a n a d e q u a t e o x y g e n s u p p l y r a t e t o
growing c e l l s i s o f t e n c r i t i c a l i n a e r o b i c f e r m e n t a t i o n s . Because
o f i t s low s o l u b i l i t y i n w a t e r * g a s e o u s o x y g e n * u s u a l l y i n t h e
f o r m o f a i r * m u s t be s u p p l i e d c o n t i n u o u s l y t o t h e f e r m e n t a t i o n
medium i n s u c h a way t h a t t h e o x y g e n a b s o r p t i o n r a t e a t least
equals the oxygen consumption r a t e of the c e l l s . Even temporary
d e p l e t i o n o f d i s s o l v e d o x y g e n c o u l d mean i r r e v e r s i b l e c e l l damage.
I n t h i s r e s p e c t * i t i s w o r t h n o t i n g t h a t t h e same m i c r o b i a l
s p e c i e s may show l a r g e v a r i a t i o n s i n i t s o x y g e n r e q u i r e m e n t s *
depending on t h e o x y g e n c o n c e n t r a t i o n t o w h i c h t h e y have b e e n
adapted.
P r e v i o u s s t u d i e s i n w h i c h t h e o x y g e n s u p p l y t o a submerged
g r o w i n g m i c r o b i a l c u l t u r e was s t o p p e d * h a v e s h o w n a l i n e a r
decrease i n oxygen c o n c e n t r a t i o n with time over a large
concentration range (Finn* 1954). Below a c e r t a i n oxygen
c o n c e n t r a t i o n * c a l l e d the " c r i t i c a l oxygen tension*** the decrease
f o l l o w e d a h y p e r b o l i c p a t t e r n compatible with Michaelis-Menton
kinetics. O f t e n d e v i a t i o n s f r o m t h e l i n e a r and h y p e r b o l i c o x y g e n
concentration decrease patterns are found. The r a t e c o n t r o l l i n g
s t e p i n a f e r m e n t a t i o n p r o c e s s may s h i f t t h e o x y g e n s u p p l y r a t e
i n t o the b u l k l i q u i d t o t h e demand r a t e i n s i d e the c e l l i f c e l l
a g g r e g a t e s a r e f o r m e d w h i c h a r e l a r g e r t h a n a few h u n d r e d t h s o f
millimeters. U s u a l l y * t h i s i s s e e n as an i n c r e a s e d v a l u e o f t h e
c r i t i c a l oxygen t e n s i o n o r t o t a l a b s e n c e o f a l i n e a r p a r t of the
oxygen c o n c e n t r a t i o n d e c r e a s e c u r v e * s h o w i n g d e p e n d e n c e o n t h e
c o n c e n t r a t i o n d r i v i n g force at the c e l l surface*
RATE-CONTROLLING STEPS
F i g u r e 1 g i v e s a g e n e r a l i z e d p h y s i c a l r e p r e s e n t a t i o n of a
b i o r e a c t o r s u b s y s t e m i n v o l v i n g two o r m o r e p h a s e s * An important
example o f t h i s r e p r e s e n t a t i o n i s an a e r o b i c f e r m e n t a t i o n i n w h i c h

NON-AQUEOUS PHASE R E A C T A N T S / AQUEOUS PHASE WITH D I S S O L V E D S O L I D PHASE R E A C T I O N
PRODUCTS REACTANTS/PRODUCTS
Sugars, M i n e r a l s , Enzymes, e t c .
Gas ( 0 , C 0 , C H , e t c . )
2 2 4 Cells
Liquid (Oils) Enzymes
Solid (Substrates) Organelles
1
Route 1 Reactant Supply and U t i l i z a t i o n
ι
Route 2 P r o d u c t Removal . and Formation
Figure 1. Generalization of biochemical reactor conditions illustrating the importance of aqueous
phase transfer steps for mass and heat. Reproduced, with permission, from Ref. 38.
Copyright 1981, Springer-Verlag.

a m i c r o b e u t i l i z e s o x y g e n ( s u p p l i e d by a i r b u b b l e s w h i c h a l s o
d e s o r b t o x i c c a r b o n d i o x i d e ) and o t h e r d i s s o l v e d n u t r i e n t s
(sugars* e t c . ) t o grow and p r o d u c e s o l u b l e e x t r a c e l l u l a r
metabolites. E i g h t r e s i s t a n c e s i n t h e mass t r a n s f e r p a t h w a y s f o r
t h e n u t r i e n t s s u p p l y and u t i l i z a t i o n and f o r m e t a b o l i t e excretion
and r e m o v a l * a r e p o s s i b l e as i n d i c a t e d i n t h e i l l u s t r a t i o n a t t h e
f o l l o w i n g l o c a t i o n s : (1) i n a g a s - f i l m (2) at the g a s - l i q u i d
i n t e r f a c e (3) i n a l i q u i d - f i l m a t t h e g a s - l i q u i d i n t e r f a c e ( 4 ) i n
b u l k l i q u i d (5) i n a l i q u i d - f i l m s u r r o u n d i n g the s o l i d (6) a t the
l i q u i d - s o l i d i n t e r f a c e ( 7 ) i n t h e s o l i d phase c o n t a i n i n g t h e c e l l s
(8) a t t h e s i t e s o f t h e b i o c h e m i c a l r e a c t i o n s . I t s h o u l d be n o t e d
t h a t a l l t h e pathways e x c e p t t h e l a s t one are purely p h y s i c a l .
F i g u r e 1 can r e p r e s e n t a wide range of o t h e r p r a c t i c a l

situations. The c o n t i n u o u s phase may be l i q u i d o r g a s * t h e l a t t e r
representing special case
(e.g., composting, t r i c k l e - b e
w h i l e t h e d i s p r s e d p h a s e may be one o r m o r e o f t h e f o l l o w i n g
phases: s o l i d ( e . g . , m i c r o b i a l c e l l s * immobi lized-enzyme
p a r t i c l e s * s o l i d s u b s t r a t e s ) ; l i q u i d (e.g., i n s o l u b l e or s l i g h t l y
s o l u b l e s u b s t r a t e s ) ; gas ( e . g . * a i r , c a r b o n d i o x i d e , m e t h a n e ) .
EFFECT OF INTERFACIAL PHENOMENA
I f we c o n s i d e r a f l u i d p a r t i c l e ( g a s o r l i q u i d ) m o v i n g
r e l a t i v e l y t o a c o n t i n u o u s l i q u i d p h a s e , t h e r e a r e two p o s s i b l e
e x t r e m e s o f i n t e r f a c i a l m o v e m e n t as c l a s s i f i e d b e l o w . (For
c o n v e n i e n c e * we w i l l c o n s i d e r a s p h e r e ) .
(a) T h e r e i s no i n t e r n a l c i r c u l a t i o n w i t h i n t h e p a r t i c l e .
These p a r t i c l e s behave e s s e n t i a l l y as i f t h e y a r e s o l i d
with r i g i d surfaces. We w i l l r e f e r t o t h e s e as p a r t i c l e s
with "rigid" surfaces.
(b) There i s a f u l l y developed i n t e r n a l c i r c u l a t i o n flow

w i t h i n t h e p a r t i c l e due t o an i n t e r f a c i a l v e l o c i t y .
The p a r t i c l e b e h a v e s as a p a r t o f an i n v i s c i d c o n t
i n u o u s phase w i t h o n l y a d e n s i t y d i f f e r e n c e . We w i l l
r e f e r t o t h e s e as p a r t i c l e s w i t h " m o b i l e " s u r f a c e s .
I l l u s t r a t i o n s of v e l o c i t y v e c t o r s f o r both k i n d s of these
p a r t i c l e s a r e i l l u s t r a t e d i n F i g u r e 2. T h i s c o n c e p t h a s been
u s e f u l i n e x p l a i n i n g many d r o p and b u b b l e phenomena. F o r e x a m p l e ,
i t has been f o u n d t h a t t r a c e amounts o f s u r f a c e - a c t i v e materials
c a n h i n d e r t h e d e v e l o p m e n t of i n t e r n a l c i r c u l a t i o n by means of a
d i f f e r e n t i a l surface pressure. S m a l l b u b b l e s r i s i n g slowly are
apt t o behave l i k e p a r t i c l e s w i t h r i g i d s u r f a c e s . T h i s phenomenon
can l e a d t o a d e c r e a s e i n k a s t h e age o f a b u b b l e increases
( L e v i c h , 1956). L a r g e r b u f e b l e s , r i s i n g more q u i c k l y * may sweep
t h e i r f r o n t s u r f a c e f r e e of t r a c e i m p u r i t i e s and t h e r e f o r e e s c a p e

15. MOO-YOUNG A N D BLANCH Biological Reactor Design 339
the c o n t a m i n a t i o n e f f e c t o f s u r f a c t a n t s as i l l u s t r a t e d i n F i g u r e
3· These e f f e c t s lead to s i g n i f i c a n t v a r i a t i o n s of k with L
&hanging b u b b l e s i z e and a g i t a t i o n power ( C a l d e r b a n k and

oo-foung. 1961). 6
In fermentation p r a c t i c e , c l e a n b u b b l e systems a r e
p r o b a b l y r a r e l y a c h i e v e d and i t i s c o n s e r v a t i v e l y s a f e t o b a s e a
d e s i g n on c o n t a m i n a t e d r i g i d i n t e r f a c e b e h a v i o r .
INTERPARTICLE TRANSFER RATES
Particles i n stagnent Environmpnts
F o r non-moving s u b m e r g e d p a r t i c l e s ( w i t h r i g i d o r m o b i l e
s u r f a c e ) i n a s t a g n a n t médium» mass t r a n s f e r o c c u r s o n l y by r a d i a l
diffusion. Re = Gr = 0
Sh = Nu = 2 (1)
As t h e l o w e r l i m i t f o r S h . we w i l l s e e t h a t t h i s v a l u e u s u a l l y
v a n i s h e s f o r b u b b l e mass t r a n s f e r , b u t i t may become s i g n i f i c a n t
when a p p l i e d t o s m a l l l i g h t p a r t i c l e s , e . g . , m i c r o b i a l c e l l s .
Pseudostagnant l i q u i d environments can exist i n viscous
fermentations and/or w i t h w e l l d i s p e r s e d s i n g l e c e l l s as
i l l u s t r a t e d i n c a s e ( a ) o f F i g u r e 3.
Moving P a r t i c l e s with Rigid Surfais
A range of these cases can occur i n packed-bed,

trickle-bed or f r e e - r i s e or f r e e - f a l l dispersed-phase reactor
s y s t e m s . F o r c r e e p i n g f l o w , Re < 1, ( e . g . . c e r t a i n p a c k e d - b e d
i m m o b i l i z e d - e n z y m e r e a c t o r s ) t h e o r y d e v e l o p d by L e v i c h (1962) and
o t h e r s show t h a t :
Sh = 0 . 9 9 R e 1 / 3
Sc 1 / 3
= 0.99 P e 1 / 3
(2)
I n t h e r e g i m e 10 < Re < 1 0 ^ ( e . g . . c e r t a i n t r i c k l e - b e d reactors)
Sh = 0 . 9 5 R e 1 / 2
Sc 1 / 3
(3)
At high a g i t a t i o n i n t e n s i t i e s , t u r b u l e n c e i s expected t o
a f f e c t t h e mass t r a n s f e r r a t e s a t s o l i d p a r t i c l e surfaces.
H o w e v e r , i n t h e s e c a s e s t h e a c t u a l p a r t i c l e v e l o c i t y i s unknown
and c o n v e n t i o n a l R e y n o l d s numbers c a n n o t be d e d u c e d .
An a p r o p r i a t e Re-number e x p r e s s i o n c h a r a c t e r i s t i c o f l o c a l
i s o t r o p i c t u r b u l e n c e c a n be d e r i v e d u s i n g t h e r o o t - m e a n - s q u a r e
f l u c t u a t i n g v e l o c i t y p o s t u l a t e d by B a t c h e l o r ( 1 9 5 1 ) .

α b e
Figure 2. Surfactant effects on bubble drop surface-flows. Key: a, zero relative

particle velocity; b, low relative particle velocity; and c, high relative particle
velocity. Reproduced, with permission, from Ref. 38. Copyright 1981, Springer-
Verlag.
Figure 3. Possible conditions of the momentum boundary layer around a sub

merged solid sphere with increasing relative velocity. Key: a, envelope of pseudo-
stagnant fluid; b, streamline flow; c, flow separation and vortex formation; d, vor
tex shedding; e, localized turbulent eddy formation. Reproduced, with permission,
from Ref. 38. Copyright 1981, Springer-Verlag.

15. MOO-YOUNG AND BLANCH Biological Reactor Design 341
4/3 2/3 1/3

Re e = d Ρ ΓΡ/Vl (4)
C a l d e r b a n k and M o o - Y o u n g ( 1 9 6 1 ) d e v e l o p e d a c o r r e l a t i o n for
r i g i d - s u r f a c e p a r t i c l e m a s s t r a n s f e r i n b i o r e a c t o r s i n terms of
the e n e r g y i n p u t t o t h e s y s t e m as f o l l o w s :
3 / 4 1 / 3
Sh = 0.13 Re Sc (5)
e
l y
where k i s s e e n t o be
L d e p e n d e n t on ( P / V ) ^ » a dependence w h i c h
may be m a s k e d by t h e e f f e c t o f p o w e r on i n t e r f a c i a l a r e a , as
discussed l a t e r .
Moving P a r t i c l e s with Mnhile Surfaces
Mobile surface f l u i
l e s s s p h e r e - l i k e t h a n t h a t of r i g i d - s u r f a c e p a r t i c l e s .
By v i s c o u s i n t e r a c t i o n w i t h t h e c o n t i n u o u s phase» o s c i l l a t i n g
shape v a r i a t i o n s o f l i q u i d d r o p s and gas b u b b l e s o c c u r * and f o r Re
1» m o b i l e s u r f a c e f l u i d p a r t i c l e s i n f r e e - r i s i n g o r falling
c o n d i t i o n s move i n a w o b b l i n g o r s p i r i a l - l i k e manner, w h i c h has a
marked i n f l u e n c e on mass t r a n s f e r r a t e s . As b e f o r e , we can a r r i v e
at d i f f e r e n t c o r r e l a t i o n s f o r d i f f e r e n t bulk flow regions. These
a r e summarized b e l o w :
1 / 2 1 / 2
Sh = 0.65( £ ) Pe (6)
^d
For h i g h e r Re-numbers. H i g b i e (1935) gave an equation which takes

the form:
1 / 2
Sh = 1.13 Pe (7)
R e f i n e m e n t s between the two extremes are possible.
I n t h i c k v i c o u s l i q u i d s (μ > 70 cp) l a r g e s p h e r i c a l - c a p
b u b b l e s are f r e q u e n t l y e n c o u n t e r e d and t h e r e l e v a n t correlation
is :
1 / 2
Sh = 1.31 Pe (8)
Non-Newtonian. F l o y Effects
When t h e l i q u i d phase e x h i b i t s n o n - N e w t o n i a n b e h a v i o r , t h e
mass t r a n s f e r c o e f f i c i e n t w i l l c h a n g e due t o a l t e r a t i o n s i n t h e
f l u i d v e l o c i t y p r o f i l e a r o u n d t h e submerged p a r t i c l e s . The t r e n d s
f o r b o t h m a s s t r a n s f e r and d r a g c o e f f i c i e n t a r e a n a l o g o u s . As
b e f o r e f o r N e w t o n i a n f l u i d s , two t y p e s o f i n t e r f a c i a l b e h a v i o r
need t o be c o n s i d e r e d .
For mobile-surface p a r t i c l e s i n power-law fluids» Hirose

and Moo-Young (1969) have o b t a i n e d a c o r r e l a t i o n f a c t o r f o r k, f o r

s i n g l e b u b b l e s , b a s e d on s m a l l p s e u d o p l a s t i c d e v i a t i o n s r r o m
N e w t o n i a n b e h a v i o r (0.7 < η < 1.0). They a l s o p r o v i d e some d a t a
on d r a g c o e f f i c i e n t s a s f u n c t i o n s o f t h e power-law and Bingham
plastic f l u i d s , with mobile i n t e r f a c e s , using perturbation
a n a l y s i s , and p r o v i d e d c o r r e l a t i o n f a c t o r s f o r t h e e n h a n c e m e n t o f
mass t r a n s f e r .
F o r r i g i d s u r f a c e b e h a v i o r , t h e mass t r a n s f e r c o e f f i c i e n t s
c a n be o b t a i n e d f r o m t h e r e s u l t s o f W e l l e k a n d H u a n g ( 1 9 7 0 ) .
M o o - Y o u n g a n d H i r o s e ( 1 9 7 2 ) showed t h a t an a d d i t i o n a l e f f e c t o f
" i n t e r f a c i a l s l i p " by a d d i t i v e s can o c c u r i n p r a c t i c e .
Effect Of Bulk Mixing PattPrns
In a d d i t i o n t
c o e f f i c i e n t , k » and t h
L
gas and l i q u i d phase mass b a l a n c e e q u a t i o n s f o r t h e s p e c i e s

t r a n s f e r r e d d e p e n d s o n t h e f l o w b e h a v i o r o f b o t h gas and l i q u i d
phase·
In low v i s c o s i t y l i q u i d s i t i s r e a s o n a b l y w e l l e s t a b l i s h e d
t h a t i n s m a l l s t i r r e d - t a n k s t h e l i q u i d phase can be c o n s i d e r e d t o
be " p e r f e c t l y m i x e d " ( W e s t e r t e r p , e t a l . . 1 9 6 3 ) . Under t h e s e
c o n d i t i o n s , t h e g a s p h a s e h a s a l s o g e n e r a l l y b e e n assumed t o be
w e l l m i x e d i n t a n k s o p e r a t i n g above c r i t i c a l i m p e l l e r s p e e d . In
l a r g e t a n k s , h o w e v e r , t h e s i t u a t i o n i s l e e s c l e a r , and c a r e must
be t a k e n t o e s t a b l i s h t h e b e h a v i o r o f b o t h p h a s e s . I n c a s e s where
the d e g r e e o f gas a b s o r p t i o n i s h i g h , t h e a s s u m p t i o n s o f w e l l
m i x e d o r p l u g - f l o w o f t h e g a s p h a s e may p r e d i c t s i g n i f i c a n t l y
d i f f e r e n t gas a b s o r p t i o n r a t e s . S h a f t l e i n and R u s s e l l ( 1 9 6 8 )
p r e s e n t d e s i g n e q u a t i o n s f o r v a r i o u s m o d e l s o f g a s and liquid
flows.
INTRAPARTICLE BIOREACTION RATES
General Concepts
In some b i o c h e m i c a l s y s t e m s t h e l i m i t i n g m a s s t r a n s f e r
s t e p s h i f t s from a g a s - l i q u i d or s o l i d - l i q u i d i n t e r f a c e (as
discussed e a r l i e r ) to the i n t e r i o r of s o l i d p a r t i c l e s . The most
i m p o r t a n t c a s e s a r e s o l i d s u b s t r a t e s and c e l l a g g r e g a t e s ( s u c h as
m i c r o b i a l f l o e s , c e l l u l a r t i s s u e s , e t c . ) and i m m o b i l i z e d enzymes
(gel-entrapped or s u p p o r t e d i n s o l i d m a t r i c e s ) . In the former,
d i f f u s i o n of o x y g e n ( o r o t h e r n u t r i e n t s ) t h r o u g h t h e p a r t i c l e
l i m i t s m e t a b o l i c r a t e s , w h i l e i n the l a t t e r , s u b s t r a t e r e a c t a n t or
p r o d u c t d i f f u s i o n i n t o o r out o f t h e enzyme c a r r i e r o f t e n l i m i t s
the o v e r a l l g l o b a l b i o r e a c t i o n r a t e s .
G e n e r a l i z e d mass b a l a n c e s f o r the a b o v e s c e n a r i o s can be

s e t up and v a r i o u s c a s e s where mass t r a n s f e r r e s i s t a n c e w i t h i n the

p a r t i c l e may become i m p o r t a n t can be i d e n t i f i e d .
Oxygen Transfer in Mold f fil l e t s
M a r s h a l l and A l e x a n d e r (1960) d i s c o v e r e d t h a t f o r s e v e r a l
p e l l e t - f o r m i n g f u n g i a "cube r o o t " g r o w t h curve f i t t h e i r data
s i g n i f i c a n t l y b e t t e r t h a n t h e s t a n d a r d e x p o n e n t i a l growth model.
I t was s u g g e s t e d t h a t t h i s was p r o b a b l y due t o t h e e f f e c t s o f
i n t r a p a r t i c l e d i f f u s i o n ; a n u t r i e n t was n o t d i f f u s i n g i n t o t h e
p a r t i c l e f a s t enough t o m a i n t a i n u n r e s t r i c t e d g r o w t h . I t was s o o n
r e a l i z e d t h a t o x y g e n was t h e l i m i t i n g n u t r i e n t .
P h i l l i p s (1966) m e a s u r e d o x y g e n d i f f u s i o n i n p e l l e t s of
ΡΡΠ i ci 11 i iim chrysogenum by f i r s t a s s u m i n g t h a t d i f f u s i o n i s t h e
mechanism that s u p p l i e
p e l l e t and t h a t t h e mas
comparatively small. Y a n o e t . a l . ( 1 9 6 1 ) and Koyayashi» e t a l .
(1973 b) d i d t h e same w i t h A s p e r g i l l u s n i g e r p e l l e t s . By t a k i n g
i n t o a c c o u n t t h e e f f e c t o f i n t r a p a r t i c l e diffusion» K o b a y a s h i and
S u z u k i ( 1 9 7 6 ) w e r e a b l e t o c h a r a c t e r i z e t h e k i n e t i c b e h a v i o r of
t h e e n z y m e g a l a c t o s i d a s e w i t h i n m o l d p e l l e t s o f HûJLLiexfillâ.
vinacea.
K o b a y a s h i , e t a l . ( 1 9 7 3 b) s t u d i e d t h r e e c a s e s : (1)
u n i f o r m r e s p i r a t i o n a c t i v i t y t h r o u g h o u t m y c e l i a l p e l l e t s * (2)
r e s p i r a t i o n a c t i v i t y as a f u n c t i o n of age d i s t r i b u t i o n w i t h i n t h e
p e l l e t * (3) r e s p i r a t i v e a c t i v i t y a d a p t a t i o n to the l o c a l oxygen
c o n c e n t r a t i o n w i t h i n the p e l l e t . I n c a s e 1» i t was f o u n d t h a t t h e
e f f e c t i v e n e s s f a c t o r ( E ) i s e q u a l t o t h e r a t i o of the s p e c i f i c
f
r e s p i r a t i o n r a t e of a p e l l e t to the r e s p i r a t i o n r a t e of w e l l
dispersed filamentous mycelia. The t h e o r e t i c a l and e x p e r i m e n t a l
r e s u l t s f o r e a c h c a s e a r e g i v e n i n F i g u r e 4. I t i s seen t h a t the
t h r e e c a s e s c o n s i d e r e d gave s i m i l a r r e s u l t s and i t i s d i f f i c u l t t o
d i s c r i m i n a t e b e t w e e n them w i t h the l i m i t e d e x p e r i m e n t a l d a t a
available.
Immobilized Enzyme Kinetics
I n t r a p a r t i c l e d i f f u s i o n c a n h a v e a s i g n i f i c a n t e f f e c t on
t h e k i n e t i c b e h a v i o r of enzymes i m m o b i l i z e d o n s o l i d c a r r i e r s o r
entrapped i n gels. In t h e i r b a s i c a n a l y s i s of t h i s p r o b l e m .
M o o - Y o u n g and K o b a y a s h i ( 1 9 7 2 ) d e r i v e d a g e n e r a l m o d u l u s and
effectiveness factor. The r e s u l t s a l s o p r e d i c t e d possible
m u l t i p l e s t e a d y - s t a t e s as w e l l as u n s t a b l e s i t u a t i o n s f o r c e r t a i n
s y s t e m s . W h i l e t h e s e r e s u l t s a r e v e r y i n t e r e s t i n g i t s h o u l d be
r e m e m b e r e d t h a t t h e y a r e p r i m a r i l y m a t h e m a t i c a l and a w a i t
extensive experimental support data.


Enzymatic D e g r a d a t i o n of Insoluble Substrates
When t h e s u b s t r a t e i n a b i o r e a c t o r i s a w a t e r - s o l u b l e
material (e.g., c e l l u l o s e , o i l s ) , the e f f e c t s of i n t r a p a r t i c l e
m a s s t r a n s f e r may be i m p o r t a n t . In such systems, e x t r a c e l l u l a r
enzymes c a n b r e a k down s u b s t r a t e e v e n t u a l l y i n t o w a t e r s o l u b l e
c o m p o n e n t s , as p r o d u c t s , o r as i n t e r m e d i a t e s f o r c o n s u m p t i o n by
m i c r o - o r g a n i s m s ( O k a z a k i and Moo-Young, 1 9 7 8 ) .
I f a s o l i d s u b s t r a t e i s s u f f i c i e n t l y p o r o u s , t h e enzyme
can d i f f u s e i n t o i t and d e g r a d a t i o n can proceed i n s i d e the
m a t e r i a l . The w a t e r - s o l u b l e s u b s t r a t e f r a g m e n t s must a l s o d i f f u s e
out o f t h e s o l i d m a t r i x t h r o u g h t h e s e same p o r e s i n t o t h e b u l k
s o l u t i o n w h e r e t h e y may be s u b j e c t t o f u r t h e r e n z y m a t i c a t t a c k .
The r e a c t i o n may c o n c u r r e n t l y p r o c e e d a t t h e e x t e r i o r o f t h e
m a t r i x s u r f a c e and f o r
much o f t h e d e g r a d a t i o n
t h e e f f e c t i v e n e s s f a c t o r and g e n e r a l m o d u l u s i s c o n v e n i e n t i n
s o l v i n g the r e l e v a n t d i f f e r e n t i a l e q u a t i o n s .
BIOREACTOR EQUIPMENT PERFORMANCE
Bubble-Columns
P n e u m a t i c a l l y a g i t a t e d g a s - l i q u i d r e a c t o r s may show w i d e
v a r i a t i o n s i n height to d i a m e t e r r a t i o s . In the p r o d u c t i o n of
baker's y e a s t , a tank-type c o n f i g u r a t i o n w i t h a r a t i o of 3 to 1 i s
common i n d u s t r i a l l y . Tower t y p e s y s t e m s have h e i g h t t o d i a m e t e r
r a t i o s of 6 to 1 or more. As w o u l d be e x p e c t e d , t h e b e h a v i o r o f
b o t h gas and l i q u i d p h a s e s may be q u i t e d i f f e r e n t i n t h e s e c a s e s .
I n g e n e r a l t h e gas phase r i s e s through the l i q u i d phase i n
p l u g - f l o w , under the a c t i o n of g r a v i t y , i n both types of system.
A v a r i e t y o f c o r r e l a t i o n s f o r k.a a r e a v a i l a b l e i n t h e
l i t e r a t u r e f o r b u b b l e - c o l u m n s . G e n e r a l l y ^ h e y a r e of t h e f o r m
n
k a
L = const. V (9)
where η i s u s u a l l y i n t h e r a n g e o f 0 . 9 t o u n i t y . Y o s h i d a and
A k i t a ( 1 9 6 5 ) c o r r e l a t e t h e o v e r a l l mass t r a n s f e r c o e f f i c i e n t s i n
b u b b l e columns i n t h e f o r m :
k
i °
a 2
„ 1/2 rri 0.62 _3 2 0.31
- V = o
- 6
^ 1
Φ - 1 ( 1 0 )
A r e c e n t r e v i e w o f b u b b l e - c o l u m n s by S c h u g e r l . e t a l .
( 1 9 7 7 . 1978) examines s i n g l e and m u l t i s t a g e columns and a v a r i e t y
of l i q u i d phases. A c o r r e l a t i o n f o r k a i s proposed f o r porous
T
and p e r f o r a t e d p l a t e s p a r g e r s (cgs u n i t s ) :

1 , 5 8
k.a = 0.0023 (V / d ) R (11)
L S D
Many o f t h e a v a i l a b l e c o r r e l a t i o n s f o r k a h a v e b e e n
L
o b t a i n e d on s m a l l s c a l e e q u i p m e n t , and have n o t t a k e n c o g n i z a n c e
o f t h e u n d e r l y i n g l i q u i d hydrodynamics. B h a v a r a j u , e t a l . (1978)
propose a design procedure b a s e d on t h e d i f f e r e n c e i n bubble s i z e
c l o s e t o t h e o r i f i c e and i n t h e l i q u i d b u l k . P r o v i d e d t h e l i q u i d
i s t u r b u l e n t * t h e e q u i l i b r i u m b u b b l e s i z e i n t h e b u l k w i l l be
independent of t h e s i z e a t f o r m a t i o n . The h e i g h t o f t h e r e g i o n
around t h e o r i f i c e where t h e bubble f o r m a t i o n p r o c e s s o c c u r s i s a
f u n c t i o n o f s p a r g e r g e o m e t r y and gas f l o w r a t e and i n l a b o r a t o r y
s c a l e e q u i p m e n t t h i s h e i g h t may be s i g n i f i c a n t f r a c t i o n o f t h e
t o t a l l i q u i d h e i g h t (up t o 3 0 % ) . I n p l a n t s c a l e e q u i p m e n t *
h o w e v e r , t h i s g e n e r a l l y r e p r e s e n t s l e s s t h a n 5% o f t h e t o t a l
l i q u i d height. Thus c o r r e l a t i o n s d e v e l o p e d on s m a l l s c a l e
a p p a r a t u s need t o be r e v i e w e
a r e a w i t h column h e i g h t
Systems w i t h S t a t i o n a r y Intpmak
Several laboratory scale devices which i n c l u d e i n t e r n a l

e l e m e n t s t o enhance mass t r a n s f e r r a t e s have a p p e a r e d . These
include d r a f t - t u b e s , m u l t i p l e sieve p l a t e s staged along the length
o f t h e c o l u m n , and s t a t i c m i x i n g e l e m e n t s .
A c o n s i d e r a b l e l i t e r a t u r e e x i s t s on d r a f t - t u b e columns*
w h e r e l i q u i d i s c i r c u l a t e d due t o a b u l k d e n s i t y d i f f e r e n c e
b e t w e e n t h e i n n e r c o r e a n d t h e s u r r o u n d i n g a n n u l a r s p a c e . The
downcoming l i q u i d i n t h e a n n u l a r s p a c e e n t r a i n s a i r b u b b l e s * and
t h u s h o l d u p i n t h e c e n t r a l c o r e and a n n u l a r r e g i o n w i l l be
different. S e v e r a l r e p o r t s o n s m a l l s c a l e a i r l i f t columns as
b i o r e a c t o r s have a p p e a r e d . C h a k r a v a r t y . e t a l . (1973) examined
gas h o l d u p a t v a r i o u s p o s i t i o n s i n a 10 cm d i a m e t e r c o l u m n . A
r e c t a g u l a r a i r l i f t h a s b e e n r e p o t e d ( G a s n e r , 1974) and a i r l i f t s
w i t h e x t e r n a l r e c i r c u l a t i o n ( K a n a z a w a . 1 9 7 5 ) have been p r o p o s e d .
The o v e r a l l mass t r a n s f e r c o r r e l a t i o n s f o r a i r l i f t d e v i c e s a r e
u s u a l l y expressed a s :
k. a = const. V (12)
L· S
I n d u s t r i a l l y , p l a n t s c a l e a i r l i f t d e v i c e s h a v e b e e n u s e d by
J a p a n e s e , B r i t i s h , F r e n c h a n d USA c o m p a n i e s m a i n l y f o r SCP
p r o d u c t i o n and w a s t e t r e a t m e n t
S t a t i c m i x i n g e l e m e n t s have been i n c o r p o r a t e d i n t o a i r l i f t
devices with the o b j e c t i v e s o f p r o v i d i n g a d d i t i o n a l m i x i n g and
h e n c e g r e a t e r mass t r a n s f e r c a p a b i l i t i e s . S t a t i c mixers are
b e c o m i n g i n c r e a s i n g l y m o r e common i n o x i d a t i o n ponds f o r

b i o l o g i c a l wastewater t r e a t m e n t . H e r e , f i n e b u b b l e s may be
produced as t h e g a s - l i q u i d m i x t u r e passes through t h e m i x i n g
element. These a r e u s u a l l y 18-24 i n c h e s i n d i a m e t e r , and a r e
placed over sparger pipes. A f a i r l y intense l i q u i d c i r c u l a t i o n
c a n be d e v e l o p e d b y s u c h m i x e r s d u e t o e n t r a i n m e n t b y t h e
g a s - l i q u i d j e t r i s i n g from the m i x i n g element.
Mpchaniral Stirred Tanks
Non-Viscous Systems.
The p r e v i o u s s e c t i o n s d e a l t w i t h g a s - l i q u i d c o n t a c t i n g
w i t h o u t m e c h a n i c a l a g i t a t i o n i n such d e v i c e s a s b u b b l e - c o l u m n s a n d
a i r l i f t towers. To o b t a i n b e t t e r g a s - l i q u i d contacting,
mechanical a g i t a t i o n i s o f t e n r e q u i r e d . The d i s c u s s i o n i s
confined to baffled sparge
A e r a t i o n by s u r f a c t i o
wastewater treatment f a c i l i t i e s ( Z l o k a r n i k , 1978).
For p a r t i c u l a t e s , such as c e l l s , i n s o l u b l e s u b s t r a t e s , o r
i m m o b i l i z e d - e n z y m e s , t h e i n t e r f a c i a l a r e a c a n be d e t e r m i n e d f r o m
d i r e c t a n a l y s e s s u c h as by m i c r o s c o p i c e x a m i n a t i o n . F o r gas
b u b b l e s and l i q u i d d r o p s , a c a n be e v a l u a t e d f r o m s e m i - e m p i r i c a l
correlations. Two c a s e s a r e i d e n t i f i e d :
(a) For "coalescing** air-water dispersions,
4
Ρ "°· 0 5
a = 0.55 φ V s- 0 5
and
-0.17 0 2 7 ,
U , Z /
d n = 0.27 C l ) V + 9 χ 10 *
Β γ s
( b ) F o r " n o n - c o a l e s c i n g " a i r - e l e c t r o l y t e s o l u t i o n dispersions»
0.7 0.3
a = 0.15 (I) V
and
B = °· Ψ 89
V
1 7
d Ρ "°· 0 17
3
I n t h e e q u a t i o n , (P/V) i s i n W a t t s / m , and V g i s i n m/s.
American Chemical
Society Library
1155 16th St., N.W.
Washington,
ACS Symposium Series; D.C. Society:
American Chemical 20036Washington, DC, 1983.
It is seen that there is a significant effect of

e l e c t r o l y t e s on t h e c o r r e l a t i o n s * E l e c t r o l y t e s and s u r f a c t a n t s
i n h i b i t bubble coalesence r e s u l t i n g i n the f o r m a t i o n of smaller
b u b b l e s and i n c r e a s e d i n t e r f a c i a l a r e a than n o r m a l l y found i n the
c l e a n water system.
A v e r y h i g h gas f l o w r a t e » l i q u i d b l o w - o u t f r o m t h e v e s s e l
may o c c u r * In a d d i t i o n , t h e above equations are applicable
p r o v i d e d t h a t t h e i m p e l l e r i s n o t f l o o d e d by t o o h i g h a g a s flow
r a t e and t h e r e i s no g r o s s s u r f a c e - a e r a t i o n due t o g a s b a c k - m i x i n g
at the surface of the l i q u i d . Equations are available to
c a l c u l a t e these c r i t e r i a (Calderbank, 1959; W e s t e r t e r p » 1963)·
The e f f i c i e n c y o f g a s - l i q u i d c o n t a c t i n g has a l r e a d y been

p r e s e n t e d i n terms o f t h e f u n d a m e n t a l s o f k. a n d a separately:
the o v e r a l l correlation
applicability. Severa
correlations. They u s u a l l y take the form
m
ρ
k a L = const. (^) V g
n
(17)
and t h e i r r a n g e s o f applicability are compared to bubble columns

and a i r l i f t d e v i c e s i n F i g u r e 5.
I n r e c e n t y e a r s , e x t e n s i v e s t u d i e s have b e e n c a r r i e d o u t
u s i n g p h y s i c a l r a t h e r than c h e m i c a l r e a c t i o n measurements f o r t h e
evaluation of k . a On t h i s b a s i s . S m i t h V a n t R i e t and M i d d l e t o n
f
(197 7) f o u n d t n a t i n g e n e r a l , t h e f o l l o w i n g c o r r e l a t i o n s a p p l y t o
a w i d e v a r i e t y o f a g i t a t o r t y p e s , s i z e s and D/T r a t i o s :
(a) For "coalescing" air-water dispersion
k a = 0.01 ^)
L ν β
0
· 4
(18)
(b) For "non-coalescing"* a i r - e l e c t r o l y t e solution dispersions
Ρ ° · 4 7 5
0 4
l ^ a = 0 . 0 2 (|) ν β
ϋ
·* (19)
-1 3
Both e q u a t i o n s a r e i n SI u n i t s k . a i n s » (P/V) i n W/m · V in g
m . The a c c u r a c y o f t h e c o r r e l a t i o n s i s ± 20% and ± 35% w i t h 95%

c o n f i d e n c e f o r t h e a b o v e two e q u a t i o n s , respectively.
From Figure 5» it is seen that for comparable power

MOO-YOUNG A N D BLANCH Biological Reactor Design 349
Figure 5. Aeration efficiencies of various gas-liquid contacting devices (air-

electrolyte systems). Reproduced, with permission, from Ref. 38. Copyright 1981,
Springer-V erlag.

i n p u t s * t h e m a g n i t u d e o f k ^ a o b t a i n e d i s a b o u t t h e same w h e t h e r
m i x i n g i s done m e c h a n i c a l l y i n s t i r r e d - t a n k s o r p n e u m a t i c a l l y i n
bubble-columns or a i r l i f t d e v i c e s . However, only mechanically
a g i t a t e d systems are capable of a t t a i n i n g h i g h v a l u e s of k a L
r q u i r e d ( e . g . * i n some a n t i b i o t i c f e r m e n t a t i o n s and t h e a c t i v a t e d
s l u d g e method o f t r e a t i n g w a s t e w a t e r ) . Non-mechanically a g i t a t e d
s y s t e m s would r e s u l t i n l i q u i d blowout b e f o r e r e a c h i n g these h i g h
aeration rates.
It s h o u l d be stressed that none of the overall

c o r r e l a t i o n s f o r k a had u n i v e r s a l a p p l i c a b i l i t y .
L The p r o b l e m i s
t h a t any s c a l e - u p p r o c e d u r e b a s e d o n e q u a l i z i n g k ^ a o f b o t h s c a l e s
a c c o r d i n g t o a g i v e n c o r r e l a t i o n may c a u s e o t h e r p h y s i c a l c r i t e r i a
t o be v i o l a t e d . A l s o * d e p e n d i n g on b i o l o g i c a l demands and
t o l e r a n c e s * o t h e r c r i t e r i a may be m o r e i m p o r t a n t . F o r example*
i n c r e a s i n g t h e k a c a n sometime
L
in highly turbulent fermentatio
Viscous Systems
Two t y p e s of viscous fermentations need t o be distinguished:
(a) m y c e l i a l fermentations ( e . g . * molds* a c t i n o m y c e t e s ) where t h e

v i s c o s i t y i s due t o t h e m i c r o b i a l n e t w o r k structure dispersed in
c o n t i n u o u s aqueous p h a s e .
(b) p o l y s a c c h a r i d e f e r m e n t a t i o n s w h e r e t h e v i s c o s i t y i s due t o
polymers i n the continuous aqueous p h a s e r e s u l t i n g i n an
e s s e n t i a l l y homogeneous* v i s c o u s l i q u i d .
The f i r s t t y p e o f f e r m e n t a t i o n b r o t h c a n be s i m u l a t e d b y
m a t e r i a l s u c h as p a p e r p u l p * w h i c h has a m a c r o s c o p i c s t r u c t u r e
analogous t o f u n g a l h y p h a e * s u s p e n d e d i n w a t e r . The second t y p e
may be s i m u l a t e d by aqueous p o l y m e r s o l u t i o n s o f known p r o p e r t i e s .
Sideman* e t a l . (1966) have p r o p o s e d t h e f o l l o w i n g form o f

a general c o r r e l a t i o n for the l i q u i d phase mass transfer
coefficient.
D L
A
<pD >
L
(
σ > (
M a > V
A d d i t i o n a l d i m i n s i o n l e s s g r o u p s be i n c o r p o r a t e d t o a c c o u n t f o r
geometric v a r i a b l e s * e . g . H / T » D/T. L T h e a b o v e e q u a t i o n c a n be
a l t e r e d t o r e l a t e k a t o t h e s p e c i f i c power i n p u t .
L
L o u c a i d e s and McManamey ( 1 9 7 3 ) e x a m i n e d r a t e s i n p a p e r
pulp supensione s i m u l a t i n g filamentous fermentation media. Tank
g e o m e t r i e s and i m p e l l e r g e o m e t r i e s were b o t h v a r i e d ; vessel

volumes ranging from 5 to 72 l i t e r s * Analogous t o the r e s u l t s f o r

n o n - v i s c o u s solutions» k a as found t o c o r r e l a t e v e i l w i t h
L
v a r i a t i o n s i n tank diameter*
k a = C j (T/H ) Ν D ζ-Τ .5
L L
(21)
By v a r y i n g the i m p e l l e r blade dimensions» v a r i a t i o n s i n power per

u n i t volume were made a t a c o n s t a n t i m p e l l e r speed* A t low power
p e r u n i t volumes t h e r e was a l i n e a r i n c r e a s e i n kâ » w h i c h
c o r r e l a t e d w i t h P/V w i t h an exponent of 0.9 t o 1.2* Hteyond the
breakpoint* the exponent r e l a t i n g the P/V dependence was 0*53* I n
both r e g i o n s , the k a dependen
t h e 0*3 power. Tfies
e a r l i e r by B l a k e b r o u g h and Sambamurthy (1966)· and Hamer and
Blakebrough (1963)* obtained i n s m a l l e r s c a l e v e s s e l s * a l s o u s i n g
paper p u l p s u s p e n s i o n s . Other r e f e r e n c e s on t h e a e r a t i o n o f
v i s c o u s non-Newtonian f e r m e n t a t i o n b r o t h a r e Banks (1977)· and
Blanch and Bhavaraju (1976).
NOMENCLATURE
a s p e c i f i c i n t e r f a c i a l area (based on d i s p e r s i o n
volume)
C Concentration of s o l u t e i n bulk l i q u i d (generally)
C Concentration of s o l u t e i n bulk media ( f o r

i n t r a p a r t i c l e d i f f u s i o n case)
D D i l u t i o n r a t e ; i m p e l l e r diameter
L i q u i d phase d i f f u s i v i t y
d Diameter of p a r t i c l e as an equi-volume sphere
Bubble diameter
Effectiveness factor
L i q u i d height i n r e a c t o r
L i q u i d phase mass t r a n s f e r c o e f f i c i e n t
C o n t i n u e d on n e x t page

V o l u m e t r i c l i q u i d phase mass t r a n s f e r coefficient
m An e x p o n e n t
Ν Speed o f agitator
Ρ Agitator power r e q u i r e m e n t s f o r u n g a s s e d systems
Ρ Agitator requirements f o r g a s - l i q u i d dispersions

g
Τ Temperature; tank diameter
V Volume o f r e a c t o r c o n t e n t s
V Superficial gas v e l o c i t y
6
VVM Volume
&£££k L e t t e r s
Ρ Viscosity ( d y n a m i c ) o f c o n t i n u o u s phase
P
a Apparent v i s c o s i t y (dynamic)
Viscosity (dynamic) of d i s p e r s e d phase
ν K i n e m a t i c v i s c o s i t y o f c o n t i n u o u s phase
Ρ D e n s i t y o f c o n t i n u o u s phase
Φ Holdup o f d i s p e r s e d phase.
A b b r e v i a t i o n s f o r D i m e n s i o n l e s s Groups
Gr G r a s h o f number f o r mass t r a n s f e r ( b a s e d o n
p a r t i c l e environment d e n s i t y d i f f e r e n c e )
Nu N u s s e l t number
Pe P e c l e t number ( s i n g l e particles)
Pr P r a n d t l number
Re R e y n o l d s number f o r p a r t i c l e s
Re R e y n o l d s numbers f o r i s o t r o p i c turbulence
e
Sh Sherwood number
Se S c h m i d t number

Literature Cited
1. Banks, G.T., in "Topics in Enzymes and Fermentation

Biotechnology", Wiseman (Ad.), John Wiley and Sons,
1977, 1, 72.
2. Batchelor, G.K., Proc. Camb. Phil. Socl., 1951, 47, 359
3. Bhavaraju, S.M., Mashelkar, R.A., Blanch, H.W., AIChE J.,

1978, 24 1063; AIChE J., 24, 1070
4. Bhavaraju, S.M., Russell, T.W.F., Blanch, H.W., AIChE J.,

1978, 24, 454
5. Blakebrough, Ν., Sambamurthy K., Biotechnol. Bioeng., 1966

8, 25.
6. Blanch, H.W., Bhavaraju, S.M., Biotechnol. Bioeng., 1976,

18. 745.
7. Calderbank, P.H., Trans. Inst. Chem. Engrs., 1959, 37, 173.
8. Calderbank, P.H., Moo-Young, M. Chem. Eng. Sci., 1961

16, 39.
9. Calderbank, P.H., Moo-Young, M., Trans. Inst. Chem. Engrs.,

1961, 39, 337.
10. Chakravarty, Y., Begum, S., Singh, A.D., Baruah, J.N.

Iyengar, M.S., Biotechnol. Bioeng. Symp., 1973, No. 4
363.
11. Finn, R.K., Bact. Rev., 1954, 18. 245.
12. Gasner, L.L., Biotechnol, Bioeng., 1974, 16, 1179
13. Hadamard, J . , Compt. Rend. Acad. Sci., 1911, 152, 1735.
14. Hamer, G., Blakebrough, Ν., J . Appl. Chem., 1963, 13, 517
15. Higbie, R., Trans. Amer. Inst. Chem. Engrs., 1935, 31, 365.
16. Hirose, T., Moo-Young, M., Can. J . Chem. Eng., 1969, 47,
265.
17. Kanazawa, M., in "SCP II," Tannenbaum and Wang (Eds.), MIT
Press, 1975.
18. Kobayashi, H., Suzuki, M., Biotechnol. Bioeng., 1976, 18, 37.
19. Kobayashi, T., Moo-Young, M., Biotechnol. Bioeng., 1973 a,

15, 47.

20. Kobayashi, T., van Dedem, Β., Moo-Young. M., Biotechnol.

Bioeng., 1973 b., 15,. 27.
21. Levich, V.G., "Physicochemical Hydrodynamics," 1962,

Prentice-Hall.
22. Loucaides, R., McManamey, W.J., Chem. Eng. Sci., 1973, 28,
2165.
23. Marshall, K.C., Alexander, M., J . Bacteriol., 1960, 80,

412.
24. Moo-Young, M., Hirose, T., Can. J . Chem. Eng., 1972, 50

128.
25. Moo-Young, M. Kobayashi,

50, 128.
26. Okazaki, M., Moo-Young. M., Biotechnol. Bioeng., 1978,

20, 637.
27. P h i l l i p s , H.H., Biotechnol. Bioeng., 1966, 8, 456.
28. Schugerl, K., Oels, U., Lucke, J., Adv. Biochem. Eng.,
1977, 7, 1.
29. Schugerl, Κ., Lucke, J., Lehman, J., Wagner, F., Adv.
Biochem. Eng., 1978, 8, 63.
30. Shaftlein, R.W., Russell, T.W.F.,Ind.Eng,Chem.,1968,

60(5), 13.
31. Sideman, S., Hortacsu, O., Fulton, J.W., Ind.Eng,Chem.,

1966, 58(7), 32.
32. Smith, J . , Van't Riet. K., Middleton, J . , Second European

Conf. on Mixing, April 1977, Preprint.
33. Wellek, R.M., Huang, C.C., Ind. Eng, Chem. Fund., 1970,
2 , 480.
34. Westerterp, Κ., Chem. Eng. Sci., 1963, 18, 157.
35. Yano, T., Kodama, T., Yamada, K., Agr. Biol. Chem., 1961,
25, 589.
36. Yoshida, F., Akita, Κ., AIChE J., 1965, 11, 9.
37. Zlokarnik, M., Adv. Biochem. Eng., 1978, 8, 133.
38. Moo-Young, Μ., Blanch, H.W., Adv. Biochem. Eng., 1981,

18, 2.

16
Reactor Design Fundamentals
Hydrodynamics,MassTransfer,HeatExchange,Control,and Scale-up
ALES PROKOP
Kuwait Institute for Scientific Research, Biotechnology Department,
P.O. Box 24885, Kuwait
For the convenience of d i s c u s s i n g the above subject

it i s h e l p f u l t
and s y n t h e s i s ) , i . e .
i n t o s e v e r a l orders of s t r u c t u r a l and f u n c t i o n a l
h i e r a r c h y , i n c l u d i n g b i o l o g i c a l and p h y s i c a l l e v e l s
(such as molecular, cellular, p o p u l a t i o n , r e a c t o r
and t e c h n o l o g i c a l l i n e ) , and to s e l e c t from a number
of elementary events systemic phenomena p l a y i n g a
d e c i s i v e r o l e a t each l e v e l of h i e r a r c h y . This
approach allows an i n t e g r a l d e s c r i p t i o n during pro-
cess synthesis while maintaining the s p e c i f i c pro-
p e r t i e s of each l e v e l . The b i o r e a c t o r l e v e l is dis-
tinguished by the f o l l o w i n g fundamental events and
f u n c t i o n s : b i o r e a c t o r hydrodynamics, mass t r a n s f e r ,
heat exchange, b i o r e a c t o r c o n t r o l and its scale-up.
This review will emphasize r e a c t o r s employed i n
m i c r o b i a l p r o t e i n production and the systems appro-
ach i s intended as a methodological a i d in process
research and development.
R e c e n t l y , t h e author advocated t h e a p p l i c a t i o n o f t h e systems

a p p r o a c h i n t h e a n a l y s i s and s y n t h e s i s o f a b i o t e c h n o l o g i c a l p r o -
c e s s ( 1 ) . The h i e r a r c h y o f a b i o l o g i c a l s y s t e m i n v o l v e s s t r u c t u -
r a l , f u n c t i o n a l , d e s c r i p t i v e , c o n t r o l a n d dynamic o r g a n i z a t i o n .
S t r u c t u r a l and f u n c t i o n a l h i e r a r c h i e s o f a b i o t e c h n o l o g i c a l p r o -
c e s s a r e d e p i c t e d i n T a b l e s I and I I . I n T a b l e I I a c o m p a r t m e n t a l
l e v e l i s o m i t t e d . T h i s i s p o s s i b l e as l o n g as t h e t r a n s l o c a t i o n
o f s u b s t a n c e s between o r g a n e l l e s and c y t o p l a s m i s n o t i m p o r t a n t .
The scheme o f f u n c t i o n s s h o u l d be u n d e r s t o o d i n b o t h d i r e c t i o n s ,
i . e . , f r o m h i g h e r l e v e l s t o l o w e r ( a n a l y s i s ) and v i c e v e r s a
( s y n t h e s i s ) , r e p r e s e n t i n g b a s i c a n d a p p l i e d r e s e a r c h . The f i r s t
On l e a v e f r o m : I n s t i t u t e o f M i c r o b i o l o g y , C z e c h o s l o v a k Academy
K I S R 620 o f S c i e n c e s , 142 20 P r a g u e 4, C z e c h o s l o v a k i a
0097-6156/83/0207-0355$06.50/0

Table I S t r u c t u r a l and F u n c t i o n a l H i e r a r c h i e s
of a B i o t e c h n o l o g i c a l Process
Level S t r u c t u r a l Elements Function
Molecular Atoms, molecules Synthesis of macromolecules
Organelles Macromolecules Duplication of organelles,

metabolic manifestation of c e l l s
Cell Organelles Translocation of substances,

complex b i o c h e m i c a l r e a c t i o n s ,
morphological d i f f e r e n t i a t i o n ,
cell cycle
Population Cells Growth and m u l t i p l i c a t i o n ,

i n t e r a c t i o n between c e l l s
Reactor Elementary zones Mass t r a n s f e r , h e a t exchange,

of r e a c t o r diffusion, etc.
B i o t e c h n o U n i t equipment Unit operations

logical
process
(line)
I n t e r n a t . J . G e n e r a l Systems 1982, j $ ( l ) , T a b l e V I I ( 1 )

16. PROKOP Reactor Design Fundamentals 357
Table I I Hierarchy of Functions of B i o t e c h n o l o g i c a l Processes
Molecular Level
1.1. C a t a b o l i c and a n a b o l i c m a n i f e s t a t i o n s o f l i v i n g systems
1.1.1. E n e r g y f o r m a t i o n
1.1.2. S y n t h e t i c p r o c e s s e s
1.1.3. M a c r o m o l e c u l a r d e c a y
1.2. C o n t r o l mechanisms
1.2.1. R e a c t i o n mechanisms
1.2.2. E p i g e n e t i
1.2.3. B i o c h e m i c a
1.2.4. R e p r o d u c t i o n mechanisms
S t r u c t u r a l H i e r a r c h y o f C e l l s (Compartments)
2.1. T r a n s l o c a t i o n o f s u b s t a n c e s
2.2. Complex b i o c h e m i c a l n e t w o r k
2.3. M o r p h o l o g i c a l d i f f e r e n t i a t i o n
2.4. C e l l c y c l e
2.5. Genetic phenomena a t l e v e l o f organelles
Population
3.1. D i s p e r s e d growth
3.1.1. P u r e c u l t u r e
3.1.2. M i x e d c u l t u r e
3.2. A g g r e g a t e d g r o w t h
B i o r e a c t o r and O t h e r E q u i p m e n t
4.1. M a c r o - and m i c r o - m i x i n g o f e q u i p m e n t c o n t e n t s
4.2. Mass t r a n s f e r
4.3. Heat e x c h a n g e
4.4. B i o r e a c t o r c o n t r o l and o f o t h e r e q u i p m e n t
4.5. S c a l e - u p
Technological Line
5.1. B a t c h p r o c e s s e s
5.2. C o n t i n u o u s p r o c e s s e s
Internat.J.General Systems 1982, 8(1), Table V I I I (1)

l e v e l ( m o l e c u l a r ) i n T a b l e I I i s c o n s i d e r e d quasihomogeneous
( r e a c t i o n s o c c u r r i n g i n a s o l u t i o n ) , the second i s represented by
a c e l l c o n s i d e r e d a s a two-phase s y s t e m , composed o f o r g a n e l l e s
h o m o g e n e o u s l y d i s p e r s e d i n t h e c y t o p l a s m . The p o p u l a t i o n l e v e l i s
m a n i f e s t e d b y d i f f e r e n t m o r p h o l o g i c a l s t a t e s ( d i s p e r s e d and a g g r e
g a t e d ) o f a p o p u l a t i o n . The l e v e l s o f t h e r e a c t o r and t e c h n o l o g i
c a l l i n e a r e d i s t i n g u i s h e d by fundamental f u n c t i o n s t y p i c a l f o r
chemico-technological processes.
The r e a c t o r l e v e l i s d i s t i n g u i s h e d b y t h e f o l l o w i n g f u n d a
m e n t a l e v e n t s and f u n c t i o n s : m a c r o - and m i c r o - m i x i n g o f r e a c t o r
( e q u i p m e n t ) c o n t e n t s and t h e c o r r e s p o n d i n g mass t r a n s f e r a n d h e a t
e x c h a n g e i n v o l v e t h e d e t e r m i n a t i o n o f e l e m e n t a r y zones o f r e a c t o r ,
mass t r a n s f e r and h e a t e x c h a n g e b e t w e e n them and a l s o b e t w e e n
i n d i v i d u a l phases ( l i q u i d , gaseous, s o l i d ) ; b i o r e a c t o r c o n t r o l
and s c a l e - u p ( 2 ) . I f n o n - h o m o g e n e i t i e s o f r e a c t o r p e r f o r m a n c e i n
s p a c e and t i m e a r e e x p e c t e d
and momentum t r a n s p o r t
As a s o l u t i o n t o momentum t r a n s p o r t ( N a v i e r - S t o k e s e q u a t i o n ) i s
u s u a l l y n o t a v a i l a b l e f o r b i o r e a c t o r v e l o c i t y f i e l d , we a r e l e f t
w i t h mass and h e a t b a l a n c e s . A l o c a l b a l a n c e d e s c r i b i n g m i x i n g ,
f l o w and i n t e r f a c e t r a n s f e r , i n c l u d i n g r e a c t i o n i s known a s t h e
d i s p e r s e d model ( 3 ) :
— — + Vc.. u + V D V C . + r..
at ij ij ij
-k-id«) \ \ ( c
iks - °ik > • ° ( i )
where s u b s c r i p t i d e n o t e s s y s t e m component, j i s t h e number o f a

phase i n t h e f - p h a s e s y s t e m , k i s a p h a s e o t h e r t h a n j , s u b s c r i p t
i k s d e n o t e s c o n c e n t r a t i o n n e a r t h e i n t e r f a c i a l a r e a on s i d e j ,
and u i s t h e l o c a l v e l o c i t y o f p h a s e s j . The l a s t t e r m d e n o t e s
the t r a n s p o r t between phases. A l i m i t e d a p p l i c a t i o n o f t h i s
s y s t e m o f p a r t i a l d i f f e r e n t i a l e q u a t i o n s was made f o r a s t i r r e d -
t a n k r e a c t o r (STR) w i t h a n e g l e c t o f t r a n s f e r t e r m (4_) as w e l l
as f o r a t u b u l a r r e a c t o r ( 5 ) .
Hydrodynamics
E l e m e n t a r y r e g i o n s o f b i o r e a c t o r . A more s i m p l e d e s c r i p t i o n
of the flow c h a r a c t e r i s t i c s o f a r e a c t o r than that by a l o c a l
mass b a l a n c e i s done b y i n t r o d u c i n g t h e r e s i d e n c e t i m e d i s t r i b u
t i o n c o n c e p t (RTD), w h i c h p r o v i d e s m a c r o - m i x i n g c h a r a c t e r i s t i c s
(determination of the elementary regions o f the r e a c t o r ) . I n
f a c t , u s i n g t h e r e s i d e n c e t i m e c o n c e p t a p a r t i c u l a r s i t u a t i o n may
be d e s c r i b e d b y m i x e d - t y p e m o d e l s c o n s i d e r i n g " t h e r e a c t o r a s

c o n s i s t i n g o f i n t e r c o n n e c t e d f l o w s b e t w e e n and a r o u n d t h e s e r e
g i o n s " ( 6 ) . The f o l l o w i n g k i n d s o f e l e m e n t a r y r e g i o n s a r e u s e d i n
t h e c o n s t r u c t i o n o f m i x e d m o d e l s : two t y p e s o f i d e a l z o n e s ( p l u g -
f l o w and p e r f e c t l y s t i r r e d = b a c k m i x e d ) , d i s p e r s e d p l u g f l o w and
deadwater. I n a d d i t i o n t o these r e g i o n s , the f o l l o w i n g k i n d s o f
f l o w may be u s e d : b y - p a s s , r e c y c l e and c r o s j s - f l o w . The above n o n -
i d e a l i t i e s i n m i x i n g may be d e s c r i b e d e i t h e r b y means o f d i s p e r
sed o r d i s c r e t e ( s t a g e - w i s e ) models, t h e l a t t e r c o n s i d e r i n g a
s e r i e s o f i d e a l l y mixed v e s s e l s .
Residence time d i s t r i b u t i o n . The above d i s t r i b u t i o n o f l i

q u i d , g a s e o u s and s o l i d ( c e l l s ) p h a s e s i s u s u a l l y m e a s u r e d b y a
stimulus-response experiment. Frequently, r a d i o a c t i v e t r a c e r s are
u s e d ( 7 , 8 ) . I n t h e a b s e n c e o f m i c r o o r g a n i s m s and f o r l i q u i d RTD,
a pH t r a c e r i s q u i t e c o n v e n i e n
t e - t y p e model f o r i n t e r p r e t i n
Micro-mixing c h a r a c t e r i s t i c s . I t i s w e l l established that

t h e RTD c o n c e p t does n o t c h a r a c t e r i z e a l l a s p e c t s o f m i x i n g . To
c l a r i f y t h i s a concept o f s e g r e g a t i o n has been i n t r o d u c e d ( 1 0 ) .
M i c r o - m i x i n g f a l l s , t h e n , b e t w e e n two l i m i t i n g c a s e s o f maximum
m i x e d n e s s and c o m p l e t e s e g r e g a t i o n , and i s t y p i c a l f o r r e a c t o r
zones s m a l l e r than the elementary r e g i o n s d e f i n e d above, u s u a l l y
a t t h e l e v e l o f i n d i v i d u a l c e l l s , b u b b l e s , m o l e c u l e s and t h e i r
c l u m p s . M i c r o - m i x i n g c h a r a c t e r i s t i c s , h o w e v e r , c a n n o t be e x t r a c t e d
f r o m RTD d a t a . F o r t u n a t e l y , t h e r e a c t o r p e r f o r m a n c e i s n o t o f t e n
too s e n s i t i v e t o m i c r o - m i x i n g . F o r example, f o r growth o f y e a s t s
on h y d r o c a r b o n s , where one c a n assume an i n f l u e n c e o f t h e d e g r e e
o f o i l d r o p and c e l l s e g r e g a t i o n , t h e d i f f e r e n c e s b e t w e e n c o m p l e
t e s e g r e g a t i o n and maximum m i x e d n e s s i s a p p r e c i a b l e o n l y f o r l o n g
e r c u l t i v a t i o n t i m e s ( 1 1 ) . Some p r o g r e s s c a n be e x p e c t e d f r o m
t h e o r e t i c a l and e x p e r i m e n t a l i n v e s t i g a t i o n o f t h e e f f e c t o f t h e
gas phase ( b u b b l e ) s e g r e g a t i o n o n mass t r a n s f e r and o x y g e n u t i l i
z a t i o n e f f i c i e n c y i n connection with d i f f e r e n t reactor designs.
F o r a l m o s t a l l p r a c t i c a l c a s e s maximum s u b s t r a t e c o n v e r s i o n i s
a c h i e v e d a t maximum m i x e d n e s s .
Mass T r a n s f e r
Mass t r a n s f e r i n v o l v e s e s t a b l i s h i n g a t r a n s f e r b e t w e e n t h e
e l e m e n t a r y r e g i o n s o f t h e r e a c t o r and b e t w e e n i n d i v i d u a l p h a s e s
( i n t e r f a c i a l mass t r a n s f e r c o e f f i c i e n t s : gas phase mass t r a n s f e r ,
l i q u i d p h a s e mass t r a n s f e r , mass t r a n s f e r w i t h r e a c t i o n , l i q u i d -
s o l i d mass t r a n s f e r ) , a s w e l l a s o t h e r e l e m e n t a r y phenomena and
p r o c e s s e s c o n n e c t e d w i t h mass t r a n s f e r : g a s p h a s e phenomena and
p r o c e s s e s ( g a s h o l d - u p , b u b b l e s i z e , i n t e r f a c i a l a r e a and b u b b l e
c o a l e s c e n c e / r e d i s p e r s i o n ) , v o l u m e t r i c mass t r a n s f e r and power
c o n s u m p t i o n d u r i n g mass t r a n s f e r ( 2 ) .

I n t e r f a c i a l mass t r a n s f e r c o e f f i c i e n t s . T h e o r e t i c a l and
e x p e r i m e n t a l i n v e s t i g a t i o n s o f many a u t h o r s h a v e shown t h a t d u r i n g
t h e a b s o r p t i o n o f s p a r i n g l y s o l u b l e g a s e s s u c h as o x y g e n , t h e gas
phase c o e f f i c i e n t ( k ) i s s e v e r a l o r d e r s i n m a g n i t u d e h i g h e r t h a n
ç
t h e l i q u i d phase mass t r a n s f e r c o e f f i c i e n t ( k _ ) . B e c a u s e o f t h e
above f a c t and a l s o due t o t h e h i g h v a l u e o f H e n r y ' s Law c o n s t a n t
f o r o x y g e n , t h e l i q u i d p h a s e mass t r a n s f e r c o e f f i c i e n t d e t e r m i n e s
t h e o v e r a l l r a t e o f mass t r a n s f e r . I n d e a l i n g w i t h i n t e r f a c i a l
phenomena, two e x t r e m e c a s e s a r e e n c o u n t e r e d : mass t r a n s f e r by
m o l e c u l a r d i f f u s i o n and c o n v e c t i o n f r o m r i g i d i m m o b i l e s p h e r e s ,
r e p r e s e n t e d by gas b u b b l e s w i t h c o n t a m i n a t e d s u r f a c e s and d e s c r i
bed by t h e F r B s s l i n g e q u a t i o n ( e . g . , i n ( 1 2 ) ) , and mass t r a n s f e r
by c o n v e c t i o n f r o m m o b i l e s p h e r e s , d e s c r i b e d by t h e B o u s s i n e s q
e q u a t i o n (e.g., i n ( 1 3 ) ) . In p r a c t i c e , the l a t t e r system i s r a r e
l y a c h i e v e d s i n c e f e r m e n t a t i o n media w i t h e l e c t r o l y t e s p r o t e i n s
p r o d u c t s o f m e t a b o l i s m an
m a t e r i a l may h i n d e r t h
gas b u b b l e s . V a r i o u s t h e o r e t i c a l and e x p e r i m e n t a l c o r r e l a t i o n s
f o r p r e d i c t i n g k^ f o r b u b b l e s w i t h i m m o b i l e i n t e r f a c e s h a v e b e e n
p r e s e n t e d i n r e v i e w s ( 1 4 , 15) i n v o l v i n g s i t u a t i o n s w i t h c r e e p i n g
f l o w ( t y p i c a l f o r a i r - l i f t r e a c t o r s ) and l o w - h i g h Re numbers, and
a l s o e f f e c t s o f n o n - N e w t o n i a n f l u i d on mass t r a n s f e r f r o m m o v i n g
b u b b l e s . These c o r r e l a t i o n s a r e o f i m p o r t a n c e i n p o l y s a c c h a r i d e
and a n t i b i o t i c p r o d u c t i o n s . The e f f e c t o f s u r f a c e - a c t i v e compounds
on k^ and a i s d i f f e r e n t i a l b e c a u s e o f t h e i r d i f f e r e n t i n f l u e n c e
on s u r f a c e t e n s i o n ( i n c r e a s e o r d e c r e a s e ) . T h e s e phenomena h a v e
not y e t been i n c o r p o r a t e d i n t o a s u f f i c i e n t l y g e n e r a l t h e o r y .
Mass t r a n s f e r w i t h r e a c t i o n . The p r e c e d i n g s i t u a t i o n i n v o l
v e d o n l y a p h y s i c a l mass t r a n s f e r b e t w e e n g a s - l i q u i d o r s o l i d - l i
q u i d i n t e r f a c e s . In the presence of microorganisms, oxygen uptake
by r e s p i r i n g c e l l s w i t h i n t h e r e l a t i v e l y s t a g n a n t l i q u i d r e g i o n
a d j a c e n t t o b u b b l e s submersed i n a c e l l s u s p e n s i o n may l e a d t o
e n h a n c e d o x y g e n t r a n s f e r r a t e s . U s i n g an enhancement c o n c e p t o f
A s t a r i t a , B l a n c h (16) p r e s e n t e d r u l e s f o r i t s p r e d i c t i o n : t h e
d i f f u s i o n t i m e f o r t h e o x y g e n o f gas b u b b l e s a s c e n d i n g t h r o u g h a
l i q u i d , e s t i m a t e d f r o m b u b b l e d i a m e t e r and a s c e n d i n g a i r v e l o c i t y ,
dg / u R , s h o u l d be g r e a t e r o r a t l e a s t o f t h e same o r d e r as t h e
r e a c t i o n t i m e , e v a l u a t e d from d i s s o l v e d oxygen c o n c e n t r a t i o n ,
o x y g e n u p t a k e r a t e and b i o m a s s d e n s i t y , C ^ / ( Q Q X ) . S o b o t k a e t a l .
(17) h a v e shown t h a t t h e c e l l f l o t a t i o n 2 mechanism c a n be
s u c c e s s f u l l y u t i l i z e d to obtain a b e t t e r f i t f o r experimental
growth d a t a , c o n s i d e r i n g g a s - s o l i d ( c e l l s ) a b s o r p t i o n of oxygen
( i t may amount up t o a h a l f o f t o t a l a b s o r p t i o n ) i n a d d i t i o n t o
the p r e v a i l i n g t y p i c a l oxygen path t o c e l l s v i a d i s s o l v e d oxygen
( F i g u r e 1 ) . C o n c l u s i o n of t h i s work, p r o v i d i n g enhanced a b s o r p t i
on ( r e d u c e d b u b b l e s l i p v e l o c i t y due t o e l e c t r o l y t e , a l c o h o l s and
o t h e r s u r f a c e - a c t i v e compounds; o x y g e n l i m i t a t i o n o f g r o w t h ; h i g h
r e s p i r a t i o n r a t e ; h i g h c e l l d e n s i t y a t the i n t e r f a c e ) are i n f u l l

Figure 1. Fitting data to one-phase (liquid) and two-phase (liquid and gas) oxygen uptake models.
2 T
Key: *, oxygen in outlet gas (y scale); O, biomass concentration (X scale); ·, dissolved oxygen
0
concentration; dashed line, one-phase model; solid line, two-phase model; X , biomass concentra-
tion at the interface. Reprinted, with permission, from Ref. 17. Copyright 1981, John Wiley &
Sons, Inc.
a c c o r d w i t h B l a n c h ' s a r g u m e n t a t i o n . Enhanced a b s o r p t i o n i s , i n
f a c t , o n l y a p p a r e n t , b e c a u s e o f t h e p a r a l l e l pathway o f o x y g e n
utilization.
I n t r a p a r t i c l e mass t r a n s f e r . F o r some p r a c t i c a l s i t u a t i o n s
mass t r a n s f e r l i m i t i n g s t e p i s l o c a l i z e d i n t h e i n t e r i o r o f a s o
l i d phase. T h i s i s t h e case f o r c e r t a i n m y c e l i a l f e r m e n t a t i o n s
where t h e o x y g e n t r a n s f e r v i a p e l l e t o r m y c e l i a l clump i n t e r i o r
may l i m i t g r o w t h o r p r o d u c t i o n p r o c e s s e s . T h i s s i t u a t i o n , e m p l o y
i n g t h e e f f e c t i v e n e s s c o n c e p t , i s r e v i e w e d b y Moo-Young and
B l a n c h ( 1 4 ) . I n t h e f o l l o w i n g , some o t h e r e l e m e n t a r y phenomena
and p r o c e s s e s c o n n e c t e d w i t h mass t r a n s f e r a r e r e v i e w e d .
Gas p h a s e phenomena and p r o c e s s e s . T h e s e i n c l u d e gas h o l d

up, b u b b l e s i z e , i n t e r f a c i a coalescence/redisper
s i o n . Gas h o l d - u p and b u b b l
physico-chemical propertie ,
c o n f i g u r a t i o n and o n o p e r a t i o n c o n d i t i o n s . The i n i t i a l b u b b l e
d i a m e t e r a t o r i f i c e i s an o r d e r o f m a g n i t u d e s m a l l e r t h a n t h e
e q u i l i b r i u m b u b b l e s i z e a n d , a t some d i s t a n c e f r o m t h e gas d i s t r i
b u t o r , t h e s i z e o f b u b b l e s i s dependent o n l y on b r o t h p r o p e r t i e s
and on l o c a l t u r b u l e n c e . I n b u b b l e c o l u m n , on t h e o t h e r h a n d ,
d i f f e r e n t r e g i o n s c a n be o b s e r v e d , w h e r e b u b b l e d i a m e t e r i s
e i t h e r determined by o r i f i c e c h a r a c t e r i s t i c s o r by a p a r t i c u l a r
t u r b u l e n c e . A d i s t i n c t i o n must be made b e t w e e n c o a l e s c i n g m e d i a
( w a t e r ) and n o n - c o a l e s c i n g m e d i a w i t h e l e c t r o l y t e s , a l c o h o l s and
o t h e r s u r f a c e - a c t i v e a d d i t i o n s . I n media w i t h a c l e a n i n t e r f a c e
( c o a l e s c i n g ) , an a v e r a g e e q u i l i b r i u m s i z e o f b u b b l e s , d e t e r m i n e d
by a n e x t e n t o f p a r a l l e l p r o c e s s e s o f c o a l e s c e n c e and r e d i s p e r
s i o n , i s h i g h e r because o f enhanced c o a l e s c e n c e . Under these
c o n d i t i o n s , t h e s e c o n d a r y b u b b l e s , a r i s i n g f r o m p r i m a r y ones a t
t h e o r i f i c e , c o a l e s c e e a s i l y a t some d i s t a n c e f r o m t h a t s p a c e .
On t h e o t h e r h a n d , d i s p e r s i o n o f gas b u b b l e s i s e n h a n c e d i n t h e
presence o f e l e c t r o l y t e s o r a l c o h o l s . T h i s e f f e c t i s e x p l a i n e d on
t h e b a s i s o f c o m p l e x phenomena c o n n e c t e d w i t h w a t e r s t r u c t u r e
changes n e a r t h e i n t e r f a c e ( 1 8 ) . The i n f l u e n c e o f e l e c t r o l y t e s
and a l c o h o l s i s d i f f e r e n t i a t e d : t h e c o n c e n t r a t i o n o f e l e c t r o l y t e s
a t t h e f r e s h i n t e r f a c e d e c r e a s e s w i t h b u b b l e age w h e r e a s t h a t o f
a l c o h o l s i n c r e a s e s . These phenomena h a v e , no d o u b t , an i m p a c t on
r e a c t o r d e s i g n a s b u b b l e p a t h and age c a n c o n s i d e r a b l y i n f l u e n c e
gas h o l d - u p and i n t e r f a c i a l a r e a ( m e c h a n i c a l l y - m i x e d r e a c t o r s v s .
v e r t i c a l m u l t i - s t a g e r e a c t o r s o r b u b b l e c o l u m n s ) . However, no
sound t h e o r e t i c a l b a s i s h a s b e e n p u t f o r w a r d f o r g e n e r a l i z i n g t h e
e f f e c t o f a l c o h o l s , e l e c t r o l y t e s and o t h e r s u r f a c e - a c t i v e com
p o u n d s . S i m i l a r l y , no q u a n t i t a t i v e d e s c r i p t i o n o f a b u b b l e b e h a
v i o u r d u r i n g mass t r a n s f e r i n t h e p r e s e n c e o f m i c r o o r g a n i s m s ,
i n c l u d i n g b u b b l e c o a l e s c e n c e and r e d i s p e r s i o n and c e l l a d s o r p t i o n
and d e s o r p t i o n , h a s b e e n s u g g e s t e d . E v e n a s i m p l e measurement o f
bubble s i z e o r o f t o t a l i n t e r f a c i a l area i n a c e l l suspension

p r e s e n t s a p r o b l e m . The same a p p l i e s t o a n a s s e s s m e n t o f c o a l e s -
c e n c e / r e d i s p e r s i o n r a t e s and mechanisms a l t h o u g h some methods f o r
c o a l e s c e n c e f r e q u e n c y measurement h a v e b e e n s u g g e s t e d ( 1 9 , 2 0 ) .
Some f u r t h e r t h e o r e t i c a l c o n s i d e r a t i o n s and c o r r e l a t i o n s f o r p r e
d i c t i n g b u b b l e s i z e , gas h o l d - u p a n d i n t e r f a c i a l a r e a were
r e v i e w e d ( 1 6 , 1 5 ) . Gas h o l d - u p and p r e s s u r e d r o p a r e o f i m p o r t a n
ce i n a r a t i o n a l d e s i g n o f a i r - l i f t r e a c t o r s ( 2 1 ) .
V o l u m e t r i c mass t r a n s f e r c o e f f i c i e n t k _ a . This represents

t h e most i m p o r t a n t d e s i g n p a r a m e t e r b e c a u s e and a a r e d i f f i
c u l t t o measure u n d e r r e a l c o n d i t i o n s . The k ^ a measurement
methods were r e v i e w e d b y S o b o t k a e t a l . ( 2 2 ) . The most r e l i a b l e
v a l u e s a r e o b t a i n e d b y b a l a n c e m e t h o d s . The model o f S i n c l a i r
and R y d e r ( 2 3 ) may s e r v e a s a good e x a m p l e o f t h e a p p l i c a t i o n o f
t h e above g e n e r a l b a l a n c e e q u a t i o n ( 1 ) w i t h a d o u b l e s u b s t r a t e
l i m i t a t i o n o f growth (carbo
f i t t i n g o f t h i s m o d e l , compose
s u b s t r a t e and b i o m a s s b a l a n c e s , t o g r o w t h d a t a p r o v i d e s a p o s s i
b i l i t y o f e s t i m a t i n g L a a s one o f s e v e r a l e v a l u a t e d model p a r a
m e t e r s . An e x t e n s i o n or* t h i s model w i t h a p r o v i s i o n f o r a n a l t e r
n a t i v e oxygen uptake from t h e gaseous phase has been proposed by
Sobotka e t a l . ( 1 7 ) .
F o r k ^ a p r e d i c t i o n i n b u b b l e c o l u m n s , an e m p i r i c a l e q u a t i o n
o f A k i t a and Y o s h i d a ( 2 4 ) , w h i c h d e s c r i b e s f a i r l y w e l l v a r i o u s
d a t a o b t a i n e d f r o m l a r g e s c a l e e q u i p m e n t , c a n be u s e d :
1 5 0 6 2 3 1
Sh ( a D c ) = 0.6 e j ' , c°' . Bo '
S . Ga°' (2)
c o n t a i n i n g d i m e n s i o n l e s s Sherwood, S c h m i d t , B o d e n s t e i n and G r a s -
h o f numbers, i n t e r f a c i a l a r e a a , b u b b l e c o l u m n d i a m e t e r D and
gas h o l d - u p ε . A s i m i l a r e q u a t i o n may be a p p l i c a b l e t o b u b b l e
c o l u m n s p r o v i d e d w i t h t h e two-phase n o z z l e ( e j e c t o r ) , a s i n d i c a
t e d f r o m k ^ a v s . ε^ dependence ( F i g u r e 2 ) . Two-phase n o z z l e s and
m u l t i - o r i f i c e spargers a r e s u i t e d f o r i n d u s t r i a l r e a c t o r s because
of h i g h v o l u m e t r i c mass t r a n s f e r c o e f f i c i e n t s . The m a i n f e a t u r e
of two-phase n o z z l e i s i n p r e s e r v i n g t h e e n e r g y o f t h e j e t , u t i
l i z e d f o r the p r o d u c t i o n o f a h i g h i n t e r f a c i a l a r e a , by suppres
s e d c o a l e s c e n c e i n s u i t a b l e m e d i a and b y a s u i t a b l e g u i d e t u b e
(momentum t r a n s f e r t u b e , ( 2 6 ) ) . The i n d u s t r i a l a p p l i c a t i o n s i n
v o l v e t h o s e i n SCP p r o d u c t i o n ( 2 7 ) a n d i n w a s t e w a t e r t r e a t m e n t
( 2 8 ) . The c o r r e l a t i o n o f t h e t y p e o f e q . ( 2 ) c a n , p e r h a p s , be o f
use i n p r e d i c t i n g k ^ a f o r s t i r r e d - t a n k r e a c t o r s , b a s e d o n a
s i n g l e m e a s u r e d v a l u e (gas h o l d - u p ) . C o r r e l a t i o n s b a s e d o n power
i n p u t and s u p e r f i c i a l v e l o c i t y a r e t h e most f r e q u e n t ( 2 9 ) .
E f f e c t o f v i s c o s i t y o n k ^ a a n d power c o n s u m p t i o n d u r i n g mass
t r a n s f e r . The e f f e c t o f v i s c o s i t y o n k ^ a i s o f i m p o r t a n c e i n
s e v e r a l h i g h l y v i s c o u s N e w t o n i a n and n o n - N e w t o n i a n f e r m e n t a t i o n s .
Some c o r r e l a t i o n s d e s c r i b i n g l ^ a d e c r e a s e w i t h v i s c o s i t y have

+ - V L = 1.70 10 ms
S
3 1
A - V L 0.86. 1Ô ms
S
S 3 1
1 ' ' 1
4 6 8 10 20 30
Gas hold-up £q {%)

Figure 2. The k a dependence on € , bubble column with two-phase nozzle (3 mm
L 0
ID), electrolyte solution plus ethanol (0.1% v/v) (25).

b e e n s u m m a r i z e d i n ( 1 5 ) . From t h i s p a p e r , t h e H e n z l e r ' s c o r r e l a
t i o n f o r 1-2% CMC s o l u t i o n s i n b u b b l e c o l u m n s ( f l o w b e h a v i o u r r e p
resentative f o r p e n i c i l l i n fermentation) i s u s e f u l :
where v ^ i s t h e s u p e r f i c i a l a i r v e l o c i t y and υ k i n e m a t i c v i s c o s i
g
t y . R e u s s e t a l . ( 3 0 ) s u g g e s t e d a much s i m p l e r e q u a t i o n f o r s t i r -
red-tank reactor:
V T Ρ
a — = f ( § ) (4)
GV
V L S > ( U
i n v o l v i n g two d i m e n s i o n l e s s g r o u p s ( i svolumetric a i r flow

rate, r e a c t o r v o l u m e ) . Power c o n s u m p t i o n i n g a s s e d s y s t e m s , Ρ ,
i s then c a l c u l a t e d from: ^
0 0 6 4 0 1 5 6 0 3 8
Ρ /Ρ = 0.0312 R e ' Fr" ' Na" ' ( A _ )<>.8 ( 5 )
g o ai
i n v o l v i n g R e y n o l d s a n d F r o u d e numbers o f i m p e l l e r , a e r a t i o n num
b e r Na and r a t i o o f t a n k a n d i m p e l l e r d i a m e t e r . The a p p l i c a b i l i t y
of eq.(4) f o r p r e d i c t i n g kâ d u r i n g f e r m e n t a t i o n o f P.chrysogenum
and A . n i g e r i s shown i n F i g u r e 3. T h u s , t h e b a s i c p h y s i c a l p r o
p e r t i e s o f c u l t i v a t i o n broths ( i n t h i s case v i s c o s i t y ) a r e i n c o r
p o r a t e d i n t o c o r r e l a t i o n s f o r t r a n s p o r t phenomena t o g e t h e r w i t h
k e y d e s i g n p a r a m e t e r s ( k ^ a and power c o n s u m p t i o n ) .
N i s h i k a w a e t a l . (31; have r e c e n t l y proposed t h e t w o - r e g i o n
model ( b u b b l i n g - and a g i t a t i o n - c o n t r o l l e d ) f o r e s t i m a t i n g k a i n T
a mixed r e a c t o r . Exponents f o r v i s c o s i t y terms a r e d i f f e r e n t f o r

d i f f e r e n t regions. P r e d i c t i o n s by dimensionless c o r r e l a t i o n s a r e
a p p l i c a b l e f o r b o t h non-Newtonian and Newtonian l i q u i d s .
F o r b u b b l e c o l u m n s f i t t e d w i t h a two-phase n o z z l e a n d e l e c
t r o l y t e s o l u t i o n s , k a c a n be p r e d i c t e d f r o m power f o r a e r a t i o n
L
Ε and f r o m power f o r l i q u i d c i r c u l a t i o n v i a n o z z l e Ε (Figure 4 ) .

a ρ
Heat Exchange
H e a t e x c h a n g e i n v o l v e s h e a t g e n e r a t i o n and t r a n s f e r i n a n d
between e l e m e n t a r y r e g i o n s o f a b i o r e a c t o r and t h a t o f i n d i v i d u a l
phases o f a r e a c t o r ( 2 ) . A r a t i o n a l s t a r t f o r these c o n s i d e r a t i
ons i s t h e g e n e r a l l o c a l h e a t b a l a n c e , s i m i l a r t o t h a t o f mass
transfer:

Figure 3. Correlation of Reuss as applied for k a estimate during mold fermen-

L
tations. Reprinted, with permission, from Ref. 30. Copyright 1981, Pergamon
Press Ltd.

PROKOP Reactor Design Fundamentals 367
Figure 4. The k/.a dependence on E and E ; bubble column with two-phase

e p
nozzles (3-5 mm ID), electrolyte solution plus ethanol (0-0.1 % v/v) (25).

3 ρ . C . T.
+ V . c .
P T . . +Vk . V T . + ΣΔΗ r..
J PJ iJ ej j n j ij
a t
f
(6)
-k-l(k«) \ \ T
< j s - T
j > - 0
where Ρ i s s p e c i f i c d e n s i t y , C i s m o l a r h e a t , Τ i s a b s o l u t e tem
perature, k i s e f f e c t i v e thermal c o n d u c t i v i t y , Δ Η i s heat of
r e a c t i o n , h i s h e a t t r a n s f e r c o e f f i c i e n t and T. i s t h e t e m p e r a
t u r e a t t h e i n t e r f a c e a r e a . The f i r s t t e r m o f tftîs b a l a n c e i s a n
a c c u m u l a t i o n term a c c o u n t i n g f o r heat c a p a c i t y ; t h e second term
i s heat t r a n s p o r t due t o c o n v e c t i o n , w h i c h i s i m p o r t a n t i n t u b u
l a r r e a c t o r s . The t h i r d t e r r e p r e s e n t h e a t b a c k m i x i n d
be i m p o r t a n t i n b i o r e a c t i o n
nes o r i n a deep l a y e r clumps
f o u r t h t e r m c h a r a c t e r i s e s h e a t g e n e r a t i o n due t o a r e a c t i o n and
the f i f t h i s t h e i n t e r f a c e h e a t t r a n s f e r . I n b i o r e a c t o r s t h a t a r e
t h o r o u g h l y m i x e d o n l y t h e a c c u m u l a t i o n and h e a t g e n e r a t i o n terms
are s i g n i f i c a n t and t h e t e m p e r a t u r e f i e l d i s u n i f o r m . The b o u n d a
ry c o n d i t i o n s o f t h i s s i m p l i f i e d e q u a t i o n a r e r e p r e s e n t e d by the
o v e r a l l heat t r a n s f e r from the c u l t i v a t i o n b r o t h t o the c o o l i n g
o r h e a t i n g ( s t e r i l i z a t i o n ) medium.
The amount o f h e a t g e n e r a t e d i n a b i o r e a c t o r c a n be e a s i l y
e v a l u a t e d f r o m t h e above s i m p l i f i e d h e a t b a l a n c e ^ r e s u l t i n g i n a A
v o l u m e t r i c rate o f heat g e n e r a t i o n , Q ( J . l t .h ) . Cooney e t a l .

f
(32) o b t a i n e d b y means o f dynamic c a l o r i m e t r y . F o r a r o u g h

e s t i m a t i o n o f Q , a p p l i c a b l e u n d e r o x y g e n l i m i t a t i o n and w i t h
f
c e l l s a s t h e s o l e r e a c t i o n p r o d u c t , M i n k e v i c h and E r o s h i n ( 3 3 )
derived a t h e o r e t i c a l formula:
y
= 818 Na = 818 k a —
T (7)
f L H
w h e r e ^ N a ^ i s t h e maximum o x y g e n t r a n s f e r o f a b i o r e a c t o r ( i n
g.lt .h ) , y i s t h e mole f r a c t i o n o f oxygen i n t h e e x i t gas
2
and H i s H e n r y *s c o n s t a n t .The c o e f f i c i e n t 818 h a s t h e m e a n i n g o f

h e a t g e n e r a t i o n p e r 1 g o f oxygen consumed. Cooney e t a l . ^ ^ u s i n g
d i r e c t m e a s u r e m e n t s , f o u n d t h i s c o e f f i c i e n t t o be 822 J . g oxy
gen. E q . ( 7 ) seems t o be u s e f u l f o r p r e d i c t i n g t h e d e s i g n o f h e a t
e x c h a n g e r s . Q i s a l s o c o r r e l a t e d t o b i o m a s s y i e l d and p r o d u c t i
f
vity ( X ) : y
k
Q
f = ( k
i " ~ T — > υ x ( 8 )
x/s
where k^ and k^ a r e c o n s t a n t s t h a t c a n be d e r i v e d f r o m t h e h e a t s
of c o m b u s t i o n o f s u b s t r a t e and c e l l s . T h u s , c a n be a s s e s s e d

for a g i v e n s u b s t r a t e and e x p e c t e d y i e l d and p r o d u c t i v i t y ( 3 4 ) .

The o v e r a l l h e a t t r a n s f e r c o e f f i c i e n t i s composed o f a m i n i
mum o f t h r e e c o n t r i b u t i o n s , t h e m a j o r o f w h i c h i s t h e c o e f f i c i e n t
of h e a t t r a n s f e r from g a s - l i q u i d phase t o h e a t exchanger-wall.
P o l l a r d and T o p i w a l a ( 3 5 ) r e p o r t e d v a l u e s o f h e a t t r a n s f e r c o e f
f i c i e n t s i n g a s - l i q u i d bioreactors f o rvarious configurations of
h e a t e x c h a n g e r ( c o i l , j a c k e t ) . These a u t h o r s q u e s t i o n e d t h e a p p l i
c a t i o n o f c u r r e n t l y used c o r r e l a t i o n s o b t a i n e d i n l i q u i d media
(36) t o g a s - l i q u i d s y s t e m s . The i n t r o d u c t i o n o f a i r a f f e c t s t h e
heat t r a n s f e r c o e f f i c i e n t . A novel theory f o r p r e d i c t i n g heat
t r a n s f e r i n b u b b l e column r e a c t o r s was p u t f o r w a r d b y Deckwer ( 3 7 ) .
B l a k e b r o u g h and McManamey ( 3 8 ) m e a s u r e d t h e h e a t t r a n s f e r o f m o l d
f e r m e n t a t i o n s i n an a i r - l i f t r e a c t o r . The e f f e c t s o f non-Newto
n i a n a e r a t e d l i q u i d s o n h e a t t r a n s f e r c o e f f i c i e n t were s h o r t l y
r e v i e w e d b y P a c e and R i g h e l a t
Process Synthesis at Reactor Level
The o v e r a l l d e s c r i p t i o n (model) o f a r e a c t o r i s o b t a i n e d
through p r o c e s s s y n t h e s i s by combining models o f r e a c t o r hydrody
n a m i c s , mass t r a n s f e r and h e a t e x c h a n g e w i t h an a p p r o p r i a t e c e l l
( s u b c e l l u l a r ) o r p o p u l a t i o n model ( 1 ) . D e s c r i p t i o n o f a p o p u l a t i o n
should take i n t o c o n s i d e r a t i o n p o s s i b l e d i s p e r s e d o r aggregated
(the d i s t i n c t morphological appearances o f a c u l t u r e : p e l l e t s ,
m y c e l i u m , f l o c k s , g r o w t h on r e a c t o r w a l l i n t h e f o r m o f m i c r o b i a l
f i l m ) forms o f p o p u l a t i o n . Biomass s u p p o r t p a r t i c l e s a r e g a i n i n g
a p p r e c i a b l e importance i n a e r o b i c (40) as w e l l as i n a n a e r o b i c
processes.
The d e s c r i p t i o n h i e r a r c h y a c q u i r e s d i f f e r e n t w a y s : p h y s i c a l
p r o c e s s e s i n mass and e n e r g y b a l a n c e s ; i n f o r m a t i o n p r o c e s s i n g and
c o n t r o l ; o p t i m i z a t i o n o f the system a t t h e r e a c t o r l e v e l o r as
a w h o l e ( 4 1 ) . The most e l a b o r a t e c o n c e p t o f d e s c r i p t i v e h i e r a r c h y ,
i n c l u d i n g a s e t o f p r o g r a m s , was p u b l i s h e d and r o u t i n e l y u s e d b y
K l i r ( 4 2 ) . As d e s c r i p t i v e h i e r a r c h y u s u a l l y f o l l o w s s t r u c t u r a l
and f u n c t i o n a l h i e r a r c h y , i t i s t h u s a s s u r e d t h a t t h e m o d e l l e d
o b j e c t p o s s e s s e s enough s t r u c t u r a l and f u n c t i o n a l f e a t u r e s , o t h e r
w i s e t h e r e a c t o r c o n t r o l b y means o f s u c h m o d e l s w o u l d n o t s a t i s
f a c t o r i l y respond t o changing c o n d i t i o n s .
Reactor Control
Reactor c o n t r o l concerns the system p r o p e r t i e s from the

c o n t r o l v i e w p o i n t , and t h e d e t e r m i n a t i o n o f c r i t e r i a o f c o n t r o l
and o p t i m i z a t i o n . S y s t e m p r o p e r t i e s i n c l u d e d y n a m i c p r o p e r t i e s
s u c h a s t h e n a t u r e o f s y s t e m r e s p o n s e t o s m a l l and l a r g e d i s t u r
b a n c e s . R e g u l a r t r a n s i e n t r e a c t o r o p e r a t i o n may be o f f u t u r e
i m p o r t a n c e e v e n f o r some p r o d u c t f o r m a t i o n p r o c e s s e s ( 4 3 ) . E l u c i
dation of input operating v a r i a b l e s a f f e c t i n g a process i s then
valuable f o r reactor c o n t r o l . In t h i s connection the parametric

s e n s i t i v i t y o f t h e s e v a r i a b l e s , d e f i n e d as a r e l a t i v e o r a b s o l u t e
change i n an o p t i m i z a t i o n c r i t e r i o n on t h e i r change ( 4 4 ) , i s r e
q u i r e d . The d e c i s i v e p a r a m e t e r s s e r i o u s l y a f f e c t i n g t h e s y s t e m
a r e d e f i n e d i n t h i s way. The c r i t e r i o n o f c o n t r o l and o p t i m i z a
t i o n i s c o n v e n i e n t l y s e t up, e.g., as t h e maximum b i o l o g i c a l
t i t r e , maximum p r o d u c t i v i t y , l o w e s t r u n n i n g c o s t s , e t c .
C o n t r o l and d y n a m i c h i e r a r c h i e s may s e r v e as a good g u i d e l i
ne f o r s e t t i n g up p r o p e r c o n t r o l . C o n t r o l h i e r a r c h y o f b i o l o g i c a l
l e v e l s i s i n T a b l e I I I , c o n t r o l l o o p s and g o a l f u n c t i o n s ( c o n t r o l
and o p t i m i z a t i o n c r i t e r i a ) o f b i o l o g i c a l l e v e l s a r e i n T a b l e I V .
The c o m p l e x i t y o f b i o l o g i c a l phenomena i s r e f l e c t e d i n t h e p r e s e n
ce o f m u l t i p l e l o c a l g o a l f u n c t i o n s o f d i f f e r e n t s u b s y s t e m s , w h i c h
may a c t i n an a c c o r d o r o p p o s i t e l y . N o t e t h a t T a b l e IV has b e e n
d e v e l o p e d f o r e c o s y s t e m s . The i m p l e m e n t a t i o n o f c o n t r o l h i e r a r c h y
i n t h e m o d e l l i n g and c o n t r o l f b i o l o g i c a l s y s t e m ha t t
b e e n c a r r i e d o u t . The c o n t r o
namic h i e r a r c h y of tim
d i v i d u a l s u b s y s t e m s ) . The r e l a x a t i o n t i m e i s c o n s i d e r e d t o be t h e
t i m e f o r a change f r o m one s t a t e t o a n o t h e r (due t o r e a c t i o n ,
d i f f u s i o n , change i n t h e a d j o i n i n g l e v e l , e t c . ) . T a b l e V p r e s e n t s
a v e r a g e r e l a x a t i o n t i m e s o f some c e l l u l a r s u b s y s t e m s . As a p p e a r s
f r o m t h i s t a b l e , s l o w (and l i m i t i n g ) p r o c e s s e s g i v e r i s e t o s t r u c
t u r a l e l e m e n t s , s e r v i n g as b a s i s f o r f a s t p r o c e s s e s . U s u a l l y ,
responses of h i g h e r l e v e l s of the system t o d i s t u r b a n c e s coming
from the s u p e r i o r l e v e l are s l o w e r than responses a t the n e x t
lower l e v e l . B e s i d e s r e l a x a t i o n times of b i o l o g i c a l subsystems of
c e l l s , t h e s e l e c t i o n o f s y s t e m i c phenomena may p r o c e e d v i a an
u t i l i z a t i o n o f , e.g., c e l l g e n e r a t i o n t i m e , r a t e o f s u b s t r a t e
c o n s u m p t i o n , r e s p i r a t i o n r a t e , o x y g e n t r a n s f e r and o f t h e homoge-
n i z a t i o n t i m e o f a r e a c t o r , t o m e n t i o n some.
The above d i s c u s s i o n may i n v o l v e b o t h c o n t r o l and d y n a m i c s
o f s t e a d y s t a t e ( s i n g l e and m u l t i p l e ) , u n s t e a d y s t a t e ( t r a n s i t i o n
b e t w e e n two s t a t e s ) and u n s t a b l e o p e r a t i o n s o f t h e b i o r e a c t o r
r e s u l t i n g from t y p i c a l n o n - l i n e a r response of b i o l o g i c a l systems.
In s p i t e of the f a c t t h a t the s t a b i l i t y a n a l y s i s of n o n - l i n e a r
systems i s q u i t e advanced, e x p e r i m e n t a l c o n f i r m a t i o n of m u l t i p l e
s t e a d y s t a t e s and i n s t a b i l i t i e s l a g s b e h i n d ( f o r a r e v i e w o f
t h e o r y and e x p e r i m e n t s see ( 4 5 ) ) . An e x c e l l e n t e x a m p l e o f e x p e r i
mental demonstration of unstable operation of a continuous
reactor i s i n (46).
Reactor Scale-Up
On t h e b a s i s o f a r e a c t o r m o d e l , i n v o l v i n g enough s t r u c t u r a l
and f u n c t i o n a l f e a t u r e s o f b i o l o g i c a l and e n g i n e e r i n g (physical)
p r o p e r t i e s o f a b i o t e c h n o l o g i c a l p r o c e s s , new c r i t e r i a f o r a
s c a l e - u p may e v o l v e . The employment o f o n l y one c r i t e r i o n , t a k e n
f r o m one a r b i t r a r y l e v e l o f a h i e r a r c h y , i n the m o d e l l i n g and
s u b s e q u e n t s c a l e - u p f r e q u e n t l y f a i l s , as i s a p p a r e n t f r o m numerous

Table I I I Control Hierarchy of B i o l o g i c a l Levels
Control Dynamic B e h a v i o u r The H i g h e s t Control

Level Mechanism
1 Mass and e n e r g y change ( s t e a d y D i r e c t and f e e d - b a c k

number o f s t r u c t u r a l e l e m e n t s control
and s t e a d y p a r a m e t e r s
2 F u n c t i o n a l dynamics (steady Adaptation

number o f s t r u c t u r a l e l e m e n t s ,
changing parameters)
3 S t r u c t u r a l dynamics Organization
( c h a n g i n g number o f s t r u c t u r a l
elements )
4 Developmental dynamics Development

(changing goal function)
Internat.J.General Systems 1982, 8 ( 1 ) , T a b l e I V ( 1 )

T a b l e IV C o n t r o l Loops and G o a l s o f B i o l o g i c a l Levels
S t r u c t u r a l H i e r a r c h y
Molecular Cellular Population Trophic
Goal Optimization of Optimization of Optimization of Optimization of

function individual subsystems o f structural energy f l o w o f each
subsystems by a c e l l by elements o f trophic level
controlling controlling population
biochemical individual
pathways subsystems
D i r e c t and Simple kinetics Stabilization of S t a b i l i z a t i o n of S t a b i l i z a t i o n of

feed-back r a t i o o f i n d i v i d u a l a number o f a number o f p o p u l a t i o n s
subsystems individuals
Adaptation Multiple kinetics Physiological Adaptation of Adaptation of trophic

adaptation of populations levels
subsystems
Organization Change-over from Change i n f u n c t i o n Change i n r a t i o o f Change i,n r a t i o o f

(selection) one t o a n o t h e r of subsystems s t r u c t u r a l elements different populations
b i o c h e m i c a l pathway

Development Change i n g o a l Change i n g o a l Change i n g o a l Change i n g o a l f u n c t i o n

function of a function of i n d i function of of ecosystem
subsystem vidual cells population
I n t e r n a t . J . G e n e r a l Systems 1982, J K 1 ) , T a b l e V (1)

Table V Dynamic H i e r a r c h
System (Subsystem) R e l a x a t i o n Time, s
. . 2 4
C e l l m u l t i p l i c a t i o n , DNA r e p l i c a t i o n 10 - 10
3 4
mRNA s y n t h e s i s on o p e r o n 10 - 10
* 1 3
T r a n s l o c a t i o n of substances i n t o c e l l s 10 - 10
1 2
P r o t e i n s y n t h e s i s i n polysomes 10 - 10
Allosteric control o f enzyme s y n t h e s i s 10°
Glycolysis i n cytoplasm 10 * - 10
-2
Oxidative phosphorylation i n mitochondria 10
—6 —3
E n z y m a t i c r e a c t i o n and t u r n o v e r 10 - 10
Bond b e t w e e n enzyme and s u b s t r a t e 10
I n t e r n a t . J . G e n e r a l Systems 1982, 8 ( 1 ) , T a b l e V I ( 1 )

examples of a p p l i c a t i o n s of e n g i n e e r i n g c r i t e r i a ( e . g . , i n ( 2 9 ) ) .
The n e g l e c t o f b i o l o g i c a l f e a t u r e s o f b i o t e c h n o l o g i c a l p r o c e s s e s
f r e q u e n t l y l e a d s t o an u n r e s o l v e d c a s e o f s i m u l t a n e o u s m a i n t e n a n
ce o f t h e n u m e r i c a l v a l u e s o f a l l d i m e n s i o n l e s s g r o u p s o f p h y s i
c a l p a r a m e t e r s (mass t r a n s f e r , power i n p u t p e r u n i t v o l u m e , m i x i n g
time, e t c . ) . For example, a s i n g l e c r i t e r i o n of constant m i x i n g
t i m e w o u l d v i o l a t e an i n v a r i a n c y o f gas p h a s e d y n a m i c s . M a c r o -
m i x i n g o f t h e f l u i d phase d e t e r m i n e s , t o a c o n s i d e r a b l e e x t e n t ,
t h e b e h a v i o u r o f t h e gas p h a s e , i . e . , t h e r e s i d e n c e t i m e d i s t r i
b u t i o n o f gas b u b b l e s . I n r e a c t o r s c a l e - u p , a c o n s i d e r a b l e d i f f e
r e n c e i n gas p h a s e r e s i d e n c e t i m e c a n be e x p e c t e d as t h e l i q u i d
f l o w p a t h s ( l o o p s ) g r o w w i t h i n c r e a s i n g r e a c t o r s c a l e . The m a i n t e
n a n c e o f m i x i n g t i m e w o u l d r e s u l t i n an i n c r e a s e i n o x y g e n u t i l i
z a t i o n e f f i c i e n c y . Some l i m i t e d e x p e r i m e n t a l d a t a f o r d i f f e r e n t
r e a c t o r designs are a v a i l a b l e i n (47)
Q u a l i t a t i v e l y new c r i t e r i a
scheme o f t h e h i e r a r c h i c a
c e s s , suggest a need f o r m a i n t a i n i n g s i m i l a r i t y between systems
o f d i f f e r e n t s i z e s o f l a b o r a t o r y and p l a n t l e v e l s , i . e . , t h e
i d e n t i t y o f v a l u e s o f p a r a m e t e r s and c o n s t a n t s (1,2) · Such a
scale-up procedure would r e q u i r e a r e p e t i t i o n of systemic f e a t u
res of d e c i s i v e l e v e l s of h i e r a r c h y i n v o l v i n g those of b i o t e c h n o
l o g i c a l and p h y s i c a l n a t u r e on t h e l a b o r a t o r y and p l a n t s c a l e
and w o u l d assume t h e i n v a r i a n c e o f t h e s y s t e m i c phenomena o f a
g i v e n l e v e l w i t h r e s p e c t t o t h e s c a l e . The m e t h o d o l o g y o f s c a l e -
up v i a m o d e l l i n g and s i m u l a t i o n i s y e t t o be d e v e l o p e d .
Conclusions
F u r t h e r d e v e l o p m e n t o f r e a c t o r d e s i g n i s hampered by a l a c k
o f a p p r o p r i a t e t h e o r y o f some e l e m e n t a r y phenomena. T h i s s i t u a
t i o n l e a d s t o e m p i r i c a l c o r r e l a t i o n s t h a t may be o f some p r a c t i
c a l u s e . Systems a p p r o a c h a i d s i n c r e a t i n g a g u i d e l i n e f o r r a t i o
n a l b i o r e a c t o r d e s i g n , e s p e c i a l l y on t h e b a s i s o f sound b i o l o g i
c a l theory.
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17
Immobilized Cells
Catalyst Preparation and Reaction Performance
J. KLEIN and K.-D. VORLOP

Technical University of Braunschweig, Institute of Chemical Technology,
Federal Republic of Germany
Immobilized cells have proven to be effective cata

lysts i n the enzymati
pounds. Such catalyst
entrapment of cells i n polymeric carriers, and the
methods of ionotropic gelation and polycondensation
of epoxids w i l l be described. Depending on enzymatic
activity and particle size the transformation may
proceed i n the reaction or diffusion controlled re
gime. Quantitative estimation of the effectiveness
factor-Thiele modulus relation w i l l be presented for
different reaction types. This includes the experi
mental determination of the catalytically active
c e l l concentration and the effective d i f f u s i v i t y i n
the porous polymeric carrier. Transport limitation
can also be a controlling factor i n the experimental
determination of the operational s t a b i l i t y of such
biocatalysts.
A large number of products i n the pharmaceutical and food

industry i s obtained from fermentation processes. Examples are
amino acids, stereoregular organic acids, antibiotics, ethanol,
etc. In a classical fermentation process the product formation
is s t r i c t l y coupled to c e l l growth resulting i n a possibly unfav
orable byproduction of biomass. Furthermore these processes are
typically performed as batch operations.
As has been shown already on an industrial scale, fermenta
tion can be substituted by heterogeneous catalysts with resting
microbial cells immobilized i n polymeric carriers. Repeated use
of the once formed biomass, continuous process operation, and elim
ination of costly separation steps of product solution from bio
mass are obvious advantages of this new technology. Some p r i n c i
pal aspects of a) immobilization methodology, b) catalyst effect
iveness, and c) operational s t a b i l i t y shall be outlined i n this
contribution.
0097-6156/83/0207Ό377$06.00/0

Polymer Entrapment
V a r i o u s methods h a v e b e e n p r o p o s e d f o r w h o l e c e l l i m m o b i l i z a
t i o n i n c l u d i n g a d s o r p t i o n and c o v a l e n t a t t a c h m e n t t o a p r e f o r m e d
c a r r i e r , c r o s s l i n k i n g , f l o c c u l a t i o n , m i c r o e n c a p s u l a t i o n , and e n
t r a p m e n t . P h y s i c a l e n t r a p m e n t i n a p o r o u s m a t r i x i s by f a r t h e most
f l e x i b l e and most commonly u s e d t e c h n i q u e . Considering the f a c t
t h a t t h e p o l y m e r n e t w o r k has t o be f o r m e d i n t h e p r e s e n c e o f t h e
f i n a l l y entrapped b i o l o g i c a l m a t e r i a l , the performance c r i t e r i a of
c h e m i c a l and p h y s i c a l n a t u r e a r e as f o l l o w s :
(1) The n e t w o r k f o r m a t i o n has t o p r o c e e d u n d e r m i l d c o n d i
t i o n s (pH and t e m p e r a t u r e ) i n an aqueous e n v i r o n m e n t ;
(2) t h e n e t w o r k has t o be c h e m i c a l l y s t a b l e u n d e r v a r i o u s r e
a c t i o n c o n d i t i o n s (pH, b u f f e r s o l u t i o n , i o n i c and n o n i o n i c s u b
strates, etc.);
(3) t h e s i z e and t h
e r a b l y as b e a d s ) has t
(4) t h e p o s s i b i l i t y f o r a l a r g e v a r i a t i o n o f b i o m a s s c o n t e n t
i n t h e c a t a l y s t s h o u l d be g i v e n ;
(5) t h e c a t a l y s t b e a d s s h o u l d be m e c h a n i c a l l y s t a b l e t o be
used i n v a r i o u s r e a c t o r c o n f i g u r a t i o n s (packed bed, f l u i d i z e d bed,
s t i r r e d tank).
A p p r o p r i a t e p o l y m e r i c c a r r i e r s c a n be o b t a i n e d f r o m p o l y m e r i c ,
o l i g o m e r i c , and monomeric p r e c u r s o r s . Due t o unwanted c h e m i c a l i n
t e r a c t i o n of such chemicals w i t h the c e l l m a t e r i a l l a r g e r s i z e of
t h e s e p r e c u r s o r s i s f a v o r a b l e . The i o n o t r o p i c g e l a t i o n , s t a r t i n g
f r o m p o l y e l e c t r o l y t e s and t h e p o l y c o n d e n s a t i o n , s t a r t i n g f r o m o l i
g o m e r i c epoxy r e s i n s , a r e t y p i c a l p r o b l e m s o l u t i o n s .
I o n o t r o p i c G e l a t i o n of P o l y e l e c t r o l y t e s
T h i s method o f n e t w o r k f o r m a t i o n i s d e f i n e d as a c r o s s l i n k i n g
r e a c t i o n of p o l y e l e c t r o l y t e s w i t h lower molecular weight m u l t i v a
lent counterions. C o n s i d e r i n g t h e p o l y m e r i c component, a n i o n i c
( e . g . , a l g i n a t e , CMC (I) o r c a t i o n i c ( c h i t o s a n (2)) substances can
be u s e d . T h i s v a r i e t y o f p o l y m e r s and t h e a p p r o p r i a t e c o u n t e r i o n s
a r e s u m m a r i z e d i n F i g u r e 1. The c h o i c e o f t h e p o l y m e r i s d e t e r
m i n e d by t h e pH r e g i o n o f t h e r e s p e c t i v e b i o c a t a l y t i c r e a c t i o n ,
s i n c e a l l i o n o t r o p i c g e l s a r e r e v e r s i b l e s t r u c t u r e s w h i c h c a n be
r e d i s s o l v e d by i n c r e a s e ( a l g i n a t e ) o r d e c r e a s e ( c h i t o s a n ) o f pH b e
yond c e r t a i n l i m i t s . A second important c r i t e r i o n i s the i o n i c
c o m p o s i t i o n o f t h e r e a c t i o n medium and t h e p o s s i b i l i t y o f i n s o l u b l e
byproduct o r complex f o r m a t i o n w i t h the network forming i o n s .
I n a t y p i c a l a l g i n a t e entrapment process the c e l l s are sus
pended i n a 3% s o d i u m a l g i n a t e s o l u t i o n and t h i s v i s c o u s s u s p e n
s i o n i s p r e c i p i t a t e d d r o p w i s e i n a 1% C a C l 2 s o l u t i o n . A f t e r 30
m i n u t e s s t a b l e C a - a l g i n a t e g e l s a r e f o r m e d where t h e c e l l s a r e i m
m o b i l i z e d i n a macroporous s t r u c t u r e . F o l l o w i n g to t h i s p r e c i p i
t a t i o n p r o c e s s a p a r t i a l d r y i n g s t e p c a n be a p p l i e d w h i c h r e s u l t s
i n a homogeneous s h r i n k i n g o f t h e p a r t i c l e s , t h u s i n c r e a s i n g c o n -

KLEIN A N D VORLOP Immobilized Cells as Catalysts 379
POLYELECTROLYTES MULTIVALENT COUNTERIONS
POLYANIONS
ALGINAT
CARBOXYMETHYL-
'""cOO^ Χ φ
CELLULOSE
Ca . Fe . Zn
2 + 2 + 2 +
...
CARBOXY-GUAR-
Al \
3
Fe 3 +
GUM
COPOLY-STYRENE-
MALEIC ACID
POLYCATIONS
CHITOSAN Fe(CN) 6
4
\ Fe(C^ 3
"
POLY-PHOSPHATE
/
^ΝΗ3 Φ
Χ θ
POLY-ALDEHYDO-CARBONIC
ACID
P0LY-1-HYDR0XY-1-
SULF0NATE-PR0PEN-2
ALGINATE
Figure 1. Summary of polymer-counterion systems to be used in ionotropic

gelation for whole cell entrapment. Reprinted, with permission, from Ref. 13. Copy
right 1982, Plenum Publishing Corp.

s i d e r a b l y t h e m e c h a n i c a l s t a b i l i t y as w e l l a s t h e p a c k i n g d e n s i t y
of the entrapped c e l l s i t s e l f . A l l these f a c t o r s are very advanta
geous f o r t h e b i o c a t a l y t i c a p p l i c a t i o n . The f l e x i b i l i t y o f t h e a l -
g i n a t e - m e t h o d c a n be d e m o n s t r a t e d a c c o r d i n g t o t h e f o l l o w i n g p a r
a m e t e r b o u n d a r y v a l u e s : p o l y m e r c o n c e n t r a t i o n f r o m 0.5 t o 8%, CaC^
c o n c e n t r a t i o n f r o m 0.05 t o 2%, c e l l c o n c e n t r a t i o n (on wet w e i g h t
b a s i s ) f r o m 0.1 t o 100%, b e a d d i a m e t e r s f r o m 0.1 t o 5 mm, and
p r e p a r a t i o n t e m p e r a t u r e s f r o m 0 t o 80° C. Cells of different
s t r u c t u r e ; e.g., a e r o b i c (1) o r a n a e r o b i c m i c r o b e s ( 3 ) , p l a n t c e l l s
( 4 ) , mammalian c e l l s ( 5 ) , c a n be e n t r a p p e d and t h u s s t a b i l i z e d
without c o n s i d e r a b l e t o x i c i t y problems.
A p r o b l e m o f p r a c t i c a l i m p o r t a n c e i s t h e s c a l e up o f t h e i m
m o b i l i z a t i o n p r o c e s s f r o m amounts o f s e v e r a l grams t o s e v e r a l
hundred l i t e r s . W h i l e s m a l l amounts c a n e a s i l y be p r e p a r e d u s i n g
one c a p i l l a r y o r i f i c e , a b u n d l e o f s u c h c a p i l l a r y i n a s i e v e p l a t e
type c o n s t r u c t i o n w i l l g i v
i f the c a p i l l a r y c h a r a c t e r i s t i c
a r e shown i n F i g u r e 2.
Polycondensation o f Epoxy Resins
I n t h i s c a s e c o v a l e n t n e t w o r k s o f h i g h m e c h a n i c a l and c h e m i
c a l s t a b i l i t y a r e o b t a i n e d as a r e s u l t o f c r o s s l i n k i n g r e a c t i o n of
e p o x i d e s w i t h m u l t i f u n c t i o n a l a m i n e s ( 6 ) . The m a i n p r o b l e m s o f
t h i s t e c h n i q u e a r e t h e t o x i c i t y o f t h e amino-component and t h e
u s u a l l y l o w p o r o s i t y o f t h e p o l y m e r i c n e t w o r k . The t o x i c i t y , mea
s u r e d by t h e v i a b i l i t y o f i m m o b i l i z e d y e a s t c e l l s , c o u l d be m i n i
m i z e d a) by p r o p e r s e l e c t i o n o f e p o x y and amino components and b)
by i n t r o d u c t i o n o f a p r e g e l l i n g t i m e i n t h e o r d e r o f 15 m i n u t e s
b e f o r e m i x i n g t h e c e l l s w i t h t h e c o n d e n s a t i n g o l i g o m e r s ( 7 ) . The
p o r o s i t y o f t h e m a t r i x i s i n t r o d u c e d by t h e i m m o b i l i z e d c e l l s i t
s e l f and by an i n t e r m e d i a t e p r e p a r a t i o n o f an i n t e r p e n e t r a t i n g
n e t w o r k w i t h an i o n o t r o p i c g e l . The i o n o t r o p i c g e l a t i o n i s a l s o
u s e d t o c o n t r o l t h e p a r t i c l e s h a p e and s i z e . A c o m p l e t e scheme o f
s u c h a n i m m o b i l i z a t i o n p r o c e s s i s shown i n F i g u r e 3. A g a i n q u i t e
h i g h c o n c e n t r a t i o n s (up t o 70% on wet w e i g h t b a s i s ) o f c e l l s c a n
be f i n a l l y i n c o r p o r a t e d i n s u c h a p o l y m e r i c s t r u c t u r e .
The v i a b i l i t y o f t h e y e a s t c e l l s , and t h u s t h e r e d u c e d t o x i c
i t y o f t h e e n t r a p m e n t m e t h o d , c a n be d e m o n s t r a t e d by c e l l g r o w t h
i n the m a t r i x , which gives r i s e to a corresponding a c t i v i t y i n
crease f o r ethanol p r o d u c t i o n from glucose ( 7 ) . This behavior i s
shown i n F i g u r e 4. The f a c t o r o f a c t i v i t y i n c r e a s e compared t o t h e
i n i t i a l v a l u e i n c r e a s e s w i t h d e c r e a s i n g i n i t i a l l o a d i n g ; however,
i t i s o b v i o u s t h a t an u p p e r l i m i t o f a c t i v i t y w i l l f i n a l l y be
reached. The r e a s o n f o r t h i s phenomenon, as w e l l as f o r t h e a c
t i v i t y d e c r e a s e w i t h i n c r e a s i n g i n c u b a t i o n t i m e , w i l l become
obvious from the d i s c u s s i o n s o f the f o l l o w i n g chapter.

PRESSURE
Figure 2. Scheme for catalyst bead formation by ionotropic gelation, including

scale-up device.
10g tpoxy resin

• 33g curing agent (30%-soUn HÔ)
* 7 ml H 0
-±
2
±
precondensotion
(15m. n.)
mixing
periodical
6g bake i*s yeast • 2ml
injection
• 20g β % alginate- sol.
, Qoooonnnnnooo 0
ο.ο
drying (24h) crosslink!ng 9r^-> ol
(20min) 2wt%CaCl2-solution
EP0XY-BEADS
alginate-dissolution (40min)
in 0.1« phosphate-buffer (pH> 6.0)
Figure 3. Process scheme for preparation of biocatalysts by cell entrapment in

epoxy beads. Reprinted, with permission, from Ref. 14. Copyright 1982, Science
and Technology Letters.

Figure 4. Dependence of biocatalytic activities for the batch fermentation of

ethanol from glucose with immobilized yeast cells as a function of incubation time
(time for cellgrowth in the carrier) for various initial cell loadings in epoxy carriers.

17. KLEIN A N D VORLOP Immobilized Cells as Catalysts 383
Effectiveness o f Immobilized C e l l Catalysts
I t i s a w e l l known f a c t i n h e t e r o g e n e o u s c a t a l y s i s , t h a t t h e
c a t a l y t i c a c t i v i t y i s generally not d i r e c t l y proportional to the
c o n c e n t r a t i o n o f a c t i v e s i t e s b u t depends a l s o o n h y d r o d y n a m i c
c o n d i t i o n s i n t h e s u r r o u n d i n g o f t h e p a r t i c l e s , on p a r t i c l e s i z e
and m a t r i x p o r o s i t y . I t i s furthermore w e l l understood, that
v a r i o u s t r a n s p o r t phenomena h a v e t o be t a k e n i n t o a c c o u n t , m a i n l y
d i f f u s i o n a l transport processes which n e c e s s a r i l y a r e preceding to
the r e a c t i o n step i t s e l f .
A d i m e n s i o n l e s s number, u s u a l l y c a l l e d T h i e l e - m o d u l u s , c a n be
used t o q u a n t i t a t i v e l y account f o r t r a n s p o r t - r e a c t i o n c o u p l i n g
phenomena. A s s u m i n g t h e v a l i d i t y o f M i c h a e l i s - M e n t e n r a t e e q u a
t i o n - which i s j u s t i f i e d f o r simple enzymatic r e a c t i o n s i n whole
c e l l s t o o - t h e f o l l o w i n g e x p r e s s i o n f o r t h e T h i e l e modulus has
been d e r i v e d ( 8 ) :
f
where R i s t h e p a r t i c l e r a d i u s , v t h e r a t e o f r e a c t i o n , the
M i c h a e l i s c o n s t a n t , S t h e s u b s t r a t e c o n c e n t r a t i o n and D the e f
e
f e c t i v e s u b s t r a t e d i f f u s i v i t y i n the porous c a t a l y s t p a r t i c l e .
On t h e o t h e r hand t h e e f f e c t i v e n e s s f a c t o r η i s d e f i n e d a s t h e
r a t i o o f t h e e f f e c t i v e r e a c t i o n r a t e ν t o t h e maximum r e a c t i o n
rate ν w h i c h w o u l d be o b s e r v e d w i t h o u t t r a n s p o r t l i m i t a t i o n
max
n = ~ - (2)
max
F o r i m m o b i l i z e d c e l l c a t a l y s t s t h e r e a r e two p o s s i b i l i t i e s t o ob
t a i n t h i s f a c t o r . F i r s t l y , t h e p a r t i c l e s o f l a r g e r r a d i u s can be
g r i n d e d down t o s u c h a s m a l l s i z e t h a t p o r e d i f f u s i o n becomes neg
ligible. I n t h i s case = v ^ + .
e Q Due t o t h e s i z e and t h e
s i m p l e entrapment o f t h e c a t a l y t i c s p e c i e s l o s s from t h e m a t r i x
may b e c o n s i d e r a b l e . Therefore, secondly, the free c e l l a c t i v i t y
can be used i n t h e denominator, i f t h e e x a c t c o n c e n t r a t i o n o f c a t -
i s
a l y t i c a l l y a c t i v e immobilized c e l l s ( X )
a c t known. Since
v.-I.ilfi (3)
i s t h e s p e c i f i c r e a c t i o n r a t e o f t h e f r e e l y suspended c e l l s , t h e
equation
ν = ν ' X = v* (4)
max act
1
h o l d s , w h i c h f u r t h e r m o r e d e f i n e s v i n E q n . ( 1 ) . B a s e d o n numer
i c a l calculations t y p i c a l functions

η - f (Φ) (5)
h a v e b e e n d e v e l o p e d , w h i c h i n t e r r e l a t e t h e two d i m e n s i o n l e s s p a r
ameters and w h i c h c a n be checked e x p e r i m e n t a l l y .
To e v a l u a t e t h e a p p l i c a b i l i t y o f E q n . ( 5 ) , t h e p a r a m e t e r s i n
Eqns. (1-4) have t o be determined i n d e p e n d e n t l y . T h i s has been
done f o r t h e c l e a v a g e r e a c t i o n o f P e n i c i l l i n G t o 6APA w i t h immo
b i l i z e d E. Qoli c e l l s i m m o b i l i z e d i n e p o x i d e b e a d s ( 9 ) .
The r a d i u s R: The r a d i u s o f p a r t i c l e s o b t a i n e d f r o m i o n o t r o p -
i c g e l a t i o n i s u s u a l l y c o n t r o l l e d w i t h i n v e r y narrow l i m i t s and
t h e s i z e c a n e a s i l y b e d e t e r m i n e d by m i c r o s c o p i c m e a s u r e m e n t s .
Reaction K i n e t i c s ; The r e a c t i o n r a t e s h a v e b e e n m e a s u r e d a t
pH = 7.8 a n d Τ = 37o C, u s i n g a 5% P e n G s u b s t r a t e c o n c e n t r a t i o n .
T i t r a t i o n w i t h 0.1 m o l a r NaOH h a s b e e n u s e d t o d e t e r m i n e t h e amount
o f p r o d u c t f o r m a t i o n . The K^j v a l u e s o f f r e e a n d i m m o b i l i z e d c e l l s
have been o b t a i n e d from
e x a m p l e s i n F i g u r e 5. F o l l o w i n
enzymes d u r i n g t h e p r o c e s s o f i m m o b i l i z a t i o n , t h e i n e q u a l i t y X £ < a C
n
^ m m o b i l . °lds. f o l l o w i n g approach has been developed f o r
the determination o f X : i n a c e r t a i n experiment, i n a f i r s t ap
a c t
proximation X a c t - Ximm.; i . e . , 100% o f a l l i m m o b i l i z e d c e l l s a r e

β
assumed t o b e a c t i v e . I n t h i s case, η 0.23 h a s b e e n d e t e r m i n e d .
I f x < η
act ^mm. » become l a r g e r , due t o t h e d e c r e a s e o f t h e
d e n o m i n a t o r i n E q n . ( 2 ) . I n t h e same way a s e r i e s o f Φ-values c a n
be c a l c u l a t e d f r o m E q n . ( 1 ) o n t h e b a s i s o f d i f f e r e n t v ' - v a l u e s .
The c o r r e s p o n d i n g s e t s o f v a l u e s f o r d i f f e r e n t a s s u m p t i o n s o f X a c t
are l i s t e d i n Table I :
Table I . C a l c u l a t e d η- a n d Φ- v a l u e s b a s e d
on d i f f e r e n t assumptions o f r e
s i d u a l c e l l a c t i v i t y a f t e r immo
bilization.
: X
act^ imm.
% η cale. Φ cale.
10 2.36 0.59
20 1.18 1.34
30 0.79 1.44
40 0.59 1.63
50 0.47 2.11
60 0.39 2.32
70 0.34 2.50
80 0.30 2.68
90 0.26 2.84
100 0.24 3.00
The t r u e v a l u e o f X a c t has t o s a t i s f y Eqn. ( 5 ) , w h i c h has been

KLEIN AND VORLOP Immobilized Cells as Catalysts 385
Figure 5. Determination of the Michaelis constant, K , for 6 ΑΡΑ formation

v
from penicillin G with immobilized E . coli cells. Conditions: pH 7.81, 37°C, 5%

penicillin G solution, epoxy carrier.

g e n e r a l l y c a l c u l a t e d ( 8 ) . P l o t t i n g a l l data from Table I together

w i t h t h e f u n c t i o n Eqn. ( 5 ) i n F i g u r e 6 g i v e s a p o i n t o f i n t e r s e c
t i o n , w h i c h d e t e r m i n e s t h e t r u e p a i r o f η/Φ-values and t h u s X « a c t
C o m p a r i s o n o f s e v e r a l i m m o b i l i z e d c e l l p r e p a r a t i o n s gave p r a c t i c
a l l y i d e n t i c a l r e s u l t s , showing that a value X a c t = 0*43X i m m #
i s a t y p i c a l one f o r t h i s e p o x i d e - i m m o b i l i z a t i o n p r o c e d u r e .
E f f e c t i v e Pore D i f f u s i v i t y : The e f f e c t i v e p o r e d i f f u s i v i t y
o f p e n i c i l l i n G has been determined e x p e r i m e n t a l l y i n a b a t c h ex
p e r i m e n t ( 1 0 ) . M i x i n g a c e r t a i n number o f c a t a l y s t b e a d s h a v i n g a
homogeneous s u b s t r a t e c o n c e n t r a t i o n S ( g / 1 ) , w i t h a c e r t a i n v o l u m e
a
o f f r e s h , n o n - c o n d u c t i n g w a t e r , t h e d i f f u s i o n p r o c e s s c a n be f o l
l o w e d by t h e c o n d u c t i v i t y a n d t h e c o n c e n t r a t i o n i n c r e a s e i n t h e
supernatent l i q u i d . I f S i s the s u b s t r a t e c o n c e n t r a t i o n i n the
e
p a r t i c l e a t t + «> a n d S t h e c o n c e n t r a t i o n a t t i m e t , R t h e p a r t i
2
c l e r a d i u s (cm) a n d D t h e e f f e c t i v e d i f f u s i v i t y ( c m / s e c ) t h e
e
d i f f u s i o n c o e f f i c i e n t ca
In (S-S )/(S -S ) vs. t
e a e
(6)
Such a p l o t i s shown i n F i g u r e 7.
Comparison o f Theory and E x p e r i m e n t : I f t h e s i m p l e model o f
t r a n s p o r t l i m i t a t i o n due t o p o r e d i f f u s i o n h o l d s , a l l e x p e r i m e n t
a l l y d e t e r m i n e d p a i r s o f η and Φ s h o u l d f a l l o n t h e n o n - d o t t e d
η = ί(Φ) - f u n c t i o n shown i n F i g u r e 6. U s i n g t h e i n d e p e n d e n t l y
d e t e r m i n e d parameters as d e s c r i b e d b e f o r e such v a l u e s have been
d e t e r m i n e d f o r a number o f c a t a l y s t p r e p a r a t i o n s u s e d i n t h e p e n
i c i l l i n G c l e a v a g e r e a c t i o n . The e x c e l l e n t agreement b e t w e e n c a l
c u l a t i o n o f Eqn. (5) a n d e x p e r i m e n t a l d e t e r m i n a t i o n c a n b e s e e n
f r o m F i g u r e 8. I n t h i s s e r i e s o f e x p e r i m e n t s two d i f f e r e n t
s t r a i n s o f E. ooli c e l l s h a v e b e e n u s e d , t h e one o f them h a v i n g
been o b t a i n e d by g e n e t i c e n g i n e e r i n g l e a d i n g t o a t e n f o l d a c t i v i t y
i n c r e a s e compared t o t h e c o n v e n t i o n a l s t r a i n ATCC 11 105 (1)·
C a t a l y s t O p t i m i z a t i o n : I n a n o t h e r r e a c t i o n example t h e a p
p l i c a t i o n o f the Thiele-modulus concept f o r the o p t i m i z a t i o n o f
c a t a l y s t c o m p o s i t i o n c o u l d be demonstrated. Here t h e o x i d a t i o n o f
g l u c o s e t o g l u c o n i c a c i d , c a t a l y z e d by A c e t o b a c t e r s i m p l e x c e l l s
has been s t u d i e d , where oxygen i s t h e r a t e l i m i t i n g s u b s t r a t e .
I n t h i s case a C a - a l g i n a t e m a t r i x has been used f o r c e l l immobil
ization. U s i n g s i m p l i f i e d e q u a t i o n s f o r t h e c a l c u l a t i o n o f D ande
furthermore assuming X a c t = Xi
m m # ( 1 2 ) , t h e r e l a t i o n between r e l a
t i v e a c t i v i t y (η i n %) a n d p a r t i c l e d i a m e t e r c o u l d be c a l c u l a t e d
for different c e l l concentrations. As c a n be shown i n F i g u r e 9,
a good agreement w i t h e x p e r i m e n t a l d a t a i s o b t a i n e d .
The a b s o l u t e a c t i v i t y f o r g l u c o n i c a c i d p r o d u c t i o n i s o b
t a i n e d i f the s p e c i f i c a c t i v i t y o f the d i f f e r e n t preparations i s
m u l t i p l i e d w i t h t h e c e l l c o n c e n t r a t i o n . The n o n - d o t t e d c u r v e i n
F i g u r e 10 i s t h e r e s u l t o f t h i s c a l c u l a t i o n , w h i c h g i v e s a good

KLEIN AND VORLOP Immobilized Cells as Catalysts 387
Figure 6. Determination of catalytically active cell concentration, X , act on the

basis of the effectiveness factor/Τhiele modulus relation.
Ο Γ
Î ln[(S - S ) / (S
e a - Se)]
•
-2 -
-4 h
i t i i i l » 1
100 200 300 400

t <··ο
Figure 7. Plot of time dependence of solution concentration from a batch diffusion
experiment for the determination of the effective substrate diffusivity, D . e

1.0
0.5
0.2h
0.1
Figure 8. Comparison of calculated and experimentally determined effectiveness

factor and Thiele modulus
immobilized E . coli cells. Conditions:
epoxy-carrier. Key: ψ , ATCO11 105; · , E . coli 5 Κ (pHM 12).
-I ι ι 1—
1 2 3 4
P a r t i c l e Diameter (mm)
Figure 9. Dependence of residual activity (effectiveness of immobilized cells) on

particle size and cell loading for the production of gluconic acid from glucose with
calcium alginate immobilized Acetobacter simplex cells.

Figure 10. Catalytic effectiveness (activity in % ) and absolute catalytic activity as

a function of cell concentration for gluconic acid production from glucose with
calcium alginate immobilized Acetobacter simplex cells. Reprinted, with permission,
from Ref. 13. Copyright 1982, Plenum Publishing Corp.

c o r r e l a t i o n t o e x p e r i m e n t a l d a t a shown i n t h e same g r a p h . The

maximum i n t h i s c u r v e g i v e s t h e optimum v a l u e o f c e l l l o a d i n g and
c a n be e x p l a i n e d by t h e i n c r e a s e d b l o c k i n g o f p o r e s p a c e f o r o x y
gen t r a n s p o r t w i t h an i n c r e a s i n g number o f i m m o b i l i z e d c e l l s . In
t h e same s e n s e t h e c u r v a t u r e o f t h e r e a c t i v i t y c u r v e s o f F i g u r e 4
s h o u l d be r e c a l l e d .
C a t a l y s t D e a c t i v a t i o n and O p e r a t i o n a l Stability
Coming b a c k t o t h e p e n i c i l l i n G c l e a v a g e r e a c t i o n , l o n g t i m e
e x p e r i m e n t s w i t h e p o x i d e - i m m o b i l i z e d E. ooli c e l l s h a v e b e e n r e
p o r t e d e a r l i e r (6) . W i t h o u t any c o r r e c t i o n an e f f e c t i v e h a l f - l i f e
v a l u e t , i 2 o f c a t a l y t i c a c t i v i t y o f 43 d a y s h a s b e e n d e t e r m i n e d .
R e p e a t e d e x p e r i m e n t s c o n f i r m e d t h e a b r u p t change o f s l o p e i n t h e
a c t i v i t y - t i m e curve of consecutive batch r e a c t i o n s . In another
e x p e r i m e n t , where t h e c e l l
t r a t i o n a t the s u r f a c e o
l i n e a c t i v i t y - t i m e f u n c t i o n has b e e n f o u n d w i t h a much s m a l l e r
v a l u e t j / 2 H d a y s (9).
=
I t t h e r e f o r e becomes o b v i o u s , t h a t
t r a n s p o r t l i m i t a t i o n due t o much h i g h e r c e l l l o a d i n g and l o n g e r
d i f f u s i o n p a t h w a y s i n a b e a d - t y p e c a r r i e r m i g h t be r e s p o n s i b l e f o r
t h e two d i f f e r e n t s l o p e s .
The e x p e r i m e n t a l d a t a a r e shown i n F i g u r e 11a, t o g e t h e r w i t h
a d o t t e d l i n e , e x t r a p o l a t i n g the r e a c t i o n c o n t r o l l e d regime to
z e r o t i m e . On t h e o t h e r h a n d some m o d e l c a l c u l a t i o n s h a v e b e e n
performed under the assumption t h a t , a t the b e g i n n i n g of the ex
periment, the r e a c t i o n r a t e i s i n the d i f f u s i o n c o n t r o l l e d regime.
Then o n l y a r e s t r i c t e d s p h e r i c a l s h e l l c l o s e t o t h e b e a d s u r f a c e
i s p a r t i c i p a t i n g i n t h e s u b s t r a t e c o n v e r s i o n . I f s u p e r i m p o s e d on
the r e a c t i o n a time dependent c a t a l y s t d e a c t i v a t i o n o c c u r s , the
reactive s h e l l s h o u l d move t o t h e c e n t e r o f t h e c a t a l y s t p a r t i
c l e where a c t i v e c e l l s a r e a v a i l a b l e t o s u b s t i t u t e f o r the d e a c t i
vated c e l l s c l o s e r to the s u r f a c e .
Assuming d i f f e r e n t r a t e e q u a t i o n s f o r the time dependent
d e a c t i v a t i o n r e a c t i o n the graphs i n F i g u r e l i b are obtained.
W i t h o u t a c h i e v i n g q u a n t i t a t i v e agreement i n such a s i m p l i f i e d c a l
c u l a t i o n , we b e l i e v e t h a t t h e good q u a l i t a t i v e agreement o f t h e
non-dotted l i n e s i n F i g u r e l i b w i t h the experimental curve i n
F i g u r e 11a s u p p o r t s o u r c o n s i d e r a t i o n s i n p r i n c i p l e . The g e n e r a l
c o n c l u s i o n i s t h a t one s h o u l d be v e r y c a r e f u l i n d e r i v i n g i n t r i n s i c
h a l f - l i f e values of immobilized c e l l s from o v e r a l l o p e r a t i o n a l
s t a b i l i t y data of b i o c a t a l y t i c conversion. D i f f u s i o n a l l i m i t a t i o n
w i l l always l e a d to a r t i c i a l l y higher v a l u e s .
Acknowledgements
The f r u i t f u l c o o p e r a t i o n w i t h P r o f e s s o r F. Wagner and h i s r e

s e a r c h g r o u p as w e l l as t h e f i n a n c i a l s u p p o r t f r o m t h e BMFT u n d e r
G r a n t No. PTB 8150 i s g r a t e f u l l y a c k n o w l e d g e d .

17. KLEIN AND VORLOP Immobilized Cell as Catalysts 391
5+
4+ experimental v
-
observation \^ 30 d
3t Λ . . .
6-ΑΡΑ 43 d
• 2
1 +
—r—
10 20 30 40
t
days « cycles
_ 100
Figure 11. Operational stability functions of E . coli cells immobilized in epoxy

carrier for consecutive batch operation in the production of 6 ΑΡΑ from penicillin
G. Top, experimental data; bottom, model calculations for the superposition of 6
ΑΡΑ formation and conversion dependence catalyst deactivation reaction in the
diffusion controlled regime.

Literature cited
1. K l e i n , J . ; Hackel,U., and Wagner,F. ACS Symposium Series,1979,

106,101
2. Vorlop,K.-D., and K l e i n , J . B i o t e c h n o l . L e t t e r s 1981,3,65
3. Scherer,P.; Kluge,M.; K l e i n , J . , and Sahm,H., B i o t e c h n o l . B i o -
eng. 1981,XXIII,1057
4. Scheurich,P.; Schnabl,H.; Zimmermann,U., and K l e i n , J . Biochem
& Biophys. Acta 1980,598,645
5. Pilwat,G.; Washausen,P.; K l e i n , J . , and Zimmermann,U., Z. Na-
turforschung 1980,35c,352
6. K l e i n , J . , and Eng,H., B i o t e c h n o l . L e t t e r s 1979,1,171
7. Kressdorf,B., Diplom T h e s i s , T e c h n i c a l U n i v e r s i t y Braunschweig
1981
8. B i s c h o f f , K.B., AICHE J 1965,11,351
9. Eng,H., PhD. T h e s i s
10. K l e i n , J . , and Washausen,P.
l a g Chemie, Weinheim 1979, p. 300
11. K l e i n , J . , and Wagner,F., Enzyme Engineering Vol.5,; H.Weetall
and G. Royer, ed., Plenum Publ. Corp., New York 1980,p.359
th
12. Vorlop, K.-D.; K l e i n , J . , and Wagner,F. 6 I n t e r n . Fermenta-
t i o n Symposium, London (Canada), 1980, paper F 12.1.12.p.
13. K l e i n , J . , and Manecke,G., Enzyme Engineering Vol.6, Plenum
Publ. Corp., New York 1982.
14. Klein,J.,and Kressdorf,B., B i o t e c h n o l . L e t t e r s 1982, 4, 375.
R E C E I V E D August 18, 1982

18
Dissolved Oxygen Contours in Pseudomonas
ovalis Colonies
H. R. BUNGAY, P. M. PETTIT, and A. M. DRISLANE

Rensselaer Polytechnic Institute, Troy, NY 12181
Pseudomonas o v a l i s colonies of various sizes grown

on agar supplemente
of dissolved oxyge
agar. Proximity to glucose from the agar determines
the demand for oxygen, so regions of a colony that
are distant from the agar tend to have ample d i s -
solved oxygen. A control colony on unsupplemented
agar was r i c h i n dissolved oxygen. As colony size
increased on agar with added glucose, regions quite
low i n dissolved oxygen developed.
Our group has investigated oxygen transfer i n a number of micro-

b i a l systems such as slimes, p e l l e t s , f l o e s , and films (1). A
l o g i c a l extension i s the study of colonies because many micro-
organisms grow attached to surfaces. Although colonies are often
bathed by nutrient media, i t seemed convenient and i n t e r e s t i n g to
work with bacteria growing on a s o l i d surface that supplied
nutrients. Studies of oxygen transfer i n microbial systems often
employ Pseudomonas o v a l i s because t h i s bacterium has a high
demand f o r oxygen as i t converts glucose to gluconic acid. We
have at times used oxygen microelectrodes drawn to t i p s one
micrometer i n diameter, but a t i p of about four micrometers i s
more rugged and i s adequate for studying colonies that are
several millimeters i n diameter.
Materials and Methods
Colonies of Pseudomonas o v a l i s Strain ATCC 8209 were grown

i n p e t r i dishes on nutrient agar (8 g. Difco nutrient broth +
15 g. Difco Bacto-agar/L.) with supplemental glucose. Agar was
poured into p e t r i dishes to a height of about 0.5 cm. Dilute
suspensions of organisms were streaked onto the agar c a r e f u l l y
to avoid d i s t o r t i n g the surface.
0097-6156/83/0207-0395$06.00/0

The a p p a r a t u s h a s b e e n r e p o r t e d p r e v i o u s l y ( 1 ) . The m i c r o
m a n i p u l a t o r had c o a r s e c o n t r o l i n t h e X and Y a x e s and a p r e c i s e
v e r n i e r f o r the Ζ a x i s . A r e f e r e n c e p l a n e was e s t a b l i s h e d by
l o w e r i n g t h e m i c r o p r o b e u n t i l e l e c t r i c a l c o n t a c t was e s t a b l i s h e d
a t p o i n t s s e v e r a l m i l l i m e t e r s away f r o m t h e c o l o n y i n a l l d i r e c
tions. The c o l o n y was t r a v e r s e d by t h e m i c r o p r o b e a t t h e c e n t e r
l i n e , a t s e v e r a l l o c a t i o n s p r o g r e s s i n g t o w a r d t h e e d g e , and a d j a
c e n t a g a r was a l s o p r o b e d . S m a l l , medium, and l a r g e c o l o n i e s
were s e l e c t e d .
R e s u l t s and Discussion
F i r s t e l e c t r i c a l c o n t a c t w i t h t h e c o l o n y was n o t r e p r o d u c
i b l e , and an u n c e r t a i n t y o f a b o u t 5 m i c r o m e t e r s e x i s t s . Further
more, t h e s i g n a l f r o m t h
e r r a t i c near the p o i n t
agar remote from the c o l o n y
proof i s o f f e r e d , i t i s thought t h a t o u t e r c e l l s or a t h i n f i l m
o f m o i s t u r e c a n d r a w away f r o m t h e e l e c t r o d e a s a r e s u l t o f
e l e c t r i c a l s t i m u l a t i o n . When a d v a n c e d a few m i c r o n s p a s t t h e
p o i n t of f i r s t e l e c t r i c a l c o n t a c t , the m i c r o e l e c t r o d e d e l i v e r e d
a stable signal.
P r o f i l e s f r o m t y p i c a l t r a v e r s e s a r e i n F i g u r e 1. From s u c h
p r o f i l e s , l o c a t i o n s o f a g i v e n o x y g e n c o n c e n t r a t i o n were t a k e n
t o c o n s t r u c t t h e c o n t o u r l i n e s f o r F i g u r e 2. C o l o n i e s were
assumed t o be s y m m e t r i c a l , so o n l y one s i d e was p r o b e d . Note
t h a t t h e a b s c i s s a c h a n g e s , and t h e c o l o n i e s f o r F i g u r e 2C and
2D a r e much l a r g e r t h a n t h o s e f o r 2A and 2B. The c o l o n i e s d i d
n o t t e n d t o s i t e x a c t l y on t h e p l a n e o f t h e a g a r ; t h i s may be
e r r o r because the p r e c i s i o n of p o s i t i o n i n g the m i c r o e l e c t r o d e i s
g r e a t compared t o p o s s i b l e bumps i n t h e a g a r s u r f a c e . However,
a s t h e shape o f t h e c u r v e f o r f i r s t e l e c t r i c a l c o n t a c t was
u s u a l l y p e c u l i a r a t the edges o f the c o l o n y , i t i s l i k e l y t h a t
g r o w t h and m e t a b o l i s m d i s t o r t t h e a g a r .
No s u p p l e m e n t a l g l u c o s e was i n t h e a g a r f o r t h e c o l o n y f o r
F i g u r e 2A. Compared t o a c o l o n y o f s i m i l a r s i z e on a g a r p l u s
g l u c o s e ( F i g u r e 2 B ) , t h e c o l o n y w i t h l i t t l e demand f o r o x y g e n
i s r i c h i n oxygen throughout. Shape o f t h e c o l o n y c h a n g e s a s i t
grows; h e i g h t remains f a i r l y c o n s t a n t as the c o l o n y spreads.
O u t e r p o r t i o n s o f a c o l o n y a r e r i c h i n o x y g e n , and s t e e p g r a d i e n t s
e x i s t t o w a r d t h e a g a r s u r f a c e . Oxygen c a n d i f f u s e t h r o u g h t h e
a i r i n t e r f a c e o f the c o l o n y o r can e n t e r the s u r r o u n d i n g agar
and d i f f u s e t o w a r d t h e c o l o n y a s t h e c o n t o u r s i n d i c a t e . Glucose
i s a l a r g e r m o l e c u l e t h a n o x y g e n and d i f f u s e s more s l o w l y . The
s h a p e o f t h e c o n t o u r l i n e s and t h e i r s t e e p n e s s n e a r t h e a g a r
i n d i c a t e regions of r a p i d metabolic a c t i v i t y . I t s h o u l d be
p o s s i b l e to r e l a t e the contours to r a t e of glucose d i f f u s i o n
through the colony.
T h i s new method f o r i n v e s t i g a t i n g c o l o n i a l g r o w t h and mass
t r a n s f e r c a n be e x t e n d e d t o v a r i o u s o r g a n i s m s and d i f f e r e n t m e d i a .

Figure 1. Dissolved oxygen profiles from typical traverses of a microelectrode through a P. ovalis
colony. Colony diameter, 1.5 mm; 40 mg/L glucose. Key: A , center line of colony; B, 0.5 mm from
center; C , 0.75 mm from center; D, 1.0 mm from center. vo
- CONTACT LINE 00
2A 2B
—
— CONTACT LINE
/ - —
-
UJ
-
' Il \ L *\ *
5
- j\ ' 7 III' ι I \ \\\\ Ν
' 'IV : !
CL ·'/'' '' · '··\
// Μ \ / t II
/ I !
/ ι
J ;\ \ο ' : ι ι
6 ν
— 1 ! ί \ £ y : ! 1
<ΪΓι I \
o o -60
ι
w
1
1
1 I » / ι
6! 1
1 1 ! \ ' ·
y -80 1 1 51 \ ' ι
LU , 1 1
-100 - 1 1 ί V S ί
ο 1 1
1 ι " — " I
1
-120 - 1
6 1
1 ι
/ I
CO \ ι
û -140
ι
- X !
-160
I /
\ 1 \ \ ι
- 1 '
-180 • ι » 1 \
• 1
-200 - 1 !
I
ι 1

-220 - i !
,! ί , ι ι ι ι ι ι
1.5 1.0 ι
.5 :ι
ι 1.0 ι
1.5 1.0 .5 1.0 1.5

DISTANCE FROM COLONY CENTER
MILLIMETERS
Figure 2A and B. Contours of dissolved oxygen in colonies of P. ovalis of different sizes. Note
that the colonies are much smaller than those shown in Figures 2C and 2D. Continued on next page.
^
Contours of dissolved oxygen in a colony of P. ovalis. Continued on next page.
Figure 2C.

DISTANCE FROM COLONY CENTER

MILLIMETERS
Figure 2D. Contours of dissolved oxygen in a colony of P. ovalis.

18. BUNGAY E TA L . Dissolved Oxygen Contours 401
The r e l e a s e o f o x y g e n f r o m c o l o n i e s o f p h o t o s y n t h e t i c o r g a n i s m
w o u l d be p a r t i c u l a r l y i n t e r e s t i n g . Measurement o f d i s s o l v e d
o x y g e n c o n t o u r s p r o v i d e s new p e r s p e c t i v e s t o t h e a n a l y s i s o f
c o l o n i a l g r o w t h ( 2 ) . F u r t h e r m o r e , b e t t e r u n d e r s t a n d i n g o f mass
t r a n s f e r t o c o l o n i e s h a s economic v a l u e f o r b e t t e r o p e r a t i o n o f
p r o c e s s e s dependent on a t t a c h e d o r g a n i s m s . F o r example, t r i c k
l i n g f i l t e r s f o r b i o l o g i c a l waste treatment, p u r i f i c a t i o n o f
streams by s l i m e s , and f o u l i n g o f c o o l i n g towers by s l i m e s i n
v o l v e c o l o n i a l growth.
Acknowledgement
T h i s p r o j e c t was s u p p o r t e d b y t h e N a t i o n a l S c i e n c e F o u n d a t i o n
t h r o u g h G r a n t //CPE-7920121.
Literature Cited
1. Whalen, W.J.; Bungay, H.R.; Sanders, W.M. Environ. Sci.

Technol. 1969, 3, 1297.
Sanders, W.M.; Bungay, H.R.; Whalen, W.J. AIChE Symp. 107
1971, 69.
Huang, M.Y.; Bungay, H.R. B i o t e c h n o l . Bioengr. 1973, 15,
1193.
Bungay, H.R.; Masak, R.D. B i o t e c h n o l . Bioengr. 1981, 23,
1155.
Bungay, H.R.; Chen, Y.S. B i o t e c h n o l . Bioengr. 1981, 23,
1893.
2. P i r t , S.J. J . Gen. M i c r o b i o l . 1967, 47, 181.
Cooper, A.L.; Dean, A.C.R.; Hinshelwood, C. Proc. Royal Soc.
1968, 175, B171.
Palumbo, S.A.; Johnson, M.G.; Rieck, V.T.; W i t t e r , L.D.
J . Gen. M i c r o b i o l . 1971, 66, 137.

19
Stochastic Growth Patterns Generated by
Phycomyces Sporangiophores
R. IGOR GAMOW and DAVID E. CLOUGH

University of Colorado, Department of Chemical Engineering, Boulder, CO 80309
The giant a e r i a l sporangiophore of the fungus Phyco-

myces blakesleeanu
out a left-handed
laboratory became f a s c i n a t e d with the nature of t h i s
spiral growth f o r it appeared to us that it must
a c c u r a t e l y r e f l e c t the molecular a r c h i t e c t u r e of the
growing plant cell w a l l . Fine s t r u c t u r e k i n e t i c
a n a l y s i s of both the r o t a t i o n a l and the e l o n g a t i o n a l
components of s p i r a l growth revealed what appeared
to be random growth irregularities. We now show by
using time s e r i e s a n a l y s i s that the apparent r o t a -
t i o n and elongation i r r e g u l a r i t i e s are t r u l y s t o -
c h a s t i c and, most s u r p r i s i n g l y , are not c o r r e l a t e d .
D u r i n g t h e p a s t 100 y e a r s a number o f l a b o r a t o r i e s h a v e
s t u d i e d the growing " a n t i c s " of the g i a n t , s i n g l e - c e l l e d sporan
g i o p h o r e o f t h e f i l a m e n t o u s f u n g u s , Phycomyces b l a k e s l e e a n u s .
" A n t i c s " i s a p p r o p r i a t e h e r e s i n c e , i n terms o f d e s c r i b i n g t h e
v a r i e t y o f growth p a t t e r n s t h a t t h e mature s p o r a n g i o p h o r e p r o
d u c e s , i t c e r t a i n l y a p p e a r s t o be t h e " c l o w n " o f t h e p l a n t w o r l d .
Even l e f t t o i t s own d e v i c e s and n o t s u b j e c t e d t o any s e n s o r y
i n p u t s , i t i s happy i n a d a r k , damp room a t a b o u t 22°C; i t s b e
h a v i o r i s s t i l l q u i t e e x t r a o r d i n a r y b e c a u s e o f i t s phenomenal
g r o w t h r a t e o f t h r e e m i l l i m e t e r s p e r h o u r (50 ym/min) s i m u l t a n e
o u s l y c o u p l e d w i t h a r o t a t i o n r a t e o f 12 t o 15 d e g r e e s p e r m i n .
Shown on t h e TV m o n i t o r i n F i g u r e 1 i s t h e u p p e r p a r t o f a m a t u r e
Phycomyces s p o r a n g i o p h o r e c o n s i s t i n g o f a c y l i n d r i c a l s t a l k , a b o u t
100 ym i n d i a m e t e r crowned w i t h a s p h e r i c a l s p o r e s a c k , a b o u t 500
pm i n d i a m e t e r .
B i o p h y s i c i s t s have l o n g b e e n f a s c i n a t e d w i t h t h i s o r g a n i s m
because of i t s unique sensory a p p a r a t u s . Both the d i r e c t i o n o f
g r o w t h and t h e r a t e o f g r o w t h a r e q u a n t i t a t i v e l y c o n t r o l l e d by a
v a r i e t y of w e l l - d e f i n e d s t i m u l i : they respond t o both the i n t e n
s i t y and t h e d i r e c t i o n o f e i t h e r v i s i b l e o r UV l i g h t , t o a v a r i e t y
o f w i n d g r a d i e n t s , and t o t o u c h . The o r g a n i s m e l i c i t s c o m p l e x
0097-6156/83/0207-0403$06.00/0

ο

5
w

S
2
w
w
Figure 1. The video recorder and TV monitor of the video microscope. On the screen of the 2
monitor is shown a mature stage IVb P h y c o m y c e s sporangiophore. §
19. GAMOW AND CLOUGH Growth of Sporangiophores 405
o l f a c t o r y r e s p o n s e s , i t grows away f r o m g r a v i t y , and f i n a l l y , i t

senses the presence o f d i s t a n t o b j e c t s and thus a v o i d growing
i n t o them. D e t a i l s c o n c e r n i n g t h e s e b e h a v i o r s h a v e b e e n d e
s c r i b e d i n a number o f r e v i e w a r t i c l e s ( 1 - 4 ) . The l a s t b e h a v i o r
l i s t e d , t h e a v o i d a n c e r e s p o n s e , a p p e a r s t o be a c o m b i n a t i o n o f
two i n d e p e n d e n t s e n s o r y i n p u t s , o l f a c t i o n and w i n d (_5, 6 ) . As a
r e s u l t o f a l l these studies concerning t h e sensory apparatus,
more i s known a b o u t t h e t w o - d i m e n s i o n a l g r o w t h p a t t e r n s p r o d u c e d
by t h e Phycomyces s p o r a n g i o p h o r e t h a n a n y o t h e r p l a n t s y s t e m . I t
i s t h u s a l l t h e more s u r p r i s i n g t h a t t h e d e t a i l e d f i n e s t r u c t u r e
of t h e s p i r a l g r o w t h i s o n l y now b e g i n n i n g t o b e known. I n g e n
e r a l , t h e o l d e r Phycomyces l i t e r a t u r e c o n s i d e r e d s p i r a l g r o w t h t o
be a n a l o g o u s t o a r o t a t i n g s c r e w , i . e . t h e f a s t e r t h e s c r e w w o u l d
r o t a t e , t h e f a s t e r i t would e l o n g a t e , and t o t h e f i r s t approxima
tion this i s true. Bu d e t a i l e d f i n f
r o t a t i o n and e l o n g a t i o
showed t h a t t h i s a n a l o g y poo (7)
i n 1958 f i r s t r e a l i z e d t h a t a m a t e r i a l s e c t i o n o f t h e g r o w i n g
z o n e , GZ, i s n o t e q u i v a l e n t t o t h e e n t i r e GZ b u t e a c h s e c t i o n o f
t h e GZ i s i n s t e a d y s t a t e f l u x c h a r a c t e r i s t i c o f t h a t p a r t i c u l a r
section. They a l s o f o u n d t h a t t h e d e g r e e o f r o t a t i o n and e l o n
g a t i o n was n o t u n i f o r m l y d i s t r i b u t e d w i t h i n t h e GZ. I n 1974
O r t e g a , H a r r i s a n d Gamow (8) d e t e r m i n e d t h e r a t i o o f r o t a t i o n t o
e l o n g a t i o n a s a f u n c t i o n o f g r o w i n g zone p o s i t i o n a n d f o u n d t h a t
t h i s r a t i o i n c r e a s e d a s measurements w e r e t a k e n t o w a r d s t h e l o w e r
edge o f t h e g r o w i n g z o n e .
M o s t r e c e n t l y , Gamow and B o t t g e r ( 9 ) , u s i n g e i t h e r h i g h
r e s o l u t i o n 35 mm p h o t o g r a p h y o r a v i d e o TV m i c r o s c o p e , h a v e f o u n d
t h a t when t h e m i n u t e - p e r - m i n u t e r o t a t i o n a l a n d e l o n g a t i o n a l
growth r a t e s were s i m u l t a n e o u s l y measured, b o t h r a t e s were q u i t e
i r r e g u l a r and " a p p e a r e d " t o b e random. I n a d d i t i o n , i t
" a p p e a r e d " t h a t l i t t l e o r no c o r r e l a t i o n e x i s t e d b e t w e e n t h e
i r r e g u l a r i t i e s i n t h e r o t a t i o n r a t e and t h e e l o n g a t i o n r a t e . I n
t h i s p a p e r we h a v e t r e a t e d t h e s e g r o w t h d a t a , b o t h r o t a t i o n a n d
e l o n g a t i o n , t o what i s p o p u l a r l y known a s t i m e s e r i e s a n a l y s i s .
We h a v e c o n c l u d e d f r o m t h e s e a n a l y s e s t h a t n o t o n l y a r e t h e
m e a s u r e d i r r e g u l a r i t i e s i n r o t a t i o n and e l o n g a t i o n p u r e l y random
white n o i s e , b u t that they a r e , i n a d d i t i o n , n o t c o r r e l a t e d . We
would expect t h a t on a m i c r o s c o p i c s c a l e , a l l growth p r o c e s s e s
w o u l d b e s t o c h a s t i c , b u t what i s s u r p r i s i n g i s t h a t t h e m a c r o
s c o p i c g r o w t h k i n e t i c s o f a Phycomyces s p o r a n g i o p h o r e i s a l s o
stochastic.
M a t e r i a l s and M e t h o d s
W i l d - t y p e Phycomyces b l a k e s l e e a n u s s p o r a n g i o p h o r e s ,
N R R L 1 5 5 5 ( - ) , o r i g i n a l l y o b t a i n e d f r o m M. D e l b r i i c k , w e r e grown i n
s h e l l v i a l s c o n t a i n i n g 5.0% p o t a t o d e x t r o s e a g a r (PDA) w i t h 1.0%
yeast extract. The s h e l l v i a l s were i n c u b a t e d u n d e r d i f f u s e i n
c a n d e s c e n t l i g h t i n a h i g h - h u m i d i t y room w i t h a t e m p e r a t u r e r a n g e

b e t w e e n 22° and 27°C. B e f o r e e a c h e x p e r i m e n t t h e s p o r a n g i o p h o r e s

were d a r k a d a p t e d i n r e d l i g h t f o r a t l e a s t 20 m i n u t e s . A l l ex
p e r i m e n t s were c a r r i e d out w i t h a w a t e r - f i l t e r e d r e d l i g h t source.
The a p p a r a t u s u s e d t o s i m u l t a n e o u s l y measure m i n u t e - b y - m i n
u t e t h e n e t r o t a t i o n and t h e n e t e l o n g a t i o n o f a s t a g e IVb GZ was
f i r s t d e s c r i b e d by Cohen and D e l b r u c k (7) and t h e n m o d i f i e d by
Gamow and B o t t g e r ( 9 ) . The m a t u r e s t a g e IVb s p o r a n g i o p h o r e i n a
g l a s s s h e l l v i a l was f i r m l y s e c u r e d t o a s t a g e t h a t r o t a t e d
c l o c k w i s e once e v e r y 60 s e c o n d s . To e n s u r e t h a t t h e GZ o f t h e
s p o r a n g i o p h o r e was v e r t i c a l ( p a r a l l e l t o t h e a x i s o f r o t a t i o n o f
t h e s t a g e ) , a d o u b l e knee was i n s e r t e d b e t w e e n t h e s t a g e and t h e
vial. The r o t a t i n g s t a g e a l l o w e d us t o measure t h e a n g u l a r v e
l o c i t y o f any p a r t i c l e s i t u a t e d a b o v e , b e l o w , o r i n t h e GZ. Be
c a u s e t h e n e t r o t a t i o n o f a m a t u r e s t a g e IVb s p o r a n g i o p h o r e i s i n
t h e same d i r e c t i o n a s t h r o t a t i n (clockwise) particl
e i t h e r above o r i n t h e
one r e v o l u t i o n . By d e t e r m i n i n g ,
a n g u l a r v e l o c i t y of the p a r t i c l e i n r e s p e c t t o the o b s e r v e r .
(For i n s t a n c e , i f a p a r t i c l e c o m p l e t e s one r e v o l u t i o n i n 58 s e c
o n d s , we c a n e a s i l y c a l c u l a t e t h a t t h e e n t i r e r e g i o n b e l o w t h e
p a r t i c l e must h a v e a t o t a l a n g u l a r v e l o c i t y o f 12°/58 s e c o r
12.4°/min.) F o r o u r p r e s e n t e x p e r i m e n t s , we u s e d a g l a s s b e a d
a p p r o x i m a t e l y 15 pm i n d i a m e t e r . The b e a d was p l a c e d on t h e
s t a l k a p p r o x i m a t e l y 50 urn b e l o w t h e s p o r a n g i u m ; t h i s r e g i o n o f
t h e s t a l k shows no e l o n g a t i o n . The GZ, w i t h t h e a t t a c h e d g l a s s
b e a d , was o b s e r v e d c o n t i n u o u s l y t h r o u g h a TV v i d e o camera
a t t a c h e d t o a l o w - p o w e r e d (^X10 m i c r o s c o p e ) . An e l e c t r o n i c t i m e r
w i t h a d i g i t a l p r i n t o u t r e c o r d s t h e t i m e w i t h a r e s o l u t i o n o f 10
milliseconds.
Experimental Controls. The r o t a t i o n a l v e l o c i t y o f a p a r

t i c l e p l a c e d j u s t b e l o w t h e GZ was m e a s u r e d i n o r d e r t o d e t e r m i n e
t h e r o t a t i o n measurement e r r o r . V e l o c i t y measurements o f a p a r
t i c l e b e l o w t h e GZ make a n e x c e l l e n t c o n t r o l s i n c e a l l t h e param
e t e r s a r e t h e same e x c e p t t h e i n n a t e r o t a t i o n o f t h e GZ i t s e l f .
We m e a s u r e d t h e r o t a t i o n r a t e f o r s u c h a p a r t i c l e o n c e e v e r y m i n
u t e f o r 56 c o n s e c u t i v e m i n u t e s and d e t e r m i n e d t h a t t h e s t a n d a r d
d e v i a t i o n was ±1.7° p e r m i n u t e ( 9 ) . The b e a u t y o f o u r r o t a t i o n -
m e a s u r i n g method i s t h a t t h e measurements a r e v i r t u a l l y i n s e n s i
t i v e t o p a r a l l a x p r o b l e m s and t o t h e h u n t i n g p r o b l e m m e n t i o n e d
below. C a l c u l a t i n g e l o n g a t i o n a l v e l o c i t i e s , on t h e o t h e r h a n d ,
i s b e s e t w i t h numerous d i f f i c u l t i e s . Although the photographs
are not n e c e s s a r y i n o r d e r t o c a l c u l a t e the a n g u l a r v e l o c i t i e s ,
t h e y a r e needed i n o r d e r t o measure t h e e l o n g a t i o n a l v e l o c i t i e s .
To d e t e r m i n e o u r e r r o r f o r t h e e l o n g a t i o n a l measurement, a n a r t i
f i c i a l s p o r a n g i o p h o r e was c o n s t r u c t e d . The a r t i f i c i a l s p o r a n g i o
p h o r e c o n s i s t e d o f a s t r a i g h t 0.5 mm d i a m e t e r w i r e a t t a c h e d t o a
m o t o r - d r i v e n micrometer screw. A s m a l l s p u r g e a r on t h e m o t o r -
s h a f t o f a s y n c h r o n o u s c l o c k m o t o r (1 rpm) d r i v e s a l a r g e r s p u r
g e a r on a m e t r i c m i c r o m e t e r head w i t h a g e a r r a t i o o f 1:8. The

m i c r o m e t e r s c r e w was a d v a n c i n g a t 63 pm/min. The a r t i f i c i a l

P h y c o was p h o t o g r a p h e d n e x t t o a c a l i b r a t e d s c a l e p l a c e d i n t h e
o c u l a r of the microscope. From a s e r i e s o f m i n u t e - p e r - m i n u t e
p h o t o g r a p h s , we d e t e r m i n e d t h a t t h e s t a n d a r d d e v i a t i o n o f o u r
e r r o r was ±1.4 ym p e r m i n ( 9 ) . T h i s p h o t o g r a p h i c method i s
h i g h l y d e p e n d e n t on u s i n g s t r a i g h t s p o r a n g i o p h o r e s t o a v o i d
parallax errors. P e r f e c t l y straight-growing sporangiophores are
n e a r l y i m p o s s i b l e t o o b t a i n s i n c e t h e y show b o t h f a s t , 5 t o 7.5
m i n , and s l o w , 30 t o 60 m i n o s c i l l a t i o n s ; a t e r m u s e d t o d e s c r i b e
these o s c i l l a t i o n s i s h u n t i n g (10). I n order to v e r i f y t h a t our
measurements w e r e n o t s i g n i f i c a n t l y i n f l u e n c e d by e i t h e r a p a r a l
l a x p r o b l e m o r s p o r a n g i o p h o r e h u n t i n g , we p l a c e d an a d d i t i o n a l
m a r k e r b e l o w t h e g r o w i n g z o n e and c a l c u l a t e d d i r e c t l y t h e change
i n l e n g t h b e t w e e n t h e two m a r k e r s . The d a t a o b t a i n e d i n t h i s
manner w e r e no d i f f e r e n
scale with reasonably straigh
of p h o t o g r a p h s t a k e n w i t h a o n e - m i n u t e i n t e r v a l , s m a l l bends
w e r e s e e n t o o c c u r i n t h e GZ, b u t s i n c e t h e bend a n g l e b e t w e e n
any p a i r o f p h o t o g r a p h s was l e s s t h a n a d e g r e e , t h e e r r o r i n t e r m s
of bending i s n e g l i g i b l e . We h a v e d e t e r m i n e d t h a t h u n t i n g c a u s e s
a b e n d i n g r a t e o f a b o u t 0.5 d e g r e e / m i n , and i n a c o n t i n u o u s s e t
of measurements l a s t i n g f o r 38 m i n u t e s , t h e b e n d i n g a n g l e w i l l n o t
v a r y more t h a n f i v e d e g r e e s f r o m t h e v e r t i c a l . From a s i n g l e p a i r
o f p h o t o g r a p h s , we c a n n o t e l i m i n a t e t h e p o s s i b i l i t y t h a t t h e grow
i n g z o n e i s b e n t d i r e c t l y t o w a r d s o r d i r e c t l y away f r o m t h e cam
e r a , b u t s i n c e we f i n d no s i g n i f i c a n t bends f r o m a l a r g e random
s a m p l e o f p h o t o g r a p h s , we h a v e d i s c o u n t e d t h i s p o s s i b i l i t y .
A TV v i d e o camera i n c o n j u n c t i o n w i t h a TV m o n i t o r and v i d e o
c a s s e t t e r e c o r d e r ( F i g u r e 1) h a s an enormous a d v a n t a g e o v e r t h e
c o n v e n t i o n a l 35 mm p h o t o g r a p h y . F i r s t l y , s i n c e an e n t i r e e x p e r i
ment c a n be s t o r e d on a TV c a s s e t t e and t h u s becomes p a r t o f a
permanent Phycomyces l i b r a r y , we c a n r e r u n any g i v e n e x p e r i m e n t ,
many l a s t i n g s e v e r a l h o u r s , a t any f u t u r e d a t e . S e c o n d l y , t h e
r e l i a b i l i t y o f measurements i s enhanced s i n c e r e p l i c a t e s may be
t a k e n and t h e t a p e d e x p e r i m e n t may be s t o p p e d o r rewound t o c h e c k
results.
E x p e r i m e n t a l R e s u l t s and S t o c h a s t i c M o d e l i n g . M e a s u r e m e n t s
o f r o t a t i o n a l and e l o n g a t i o n a l g r o w t h r a t e s f o r a t y p i c a l e x p e r i
ment a r e shown i n F i g u r e 2. The s a m p l e i n t e r v a l i s one m i n u t e ,
and t h e r e a r e 110 m e a s u r e m e n t s . I t c e r t a i n l y a p p e a r s t h a t b o t h
the g r o w t h and r o t a t i o n s e r i e s a r e h i g h l y i r r e g u l a r i n t e r m s o f
t h e i r growth r a t e s . I t i s d i f f i c u l t t o s e e any s y s t e m a t i c b e h a v
i o r and t h e r e i s l i t t l e a p p a r e n t c o r r e l a t i o n b e t w e e n t h e two.
These q u a l i t a t i v e o b s e r v a t i o n s must be q u a n t i f i e d .
E s t i m a t e s o f t h e a u t o c o r r e l a t i o n f u n c t i o n and t h e p a r t i a l
a u t o c o r r e l a t i o n s f o r the r o t a t i o n a l growth curve i n F i g u r e 2 a r e
p r e s e n t e d i n F i g u r e s 3 and 4. The a u t o c o r r e l a t i o n f u n c t i o n o f t e n
d e p i c t s d e p e n d e n c e o f a measurement v a l u e on r e c e n t p r e v i o u s
v a l u e s w h e r e a s t h e p a r t i a l a u t o c o r r e l a t i o n s show t h e s i g n i f i c a n c e

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s:
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c:
2.5
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i l
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s: ο
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s:
"a
.1

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LRC LPG
Figure 3. A utocorrelation function of a rotational growth series. Figure 4. Partial autocorrelation of a rotational growth series.
of adding a d d i t i o n a l terms t o an a u t o r e g r e s s i v e model o f t h e

series. I n F i g u r e 3, t h e r e i s l i t t l e s i g n i f i c a n c e and no r e a l
p a t t e r n apparent. Two s t a n d a r d - e r r o r l i m i t s f o r t h i s s a m p l e s i z e
a r e a b o u t ±0.2, and o n l y t h e a u t o c o r r e l a t i o n e s t i m a t e a t l a g 7
exceeds these l i m i t s . The same o b s e r v a t i o n s c a n be made o f F i g
u r e 4. The i m p l i c a t i o n i s t h a t t h e v a r i a t i o n s i n r o t a t i o n a l
g r o w t h a r e " w h i t e , " t h a t i s , random and u n c o r r e l a t e d ( w i t h e a r
lier variations). To c o n f i r m t h i s , a c u m u l a t i v e p e r i o d o g r a m o f
t h e f r e q u e n c y s p e c t r u m was computed and i s p r e s e n t e d i n F i g u r e 5.
S i n c e t h e s o l i d c u r v e f o l l o w s t h e 45°-dashed-line, t h i s p r o v e s
that the rotational series i s e s s e n t i a l l y white, quantifying the
c o n j e c t u r e made i n a p r e v i o u s p a p e r ( 9 ) .
The a u t o c o r r e l a t i o n f u n c t i o n and p a r t i a l a u t o c o r r e l a t i o n f o r
t h e e l o n g a t i o n a l g r o w t h s e r i e s a r e p r e s e n t e d i n F i g u r e s 6 and 7.
Systematic behavior i evident i th regula patter fth
autocorrelation functio
autocorrelations at lag y suggest
s e c o n d - o r d e r a u t o r e g r e s s i v e model i s a p p r o p r i a t e f o r t h i s s e r i e s
(11). The m o d e l f o r m i s
g + a + a = e
k l*k-l 2*k-2 k
t h
where g£ i s t h e i element o f t h e s e r i e s ,
a ^ , a£ a r e m o d e l p a r a m e t e r s , and
e± i s t h e i t n
model e r r o r o r r e s i d u a l .
L e a s t s q u a r e s e s t i m a t i o n was a p p l i e d t o t h i s s e r i e s f o r t h e
s e c o n d - o r d e r m o d e l a b o v e , and t h e f o l l o w i n g p a r a m e t e r v a l u e s w e r e
determined:
a x = 0.570
and
a 2 = 0.412
Such a m o d e l d e s c r i b e s a n underdamped o s c i l l a t o r y b e h a v i o r w i t h a
s i n u s o i d a l p e r i o d o f 11 m i n u t e s and damping c o e f f i c i e n t o f 0.44.
This p e r i o d o f o s c i l l a t i o n corresponds t o the "hunting" swings o f
the sporangiophore; t h e r e f o r e , i t appears t h a t o u r model accounts
f o r t h e g r o s s m e c h a n i c a l b e h a v i o r o f Phycomyces and i t s i n t e r
a c t i o n w i t h o u r measurement s y s t e m .
I t i s p o s s i b l e t o remove t h i s s y s t e m a t i c b e h a v i o r f r o m t h e
d a t a and examine l o c a l v a r i a t i o n s i n e l o n g a t i o n a l growth r a t e by
s t u d y i n g t h e r e s i d u a l s e r i e s o f t h e above model. T h i s r e s i d u a l
s e r i e s was a n a l y z e d and f o u n d t o be e s s e n t i a l l y w h i t e : t h e cumu
l a t i v e p e r i o d o g r a m i s p r e s e n t e d i n F i g u r e 8. T h i s i s i n c o n t r a s t
to t h e p e r i o d o g r a m o f t h e o r i g i n a l e l o n g a t i o n a l s e r i e s , s e e F i g
u r e 9, w h i c h shows s i g n i f i c a n t d e v i a t i o n f r o m t h e 45° l i n e . We
c a n summarize t h e a n a l y s i s o f b o t h g r o w t h s e r i e s b y s t a t i n g t h a t
l o c a l g r o w t h b e h a v i o r e x h i b i t e d b y t h e s p o r a n g i o p h o r e i s random

GAMOW AND CLOUGH Growth of Sporangiophores
FREQUENCY
Figure 5. Cumulative periodogram of a rotational growth series.

to

9
w

S
Figure 6. Autocorrelation function of an elongational growth series. Figure 7. Partial autocorrelation of an elongational growth series.
Figure 8. Cumulative periodogram of an elongational residual series.


and u n c o r r e l a t e d when t h e e l o n g a t i o n a l r a t e and r o t a t i o n a l r a t e

s e r i e s are studied separately.
I t i s tempting to r e l a t e the n o i s y growth p a t t e r n s s i n c e
s i m i l a r mechanisms may u n d e r l i e e l o n g a t i o n and r o t a t i o n . A p l o t
of the c r o s s - c o r r e l a t i o n f u n c t i o n between the r o t a t i o n a l s e r i e s
and t h e e l o n g a t i o n a l r e s i d u a l s e r i e s i s shown i n F i g u r e 10. The
r e s u l t i s p e r h a p s u n e x p e c t e d , s i n c e t h e r e i s no s i g n i f i c a n t c o r r e
l a t i o n b e t w e e n t h e two s e r i e s , t h a t i s , t h e n o i s y g r o w t h p a t t e r n s
a p p e a r i n d e p e n d e n t . The l i m i t s shown on t h e f i g u r e a r e f o r one
s t a n d a r d e r r o r , and one w o u l d e x p e c t a s i g n i f i c a n t c r o s s - c o r r e l a
t i o n t o e x c e e d two s t a n d a r d e r r o r s .
Conclusions
We h a v e shown t h a
and e l o n g a t i o n a l g r o w t
correlated. S y s t e m a t i c b e h a v i o r i n e l o n g a t i o n a l g r o w t h was
a t t r i b u t e d to the slower " h u n t i n g " motion of the sporangiophore.
The i r r e g u l a r p a t t e r n s i n r o t a t i o n a l and e l o n g a t i o n a l g r o w t h do
n o t a p p e a r t o be i n t e r d e p e n d e n t n o r r e l a t e d t o t h e same m a j o r
source or stimulus.
Discussion
A l l o r g a n i s m s t h a t p o s s e s s a r i g i d e x o s k e l e t o n such as the
a r t h r o p o d s , i n s e c t s and c r u s t a c e a n s must h a v e e v o l v e d one o f many
p o s s i b l e s t r a t e g i e s i n order to increase i n s i z e , i . e . to uniform
l y i n c r e a s e i n v o l u m e . The a r t h r o p o d s s i m p l y m o l t t h e i r o l d e x o
s k e l e t o n and t h e n r e d e p o s i t a new one a f t e r a s h o r t b u r s t o f i n
t e n s i v e growth. P l a n t s i n g e n e r a l , but f u n g i i n p a r t i c u l a r , have
d e v i s e d a s l i g h t l y d i f f e r e n t s t r a t e g y t o e n a b l e them t o grow e v e n
though they are a l s o encased i n a r a t h e r r i g i d c e l l w a l l . The
new s y n t h e s i z e d c e l l w a l l , t e r m e d t h e p r i m a r y w a l l , a p p e a r s as an
end r e s u l t o f g i a n t c a r b o h y d r a t e m i c r o f i b r i l s b e i n g d e p o s i t e d on
the i n n e r s u r f a c e of the e x i s t i n g p r i m a r y c e l l w a l l . In a d d i
t i o n , i t has b e e n shown t h a t t h e l o n g i t u d i n a l a x i s o f t h e s e n e w l y
l a i d down p o l y m e r s i s p e r p e n d i c u l a r t o t h e n e t d i r e c t i o n o f
growth of the c e l l w a l l i t s e l f . Cell wall thickening resulting
from newly deposited m i c r o f i b r i l s i s c o n s t a n t l y b e i n g counter
b a l a n c e d by c e l l w a l l e x t e n s i o n . The d r i v i n g mechanism o f c e l l
w a l l e x t e n s i o n i s the l a r g e pressure d i f f e r e n c e , the c e l l ' s
t u r g o r p r e s s u r e , b e t w e e n t h e i n s i d e o f t h e c e l l w a l l and i t s
e x t e r n a l e n v i r o n m e n t . B e c a u s e t h e p o l y m e r s a r e l a i d down p e r p e n
d i c u l a r l y to i t s net d i r e c t i o n of growth, during c e l l w a l l exten-
t i o n they are p a s s i v e l y r e o r i e n t e d towards the c e l l ' s l o n g i t u d i
n a l a x e s , and i n a d d i t i o n , t r a n s p o r t e d t o w a r d t h e o u t e r s u r f a c e
o f t h e c e l l w a l l . The m o d e l d e s c r i b e d a b o v e i s b a s i c a l l y known
as t h e m u l t i n e t t h e o r y o f c e l l w a l l g r o w t h f i r s t p r o p o s e d by
R o e l o f s e n and Houwink (12) and e x p e r i m e n t a l l y v e r i f i e d i n N i t e l l a
by G e r t e l and G r e e n ( 1 3 ) .

CROSSCORRELflTION FUNCTION r(k)
r(k)
.5
-.5
-3 -2 -1 θ 1 2 3 4 5 6 7 8 9 18 11 12 13 14 15 16 17

Figure 10. Cross-correlation between local variations in rotational and elongational growth.

18. GAMOW AND CLOUGH Growth of Sporangiophores All
S i n c e t h e s e n e w l y l a i d down m i c r o f i b r i l s a r e i n c o n s t a n t
m o t i o n , t h e p r i m a r y c e l l w a l l b e h a v e s much l i k e a v i s c o - e l a s t i c
fluid. B a r t n i c k i - G a r c i a (14) i n 1970 p r o p o s e d t h a t t h e n e w l y
s y n t h e s i z e d c e l l w a l l i s i n a constant s t a t e of f l u x i n terms of
s y n t h e s i s and d e g r a d a t i o n r e s u l t i n g f r o m t h e p r e s e n c e o f s y n t h e
s i z i n g and l y t i c enzymes, r e s p e c t i v e l y . I n 1974 O r t e g a and Gamow
(15) p r o p o s e d t h a t t h e o r i e n t a t i o n o f m i c r o f i b r i l s , o r b u n d l e s o f
m i c r o f i b r i l s , c o u l d a c c o u n t f o r b o t h c e l l e l o n g a t i o n and c e l l
rotation. They a l s o d e d u c e d t h a t t h e m a g n i t u d e o f t h e r a t i o o f
r o t a t i o n t o e l o n g a t i o n must be a f u n c t i o n o f t h e m i c r o f i b r i l
a n g l e , the a n g l e b e i n g measured i n r e s p e c t to the l o n g i t u d i n a l
a x i s of the growing c e l l w a l l . T h i s model p r e d i c t e d t h a t the
f i b r i l s l o c a t e d i n the lower r e g i o n of the growing zone, those
which would have a l r e a d y m a x i m a l l y r e o r i e n t a t e d towards the l o n g
i t u d i n a l a x i s , w o u l d show maximum r o t a t i o n t o e l o n g a t i o n r a t i o s ,
and t h i s p r e d i c t i o n was
Our p r e s e n t r e s u l t
t h e o r y as p r e s e n t e d a b o v e s i n c e i t c l e a r l y p r e d i c t s t h a t any
i r r e g u l a r i t y i n t h e g r o w t h r a t e w o u l d be d i r e c t l y c o u p l e d w i t h
an i r r e g u l a r i t y i n t h e r o t a t i o n r a t e . Our p r e s e n t d a t a w o u l d be
c o n s i s t e n t w i t h a r e o r i e n t a t i o n m o d e l i n w h i c h t h e maximum r o t a
t i o n and e l o n g a t i o n o c c u r i n d i f f e r e n t r e g i o n s o f t h e GZ. Recent
f i n e s t r u c t u r e s t u d i e s i n our l a b o r a t o r y have c o n f i r m e d the 1958
r e p o r t by Cohen and D e l b r u c k t h a t t h e maximun e l o n g a t i o n r a t e
o c c u r r i n g i n the r e g i o n of the growing zone i s d i s t i n c t l y d i f f e r
e n t f r o m t h a t o c c u r r i n g i n t h e r e g i o n o f maximum r o t a t i o n r a t e .
Our p r e s e n t a n a l y s i s i s c o n s i s t e n t w i t h t h i s f i n d i n g b e c a u s e i f
b o t h r o t a t i o n and e l o n g a t i o n a r e s t o c h a s t i c i n t e r m s o f g r o w t h
i r r e g u l a r i t i e s , we w o u l d n o t e x p e c t t o f i n d a c o r r e l a t i o n i f t h e
r o t a t i o n and e l o n g a t i o n o c c u r r e d i n d i f f e r e n t and i n d e p e n d e n t
r e g i o n s of the growing zone.
L a s t l y , we a r e i n t r i g u e d by t h e s i n u s o i d a l b e h a v i o r , h a v i n g
a p e r i o d o f 11 m i n u t e s , i n t h e e l o n g a t i o n r a t e . A t t h i s t i m e we
h a v e no way o f d e t e r m i n i n g w h e t h e r t h i s o s c i l l a t i o n i s r e a l i n
the sense t h a t the net r a t e of growth o s c i l l a t e s w i t h t h i s p e r i o d
o r w h e t h e r we a r e o b s e r v i n g t h e w e l l - k n o w n h u n t i n g b e h a v i o r ( 1 0 ) ;
Phycomyces s p o r a n g i o p h o r e s show b o t h a f a s t , 5 t o 7.5 m i n , and a
s l o w , 30 t o 60 m i n , o s c i l l a t i o n . Our g u e s s w o u l d be t h a t t h e
1 1 - m i n u t e p e r i o d we h a v e o b s e r v e d i s a d i r e c t r e s u l t o f t h e mea
1
s u r e m e n t e r r o r c a u s e d by t h e s p o r a n g i o p h o r e s h u n t i n g behavior.
I n o r d e r t o c o n f i r m t h i s s p e c u l a t i o n , we w o u l d r e q u i r e f a s t e r
s a m p l i n g r a t e s f o r b o t h e l o n g a t i o n and r o t a t i o n a l g r o w t h . Such
an i n c r e a s e i n s a m p l i n g r a t e w o u l d a l s o f a c i l i t a t e more d e t a i l e d
study of the s t o c h a s t i c growth b e h a v i o r . We b e l i e v e an e x t e n s i o n
o f o u r p r e s e n t measurement t e c h n i q u e w o u l d a l l o w f o r t h i s . This
w o u l d be t o c o u p l e a c o m p u t e r - b a s e d p a t t e r n r e c o g n i t i o n s y s t e m t o
o u r m i c r o s c o p i c v i d e o e q u i p m e n t . By h a v i n g t h e computer f o l l o w
motion of s e v e r a l t r a c k i n g p a r t i c l e s , a high r e s o l u t i o n p r o f i l e
o f b o t h g r o w t h r a t e s i s f e a s i b l e . Such an e x t e n s i o n t o o u r p r e s
e n t a p p a r a t u s w o u l d r e q u i r e l e s s t h a n $100,000 i n c a p i t a l f u n d i n g .

A p p e n d i x : A B r i e f D e s c r i p t i o n o f Time S e r i e s A n a l y s i s
The t e c h n i q u e s o f t i m e s e r i e s a n a l y s i s h a v e b e e n a p p l i e d
a c r o s s a b r o a d s p e c t r u m o f f i e l d s , n o t a b l y b u s i n e s s and e c o n o m i c s ,
e n g i n e e r i n g , and m e t e o r o l o g y . They h a v e g a i n e d w i d e a c c e p t a n c e
p a r t l y due t o t h e h i g h y i e l d o f m o d e l i n g i n f o r m a t i o n one a c h i e v e s
t h r o u g h t h e a p p l i c a t i o n o f a s t r a i g h t - f o r w a r d , e a s y - t o - u s e method
ology. T h e r e a r e two common s c e n a r i o s :
1) a n a l y s i s o f a s i n g l e t i m e s e r i e s , u s u a l l y a s a m p l e d mea
s u r e m e n t s i g n a l , t o d e t e r m i n e i t s s y s t e m a t i c and random
character
2) a n a l y s i s o f two o r more t i m e s e r i e s t o a s s e s s and q u a n
t i f y c o n n e c t i v e r e l a t i o n s h i p s (one must be c a r e f u l w i t h
claims of "cause-and-effect" h e r e — a d d i t i o n a l informa
t i o n on s t r u c t u r
I t i s d e s i r a b l e , from a
i n as s i m p l e a form as p o s s i b l e . Such a r e l i n e a r models w i t h a
m i n i m a l number o f p a r a m e t e r s . T h e r e a r e two p r i m a r y s t e p s t o t h e
m o d e l i n g p r o c e s s : i d e n t i f i c a t i o n o f t h e m o d e l f o r m and e s t i m a t i o n
of the model's parameters. These s t e p s a r e a p p r o p r i a t e l y f o l l o w e d
by t e s t i n g o f t h e m o d e l ' s a b i l i t y t o f i t o r p r e d i c t new d a t a .
I t i s t y p i c a l o f a s i n g l e t i m e s e r i e s t h a t measurements w i l l
show some dependence on t h e i r r e c e n t p a s t h i s t o r y — m a n y p r o c e s s e s
are i n e r t i a l i n behavior. The a u t o c o r r e l a t i o n f u n c t i o n ( o r i t s
e s t i m a t e f r o m t h e s e r i e s ' d a t a ) w i l l r e v e a l s u c h d e p e n d e n c e , and
t h e p a r t i a l a u t o c o r r e l a t i o n s w i l l h e l p show how f a r i n t o t h e p a s t
t h i s " a u t o r e g r e s s i v e " m o d e l must e x t e n d . The m o d e l t h u s s e l e c t e d
c a n be f i t t o t h e d a t a by one o f many r e g r e s s i o n t e c h n i q u e s , e.g.
l i n e a r l e a s t squares. The r e s i d u a l s , t h o s e components o f t h e
d a t a w h i c h a r e n o t e x p l a i n e d by t h e a u t o r e g r e s s i v e m o d e l , f o r m
another s e r i e s . I f the r e s i d u a l s e r i e s i s u n c o r r e l a t e d ( t o i t
s e l f ) , t h i s i n d i c a t e s a random b e h a v i o r w h i c h we c a n n e i t h e r p r e
d i c t n o r r e g u l a t e — w e j u s t have t o l i v e w i t h i t as u n c e r t a i n be
havior. O f t e n , however, i t i s p o s s i b l e t o model t h e r e s i d u a l s
a l s o i n a s i m i l a r , a u t o r e g r e s s i v e manner—which would i n d i c a t e
t h a t a l t h o u g h we c a n n o t r e g u l a t e t h e s e q u a n t i t i e s , we c a n do some
prediction. This s t r u c t u r e o f the r e s i d u a l s e r i e s i s u s u a l l y
b u i l t b a c k i n t o t h e o r i g i n a l m o d e l , and t h e t e r m i n o l o g y u s e d f o r
t h e r e s i d u a l d e s c r i p t i o n i s "moving a v e r a g e . " I t i s then best t o
r e f i t t h e e n t i r e m o d e l by r e g r e s s i o n . A g e n e r a l f o r m f o r t h e
a u t o r e g r e s s i v e - m o v i n g average model i s
g. + a-g. -+...+ a g.
&
= e. + c-e. - +...+
ô
c e.
k l k-l n k-n k 1 k-1 ρ k-p
where e^ i s now an u n c o r r e l a t e d r e s i d u a l . The a b o v e m o d e l may

a p p e a r i m p o s i n g ; h o w e v e r , t h e a p p r o p r i a t e v a l u e s f o r η and ρ a r e
u s u a l l y l e s s than t h r e e .
C o n n e c t i o n b e t w e e n two s e r i e s i s m a n i f e s t e d i n t h e c r o s s -
c o r r e l a t i o n f u n c t i o n ( o r i t s e s t i m a t e ) . When a c a u s e - a n d - e f f e c t ,

19. GAMOW A N DCLOUGH Growth of Sporangiophores 419
" i n p u t - o u t p u t " r e l a t i o n s h i p i s j u s t i f i e d , a model o f t h e form
g, + a- g, -+...+ a g,
6 ô
= b χ, , + b-x. ,-+...+ b χ. ,
&
k l k-l n k-n ο k-d 1 k-d-1 m k-d-m
+ e. + c-e. ,+...+ c e.
k 1 k-1 ρ k-p
may b e p r o p o s e d w h i c h i n c l u d e s t h e a u t o r e g r e s s i v e d e p e n d e n c e o f
the o u t p u t v a r i a b l e ( g ^ ) , a " c a u s e " dependence o n v a l u e s o f t h e
i n p u t v a r i a b l e (x^) and a d e s c r i p t i o n o f r e s i d u a l (e^) b e h a v i o r
as b e f o r e . S e l e c t i o n o f m o d e l f o r m ( w h i c h may i n c l u d e a d e l a y o f
d sample i n t e r v a l s b e t w e e n t h e i n p u t a n d o u t p u t v a r i a b l e s ) i s
f a c i l i t a t e d b y s t u d y o f t h e c r o s s - c o r r e l a t i o n f u n c t i o n and i m p u l s e
response f u n c t i o n estimates i n a d d i t i o n t o the a u t o c o r r e l a t i o n
f u n c t i o n and p a r t i a l a u t o c o r r e l a t i o n s
selected, regression technique
Two m o d e l - c h e c k i n g methods a r e common, and t h e y a r e o f t e n
a p p l i e d t o a "new" s e t o f d a t a . F i r s t , t h e r e s i d u a l s a r e a n a
l y z e d f o r l a c k o f c o r r e l a t i o n o r " w h i t e n e s s . " White n o i s e has
the c h a r a c t e r i s t i c t h a t a l l f r e q u e n c i e s c o n t a i n e d i n i t s h o u l d be
present i n equal strength. The c u m u l a t i v e p e r i o d o g r a m i s t h e
i n t e g r a l o f a spectrum e s t i m a t e ; t h e r e f o r e , f o r w h i t e n o i s e , i t
s h o u l d b e t h e i n t e g r a l o f a c o n s t a n t o r a l i n e a r ramp f u n c t i o n .
The " w h i t e n e s s " o f t h e r e s i d u a l s c a n b e a n a l y z e d b y p l o t t i n g
t h e i r c u m u l a t i v e p e r i o d o g r a m and o b s e r v i n g c l o s e n e s s t o t h e i d e a l
ramp l i n e . A n o t h e r common d i a g n o s t i c t e c h n i q u e i s t o d e t e r m i n e
the s t a t i s t i c a l e f f i c i e n c y o f a d d i n g one o r two p a r a m e t e r s ( a n d
terms) t o t h e model. T h i s w i l l check i f t h e minimal p a r a m e t r i c
m o d e l c h o s e n i s t r u l y p a r s i m o n i o u s . The s t a t i s t i c a l F - t e s t b a s e d
on t h e e x t r a - s u m - o f - s q u a r e s p r i n c i p l e i s u s e f u l f o r t h i s e f f i
ciency test.
Literature Cited
1. Bergman, K.; Burke, P. V.; Cerda-Olmedo, E.; David, C. N.;

Delbrück, M.; F o s t e r , K. W.; Goodell, E. W.; Heisenberg, M.;
Meissner, G.; Zalokar, M.; Dennison, D. S.; Shropshire, W.;
Jr. B a c t e r i a l Rev. 1969, 33, 97-157.
2. Shropshire, W., J r . Phys. Rev. 1963, 43, 38-67.
3. Cerda-Olmedo, E. Ann. Rev. M i c r o b i o l . 1977, 31, 535-47.
4. Foster, K. W. Ann. Rev. Biophys. Bioeng. 1977, 6, 419-43.
5. Cohen, R. J . ; F r i e d , M. G.; Atkinson, M. M. Biochem. and
Biophys. Res. Comm. 1979, 86, 877-84.
6. Gamow, R. I . ; Böttger, B. J . Gen. P h y s i o l . , i n p r e s s .
7. Cohen, R.; Delbruck, M. J . Cell Comp. P h y s i o l . 1958, 52,
361-88.
8. Ortega, J . Κ. E.; H a r r i s , J . ; Gamow, R. I . Plant P h y s i o l .
1974, 53, 485-90.
9. Gamow, R. I . ; Böttger, B. J . Gen. P h y s i o l . 1981, 77, 65-75.

10. Dennison, D. S. Science 1959, 129, 775-7.

11. Box, G. E. P.; Jenkins, M. "Time S e r i e s A n a l y s i s : F o r e c a s t
ing and C o n t r o l " ; Revised E d i t i o n ; Holden-Day: San Francisco,
CA, 1976.
12. Roelofsen, P. Α.; Houwink, A. L. Acta. Bot. N e e r l . 1953, 2,
218-25.
13. G e r t e l , E. T.; Green, P. B. P l a n t P h y s i o l . 1977, 60, 247-54.
14. B a r t n i c k i - B a r c i a , S. In "Phytochemical Phylogeny;" Hasborne,
J . B., Ed.; Academic Press, Inc.; New York, NY, 1970; p. 81.
15. Ortega, J . Κ. E.; Gamow, R. I. J . Theoret. B i o l . 1974, 47,
317-32.
R E C E I V E D June 1, 1 9 8 2

20
Growth Characteristics of Microorganisms in

Solid State Fermentation for Upgrading of Protein
Values of Lignocelluloses and Cellulase Production
D. S. C H A H A L
Devinder Chahal Enterprises, Inc., 312-1800 Baseline Road,
Ottawa, Ontario Canada K2C 3N1
Solid state fermentation (SSF) is considered to

r e q u i r e no comple controls d t hav
advantages over l i q u i
of p r o t e i n value lignocellulose
production by SSF holds great promise. Pretreatment
of l i g n o c e l l u l o s e s with alkali o r steam i s necessary
to break lignin and carbohydrate bonds f o r s u c c e s s f u l
growth o f microorganisms i n SSF. Filamentous fungi
seem to be the most s u i t a b l e organisms f o r SSF.
Fungal hyphae can penetrate i n t o plant c e l l lumina
through p i t s , c r a c k s , o r by boring holes through the
cell w a l l . Once i n s i d e the cell lumen, the fungus
utilizes the cell w a l l from i n s i d e . I t is postulated
that sequence o f synthesis o f c e l l u l a s e s i n the fungal
hyphal t i p is t r i g g e r e d by p h y s i c a l s i g n a l s sent by
cellulose. Finally, the substrate is completely
utilized to produce fungal biomass rich in p r o t e i n
and/or c e l l u l a s e s , depending on the microorganism.
" S o l i d S t a t e F e r m e n t a t i o n " (SSF) i s defined as a process

whereby a n i n s o l u b l e s u b s t r a t e i s fermented with sufficient
moisture, but without f r e e water. I n the l i q u i d s t a t e o r s l u r r y
s t a t e f e r m e n t a t i o n , on the o t h e r hand, t h e s u b s t r a t e i s s o l u b i l i z e d
o r suspended a s f i n e p a r t i c l e s i n a l a r g e v o l u m e o f w a t e r . I n
most l i q u i d s t a t e f e r m e n t a t i o n s ( L S F ) o r submerged f e r m e n t a t i o n s
s u b s t r a t e c o n c e n t r a t i o n s r a n g i n g f r o m 0.5 t o 5% a r e u s e d . Now, i n
a number o f f e r m e n t a t i o n s t h e c o n c e n t r a t i o n o f t h e s u b s t r a t e h a s
gone up t o 1 0 % t o i n c r e a s e t h e p r o d u c t i v i t y p e r u n i t t i m e .
Solid s t a t e f e r m e n t a t i o n i s c o n s i d e r e d t o r e q u i r e no complex
c o n t r o l s and t o h a v e many a d v a n t a g e s o v e r t h e L S F (J.) · The SSF
for upgrading the p r o t e i n values of lignocelluloses,
( a g r i c u l t u r a l , f o r e s t r y and a n i m a l w a s t e s ) i s b e c o m i n g a f o c u s o f
a c t i v i t y f o r some r e s e a r c h e r s ( 2 , 3 , 3 a , 4 ) . R e c e n t l y i t has been
calulated (5) that high c e l l u l a s e activity p e r u n i t volume o f
f e r m e n t a t i o n b r o t h i s t h e most i m p o r t a n t f a c t o r i n o b t a i n i n g s u g a r
0097-6156/83/0207-0421$06.50/0

c o n c e n t r a t i o n s o f 20-30% from h y d r o l y s i s o f c e l l u l o s e i n a p r o c e s s
for ethanol p r o d u c t i o n f r o m c e l l u l o s i c m a t e r i a l s . I t has a l s o
been c o n f i r m e d ( 6 ) t h a t c e l l u l a s e a c t i v i t y per u n i t volume can be
i n c r e a s e d by i n c r e a s i n g t h e c e l l u l o s e c o n c e n t r a t i o n i n t h e medium.
But i t i s n o t p o s s i b l e to handle more t h a n 6% cellulose in
c o n v e n t i o n a l fermenter because o f r h e o l o g i c a l problems. In o r d e r ,
t h e r e f o r e , t o i n c r e a s e t h e c e l l u l o s e c o n c e n t r a t i o n h i g h e r t h a n 6%,
SSF seems t o be t h e most a t t r a c t i v e a l t e r n a t i v e (5)· To yam a (_7)
has a l r e a d y shown t h a t s u g a r s y r u p o f 22% c o n c e n t r a t i o n can be
o b t a i n e d by h y d r o l y z i n g d e l i g n i f i e d r i c e s t r a w o r b a g a s s e w i t h t h e
c o n c e n t r a t e d c e l l u l a s e s o b t a i n e d by T r i c h o d e r m a r e e s e i i n SSF.
R e c e n t l y , i n t e r e s t i n t h e p r o d u c t i o n o f a m y l a s e s and c e l l u l a s e s b y
SSF i s i n c r e a s i n g b e c a u s e a g r e a t demand o f t h e s e enzymes i s
envisaged i n t h e n e a r f u t u r e . These enzymes a r e r e q u i r e d t o
c o n v e r t s t a r c h and c e l l u l o s e i n t o g l u c o s e f o r f u r t h e r f e r m e n t a t i o n
i n t o e t h a n o l to a l l e v i a t
0 5 ) . But t h e s u r v e y o
is known a b o u t t h e g r o w t h c h a r a c t e r i s t i c s and behavior of
m i c r o o r g a n i s m s i n SSF.
O r i g i n of S o l i d S t a t e Fermentation
The o r i g i n o f SSF i s l o s t i n the m i s t o f a n t i q u i t y when

r e l a t i o n s h i p of microorganism were e s t a b l i s h e d w i t h t h e i r h o s t
(animals or plants; l i v i n g or dead). In nature microorganisms
grow i n c l o s e a s s o c i a t i o n w i t h s o l i d s u b s t r a t e s t o o b t a i n t h e i r
n u t r i t i o n s a p r o p h y t i c a l l y or p a r a s i t i c a l l y . One o f the earliest
records of SSF t r a c e d out b y Chang ( 8 ) was t h e c u l t i v a t i o n o f
paddy s t r a w mushroom, V o l v a r i e l l a v o l v a c e a , i n t h e Canton r e g i o n
o f C h i n a ' s Kwangtung p r o v i n c e d u r i n g the Chow D y n a s t y , a b o u t 3000
y e a r s ago. However, u n t i l 20 years ago, almost no scientific
r e s e a r c h has b e e n done on t h i s s p e c i e s . The o t h e r e a r l y r e c o r d o f
SSF was g r o w i n g o f " S h i i t a k e " ( L e n t i n u s edodes) by t h e C h i n e s e and
J a p a n e s e on wood l o g s a b o u t 20 c e n t u r i e s ago (9)· The s p o r e s a r e
i n o c u l a t e d i n wood l o g s and a r e l e f t t o i n c u b a t e f o r many months
before the mushrooms are harvested for eating. Agaricus
c a m p e s t r i s ( A . b i s p o r u s ) , a commonly c u l t i v a t e d mushroom i n Europe
and W e s t e r n c o u n t r i e s , h a s b e e n grown i n c a v e s i n F r a n c e s i n c e t h e
t i m e o f L o u i s XLV ( 1 6 8 3 - 1 7 1 5 ) ( 1 0 ) . Of g r e a t h i s t o r i c a l r e l e v a n c e
t o modern t e c h n o l o g y i s t h e J a p a n e s e " K o j i " p r o c e s s , i . e . g r o w i n g
o f A s p e r g i l l u s o r y z a e on r i c e ( o r o t h e r c e r e a l s ) i n s o l i d state.
P r o d u c t i o n o f " K o j i " has been i n p r a c t i c e i n J a p a n a t l e a s t s i n c e
t h e e i g h t h c e n t u r y f o r p r o d u c t i o n o f Saké - the m o s t traditional
alcoholic d r i n k i n J a p a n . The K o j i p r o c e s s was i n t r o d u c e d t o t h e
W e s t e r n W o r l d by Takamine i n 1891 f o r t h e production of fungal
diastase on a l a r g e s c a l e (11)· The K o j i p r o c e s s i s now b e i n g
e x p l o i t e d f o r t h e p r o d u c t i o n o f a m y l a s e s and c e l l u l a s e s ( 7 , 1 2 ) .
A n o t h e r e a r l y s o l i d s t a t e f e r m e n t a t i o n was t h e d i s c o v e r y o f
g a l l i c a c i d p r o d u c t i o n i n g a l l n u t s p i l e d i n a heap and moistened
w i t h water. I t was v a n Teigham ( 1 8 6 7 ) who f i r s t e s t a b l i s h e d t h a t
A s p e r g i l l u s n i g e r was r e s p o n s i b l e f o r t h i s f e r m e n t a t i o n ( 9 ) .

20. CHAHAL Solid State Fermentation 423
The records f o r the production of R o q u e f o r t cheese from

s h e e p ' s m i l k i n c a v e s o f S o u t h e r n F r a n c e go b a c k a b o u t a thousand
years. It was established by Thorn ( 1 3 ) t h a t t h e s p e c i a l
c h a r a c t e r i s t i c s o f R o q u e f o r t c h e e s e was due t o the growth o f a
f u n g u s , Pénicillium r o q u e f o r t i . T h i s o r g a n i s m grows d e e p i n t o
c h e e s e b l o c k s u n d e r l i m i t e d 0^ s u p p l y - a p e r f e c t e x a m p l e o f s o l i d
state fermentation. The s p e c i a l f l a v o u r o f Camembert c h e e s e i s
due t o t h e g r o w t h o f Pénicillium c a m e m b e r t i on the surface of
c h e e s e blocks·
Nature of the Substrate
The SSF can o n l y be a p p l i e d to i n s o l u b l e s u b s t r a t e s l i k e

c e r e a l g r a i n s , o i l s e e d s , and l i g n o c e l l u l o s e s ( a g r i c u l t u r a l crop
residues and f o r e s t r y wastes) The c e r e a l g r a i n s and o i l s e e d s
a r e composed o f e a s i l y a v a i l a b l
proteins, minerals, etc
able t o g r o w on these substrates in SSF. However, mild
p r e t r e a t m e n t s a r e s t i l l r e q u i r e d t o make them s u i t a b l e s u b s t r a t e s .
C e r e a l s l i k e r i c e g r a i n s a r e used a s s u c h w i t h o u t any treatment
whereas wheat g r a i n s require p e a r l i n g before use. During t h i s
p r o c e s s t h e s u r f a c e o f wheat g r a i n s i s a b r a d e d t o p r o v i d e s u i t a b l e
s i t e s f o r c o l o n i z a t i o n by t h e m i c r o o r g a n i s m s . I n c a s e o f c o r n and
s o y b e a n the g r a i n s a r e b r o k e n i n t o f i v e o r s i x p i e c e s to provide
s u i t a b l e s u r f a c e f o r the growth o f the m i c r o o r g a n i s m s , as the hard
seed c o a t s a r e v e r y d i f f i c u l t f o r many m i c r o o r g a n i s m s t o c o l o n i z e .
O a t s h a v e t o be d e h u l l e d f o r b e t t e r g r o w t h . A l l t h e s e g r a i n s a r e
very s u i t a b l e f o r the production o f a f l a t o x i n s and various
fermented o r i e n t a l foods ( 1 ) .
The most s e r i o u s problem encountered i n SSF of grains,
especially rice, i s that t h e s u b s t r a t e s become s t i c k y and form
compact m a s s e s i n w h i c h movement o f a i r i s r e s t r i c t e d . The g r a i n s
a l s o a g g l o m e r a t e i n t o compact m a s s e s due t o f u n g a l g r o w t h o n t h e i r
s u r f a c e s . T h e r e f o r e , t i m e l y and p r o p e r a g i t a t i o n i s r e q u i r e d to
b r e a k s u c h a g g l o m e r a t e s and p r o v i d e b e t t e r a e r a t i o n .
P r o d u c t i o n o f a f l a t o x i n s and fermented oriental foods on
grains i s much e a s i e r than the use of l i g n o c e l l u l o s e s . The
carbohydrates of l i g n o c e l l u l o s e s are not easily available and
d r a s t i c pretreatments are r e q u i r e d . Moreover, l i g n o c e l l u l o s e s are
a l s o n o t a s r i c h i n p r o t e i n s and o t h e r n u t r i e n t s a s are grains.
L i g n o c e l l u l o s e s are very c o m p l e x s u b s t r a t e s and a r e composed o f
t h r e e m a j o r c o m p o n e n t s : h e m i c e l l u l o s e s , c e l l u l o s e , and lignin.
Only a few microorganisms, l i k e white-rot fungi (Polyporus
v e r s i c o l o r ) , are able t o g r o w on such m a t e r i a l s (14). The
white-rot f u n g i h a v e c o m p l e t e enzyme s y s t e m s t o u t i l i z e a l l t h r e e
c o m p o n e n t s . S i m i l a r l y , i t has b e e n shown t h a t P l e u r o t u s o s t r e a t u s
(15 ) and Stropharia rugosoannulata (4) can u t i l i z e a l l three
components o f wheat s t r a w . On the o t h e r h a n d , many m i c r o o r g a n i s m s
c a n n o t u t i l i z e h e m i c e l l u l o s e s ; o f t h o s e t h a t c a n , most a r e u n a b l e
to metabolize c e l l u l o s e .

L i g n i n i n the p l a n t c e l l w a l l n o t o n l y e n c r u s t s t h e c e l l u l o s e
m i c r o f i b r i l s i n a s h e a t h - l i k e manner, b u t i s bonded p h y s i c a l l y and
c h e m i c a l l y t o t h e p l a n t p o l y s a c c h a r i d e s (16)« Lignin-carbohydrate
bonds form m e t a b o l i c b l o c k s that greatly limit the a c t i o n o f
m i c r o b i a l h e m i c e l l u l a s e s and c e l l u l a s e s . P h y s i c a l l y , l i g n i n f o r m s
a b a r r i e r s u p p r e s s i n g t h e p e n e t r a t i o n by p o l y s a c c h a r i d e - d i g e s t i n g
enzymes ( 17 ) . U n l e s s t h e l i g n i n i s d e p o l y m e r i z e d , s o l u b i l i z e d , o r
removed, t h e cellulose and the hemicelluiοses cannot be
metabolized by most microorganisms· As m e n t i o n e d e a r l i e r , o n l y
w h i t e - r o t and b r o w n - r o t f u n g i ( B a s i d i o m y c e t e s ) a r e a b l e t o u t i l i z e
carbohydrates from l i g n o c e l l u l o s e s but t h e y a r e v e r y s l o w growing
(4,14,15,18,19). Therefore, these f u n g i are not v e r y promising,
from the economic p o i n t o f v i e w as candidates to ferment
l i g n o c e l l u l o s e s f o r the p r o d u c t i o n o f a n i m a l feed or cellulases.
The case is s i m i l a r with other basidiomycetes especially
mushrooms. A g a r i c u s bisporus
v o l v a c e a , and P l e u r o t u
p r o d u c e f r u i t i n g b o d i e s ( m u s h r o o m s ) . Mushroom g r o w i n g , however,
i s j u s t i f i e d b e c a u s e o f t h e i r economic v a l u e a s f o o d d e l i c a c i e s .
Pretreatment of L i g n o c e l l u l o s e s
Pretreatment o f l i g n o c e l l u l o s e s i s the f i r s t requirement f o r

t h e g r o w t h o f t h e m i c r o o r g a n i s m s w h i c h a r e u n a b l e t o g r o w on the
untreated s u b s t r a t e but w h i c h , o t h e r w i s e , g r o w r a p i d l y and make
c e r t a i n products i . e . Trichoderma r e e s e i f o r c e l l u l a s e s ; and
Chaetomium cellulolyticum for single-cell protein (SCP)
production. Pretreatments t h a t i n c r e a s e the d i g e s t i b i l i t y of
lignocelluloses f o r production of SCP and c e l l u l a s e s h a v e b e e n
d i s c u s s e d i n d e t a i l by v a r i o u s w o r k e r s ( 2 0 , 2 1 , 2 2 ) . Only those
p r e t r e a t m e n t s w h i c h c o u l d make t h e s u b s t r a t e most s u i t a b l e f o r SSF
are d i s c u s s e d v e r y b r i e f l y below:
Grinding/Ball-Milling. Grinding/ball-milling of ligno

celluloses to a v e r y s m a l l p a r t i c l e s i z e r e s u l t s i n the exposure
o f more s u r f a c e a r e a f o r the g r o w t h o f m i c r o o r g a n i s m s and also
r e d u c e s c r y s t a l l i n i t y (23 ) . The m a i n p r o b l e m i n u s i n g f i n e powder
o f l i g n o c e l l u l o s e f o r SSF i s t o s u p p l y good a e r a t i o n , a s t h e m o i s t
f i n e powder o f l i g n o c e l l u l o s e w o u l d f o r m a compact mass b y i t s own
weight. Improper a e r a t i o n w i l l reduce the growth o f the organism
considerably. Frequent agitation and f o r c e d a e r a t i o n may be
n e c e s s a r y t o k e e p up t h e g r o w t h o f t h e o r g a n i s m .
Alkali. Sodium h y d r o x i d e and aqueous ammonia c a u s e e x t e n s i v e
s w e l l i n g and s e p a r a t i o n o f s t r u c t u r a l e l e m e n t s , and lead to the
f o r m a t i o n o f c e l l u l o s e I I whose X - r a y p a t t e r n d i f f e r s c o n s i d e r a b l y
t h a t o f f r o m c e l l u l o s e I . F i v e t o s i x grams o f sodium hydroxide
per 100 grams o f wood seem t o be n e c e s s a r y f o r t h e maximum e f f e c t
(24,25). This l e v e l of a l k a l i i s e s s e n t i a l l y e q u i v a l e n t to the
combined a c e t y l and c a r b o n y l c o n t e n t s o f wood. T h i s l e a d s t o the

p o s t u l a t e t h a t the main consequence o f a l k a l i treatment is the

saponification of i n t e r m o l e c u l a r e s t e r b o n d s , t h u s p r o m o t i n g the
s w e l l i n g o f wood beyond w a t e r - s w o l l e n d i m e n s i o n s and favouring
increase enzymatic and m i c r o b i o l o g i c a l penetrati> η into the
c e l l - w a l l f i n e s t r u c t u r e ( 2 2 ) . B o t h s o d i u m h y d r o x i d e and ammonia
treatments proved t o be v e r y u s e f u l i n i n c r e a s i n g the i n v i t r o
d i g e s t i b i l i t y o f l i g n o c e l l u l o s e s ( 2 1 ) as w e l l a s t h e i r u t i l i z a t i o n
as carbon source f o r SCP production i n SSF (26 ) . These
pretreatments a l s o r e t a i n the f i b r o u s s t r u c t u r e of c e l l u l o s e . The
fibrous structure i s very conducive f o r the growth o f the
o r g a n i s m s because o f e a s y p e n e t r a t i o n o f enzymes, f u n g a l hyphae
and a i r i n t h e s u b s t r a t e .
S t e a m . Steam treatment of l i g n o c e l l u l o s e s under high
p r e s s u r e ( 2 7 - 3 0 ) i s now b e c o m i n g an i m p o r t a n t p r e t r e a t m e n t t o make
t h e s u b s t r a t e e a s i l y a c c e s s i b l e t o h y d r o l y t i c enzymes (31-33).
The s u b s t r a t e i s a l s o s t e r i l i z e d
A fibrous substrate i s
The chemical changes i n steam-treated wood depend on t h e
temperature, pressure, and time of exposure to steam.
Hemicelluloses a r e h y d r o l y z e d to s o l u b l e sugars by o r g a n i c a c i d s ,
m a i n l y a c e t i c a c i d d e r i v e d from a c e t y l a t e d p o l y s a c c a r i d e s present
in wood ( 2 8 ) . Under more d r a s t i c c o n d i t i o n s , s e c o n d a r y r e a c t i o n s
occur which r e s u l t i n the formation of furfural, hydroxymethyl
f u r f u r a l , and their precursors by d e h y d r a t i o n o f p e n t o s e s and
hexoses, respectively. It has been reported (34 ) that
phenolic-like compounds increased from 0.43 t o 5.3% in
steam-pretreated bagasse. Since phenolic-like compounds and
f u r f u r a l s are u s u a l l y t o x i c t o most m i c r o o r g a n i s m s (35 , 3 6 ) , s u c h
t r e a t e d l i g n o c e l l u l o s e s may not be good substrates for SSF,
because the toxic compounds a r e i n a c o n c e n t r a t e d f o r m when t h e
s u b s t r a t e i s i n m o i s t f o r m . T h e r e f o r e , w a s h i n g out o f the toxic
compounds f r o m s t e a m - t r e a t e d s u b s t r a t e s w i l l be n e c e s s a r y b e f o r e
u s i n g them f o r SSF. Toxic compounds, on the other hand, are
diluted w i t h water i n s l u r r y s t a t e f e r m e n t a t i o n and have l i t t l e
e f f e c t on t h e g r o w t h o f m i c r o o r g a n i s m s . T h e r e was no adverse
effect of these toxic compounds o n Τ. reesei for cellulase
p r o d u c t i o n i n s l u r r y o f 4% steam t r e a t e d wood ( 3 2 ) .
Sodium Chlorite ( N a C l O ^ ) . Sodium chlorite, a strong
o x i d i z i n g a g e n t , h a s b e e n used f o r removing l i g n i n during the
preparation of " H o l o c e l l u l o s e " , the t o t a l carbohydrate p o r t i o n of
l i g n o c e l l u l o s e (37 ) . G e o r i n g and Van S e o t ( 3 8 ) d e m o n s t r a t e d that
in vitro digestibility of straws i s increased with NaClO^
treatment. Chahal et a l . (39) reported t h a t p r o t e i n p r o d u c t i v i t y
increased c o n s i d e r a b l y on NaC102 ~ d e l i g n i f i e d wheat straw
fermented w i t h C o c h l i o b o l u s s p e c i f e r . T h i s t r e a t m e n t , by removing
lignin, exposes the s u r f a c e o f h e m i c e l l u l o s e s and c e l l u l o s e f o r
e n z y m a t i c a t t a c k and a l s o c r e a t e s c a p i l l a r i e s i n the substrate
cell w a l l f o r d e e p p e n e t r a t i o n o f enzymes and h y p h a e . However,
t h i s process of d e l i g n i f i c a t i o n i s not economically attractive
because of the high cost of chemicals involved (38). Sodium

c h l o r i t e t r e a t e d s u b s t r a t e , on t h e other h a n d , i s composed of
h e m i c e l l u l o s e s and c e l l u l o s e , w h i c h w o u l d be a good s u b s t r a t e f o r
the production of hemicellulases ( e s p e c i a l l y xylanases) and
cellulases s i m u l t a n e o u s l y i n m o n o c u l t u r e or mixed c u l t u r e . There
is a great future f o r s u c h an enzyme s y s t e m ( m i x t u r e of
h e m i c e l l u l a s e s and c e l l u l a s e s ) f o r the complete h y d r o l y s i s o f
l i g n o c e l l u l o s e s i n t o monomer s u g a r s .
Choice of Microorganism
In n a t u r e , b a c t e r i a g r o w b e s t o n l y when i n a l i q u i d p h a s e , o r
at least when t h e n u t r i e n t s are i n free water. Likewise,
s i n g l e - c e l l e d f u n g i , t h e y e a s t s , g r o w w e l l when t h e n u t r i e n t s are
in a s o l u b l e f o r m . Such m i c r o o r g a n i s m s may n o t be a b l e t o g r o w
s u c c e s s f u l l y i n SSF w h e r e s u b s t r a t e c a r b o n i s n o t available in
soluble form. A limite
m a t e r i a l s i n t o animal fee
Alcaligenes faecalis) or yeasts ( A u r e o b a s i d i u m p u l l u l a n s and
C a n d i d a u t i l i s ) has b e e n r e p o r t e d i n s e m i - s o l i d s t a t e f e r m e n t a t i o n
( 3 , 3a) . Low protein yields i n the final product m i g h t be
a t t r i b u t e d to the fact that i n such system the unicellular
o r g a n i s m s ( b a c t e r i a and y e a s t s ) were u n a b l e t o p e n e t r a t e d e e p i n t o
the t i s s u e f o r complete u t i l i z a t i o n o f the s u b s t r a t e .
Streptomycetes have not been t r i e d f o r SSF so f a r but
H e s s e l t i n e (_1) has r e p o r t e d t h a t he has seen a preparation in
w h i c h S t r e p t o m y c e s a u r e o f a c i e n s was grown on m i l l e t s e e d s , t o be
used i n s w i n e f e e d m i x e s i n t h e U.S.S.R.
On the o t h e r h a n d , f i l a m e n t o u s f u n g i t y p i c a l l y g r o w i n n a t u r e
on s o l i d s u b s t r a t e s , s u c h a s , wood, s e e d s , s t e m s , r o o t s and l e a v e s
of p l a n t s , w i t h o u t t h e p r e s e n c e o f f r e e w a t e r (_1) . H i g h p r o t e i n
c o n t e n t s o f 20-24% i n t h e f i n i s h e d p r o d u c t h a v e b e e n r e c o r d e d by
Chahal et a l . (2) i n SSF of corn stover with Chaetomium
cellulolyticum. High p r o t e i n content i n the final product has
b e e n a t t r i b u t e d t o t h e f a c t t h a t t h e hyphae o f C. c e l l u l o l y t i c u m
h a v e t h e power t o p e n e t r a t e deep i n t o the intercellular and
i n t r a c e l l u l a r spaces f o r b e t t e r u t i l i z a t i o n o f the s u b s t r a t e ( 4 0 ) .
Most f i l a m e n t o u s f u n g i h a v e s u c h i n t r u s i o n power. A i s t (41) has
given a good r e v i e w o f t h e p e n e t r a t i o n o f f u n g a l h y p h a l t i p s i n t o
p l a n t c e l l w a l l s . He concluded that t h e mechanism o f fungal
penetration i n t o s u b s t r a t e s c o u l d be m e c h a n i c a l a n d / o r e n z y m a t i c .
I t i s , t h e r e f o r e , e v i d e n t t h a t the c h o i c e of the microorganisms
f o r s u c c e s s f u l SSF i s l i m i t e d t o f i l a m e n t o u s o r g a n i s m s - f u n g i and
actinomycetes.
Mechanism o f F u n g a l G r o w t h D u r i n g Solid State Fermentation
F u n g a l hyphae a r e s e p t a t e , t u b u l a r , u n i s e r i a t e f i l a m e n t s , on
a v e r a g e 10-15 ym i n d i a m e t e r . The g r o w t h i n l e n g t h o c c u r s a t t h e
t i p and i s c o n f i n e d t o t h i s a r e a , so t h a t i n s e p t a t e hyphae when a
cell i s cut o f f from the apex i t i s no l o n g e r c a p a b l e o f a n y

s i g n i f i c a n t increase i n length. T h e r e i s t h u s no increase in

i n t e r s e p t a l d i s t a n c e s , a l t h o u g h t h e r e may be i n c r e a s e s i n d i a m e t e r
and w a l l t h i c k n e s s . The o l d e r p o r t i o n o f the hypha d i s i n t e g r a t e s ,
whereas the apex c o n t i n u e s t o g r o w i n t o new sites in the
substrate. A u t o r a d i o g r a p h s made o f hyphae w h i c h h a v e b e e n fed
with b r i e f pulses o f t r i t i a t e d w a l l precursors demonstrated that
i n c o r p o r a t i o n i s h i g h e s t i n t h e a p i c a l 1 ym and f a l l s o f f r a p i d l y
after the first 5 ym, however, there is still appreciable
i n c o r p o r a t i o n f r o m 5-75 ym b e h i n d the t i p ( 4 2 ) .
H y p h a l w a l l s a r e u s u a l l y two l a y e r e d w i t h an i n n e r l a y e r o f
m i c r o f i b r i l l a r m a t e r i a l s , u s u a l l y c h i t i n o r c e l l u l o s e , o r b o t h and
an outer l a y e r o f amorphous g l u c a n . The c h i t i n i s composed o f
3-1, 4 l i n k e d N - a c e t y l glucosamine residues and cellulose i s
composed o f (3-1,4 l i n k e d g l u c o s e u n i t s . The g l u e an s o f t h e o u t e r
l a y e r s o f c e l l w a l l s a r e composed o f h i g h l y b r a n c h e d (3-1, 3 l i n k e d
g l u c a n w i t h (3-1, 6 l i n k e
c e l l w a l l s t o the h y p h a e
apex and the complex system of b r a n c h i n g ensure t h a t the o l d e r
r e a r w a r d p a r t o f t h e hypha i s f i r m l y a n c h o r e d i n t h e s u b s t r a t e and
enable the hyphal t i p to e x e r t very considerable forward
m e c h a n i c a l p r e s s u r e a s i t e x t e n d s u n d e r t u r g o r (42 ) . C o u p l e d w i t h
the production o f e x t r a c e l l u l a r enzymes and the much b r a n c h e d
hyphal system, the f u n g i can thus c o m p l e t e l y permeate even the
hardest substrate.
Fungal Growth i n S o l i d Substrate i n N a t u r e . In n a t u r e ,

a c c o r d i n g to t h e s t u d y o f C o w l i n g (43 ) , f u n g a l g r o w t h on solid
substrate can be of three general t y p e s e x h i b i t e d by
wood-inhabiting fungi:
Wood-Staining F u n g i . These f u n g i a r e o f two t y p e s :
(a) s u r f a c e m o l d s , s u c h a s , T r i c h o d e r m a l i g n o r u m , t h a t d e v e l o p on
t h e e x t e r n a l s u r f a c e o f t h e s u b s t r a t e ; and
(b) p e n e t r a t i n g f u n g i , C e r a t o c y s t i s spp. t h a t d e v e l o p deep w i t h i n
wood, most c o n s p i c u o u s l y i n r a y p a r e n c h y m a . These f u n g i j u s t g r o w
on r e s e r v e f o o d and do n o t a f f e c t t h e s t r e n g t h of the substrate
cell w a l l s . They m e r e l y p r o d u c e s t a i n s i n wood due t o the
presence o f pigments i n t h e i r hyphae.
Soft-Rot Fungi. These f u n g i a t t a c k t h e e x p o s e d s u r f a c e s o f
wood and wood p r o d u c t s t h a t a r e more o r l e s s c o n s t a n t l y saturated
with w a t e r . These f u n g i a r e confined to c y l i n d r i c a l c a v i t i e s
c r e a t e d by t h e m s e l v e s w i t h i n t h e s e c o n d a r y w a l l s o f wood cells.
B a i l e y and Vestal (44) reported f o r the f i r s t time t h a t the
s o f t - r o t f u n g i a t t a c k t h e wood p a r a l l e l to the o r i e n t a t i o n of
cellulose molecules in the secondary w a l l s of the cell.
S i m i l a r l y , s p i r a l c r a c k i n g along the angle of the fibrils of
cellulose i n t h e s e c o n d a r y w a l l o f D o u g l a s f i r wood c e l l s c a u s e d
by Fomes p i n i was o b s e r v e d b y P r o c t o r ( 4 5 ) . Soft-rot fungi such
as Chaetomium spp. and X y l a r i a s p p . a r e a l i t t l e more a g g r e s s i v e
than wood-stain f u n g i , i n that they are able to penetrate deeper
into the s u b s t r a t e , but they remain r e s t r i c t e d along the

o r i e n t a t i o n o f the c e l l u l o s e f i b r i l s . They a r e unable to cause

complete decay o f the a f f e c t e d t i s s u e because o f t h e i r poor
c e l l u l a s e systems.
T y p i c a l Wood-Destroying F u n g i . Two g r o u p s o f t h e s e f u n g i a r e
d i s t i n g u i s h e d on the b a s i s o f c o l o r and texture of the decayed
wood: w h i t e - r o t f u n g i , s u c h as P o l y p o r u s v e r s i c o l o r , produce
l i g h t c o l o r e d wood m a i n l y b e c a u s e o f the utilization of lignin
along with p o l y s a c c h a r i d e s , w h i l e brown-rot f u n g i , such as, P o r i a
monticola, p r o d u c e brown c o l o r e d wood mainly because of
utilization of polysaccharides although some l i g n i n i s also
degraded ·
B o t h w h i t e - and b r o w n - r o t f u n g i i n h a b i t t h e wood c e l l l u m i n a
and p e n e t r a t e f r o m one c e l l t o a n o t h e r , e i t h e r through n a t u r a l
o p e n i n g s , s u c h as pits, or d i r e c t l y by b o r i n g a h o l e . Proctor
( 4 5 ) r e p o r t e d t h a t t h e f u n g a l hyphae p e n e t r a t e t h e wood c e l l wall
by a n e n z y m a t i c mechanis
( 4 1 ) s u p p o r t e d the i d e
into plant c e l l wall could be a m e c h a n i c a l and/or enzymatic
m e c h a n i s m . The b o r e - h o l e s a r e formed b e c a u s e o f t h e c l o s e c o n t a c t
of surface of hyphal t i p a g a i n s t wood c e l l w a l l s u r f a c e . The
c e l l u l a s e system produced a t the t i p i s a b l e to solubilize wood
cell w a l l f i b e r s , t h e r e b y m a k i n g room f o r f u r t h e r p e n e t r a t i o n by
the hyphal t i p . T h i s process c o n t i n u e s t i l l the hyphal t i p e n t e r s
i n t o the n e x t c e l l l u m e n . The f u n g a l a t t a c k g o e s on f r o m one c e l l
to t h e o t h e r t i l l the w h o l e t i s s u e i s d e c a y e d .
The highly localized nature o f the d i s s o l u t i o n i n v o l v e d i n
the formation of these bore-holes by the white-rot fungus,
P o l y p o r u s v e r s i c o l o r , i s i l l u s t r a t e d i n F i g u r e 1 ( 4 3 ) . The hyphae
r e s p o n s i b l e f o r f o r m a t i o n of the h o l e s are a u t o l y z e d as soon as
t h e t i p o f t h e hypha r e a c h e s t h e lumen where i t g e t s more s u i t a b l e
c o n d i t i o n s f o r f u r t h e r d e v e l o p m e n t . That c o u l d be t h e m a i n r e a s o n
why t h e d i s s o l u t i o n o f wood c e l l w a l l r e m a i n s r e s t r i c t e d t o a s i z e
a l i t t l e b i g g e r t h a n t h e d i a m e t e r o f p e n e t r a t i n g h y p h a . Where t h e
hypha s t a y s f o r long t i m e , d i s s o l u t i o n beyond the b o r e - h o l e i s
n o t i c e d , a s shown i n F i g u r e 2 (43 ) . The r e m n a n t s o f hypha are
also seen i n the b o r e - h o l e . Figure 2 shows t h a t d i s s o l u t i o n
e x t e n d s more l o n g i t u d i n a l l y than t r a n s v e r s a l l y . This i n d i c a t e s
that i t i s e a s i e r f o r the c e l l u l a s e s y s t e m t o move a l o n g the
o r i e n t a t i o n o f the f i b r e s i n t h e c e l l w a l l . Figure 2 also shows
more d i s s o l u t i o n on t h e e x t r e m e l e f t and e x t r e m e r i g h t s i d e s o f
c e l l w a l l s , the s u r f a c e s o f the lumina of two adjacent cells.
This d i s s o l u t i o n i s due t o the c e l l u l a s e s y s t e m p r o d u c e d b y t h e
hyphae g r o w i n g i n t h e cell lumina. The development o f hyphae
within the wood c e l l lumen shown i n F i g u r e 3 ( 4 6 ) a l s o shows
d i s s o l u t i o n of secondary w a l l along the hyphae f o r m i n g erosion
troughs, which i n d i c a t e s that i n i t i a l d i s s o l u t i o n of c e l l u l o s e
o c c u r s v e r y c l o s e to the s u r f a c e o f f u n g a l hyphae. A s i m i l a r type
o f g r o w t h b e h a v i o r ( p e n e t r a t i o n o f hyphae t h r o u g h c e l l w a l l , v i a
p o r e s o f t r a c h a i d s and ray c e l l s ) was also shown by another
wood-inhabiting fungus, Phanerochaete chrysosporium ( 4 7 ) .

I t i s c l e a r from these o b s e r v a t i o n s (43,45,46) t h a t the most

active cellulase s y s t e m i s p r o d u c e d on t h e s u r f a c e o f t h e f u n g a l
hypha w h i c h i s a b l e t o p r o d u c e a b o r e - h o l e when i t i s p e n e t r a t i n g
the cell w a l l t r a n s v e r s e l y and where t h e enzyme s y s t e m has
r e s t r i c t e d movement, and p r o d u c e s e r o s i o n t r o u g h s i n i t i a l l y along
the hyphae when a t t a c k i n g the i n n e r s u r f a c e o f t h e c e l l l u m e n .
But i n t h e l a t t e r s i t u a t i o n t h e enzyme s y s t e m i s a b l e t o p e n e t r a t e
q u i c k l y and deeply along the o r i e n t a t i o n of fibrils i n the
s e c o n d a r y w a l l s , ahead of the hyphal surface and to cause
c o n s i d e r a b l e d e c a y . The t a n g e n t i a l s e c t i o n i n F i g u r e 4 shows
a l m o s t c o m p l e t e d i s s o l u t i o n o f s e c o n d a r y w a l l s o f t h e two a d j a c e n t
fiber trachied c e l l s , l e a v i n g behind o n l y the middle l a m e l l a ,
which are r e s i s t a n t to c e l l u l a s e system, being highly lignified
( 4 3 ) . The f u n g a l hypha i n c r o s s s e c t i o n i s a l s o s e e n on t h e r i g h t
s i d e o f the F i g u r e 4. Figure 4 clearly i n d i c a t e s that i t is
e a s i e r f o r the o r g a n i s m
i n s i d e surface of the c e l
lamella.
It i s inferred from the growth b e h a v i o r of the typical
wood-inhabiting fungi that t h e m o s t a c t i v e c e l l u l a s e s y s t e m was
p r o d u c e d on t h e s u r f a c e o f f u n g a l hypha when i t was i n close
contact with c e l l u l o s e . The c e l l u l a s e s y s t e m p r o d u c e d under s u c h
c o n d i t i o n s was able to hydrolyze native cellulose with high
c r y s t a l l i n i t y , on t h e o t h e r hand t h e enzyme s y s t e m p r o d u c e d by Τ.
r e e s e i i n LSF was n o t very effective i n hydrolyzing cellulose
until t h e c r y s t a l l i n i t y was reduced by v a r i o u s physical or
chemical pretreatments as i n d i c a t e d i n the recent evaluation of
enzymatic h y d r o l y s i s o f c e l l u l o s e 05)· I n LSF t h e f u n g a l hyphae
and c e l l u l o s e p a r t i c l e s do n o t r e m a i n i n c l o s e c o n t a c t with each
other f o r long t i m e due to h i g h a g i t a t i o n i n the c o n v e n t i o n a l
fermenter. That m i g h t be t h e r e a s o n t h a t enzyme p r o d u c e d in LSF
is not so e f f e c t i v e f o r h y d r o l y s i s o f c r y s t a l l i n e c e l l u l o s e . It
i s , t h e r e f o r e , l i k e l y t h a t c e l l u l a s e s y s t e m p r o d u c e d i n SSF will
not o n l y h a v e h i g h c e l l u l a s e a c t i v i t y p e r u n i t o f f e r m e n t a t i> η
b r o t h 05), b u t will a l s o be m o s t a c t i v e i n h y d r o l y z i n g the
crystalline cellulose.
Observations on the Fungal Growth in Solid State

Fermentation. F u n g a l g r o w t h i n s o l i d s t a t e fermentâti> η o f sodium
hydroxide-treated corn s t o v e r has b e e n r e p o r t e d by C h a h a l e t a l .
(40) i n d e t a i l . They h a v e r e p o r t e d t h a t Chaetomium c e l l u l o l y t i c u m
p r o v e d t o be t h e b e s t o r g a n i s m f o r u p g r a d i n g t h e p r o t e i n v a l u e s o f
c o r n s t o v e r f o r a n i m a l f e e d i n g . The o r g a n i s m g r e w p r o f u s e l y on
corn stover w i t h i n f i v e days. V i s u a l examination i n d i c a t e d that
t h e m y c e l i u m i m p r e g n a t e d the e n t i r e s u b s t r a t e .
Microscopic examination of the s u b s t r a t e p a r t i c l e s showed
t h a t c e l l s o f t h e s u b s t r a t e t i s s u e were l o o s e l y h e l d t o g e t h e r and
tended t o s e p a r a t e e a s i l y when p r e s s e d . I t i n d i c a t e d t h a t sodium
h y d r o x i d e t r e a t m e n t s o l u b i l i z e d l i g n i n and weakened the b o n d s of
adjacent c e l l s . F i g u r e 5 shows b r o k e n e n d s o f c e l l s on t h e s i d e s

Figure 1. Bore holes in spruce tracheid made by the hyphae of white rot fungus
P o l y p o r u s versicolor (X 20,250). Reproduced, with permission, from Ref. 43.
Copyright 1965, Syracuse University Press.
Figure 2. Advanced stage of decay around bore hole and inner side of secondary
wall of two fiber tracheids of sweetgum sapwood, X 2700. Reproduced, with
permission, from Ref. 43. Copyright 1965, Syracuse University Press.

Figure 3. Trough formed by decaying of secondary wall of birch vessels in the

vicinity of the fungal hyphae of Polystictus versicolor. Reproduced, with permis-
sion, from Ref. 46.
Figure 4. Almost complete utilization of secondary walls of two adjacent cells

leaving middle lamella intact. Reproduced, with permission, from Ref. 43. Copy-
right 1965, Syracuse University Press.

a s w e l l as on t h e e n d s o f t h e s u b s t r a t e p a r t i c l e . Such b r o k e n and
exposed c e l l s were t h e f i r s t s i t e s t o be a t t a c k e d by t h e o r g a n i s m .
F i g u r e 6 shows m y c e l i a l g r o w t h on s u c h b r o k e n e x p o s e d c e l l s o n t h e
l a t e r a l s i d e o f the s u b s t r a t e p a r t i c l e .
When s u c h s u b s t r a t e p a r t i c l e s , c o l o n i z e d by the organism,
were p r e s s e d slightly under a cover s l i p , they d i s i n t e g r a t e d
easily. The d i s i n t e g r a t e d m a t e r i a l c o n t a i n e d some s u b s t r a t e c e l l s
s h o w i n g p e n e t r a t i o n by hyphae and d e v e l o p m e n t o f m y c e l i a l g r o w t h i n
the c e l l lumina a t v a r i o u s s t a g e s . In F i g u r e 7 p e n e t r a t i o n of a
s i n g l e hypha i n t o the l u m e n o f a t h i n l o n g i t u d i n a l f i b e r c e l l i s
c l e a r l y s e e n . As s o o n a s t h e hypha e s t a b l i s h e s i t s e l f in the
lumen a t h i c k m y c e l i a l mass i s d e v e l o p e d w i t h i n t h e c e l l ( F i g u r e
8). I t was c o n c l u d e d f r o m t h e s e o b s e r v a t i o n s t h a t t h e hyphae of
C. c e l l u l o l y t i c u m entered i n t o the c e l l lumen t h r o u g h n a t u r a l
openings, mechanical breaks, or spaces ( c r e a t e d by s o l u b i l i z a t i o n
o f hemic e l l u i ο se s and l i g n i
the c e l l w a l l of the substrat
lumen, the hypha d i g e s t e d the c e l l w a l l s t a r t i n g f r o m the i n s i d e
towards the o u t s i d e . U l t i m a t e l y , the cell collapsed leaving
behind m y c e l i a l biomass r i c h i n p r o t e i n . Reese ( 4 8 ) has a l s o
r e p o r t e d t h a t i n a t t a c k i n g c o t t o n , many f u n g i p e n e t r a t e d through
t h e f i b e r w a l l i n t o t h e lumen and d i d most o f t h e i r d i g e s t i n g f r o m
within (Figure 9). S i m i l a r l y B r a v e r y ( 4 6 ) has r e p o r t e d the g r o w t h
o f P o l y s t i c t u s v e r s i c o l o r i n t h e wood c e l l lumen and has shown
t h a t d i g e s t i o n of the c e l l i s i n i t i a t e d from i n s i d e to outside
(Figure 3).
P o s t u l a t e d Mechanism o f F u n g a l G r o w t h i n S o l i d State
M i c r o o r g a n i s m s may posses the p o t e n t i a l a b i l i t y to perform

many m e t a b o l i c activities which are not obligatory for the
maintenance of c e l l u l a r f u n c t i o n and w h i c h come i n t o p l a y o n l y
u n d e r c e r t a i n s p e c i a l e n v i r o n m e n t a l c o n d i t i o n s . Such activities
are t y p i c a l l y concerned with energy-yielding metabolism ( 4 9 ) .
L i b e r a t i o n o f h y d r o l y t i c enzymes i s an a c t i v e f u n c t i o n o f living
fungal cells (50). D u r i n g g r o w t h on c e l l u l o s e , enzymes are
liberated, c h i e f l y at the hyphal t i p . They d i f f u s e t o the
substrate and d i g e s t i t . The h y d r o l y s i s p r o d u c t s e n t e r i n t o t h e
f u n g u s c y t o p l a s m . The hypha t h e n grows i n t o the d i g e s t e d region
and m a i n t a i n s c o n t i n u a l i n t i m a t e c o n t e n t w i t h t h e s u b s t r a t e ( 4 8 ) .
Whether c e l l u l a s e s a r e a d a p t i v e o r c o n s t i t u t i v e enzymes has
n o t b e e n r e s o l v e d so f a r . I t has b e e n r e p o r t e d t h a t T. r e e s e i c a n
p r o d u c e c e l l u l a s e s i n t h e p r e s e n c e o f i n d u c e r s s u c h as c e l l o b i o s e ,
s o p h o r o s e , c e l l u l o s e ( 5 1 , 5 2 , 5 3 ) . On the o t h e r hand, r e c e n t l y a
small quantity of c e l l u l a s e production has been r e p o r t e d by
hypereelluiase m u t a n t s o f Τ. reesei while growing i n glucose
medium u n d e r n o r m a l g r o w t h c o n d i t i o n s (54 , 5 5 ) * There i s e v i d e n c e
(48) t h a t g l u c o s e i s n o t a t r u e i n d u c e r but i s m e t a b o l i z e d t o an
inducer, probably a $-glucoside. I t appears that the 3-1, 4
glucosidic l i n k a g e must be p r e s e n t i n s o l u b l e compounds t h a t a c t

a s i n d u c e r s o f c e l l u l a s e . But i n n a t u r e when t h e f u n g a l t i p makes

i t s c o n t a c t w i t h c e l l u l o s e f o r the f i r s t t i m e , no such solutes
(inducers) are a v a i l a b l e to t r i g g e r the s y n t h e s i s o f c e l l u l a s e
w i t h i n the c e l l . I t i s known ( 4 9 ) t h a t the enzymatic machinery
for the performance o f these f a c u l t a t i v e metabolic a c t i v i t i e s i s
u s u a l l y s y n t h e s i z e d by c e l l s o n l y i n response to a specific
c h e m i c a l s i g n a l from the e n v i r o n m e n t . C e l l u l o s e , b e i n g i n s o l u b l e ,
c a n n o t e n t e r i n t o t h e c e l l t o send any c h e m i c a l s i g n a l s f o r the
synthesis of c e l l u l a s e s . Under s u c h c o n d i t i o n s ( i n t h e a b s e n c e
o f s o l u b l e i n d u c e r s ) , i t i s p l a u s i b l e t h a t a mere p h y s i c a l c o n t a c t
of cellulose with the f u n g a l h y p h a l t i p i s e n o u g h t o send some
s o r t o f p h y s i c a l ( i n s t e a d of chemical) s i g n a l s through the w a l l of
the fungal cell to the nucleus t o s y n t h e s i z e s p e c i f i c RNA t o
produce the r e q u i r e d i n d u c e r ( p r o b a b l y a B - g l u c o s i d e ) as mentioned
earlier (48). I t has a l s o been p o i n t e d out e a r l i e r t h a t a c l o s e
c o n t a c t between c e l l u l o s
c e l l u l a s e synthesis (56,57)
b y M a n d e l s and Reese ( 5 2 ) t h a t s o l u b l e p r o d u c t s o f enzyme a c t i o n
are natural inducers of the enzymes t h a t a t t a c k i n s o l u b l e
s u b s t r a t e s . On t h i s t h e o r y t h e y assumed t h a t s m a l l amounts of
i n d u c i b l e enzymes ( c e l l u l a s e s ) a r e p r o d u c e d even i n t h e a b s e n c e o f
i n d u c e r ( c e l l o b i o s e ) . When t h e i n s o l u b l e s u b s t r a t e ( c e l l u l o s e ) i s
p r e s e n t , i t i s h y d r o l y z e d and the s o l u b l e products (especially
cellobiose) thus e n t e r the cell and induce more enzymes
(cellulases). The above assumption a l s o supports the p o s t u l a t e
t h a t a mere p h y s i c a l c o n t a c t o f t h e o r g a n i s m w i t h c e l l u l o s e may be
r e s p o n s i b l e t o t r i g g e r the s y n t h e s i s o f c e l l u l a s e s .
The p o s t u l a t e d mechanism o f m e t a b o l i s m of c e l l u l o s e by a
c o m p l e t e c e l l u l a s e complex p r o d u c e d a t t h e t i p o f t h e f u n g a l hypha
is based on the observation recorded by various workers
( 4 0 - 5 0 , 5 2 , 5 6 , 5 7 , 5 8 ) . The mechanism given i n Figure 10 is
e x p l a i n e d as f o l l o w s :
1. P l a u s i b l y p h y s i c a l s i g n a l s a r e t r a n s m i t t e d from c e l l u l o s e to
the f u n g a l c e l l n u c l e u s t o t r i g g e r the s y n t h e s i s o f the f i r s t
enzyme ( E ) , w h i c h
1 causes splitting of fibrils and
microfibrils f r o m the c r y s t a l l i n e p o r t i o n o f c e l l u l o s e f i b e r .
The a c t i o n o f E- h e r e i s t h e same a s e x p l a i n e d by Reese (48)
for C^. The is still a c o n t r o v e r s i a l enzyme and i s
c o n f u s e d w i t h e x o - 1 , 4 - $ - D - g l u c a n c e l l o b i o h y d r o l a s e . The most
important r o l e of C^ to s p l i t g l u c o s e polymer c h a i n s from
c r y s t a l l i n e c e l l u l o s e p r o p o s e d i n 1950 ( 5 9 ) i s s t i l l retained
here as w e l l as b y Reese ( 6 0 ) . T h e r e f o r e , t h e r e i s e v e r y
p o s s i b i l i t y t h a t c l o s e c o n t a c t o f c e l l u l o s e and the organism
i s most i m p o r t a n t t o p r o d u c e C^(E^) enzyme, i f n o t , f o r o t h e r
components o f c e l l u l a s e s ( e x o - and e n d o - g l u c o n a s e s ) .
2. Subsequent s y n t h e s i s o f the s e c o n d enzyme ( E ~ ) i s triggered.

E~ i s composed o f t h r e e c o m p o n e n t s : ( i ) Endo 1, 4 - $ - D - g l u c a n
glueanohydrolase ( e n d o - g l u e a n a s e ) w i t h random attack on

Figure 5. Growth of C h a e t o m i u m cellulolyticum in NaOH-treated corn stover.

Substrate particles showing broken cells, first sites for attack by the organism
(X 94.5). From Ref. 40.
Figure 6. Hyphal growth on sites like those shown in Figure 5. From Ref. 40.

Figure 7. Penetration by a single hyphae into cell lumen through the broken end
(X 94.5). From Ref. 40.
Figure 8. Mycelial biomass developed in the cell lumen (X 94.5). From Ref. 40.

Figure 9. M e m n o n i e l l a echinata in the lumen of cotton fiber showing utilization

of secondary wall from inside towards outside. Reproduced, with permission, from
Ref. 49. Copyright 1959, University of Washington Press.
Figure 10. Utilization of secondary wall from the inside of the lumen of wood
cell, as explained in the text.

glucose polymer c h a i n s to break I t Into smaller chains

(oligomers). Some g l u c o s e u n i t s a r e a l s o r e l e a s e d d u r i n g t h i s
reaction, ( i i ) Exo-1, 4-3-D-glucan glueοhydrolase
( e x o - g l u e a n a s e ) removes g l u c o s e u n i t s f r o m the non-reducing
end of glucose polymer c h a i n s , ( i i i ) Exo-1,4-3-D-glucan
cellobiohydrolase ( e x o - g l u e a n a s e ) removes c e l l o b i o s e from
non-reducing ends o f g l u c o s e polymer c h a i n s . T h i s compound
enzyme releases a mixture of oligomers, c e l l o b i o s e and
glucose. The s e q u e n c e o f s y n t h e s i s o f t h e s e t h r e e components
o f E^ i s a n o t h e r c o n t r o v e r s i a l p o i n t i n c e l l u i a s e s y s t e m .
3. C e l l o b i o s e i s a b s o r b e d b y t h e f u n g a l c e l l s where i t t r i g g e r s
t h e s y n t h e s i s o f t h e t h i r d enzyme, 3 - g l u c o s i d a s e ( E ~ ) t o b r e a k
c e l l o b i o s e into glucose u n i t s . The E^ i s a n i n t r a c e l l u l a r
enzyme b u t a s m a l l q u a n t i t y i s a l s o s e c r e t e d o u t s i d e o f t h e
fungal c e l l . Larg
the older cells o
Thus hydrolyzes c e l l o b i o s e into glucose u n i t s i n s i d e as
w e l l as o u t s i d e the fungal c e l l s .
Finally some o f t h e g l u c o s e released from c e l l u l o s e i s

catabolized to release e n e r g y needed f o r various metabolic
processes and some i s m e t a b o l i z e d t o s y n t h e s i z e f u n g a l b i o m a s s ,
e x t r a c e l l u l a r c e l l u l a s e s and 3 - g l u c o s i d a s e . The f i n a l p r o d u c t may
be a f u n g a l b i o m a s s r i c h i n p r o t e i n ( t o be used a s SCP) a s i n t h e
c a s e o f C. c e l l u l o l y t i c u m o r i t may b e v e r y little biomass with
more e x t r a c e l l u l a r cellulases ( t o b e used f o r hydrolysis of
c e l l u l o s i c m a t e r i a l s i n t o g l u c o s e ) a s i n t h e c a s e o f T. r e e s e i .
Physiological Aspects
T e m p e r a t u r e . D u r i n g SSF t h e t e m p e r a t u r e i n t h e s u b s t r a t e
rises due to heat generated b y m e t a b o l i s m . The r i s e i n
t e m p e r a t u r e i s d i r e c t l y r e l a t e d t o t h e d e p t h o f t h e s u b s t r a t e and
the m e t a b o l i c a c t i v i t i e s o f t h e organisms. I t has been recorded
(61) that d u r i n g composting o f straw f o r mushroom g r o w i n g , t h e
temperature o f the outer l a y e r o f t h e compost h e a p ( o n e m e t e r
h i g h ) i s a r o u n d 37 C, w h i l e i n t h e i n n e r p o r t i o n t h e t e m p e r a t u r e
rises as high a s 60-77 C. On t h e o t h e r h a n d , i n t h e i n n e r m o s t
region the temperature reaches o n l y 32-49 C b e c a u s e o f l o w
m i c r o b i a l a c t i v i t y , due t o a n a e r o b i c c o n d i t i o n s i n t h a t r e g i o n .
Edwards ( 6 2 ) had r e p o r t e d t h a t o n e K g ( d r y w e i g h t ) o f o r g a n i c
matter ( c e l l u l o s e ) when consumed b y m i c r o o r g a n i s m p r o d u c e d 0.597
K g o f w a t e r , 1.465 K g o f C 0 a n d 14,960 ΒTU. Some o f t h e h e a t
2
(about 59%) i s used to evaporate water produced d u r i n g the

metabolic a c t i v i t y while the remaining (41%) i s l e f t i n the
s u b s t r a t e t o b e d i s s i p a t e d . T h i s amount o f e n e r g y , i f n o t
dissipated immediately, as i t i s released, w i l l reduce the
p r o d u c t i v i t y o r may k i l l t h e o r g a n i s m .

A e r a t i o n . A e r a t i o n has t h e f o l l o w i n g f u n c t i o n s i n SSF:
( i ) To s u p p l y 0^ f o r a e r o b i c m e t a b o l i s m .
( i i ) To c o n t r o l the t e m p e r a t u r e .
(iii) To remove CO^, w a t e r v a p o u r s , and v o l a t i l e m e t a b o l i t e s
produced d u r i n g metabolism.
The problem of a e r a t i o n w i l l i n c r e a s e c o r r e s p o n d i n g l y w i t h
the i n c r e a s e i n the thickness of the substrate layer. Proper
aeration i n the SSF a l s o d e p e n d s o n t h e a i r s p a c e s a v a i l a b l e i n
t h e s u b s t r a t e . F i b r o u s m a t e r i a l s c a n p r o v i d e more a i r s p a c e s t h a n
finely powdered s u b s t r a t e . A e r a t i o n seems t o be a v e r y c r i t i c a l
requirement. O c h r a t o x i n p r o d u c t i o n by A s p e r g i l l u s o c h r a c e u s i n a
r o t a t i n g drum f e r m e n t e r s t o p p e d when t h e a i r f l o w r a t e was g r e a t e r
t h a n 0.1 l i t e r / K g / m i n u t e ( 6 3 ) . I n c o n t r a s t , t h e increase in air
flow rate through a column of c o r n i n o c u l a t e d w i t h A s p e r g i l l u s
f l a v u s i n c r e a s e d the r a t e o f production of a f l a t o x i n (64-65).
S i m i l a r l y i t was reporte
p r o d u c t i o n by A s p e r g i l l u
c o n s i d e r a b l y when t h e a e r a t i o n was i n c r e a s e d .
Moisture. In n a t u r e , vegetables and fruits contain about
60-80% m o i s t u r e . T h i s much m o i s t u r e i s s u f f i c i e n t t o e n c o u r a g e
t h e g r o w t h o f m i c r o o r g a n i s m s (66,67 ) . But Christensen (68)
reported t h a t E u r o t i u m h a l o p h i l i c u m c a n g r o w on wheat g r a i n s a t
13-14% m o i s t u r e . M o r t o n and E g g i n s ( 6 9 ) have a l s o r e p o r t e d that
some fungi like A b s i d i a corymbifera, Fusarium s o l a n i and
Chaetomium trilatérale can g r o w and p e n e t r a t e i n wood a t a 14%
m o i s t u r e l e v e l a t 35°C.
Growth o f s o f t - r o t f u n g i i n s i t u a t i o n s o f e x t r e m e d r y n e s s has
b e e n r e c o r d e d by Duneun and E s l y n ( 7 0 ) . A b a s i d i o m y c e t e , S e r p u l a
l a c r y m a n s i s w e l l known t o c o l o n i z e d r y timber i n buildings
causing the c o n d i t i o n s known as " d r y r o t " . T h i s i s a c h i e v e d by
the fungus i n i t i a t i n g growth i n timber w i t h a h i g h water content
and then using the e n e r g y and n u t r i e n t s gained to produce
r h i z o m o r p h s w h i c h c a n e x p l o r e d r y t i m b e r many m e t e r s away f r o m the
initial colony. Though s u c h r h i z o m o r p h s c a n transport water,
f u r t h e r water i s produced a t the s i t e o f a c t i o n by the u t i l i z a t i o n
o f t h e t i m b e r w i t h t h e e v o l u t i o n o f CO- ( 7 1 ) .
V a r i o u s l e v e l s o f m o i s t u r e i n SSF have been r e p o r t e d for
different products. About 7 5 % m o i s t u r e i n s t r a w was used f o r SCP
p r o d u c t i o n (2 ,3,3a) w h e r e a s f o r a f l a t o x i n p r o d u c t i o n i t was 33.3%
in rice and 48.4% i n s t r a w (1). Thus m o i s t u r e l e v e l i n t h e SSF
d e p e n d s on t h e n a t u r e o f t h e s u b s t r a t e , o r g a n i s m and the type of
end product.
pH. M o s t f u n g i a r e a b l e t o g r o w i n a w i d e pH r a n g e o f 5-8.
A d j u s t m e n t o f pH d u r i n g t h e g r o w t h i n SSF i s v e r y d i f f i c u l t . Some
o f t h e s u b s t r a t e ( s t r a w s , g r a i n s ) have good b u f f e r i n g c a p a c i t y and
t h e r e may not be any need t o a d j u s t t h e pH d u r i n g SSF. But
maintenance of pH a r o u n d 4.5 is very crucial for cellulase
p r o d u c t i o n w i t h T. r e e s e i (_5 ) .
Osmotic P r e s s u r e . Raising the osmotic potential of a
material by a d d i t i o n of s a l t or sugar or both to a l e v e l higher
t h a n t h a t o c c u r i n g i n the c y t o p l a s m i s a w e l l t r i e d method of

microbial i n h i b i t i o n and i s w i d e l y u s e d i n p r e s e r v a t i o n o f f o o d s ,
s u c h a s meats and f r u i t s . Sugar c o n c e n t r a t i o n s i n t h e r e g i o n s of
50-70% a r e u s u a l l y a d e q u a t e ; s a l t i s added t o 2 0 - 2 5 % l e v e l ( 7 1 ) .
However, t h e r e are some o s m o p h i l i c and halophyllic organisms
( S a c c h a r o m y c e s r o u x i i , S. m e l l i s and A s p e r g i l l u s g l a u c u s s e r i e s )
which can grow i n c o n c e n t r a t e d sugar solutions (71 ) . High
concentrations of salts ( n u t r i e n t s ) are used i n SSF f o r SCP
p r o d u c t i o n from s t r a w ( 2 ) . T h e r e f o r e , t h e m i c r o o r g a n i s m s capable
o f w i t h s t a n d i n g h i g h o s m o t i c p r e s s u r e w i l l be more s u i t a b l e under
s u c h c o n d i t i o n s , o t h e r w i s e , t h e r e q u i r e d n u t r i e n t s a r e t o be added
in frequent s m a l l doses to a v o i d h i g h osmotic p o t e n t i a l i n the
substrate.
C h a r a c t e r i s t i c s o f an O r g a n i s m f o r SCP and C e l l u l a s e P r o d u c t i o n
P r o d u c t i o n o f SCP an
depend on t h e t h o r o u g h u n d e r s t a n d i n
b e h a v i o r o f SCP and cellulase producing organisms under such
conditions before the a p p l i c a t i o n o f b i o c h e m i c a l e n g i n e e r i n g to
s c a l e up t h e p r o c e s s e s . The o r g a n i s m f o r s u c c e s s f u l SSF should
have t h e f o l l o w i n g p r i m a r y q u a l i t i e s :
1. I t i s a b l e to u t i l i z e mixtures o f v a r i o u s p o l y s a c c h a r i d e s .
2. I t s h o u l d have c o m p l e t e enzyme s y s t e m s t o s w i t c h over i t s
metabolism f r o m one p o l y s a c c h a r i d e t o a n o t h e r p o l y s a c c h a r i d e
( f o u n d i n c o m p l e x s u b s t r a t e s ) w i t h o u t any l a g p h a s e .
3. It i s able to penetrate deep i n t o the t h i c k l a y e r o f the
s u b s t r a t e as w e l l as i n t o t h e c e l l s o f t h e s u b s t r a t e .
4. I t i s a b l e to grow i n h i g h c o n c e n t r a t i o n s o f n u t r i e n t s .
5. I t grows i n a v e g e t a t i v e f o r m and d o e s n o t sporulate during
the f e r m e n t a t i o n t i m e .
6. I t s h o u l d be f a s t g r o w i n g t o a v o i d c h a n c e s o f c o n t a m i n a t i o n by
other fast-growing organisms.
7. I t i s a b l e to grow i n low m o i s t u r e content o f the s u b s t r a t e .
8. It i s able t o g r o w i n t h e p r e s e n c e o f p h e n o l i c and t o x i c
compounds p r o d u c e d d u r i n g t h e p r e t r e a t m e n t o f t h e s u b s t r a t e .
Acknowledgement
The a u t h o r i s v e r y g r a t e f u l t o D r . D.J. K u s h n e r , p r o f e s s o r o f
Microbiology, University of O t t a w a , O t t a w a , O n t a r i o , Canada f o r
h i s i n v a l u a b l e s u g g e s t i o n s and f o r e d i t i n g t h e m a n u s c r i p t .
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21
Molecular Size Distribution of Starch During
Enzymatic Hydrolysis
1
J. E. ROLLINGS , M. R. OKOS, and G. T. TSAO
Purdue University, West Lafayette, IN 47907
An appropriate means of determining the mole

c u l a r s i z e and molecula
during h y d r o l y s i
chromatography. This method employs a strong a l k a
l i n e mobile phase and porous s t a t i o n a r y gel supports
which are s t a b l e to the basic s o l u t i o n s . The
separation range of molecular s i z e s i s extended to
7 2
span from 2 x 10 to 5 x 10 hydrodynamic volume
(dl/mole) by coupling s i z e e x c l u s i o n chromatographic
columns i n a manner that will maintain both l i n e a r
i t y i n molecular s i z e separation with e l u t i o n volume
and high separation e f f i c i e n c y .
This method i s used to e s t a b l i s h that molecular
s i z e d i s t r i b u t i o n , r e a c t i o n extent and r e a c t i o n rate
s t r o n g l y depend upon the c r y s t a l l i n e state of the
substrate and the a c t i v i t y of the enzyme. The
s u s c e p t i b i l i t y of starch to h y d r o l y s i s by α-amylase
i s c l o s e l y r e l a t e d to the c r y s t a l l i n e states of the
substrate. Starch c r y s t a l s are present both i n
the n a t u r a l granule s t r u c t u r e and i n retrograded
starch. Due to thermal d e a c t i v a t i o n , the highest
o v e r a l l conversion achieved f o r a given amount of
enzyme i s obtained at temperatures below the upper
l i m i t s of the g e l a t i n i z a t i o n range of s t a r c h . The
initial rate of the r e a c t i o n i s greatest at temper
atures above the g e l a t i n i z a t i o n range.
From these r e s u l t s , a p l a u s i b l e p h y s i c a l model
of starch s t r u c t u r e during h y d r o l y s i s i s presented.
The model states that various c r y s t a l l i n e states of
the constituent starch molecules are present through
out the course of degradation. These c r y s t a l l i n e
states are associated with the n a t u r a l s t a t e of the
1
Current address: Worcester Polytechnic Institute, Worcester, MA 01609
0097-6156/83/0207-0443$06.00/0

starch granule as w e l l as with r e c r y s t a l l i z e d states

caused by r e t r o g r a d a t i o n . In order to properly
describe enzymatic s t a r c h l i q u e f a c t i o n , these
c r y s t a l l i n e states must be accounted f o r .
R e c e n t l y , much a t t e n t i o n has been g i v e n t o t h e

p r o d u c t i o n o f l i q u i d s w e e t e n e r s as an a l t e r n a t i v e t o
cane s u g a r u s i n g i n e x p e n s i v e s t a r c h - c o n t a i n i n g
n a t u r a l m a t e r i a l s as t h e p r i m a r y f e e d s t o c k . This
s i t u a t i o n e x i s t s i n t h e U n i t e d S t a t e s as t h i s
c o u n t r y i s not s e l f s u f f i c i e n t i n the p r o d u c t i o n o f
c a n e , b u t must r e l h e a v i l importatio mainl
from South Americ
source of s t a r c
c o r n ( Z e a mays) and t h e l i q u i d s w e e t e n e r c o m m e r c i
a l l y produced from t h i s m a t e r i a l i s c a l l e d h i g h
f r u c t o s e corn syrup (HFCS). The c u r r e n t method o f
p r o d u c t i o n o f HFCS i s v i a wet m i l l i n g w h i c h e x p l o i t s
the p h y s i c a l p r o p e r t i e s of the whole corn c o n s t i
t u e n t s ( o i l , s t a r c h , g l u t e n , and f i b e r ) f o r t h e i r
s e p a r a t i o n coupled w i t h enzymatic h y d r o l y s i s o f the
starch f r a c t i o n to monosaccharides.
The m a j o r i t y o f r e p o r t e d r e s e a r c h a r t i c l e s
h a v e d e a l t w i t h low m o l e c u l a r w e i g h t s t a r c h c o n v e r
s i o n r e a c t i o n s s u c h as g l u c o s e i s o m e r i z a t i o n and
" l i m i t " dextranization (1-4). O n l y a few r e p o r t s
have d e a l t w i t h e n z y m a t i c h y d r o l y s i s o f h i g h
m o l e c u l a r w e i g h t s t a r c h (5-8) i n s p i t e o f the
o b v i o u s i m p o r t a n c e o f t h i s s t e p o f the p r o c e s s ;
starch liquefaction. A number o f t e c h n i c a l d i f f i
c u l t i e s have prevented q u a n t i t a t i v e m o n i t o r i n g of
changes i n t h e m o l e c u l a r p r o p e r t i e s o f s t a r c h
undergoing h y d r o l y s i s . Bulk chemical or p h y s i c a l
measurements w i l l n o t r e v e a l t h e e s s e n t i a l changes
o c c u r r i n g d u r i n g s t a r c h l i q u e f a c t i o n or provide
s u f f i c i e n t i n f o r m a t i o n f o r m o d e l i n g the o p e r a t i o n .
R e c e n t l y , a s i z e e x c l u s i o n c h r o m a t o g r a p h i c (SEC)
a p p a r a t u s has been d e s c r i b e d w h i c h i s c a p a b l e o f
t
p r o v i d i n g the r e q u i r e d i n f o r m a t i o n (9,10) * *
i s the aim o f t h i s r e p o r t t o demonstrate t h a t
aqueous SEC can be employed t o s t u d y e n z y m a t i c
s t a r c h l i q u e f a c t i o n and p r o v i d e a c l e a r p i c t u r e o f
the p h y s i c a l and c h e m i c a l e v e n t s o c c u r r i n g d u r i n g
t h i s important depolymerization process.

ROLLINGS E T AL. Enzymatic Hydrolysis
Experimental
P u r i f i e d c o r n s t a r c h p r e p a r e d by wet m i l l i n g
was o b t a i n e d f r o m Sigma C h e m i c a l Company, S t .
L o u i s , MO ( l o t no. 68C-0244) and used as s u b s t r a t e .
A c o m m e r c i a l g r a d e , e n d o - a c t i n g α-amylase used t o
h y d r o l y s e the s t a r c h s u b s t r a t e was a g i f t o f Novo
L a b o r a t o r i e s , W i l t o n , CT (Thermamy 1 - h i g h temper
a t u r e s t a b l e enzyme). A s t o c k enzyme s o l u t i o n
was p r e p a r e d by d i l u t i n g th e c o m m e r c i a l g r a d e
l i q u i d enzyme ( 1 : 1 9 ) w i t h 0.01 M a c e t a t e b u f f e r
pH 6.0 and s t o r e d a t 40°C. T h i s enzyme s o l u t i o n
was u s e d f o r t h e h y d r o l y s i s e x p e r i m e n t s as
d e s c r i b e d below. A l l c h e m i c a l s used i n t h i s s t u d y
were r e a g e n t g r a d
The r e a c t o
s t a n d a r d two l i t e r Ace G l a s s w a r e , f o u r - p o r t b a t c h
reactor. The c e n t r a l p o r t was u s e d t o h o u s e t h e
a g i t a t o r s h a f t i n a j a c k e t e d column. The a g i t a t o r
was powered by a v a r i a b l e speed m o t o r o p e r a t i n g a t
140 rpm. The t h r e e s i d e p o r t s w e r e u s e d t o h o u s e
t h e i n t e r n a l t h e r m i s t o r , a NBS t h e r m o m e t e r , and a
t e f l o n p l u g f o r the sampling p o r t . The r e a c t o r
was t h e r m o s t a t e d b o t h i n t e r n a l l y and e x t e r n a l l y i n
a well-mixed o i l bath. T h i s thermostated system
was f o u n d t o a c h i e v e a d e s i r e d t e m p e r a t u r e w i t h i n
t h r e e h o u r s and t o m a i n t a i n t h e t e m p e r a t u r e w i t h i n
± 0.3°C c o n t i n u o u s l y t h e r e a f t e r . A schematic
r e p r e s e n t a t i o n o f t h e a p p a r a t u s i s shown i n F i g u r e 1
S t a r c h h y d r o l y s a t e s a m p l e s were e x t r a c t e d f r o m
t h e b a t c h r e a c t o r a t t i m e d i n t e r v a l s and a s s a y e d by
e i t h e r t r a d i t i o n a l end g r o u p a n a l y s i s as d e s c r i b e d
p r e v i o u s l y (JLl) o r by SEC. The SEC a p p a r a t u s u s e d
i n t h e s e s t u d i e s i s shown i n F i g u r e 2. No s i n g l e
c h r o m a t o g r a p h i c r e s i n column i s c a p a b l e o f r e s o l v
i n g more t h a n two and one h a l f d e c a d e s i n m o l e c u l a r
w e i g h t f o r random c o i l p o l y m e r s (_12). During
l i q u e f a c t i o n , s t a r c h m o l e c u l e s range i n m o l e c u l a r
s i z e f r o m a p p r o x i m a t e l y 10** dJl/g t o a p p r o x i m a t e l y
10^ dA/g (9). I n o r d e r t o extend the m o l e c u l a r s i z e
r a n g e and t h e r e b y o b t a i n a m a x i m a l amount o f
m o l e c u l a r i n f o r m a t i o n f r o m each e x p e r i m e n t , a two-
column SEC s y s t e m was d e v e l o p e d w h i c h i s c a p a b l e o f
s e p a r a t i n g 4.5 - 5.0 d e c a d e s i n m o l e c u l a r s i z e f o r
water s o l u b l e polymers ( 1 0 ) . T h i s system c o n s i s t s
o f a 25.6 cm l o n g S e p h a r o s e CL-6B column c o u p l e d
w i t h a 17.85 cm l o n g S e p h a r o s e CL-2B c o l u m n . Each
column was 8 mm i n i n s i d e d i a m e t e r . The c h r o m a t o
g r a p h i c r e s i n s p a c k e d i n t h e s e columns were f r a c t i o n
a t e d by a wet s i e v i n g t e c h n i q u e t o o b t a i n b e a d s o f

Motor
Oil Bath Batch

Reactor
Figure 1. Schematic diagram of batch reactor.

21. ROLLINGS E T A L . Enzymatic Hydrolysis 447
Positive
Displacement w
Solvent
Filter
Conical Flask
Solvent Reservoir
Rheodyne Loop
Injector
Chromatographic
Column
Chart Recorder Discharge

Differential
Refractometer
Waters 401
Figure 2. Size exclusion chromatographic (SEC) apparatus.
American Chemical
Society Library
1155 16th St., N-W.
Washington,
ACS Symposium Series; U.C. 20036
40-60 μπι i n s i z e ( 9 0 . T h i s was done t o i n c r e a s e t h e

f r a c t i o n a t i o n e f f i c i e n c y t o a p p r o x i m a t e l y 3000
t h e o r e t i c a l p l a t e s p e r m e t e r and s h o r t e n t h e e l u t i o n
t i m e t o l e s s t h a n two h o u r s . The e l u e n t and s t a r c h
s o l v e n t was 0.5 Ν NaOH. A constant eluent flow
r a t e o f 6.5 ml p e r h o u r was m a i n t a i n e d u s i n g a s l o -
f l o w m i n i pump ( M i l t o n Roy: L a b o r a t o r y D a t a C o n t r o l ,
R i v i e r a Beach, F L ) .
The SEC columns were c a l i b r a t e d u s i n g n a r r o w
m o l e c u l a r weight d i s t r i b u t i o n water s o l u b l e polymer
s t a n d a r d s ; s o d i u m p o l y s t y r e n e s u l f o n a t e , NaPSS
( P r e s s u r e C h e m i c a l Company, P i t t s b u r g h , PA) and
dextrans (Pharmacia Fine Chemicals, Piscataway, NJ).
The u n i v e r s a l c a l i b r a t i o n c u r v e p r o p o s e d by G r u b i s i c
e t a l . (1J3) was f o u n d t t b applicabl fo thes
polymer standards
procedure of C o l
p o r a t e s e x c l u d e d volume e f f e c t s i n t h e n u m e r i c a l
v a l u e o f t h e F l o r y p a r a m e t e r was f o u n d t o be a p p l i
c a b l e f o r t h i s s y s t e m ( 1 5 ) . The c a l i b r a t i o n c u r v e
f o r t h e two-column n e t w o r k s y s t e m d e s c r i b e d above
i s shown i n F i g u r e 3. The K^y p a r a m e t e r u s e d on
the a b s c i s s a o f F i g u r e 3 i s a n o r m a l i z e d e l u t i o n
volume p a r a m e t e r d e f i n e d by E q u a t i o n 1,
V - V
K U
AV V - V
t ο
where V e i s t h e peak r e t e n t i o n volume o f t h e s a m p l e ,

VQ i s t h e v o i d volume o f t h e c o l u m n , and V i s the
t
t o t a l i n t e r s t i t i a l volume o f t h e c o l u m n .
In o r d e r t o d e s c r i b e the k i n e t i c s o f enzymatic
s t a r c h d e p o l y m e r i z a t i o n , i n f o r m a t i o n on r e a c t i o n
r a t e , r e a c t i o n e x t e n t , and p r o d u c t d i s t r i b u t i o n
p r o f i l e s are r e q u i r e d . T r a d i t i o n a l end-group ana
l y s i s can be u s e d t o a l i m i t e d e x t e n t i n t h e f i r s t
two a r e a s , b u t w i l l n o t p r o v i d e i n f o r m a t i o n a b o u t
the l a s t important s u b j e c t . H e n c e , SEC p r o f i l e s
can p r o v i d e s u f f i c i e n t i n s i g h t i n t o t h e mechanism
of starch degradation.
E f f e c t o f T e m p e r a t u r e on R e a c t i o n R a t e . The
i n i t i a l r a t e o f s t a r c h h y d r o l y s i s was i n v e s t i g a t e d
a t f i v e t e m p e r a t u r e s (40°C, 57.5°C, 67.5°C, 80°C
and 95°C) u s i n g t h r e e d i f f e r e n t s u b s t r a t e c o n c e n
t r a t i o n s ( 0 . 4 4 % , 0.88%, and 1.76% w t / v o l ) by

21. ROLLINGS E T A L . Enzymatic Hydrolysis 449
τ 1
r—— 1
r
Figure 3. C-P calibration curve of SEC column network.

m o n i t o r i n g the p r o d u c t i o n of r e d u c i n g sugars
( d e x t r o s e e q u i v a l e n c e , D.E.). At each i s o t h e r m ,
the i n i t i a l r a t e d a t a were shown t o f o l l o w
M i c h a e l i s - M e n t e n k i n e t i c s (11) c o n s i s t e n t w i t h t h e
o b s e r v a t i o n s o f p r e v i o u s r e p o r t s (.16,12)· Hence,
L i n e w e a v e r - B u r k p l o t s c o u l d be g e n e r a t e d f r o m the
d a t a y i e l d i n g v a l u e s o f a p p a r e n t maximum i n i t i a l
v e l o c i t i e s a t e a c h o f the t e m p e r a t u r e s . This
i n f o r m a t i o n i s p l o t t e d i n F i g u r e 4 w i t h an assumed
A r r h e n i u s dependency, which f o r t h i s system c l e a r l y
does n o t e x i s t . The s a l i e n t f e a t u r e s o f t h i s
study are that there e x i s t s n e a r l y a three f o l d
o r d e r o f m a g n i t u d e i n c r e a s e i n t h e a p p a r e n t maximum
v e l o c i t y as t h e s t a r c h s u b s t r a t e i s m o d i f i e d due t o
the g e l a t i n i z a t i o phenomena A t t h h i g h e s t tem
perature tested,
T h i s i s due t o t h e r m a enzyme
T h i s l a r g e i n c r e a s e i n the r e a c t i o n r a t e i n d i c a t e s
t h a t t h e o v e r r i d i n g e f f e c t i n v o l v e s the p h y s i c a l
s t a t e o f the s t a r c h g r a n u l e . Non-gelatinized
s t a r c h i s h i g h l y c r y s t a l l i n e (18) and t h u s t h e
s u s c e p t i b i l i t y o f s t a r c h t o be e n z y m a t i c a l l y
h y d r o l y z e d u n d e r t h e s e c o n d i t i o n s i s much l o w e r
t h a n f o r s t a r c h g r a n u l e s t h a t have been d i s r u p t e d
by h e a t i n g i n aqueous s u s p e n s i o n . Through t h e
p r o c e s s o f g e l a t i n i z a t i o n , the o r i g i n a l i n t e r n a l
o r d e r i n g of the s t a r c h m o l e c u l e s w i t h i n the g r a n u l e s
i s d i s r u p t e d , the g r a n u l e i m b i b e s l a r g e amounts o f
w a t e r and s w e l l s . E v e n t u a l l y due t o combined
a c t i o n o f s h e a r i n g w i t h i n t h e r e a c t o r and o s m o t i c
s w e l l i n g , granule i n t e g r i t y i s completely lost.
L a r g e s t a r c h m o l e c u l e s may t h e n r e a s s o c i a t e ( r e t r o
grade) producing a g e l m a t r i x . I t c a n n o t be
assumed t h a t t h e p r o g e s s o f t h e r e a c t i o n w i l l f o l l o w
the same s e q u e n c e i f t h e s u b s t r a t e i s i n v a r i o u s
physical states. I t i s therefore of i n t e r e s t to
f o l l o w the m o l e c u l a r w e i g h t d i s t r i b u t i o n p r o f i l e s
d u r i n g the c o u r s e of the r e a c t i o n at v a r i o u s s t a t e s
of macromolecular aggregation. The aqueous SEC
a p p a r a t u s d e s c r i b e d above was s p e c i f i c a l l y d e v e l o p e d
for t h i s purpose. T h i s t e c h n i q u e was shown t o be
a p p r o x i m a t e l y 10^ t i m e s more a c c u r a t e t h a n r e d u c i n g
s u g a r measurements f o r low d e g r e e o f c o n v e r s i o n
starch h y d r o l y s i s reactions (10).
E f f e c t o f T e m p e r a t u r e on H y d r o l y s i s E x t e n t and
P r o d u c t D i s t r i b u t i o n . I t was shown above t h a t s l o w
i n i t i a l r a t e of r e a c t i o n i s c h a r a c t e r i s t i c of opera
t i n g a t low t e m p e r a t u r e s ( b e l o w t h e g e l a t i n i z a t i o n
range of s t a r c h ) whereas r a p i d i n i t i a l r a t e s are

ROLLINGS E T A L . Enzymatic Hydrolysis
TEMPERATURE ( Ο β
95° 80 e
72 e
62° 50° 40°
1 1 1 J
20 i 1
10
! ι
• η
ABOVE Ι \ 1 BELOW
GELATINIZATION \, GELATINIZATION
1.0 _RANGE 1 y RANGE
.1
.03
1 1 II 1
2.7 2.8 2.9 3.0 3.1 3.2
l/T K"'xlO s
Figure 4. A rrhenius plot of • max app.

o b s e r v e d a t h i g h t e m p e r a t u r e s ( a b o v e the g e l a t i n i z a
t i o n of starch). However, t h e d i s t r i b u t i o n s o f
m a c r o m o l e c u l a r components d u r i n g h y d r o l y s i s a t
v a r i o u s t e m p e r a t u r e s have n o t b e e n r e p o r t e d and a r e
thus o f i n t e r e s t .
SEC p r o f i l e s o f h y d r o l y s i s p r o d u c t s o f enzyme-
t r e a t e d s t a r c h as a f u n c t i o n o f r e a c t i o n t i m e a t
60°C and 80°C a r e shown i n F i g u r e s 5 and 6 r e s p e c
tively. I n b o t h c a s e s , a common enzyme dose o f
one ml o f t h e 1:19 s t o c k Novo Thermamyl s o l u t i o n was
m i x e d w i t h a s l u r r y o f 15 gms s t a r c h d r y b a s i s i n
75 ml w a t e r and added t o t h e r e a c t o r . The f i n a l
volume was 1500 m l .
A t 60°C, b e l o w t h e g e l a t i n i z a t i o n ( m e l t i n g )
range o f the g r a n u l e s distributio i
e x t r e m e l y non-rando
the i n i t i a l stage y significan
amount o f i n t e r m e d i a t e m o l e c u l a r s i z e p r o d u c t s
( i . e . , b e t w e e n h y d r o d y n a m i c volume 10^ and 10^
dl/mole) detected. Throughout the time course of
l i q u e f a c t i o n , the m a j o r i t y o f t h e m o l e c u l a r s p e c i e s
present are e i t h e r very h i g h i n molecular weight
( e x c l u d e d from t h e SEC column) o r low m o l e c u l a r
w e i g h t end p r o d u c t s . Low m o l e c u l a r w e i g h t p r o d u c t s
c o n t i n u o u s l y accumulate w h i l e h i g h m o l e c u l a r weight
m a t e r i a l s d i s a p p e a r w i t h o u t d e t e c t i o n o f any s i g n i
f i c a n t amount o f i n t e r m e d i a t e m o l e c u l a r w e i g h t
material. T h i s s i t u a t i o n c o u l d r e s u l t i f t h e enzyme
i s a l l o w e d t o a c t o n l y on r e l a t i v e l y s h o r t , e x p o s e d
c h a i n s of s t a r c h present i n the c r y s t a l l i n e m a t r i x
o f the g r a n u l e , o r i f l o n g e r c h a i n m o l e c u l e s f r e e d
f r o m t h e g r a n u l e a r e more s u s c e p t i b l e t o enzyme
h y d r o l y s i s than the molecules a s s o c i a t e d w i t h the
g r a n u l e and a r e t h e r e f o r e p r e f e r e n t i a l l y d e g r a d e d .
A l t e r n a t i v e l y t h i s s i t u a t i o n c o u l d a l s o a r i s e i f the
a v a i l a b l e s t a r c h i s s i m p l y o v e r l o a d e d w i t h enzyme.
Other authors suggest that p r e f e r e n t i a l " p i t t i n g "
may o c c u r due t o d i f f e r i n g d e g r e e s i n s u b s t r a t e
susceptibility. The r e a c t i o n u n d e r t h e s e c o n d i t i o n s
does procède t o c o m p l e t i o n a l t h o u g h r e q u i r i n g a p p r o x
i m a t e l y ten hours f o r the c o n v e r s i o n .
Above t h e g e l a t i n i z a t i o n r a n g e (80°C, see
F i g u r e 6) t h e p r o d u c t d i s t r i b u t i o n d u r i n g r e a c t i o n
i s c o n s i d e r a b l y d i f f e r e n t t h a n 60°C. A g a i n , the
d i s t r i b u t i o n o f m o l e c u l a r s i z e s i s n o t random.
D u r i n g t h e e a r l y s t a g e s o f the r e a c t i o n , i n a d d i t i o n
t o t h e v e r y h i g h m o l e c u l a r w e i g h t and low m o l e c u l a r
weight products, a large f r a c t i o n of intermediate
m o l e c u l a r weight d e x t r i n s (between hydrodynamic
v a l u e 10^ and 10·* d l / m o l e , see F i g u r e 5) a r e p r e s e n t .

Figure 5. Chromatograms of starch hydrolyzed at 60°C at indicated times during
reaction. Top curve, 17 min: 26 s.

1 I I I I I
0.0 02 0.4 0.6 0.8 1.0

K
AV
Figure 6. Chromatograms of starch hydrolyzed at 80°C at indicated times during
reaction. Times indicated as min: s.

ROLLINGS E T A L . Enzymatic Hydrolysis
A t one and o n e - h a l f m i n u t e s i n t o t h e r e a c t i o n , two

d i s c r e t e i n t e r m e d i a t e m o l e c u l a r weight peaks a r e
detected at K A V v a l u e s o f 0.25 and 0.60. These
values correspond t o approximate m o l e c u l a r weights
o f 5 χ 10^ and 10^ r e s p e c t i v e l y (9). Throughout
the r e m a i n i n g time course o f t h e r e a c t i o n these
peaks p e r s i s t , t h o u g h t h e i r K A V values increase
s l i g h t l y ( t o 0.275 and 0.65 r e s p e c t i v e l y ) w i t h
i n c r e a s i n g time c h a r a c t e r i s t i c o f a r e d u c t i o n i n
t h e i r molecular weight v a l u e s . This i s apparently
due t o g r a d u a l e n d - w i s e d e p o l y m e r i z a t i o n o f t h e s e
intermediates. T h i s i n d i c a t e s t h a t under these
r e a c t i o n c o n d i t i o n s , c e r t a i n intermediate products
a r e more r e s i s t a n t t o e n z y m a t i c a t t a c k , and t h e r e
f o r e a p p e a r as d i s c r e t e c h r o m a t o g r a p h i c p e a k s
D i s c r e t e peaks c o u l
s i z e starch molecule
produced (as b y - p r o d u c t s ) o r a r e r e s i s t a n t t o
e n z y m a t i c a t t a c k , and t h e r e f o r e a p p e a r as d i s c r e t e
chromatographic peaks. P r e v i o u s r e p o r t s o f such
i n t e r m e d i a t e s d u r i n g s t a r c h l i q u e f a c t i o n have n o t
been f o u n d .
No f u r t h e r changes i n t h e c h r o m a t o g r a p h i c
t r a c i n g s a r e seen beyond e l e v e n and o n e - h a l f m i n u t e s
a t 80°C, w h i c h shows t h a t t h e r m a l d e a c t i v a t i o n o f
t h e enzyme h a s s t o p p e d t h e p r o g r e s s o f t h e r e a c t i o n .
The r e a c t i o n does n o t p r o c e e d t o c o m p l e t i o n u n d e r
these c o n d i t i o n s .
The f i n a l p r o d u c t d i s t r i b u t i o n o f a s t a r c h
s o l u t i o n h y d r o l y z e d a t 95°C u n d e r t h e same c o n d i
t i o n s as t h e o t h e r two i s o t h e r m s was d e t e r m i n e d and
i s shown t o g e t h e r w i t h t h e s i m i l a r d a t a a t 80°C,
65°C, and 60°C i n F i g u r e 7. F o u r d i s t i n c t peaks
a r e o b s e r v e d : one a t t h e low m o l e c u l a r w e i g h t l i m i t ,
and two i n t e r m e d i a t e m o l e c u l a r w e i g h t p e a k s a t K^y
v a l u e s o f 0.2 and 0.6 r e s p e c t i v e l y . This i s the
same t y p e o f c h r o m a t o g r a p h i c b e h a v i o r o b s e r v e d
d u r i n g t h e i n i t i a l s t a g e s o f r e a c t i o n a t 80°C.
Under a l l t h e r m a l s t a t e s t e s t e d , a non-random
distribution exists.
E f f e c t o f S t a r c h R e c r y s t a l l i z a t i o n on H y d r o
l y s i s E x t e n t , P r o d u c t D i s t r i b u t i o n , and R e a c t i o n
Rates. Since retrograded ( r e c r y s t a l l i z e d ) starch
i s t h o u g h t t o be more r e s i s t a n t t o e n z y m a t i c d e p o l y
m e r i z a t i o n t h a n u n r e t r o g r a d e d s t a r c h (.22,22), i t i s
o f i n t e r e s t t o compare t h e h y d r o l y s i s o f r e t r o g r a d e d
starch to non-retrograded starch. Extremely r e t r o
g r a d e d s t a r c h was p r e p a r e d by f r e e z e - d r y i n g p r e g e l a -
t i n i z e d s t a r c h , and b a l l - m i l l i n g t h i s m a t e r i a l t o a

Figure 7. Comparison of final product distribution of starch hydrolyzed at 95°C,

80°C, 65°C, and60°C.

ROLLINGS E T AL. Enzymatic Hydrolysis
f i n e powder. T h i s m a t e r i a l was e n z y m a t i c a l l y
h y d r o l y z e d a t 80°C, and t h e m o l e c u l a r s i z e d i s t r i
b u t i o n was d e t e r m i n e d by s i z e e x c l u s i o n c h r o m a t o
graphy. The c h r o m a t o g r a p h i c p r o f i l e s a r e shown
i n F i g u r e 8. The c h r o m a t o g r a p h i c p r o f i l e o f
unreacted, p r e g e l a t i n i z e d , f r e e z e - d r i e d , b a l l -
m i l l e d s t a r c h ("0" t i m e c h r o m a t o g r a m ) shows t h a t
s i g n i f i c a n t d e p o l y m e r i z a t i o n o c c u r r e d d u r i n g the
b a l l - m i l l i n g operation. D u r i n g the course o f
enzymatic d e p o l y m e r i z a t i o n of t h i s r e c r y s t a l l i z e d
s t a r c h , f o u r d i s t i n c t p e a k s a r e p r e s e n t : one n e a r
t h e e x c l u s i o n l i m i t o f t h e column s e t , one a t the
end p r o d u c t s , and two i n t e r m e d i a t e p e a k s . These
i n t e r m e d i a t e p e a k s a r e d e t e c t a b l e as e a r l y as two
and o n e - h a l f m i n u t e s i n t o t h e r e a c t i o n The p e a k s
appeared at K y v a l u e
A
0.6, t h e same v a l u e
s t a r c h was r e a c t e d a t 80°C. These values for
t h e two i n t e r m e d i a t e p e a k s i n c r e a s e d s l i g h t l y w i t h
i n c r e a s i n g r e a c t i o n time, a g a i n c o n s i s t e n t w i t h the
r e s u l t s shown a t 80°C f o r n o n - p r e g e l a t i n i z e d s t a r c h .
Thus t h e r e s i s t a n t , i n t e r m e d i a t e m o l e c u l a r s i z e
s t a r c h p r o d u c t s formed a f t e r s e v e r e rétrogradation
d u r i n g f r e e z i n g are q u i t e s i m i l a r t o the groups
formed when a n o n - p r e g e l a t i n i z e d s t a r c h i s enzyma
t i c a l l y d e p o l y m e r i z e d u n d e r t h e same c o n d i t i o n s .
No change i n t h e c h r o m a t o g r a p h i c p r o f i l e s i s
d e t e c t e d a f t e r e l e v e n m i n u t e s and the r e a c t i o n does
not proceed to completion.
Conclusion
The p r e s e n t i n v e s t i g a t i o n has p r o v i d e d new

i n s i g h t i n t o the p h y s i c a l e v e n t s o c c u r r i n g d u r i n g
enzymatic s t a r c h d e p o l y m e r i z a t i o n . Degradation of
s t a r c h u n d e r t h e c o n d i t i o n s t e s t e d does n o t p r o c e e d
by a random p r o c e s s . Granule s t r u c t u r e a f f e c t s the
m o l e c u l a r weight d i s t r i b u t i o n p r o f i l e s of the s t a r c h
hydrolysates. S t a r c h h y d r o l y s a t e s produced at
temperatures below the g e l a t i n i z a t i o n range o f the
s u b s t r a t e a r e composed m a i n l y o f h i g h m o l e c u l a r
w e i g h t m a t e r i a l o r low m o l e c u l a r w e i g h t end p r o d u c t s .
A t 80°C, above t h e m e l t i n g r a n g e o f t h e g r a n u l e s , a
l a r g e amount o f i n t e r m e d i a t e m o l e c u l a r s i z e compon
e n t s e x i s t i n a d d i t i o n t o t h e h i g h and low m o l e c u l a r
weight groups. These i n t e r m e d i a t e p r o d u c t s a r e
a p p r o x i m a t e l y 5 χ 10^ and 10^ i n m o l e c u l a r w e i g h t
and a r e t h e same s i z e components s e e n i n t h e h y d r o
l y s i s o f h i g h l y r e c r y s t a l l i z e d s t a r c h r e a c t e d at the
same i s o t h e r m . T h i s suggests t h a t the r e c r y s t a l l i -

Figure 8. Chromatograms of pregelatinized starch hydrolyzed at 80°C at indicated
times (min: s) during reaction.

21. ROLLINGS ET AL. Enzymatic Hydrolysis 459
z a t i o n p r o c e s s w i l l o c c u r even a t h i g h t e m p e r a t u r e
operation.
Although the i n i t i a l r a t e o f the r e a c t i o n i s
g r e a t e s t a t t e m p e r a t u r e s above t h e g e l a t i n i z a t i o n
range o f the s u b s t r a t e , the f i n a l e x t e n t o f con
v e r s i o n i s g r e a t e s t a t low t e m p e r a t u r e o p e r a t i o n
f o r a common amount o f enzyme l o a d i n g . This
s i t u a t i o n e x i s t s due t o t h e r m a l d e a c t i v a t i o n o f t h e
catalyst. Optimal operation of a starch l i q u e
f a c t i o n o p e r a t i o n must a c c o u n t f o r b o t h t h e r a t e o f
i n c r e a s e d s u b s t r a t e s u s c e p t i b i l i t y c a u s e d by t h e
d i s r u p t i o n o f the i n t e r n a l macromolecular o r d e r i n g
w i t h i n t h e g r a n u l e and t h e l o s s o f e n z y m a t i c
a c t i v i t y due t o t h e r m a l d e a c t i v a t i o n .
A p h y s i c a l model f starch gelatinization
rétrogradation, an
from these r e s u l t s
r e c r y s t a l l i z a t i o n v i a rétrogradation p r o c e s s w i l l
occur quite r a p i d l y f o r p r e g e l a t i n i z e d starch. I t
i s t h e r e f o r e not p o s s i b l e to study the enzymatic
hydrolysis of starch without considering r e t r o -
gradation e f f e c t s . The p h y s i c a l p i c t u r e f o r t h e
o v e r a l l p r o c e s s i s summarized i n F i g u r e 9. The
i n i t i a l p o p u l a t i o n o f s t a r c h g r a n u l e s i s suspended
i n aqueous s o l u t i o n . When s u b j e c t e d f o r a p e r i o d
o f t i m e a t o r above t h e g e l a t i n i z a t i o n t e m p e r a t u r e
r a n g e o f t h e m a t e r i a l , some o f t h e s t a r c h g r a n u l e s
melt. The m o l e c u l a r components o f t h e g r a n u l e
w i l l then r e a s s o c i a t e t o form c r y s t a l l i n e b o d i e s ,
some o f w h i c h may be more o r l e s s r e s i s t a n t t o
enzymatic h y d r o l y s i s than other c r y s t a l types. The
enzyme w i l l h y d r o l y z e s u s c e p t i b l e bond l i n k a g e s t o
low m o l e c u l a r w e i g h t o l i g o s a c c h a r i d e s as a f u n c t i o n
o f t i m e and c o n c e n t r a t i o n .

Population of Granules Mixture of ungelatin- Mixture of ungelatin- Mixture of gelatinized
izedjgelatinized gran- ized, gelatinized gran- granules, retrograded
ules + soluble ules, retrograded mat- materials + soluble
components erial + solublecompon- components
ents
····· „*oo*~ $L%> ,o~e

T , C
• · · · Tt · ° · ° ' ' s~°S£~f' T.t.Cs
,E(t),t
+ hydrolyis products + hydrolysis products

Figure 9. Physical model of gelatinization, rétrogradation, and liquefaction.

ROLLINGS E TA L . Enzymatic Hydrolysis
Literature Cited
1. Marsh, D.R.; Lee, Y.Y.; Tsao, G.T. B i o t e c h n o l .

Bioeng. 1973, 15, 483.
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E.V.; Tsao, G.T. B i o t e c h n o l . Bioeng. 1976,
18, 253.
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1973, 2, 231.
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P r i n c e t o n , NJ, 1976.
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Bioeng. 1978
8. B o n d e t s k i i , K.M. and Yarovenko, V.L. Bioorgan.
Khimiia 1975,1.,614.
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L a f a y e t t e , IN, 1978.
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Tsao, G.T. J. Appl. Polym. S c i . (accepted).
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L a f a y e t t e , IN, 1978.
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"Modern Size E x l u s i o n Chromatography" ; John
Wiley and Sons, NY, 1979, 97.
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S c i . Part Β 1967, 5, 753.
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Okos, M.R.; and Tsao, G.T. J. Applied Polym.
S c i . (accepted).
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Die Starke 1979, 31, 368.
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1979, 31, 338.
18. S t e r l i n g , C. J. Texture Studies 1978, 9, 225.
19. Chabat, J.F.; A l l e n , A.E.; and Hood, L.F.
J . Food S c i . 1978, 43, 727.
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Biopolymers 1975, 14, 265.
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1968, 45, 162.
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Chemistry, Vo. IV" R.L. W h i s t l e r , ed.;Academic
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1979, 31, 149.

22
Thermochemical Optimization of Microbial
Biomass-Production and Metabolite-Excretion
Rates
ALICIAN V. QUINLAN
Massachusetts Institute of Technology, Department of Mechanical Engineering,
Cambridge, MA 02139
The temperatur
many m i c r o b i a l processe
composition. As a r e s u l t , the o p e r a t i n g temperature
of b i o l o g i c a l r e a c t o r s should be v a r i e d to keep such
process r a t e s maximal as t h e i r chemical m i l i e u x vary.
The laws r e l a t i n g optimum operating temperature to
medium composition are not w e l l known. T h i s r e l a t i o n
was t h e o r e t i c a l l y i n v e s t i g a t e d f o r processes whose
r a t e s s a t u r a t e in s u b s t r a t e c o n c e n t r a t i o n . The
Michaelis-Menten r e a c t i o n mechanism was modified to
describe m i c r o b i a l biomass production and metabolite
e x c r e t i o n i n both batch and continuous r e a c t o r s .
Monod r a t e laws whose m a c r o - c o e f f i c i e n t s depend on
d i l u t i o n r a t e as w e l l as temperature were d e r i v e d .
Conditions f o r which these c o e f f i c i e n t s may fall,
fall, or show a minimum with rising temperature and
d i l u t i o n r a t e were enumerated. The optimum tempera-
ture was shown to vary with both s u b s t r a t e concentra-
t i o n and d i l u t i o n r a t e . The p a t t e r n of v a r i a t i o n was
explained in terms of the thermal sensitivities of
the c o e f f i c i e n t s . The validity of the t h e o r e t i c a l
r e s u l t s was discussed r e l a t i v e to published data and
found promising but i n need o f experimental t e s t i n g .
The r a t e s o f m i c r o b i a l l y m e d i a t e d p r o c e s s e s t y p i c a l l y show
a s i n g l e r a t e maximum a s t e m p e r a t u r e r i s e s ( 1 - 7 ) . Changes i n a
microorganism's chemical environment can s h i f t the temperature
a
a t w h i c h t h e t h e r m a l r a t e maximum o c c u r s (3-7)· As c o n s e q u e n c e ,
given the chemically changing m i l i e u x o f b i o l o g i c a l r e a c t o r s , t h e i r
o p e r a t i n g t e m p e r a t u r e s w o u l d h a v e t o be v a r i e d t o o p t i m i z e p r o c e s s
rates (8,9). In b i o l o g i c a l reactors, substrate concentration i s
t h e most i m p o r t a n t c h e m i c a l v a r i a b l e . So, i t ' s most d e s i r a b l e
t o know how t h e optimum o p e r a t i n g t e m p e r a t u r e depends on s u b s t r a t e
concentration.
0097-6156/83/0207-0463$07.25/0

Q u i n l a n (8-10) has a n a l y z e d how c h a n g e s i n s u b s t r a t e c o n

c e n t r a t i o n a f f e c t t h e optimum t e m p e r a t u r e o f p r o c e s s e s t h a t s a t u
rate i n substrate concentration according to a generic M i c h a e l i s -
M e n t e n r e a c t i o n mechanism. F o r s u c h p r o c e s s e s , t h e optimum tem
p e r a t u r e was shown t o v a r y l i n e a r l y w i t h t h e l o g a r i t h m o f s u b
strate concentration. T h i s l o g a r i t h m i c r e l a t i o n r e s u l t s when
b o t h t h e m a x i m u m - v e l o c i t y and h a l f - s a t u r a t i o n c o e f f i c i e n t s show
Arrhenius temperature dependencies, w i t h the l a t t e r ' s f a l l i n g
faster. Such d e p e n d e n c i e s a r e commonly s e e n i n b a t c h r e a c t o r s
(11-14), but not always (13,14).
A l t h o u g h a p p l i c a b l e t o many p r o c e s s e s , t h i s a n a l y s i s (8-10)
i s not g e n e r a l l y a p p l i c a b l e t o m i c r o b i a l l y mediated p r o c e s s e s .
I t ' s r e s t r i c t e d t o b a t c h r e a c t o r s and c a n ' t a c c o u n t f o r t h e i n
f l u e n c e o f mass i n f l o w and o u t f l o w i n c o n t i n u o u s r e a c t o r s . I t
a l s o c a n ' t e x p l a i n why som
c o e f f i c i e n t s determine
A r r h e n i u s t e m p e r a t u r e d e p e n d e n c i e s (15-19) and why o t h e r s seem
to v a r y w i t h d i l u t i o n r a t e (20,21).
I n t h i s p a p e r , t h e M i c h a e l i s - M e n t e n r e a c t i o n mechanism w i l l
be m o d i f i e d t o e l i m i n a t e t h e s e and o t h e r s h o r t f a l l s . The m o d i f i e d
r e a c t i o n mechanism w i l l t h e n be u s e d t o e x p l o r e t h e i n f l u e n c e o f
s u b s t r a t e c o n c e n t r a t i o n on t h e optimum t e m p e r a t u r e o f b i o m a s s -
p r o d u c t i o n and m e t a b o l i t e - e x c r e t i o n r a t e s . The i n f l u e n c e o f
d i l u t i o n r a t e w i l l a l s o be e x a m i n e d . The s c o p e o f a n a l y s i s w i l l
s t i l l be c o n f i n e d t o p r o c e s s e s whose r a t e s s a t u r a t e i n s u b s t r a t e
concentration.
Reaction Mechanism
The M i c h a e l i s - M e n t e n r e a c t i o n mechanism d e s c r i b e s t h e enzyme-

c a t a l y z e d c o n v e r s i o n o f s u b s t r a t e i n t o p r o d u c t a s f o l l o w s ( 2 , 22-
24):
kj k3
Ε + S ^ ES • Ε + Ρ
T h i s r e a c t i o n mechanism assumes one m o l e c u l e e a c h o f s u b s t r a t e

(S) and f r e e enzyme (E) r e v e r s i b l y c o m b i n e 0 ^ = k / k j ) to form
2
( k ) p r o d u c t (P) w i t h a c o n c o m i t a n t r e c y c l i n g o f f r e e enzyme.
3
I n b i o l o g i c a l r e a c t o r s , m i c r o b i a l biomass p l a y s a r o l e a n a l o
gous t o t h e enzyme's r o l e . L e t Y be t h e a v e r a g e number o f s u b
s t r a t e consumption s i t e s per microorganism. Then,
Ύ Ξ C/N (1)
where C i s t h e c o n c e n t r a t i o n o f c o n s u m p t i o n s i t e s i n t h e m i c r o b i a l
p o p u l a t i o n and Ν i s t h e c o n c e n t r a t i o n o f m i c r o o r g a n i s m s i n t h e
population. S i m i l a r l y , l e t β be t h e a v e r a g e b i o m a s s p e r m i c r o
organism, w i t h
β Ξ Β/Ν (2)

22. QUINLAN Thermochemical Optimization 465
where Β i s t h e biomass c o n c e n t r a t i o n o f t h e p o p u l a t i o n . Solving

e q u a t i o n s (1) and (2) f o r Ν and e q u a t i n g t h e r e s u l t i n g e x p r e s s i o n s
yields
Β = (0/Y)C (3)
E q u a t i o n (3) s t a t e s t h a t t h e b i o m a s s c o n c e n t r a t i o n o f a m i c r o b i a l
p o p u l a t i o n i s d i r e c t l y p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f sub
s t r a t e consumption s i t e s i n the p o p u l a t i o n .
As f o r t h e enzyme, t h e s u b s t r a t e c o n s u m p t i o n s i t e s may be
p a r t i t i o n e d i n t o two p o r t i o n s : t h o s e c a p a b l e o f c o n s u m i n g s u b
s t r a t e ( C ) a n d t h o s e n o t c a p a b l e ( C ) . An e q u i v a l e n t b i o m a s s
c n
may b e a s s i g n e d t o e a c h p o r t i o n t h r o u g h e q u a t i o n ( 3 ) :
B = (6/Y)C (4a)
B = (B/Y)C (4b)
The t o t a l b i o m a s s o f a m i c r o b i a l p o p u l a t i o n c a n t h e n a l s o be
p a r t i t i o n e d i n t o two p o r t i o n s s o t h a t
Β = B c + B n (5)
I n t h i s c o n t e x t , B p l a y s a r o l e a n a l o g o u s t o f r e e enzyme, a n d
c
B , t o enzyme-substrate complex.
n
I n conformance w i t h t h i s i n t e r p r e t a t i o n o f B , s u b s t r a t e i s
c
assumed t o be consumed a t a r a t e j o i n t l y p r o p o r t i o n a l t o i t s c o n
c e n t r a t i o n S a n d B , i . e . , a t a mass f l o w r a t e c S B .
c c Similarly,
t h e m e t a b o l i t e o f i n t e r e s t i s assumed t o be e x c r e t e d a t a r a t e
p r o p o r t i o n a l o n l y t o B , i . e . , a t a mass f l o w r a t e e B . I n t h i s
n n
case, t h e m i c r o s c o p i c r a t e c o e f f i c i e n t s c and e p l a y r o l e s a n a l o
gous t o t h o s e o f k a n d k . r e s p e c t i v e l y .
x 2 >
F u r t h e r m o r e , s a t u r a t e d and u n s a t u r a t e d consumption s i t e s a r e
assumed t o c y c l e a s c o m p l e x e d a n d f r e e enzyme d o , b u t w i t h no
c o n c o m i t a n t r e l e a s e o f s u b s t r a t e o r m e t a b o l i t e i n t o t h e medium.
S a t u r a t e d s i t e s a r e assumed t o u n s a t u r a t e a t a r a t e p r o p o r t i o n a l
o n l y t o t h e i r own c o n c e n t r a t i o n , i . e . , a t a mass f l o w r a t e u B . n
On t h e o t h e r h a n d , u n s a t u r a t e d s i t e s a r e assumed t o s a t u r a t e a t
a r a t e j o i n t l y p r o p o r t i o n a l t o s u b s t r a t e c o n c e n t r a t i o n and t h e i r
own, i . e . , a t a mass f l o w r a t e s S B . c I f each consumption s i t e
becomes s a t u r a t e d when o n e s u b s t r a t e m o l e c u l e o c c u p i e s i t , t h e n
the m i c r o s c o p i c s a t u r a t i o n and consumption c o e f f i c i e n t s a r e e q u a l .
T h i s i s t h e c a s e assumed by t h e M i c h a e l i s - M e n t e n r e a c t i o n mech
a n i s m . However, i f η m o l e c u l e s a r e r e q u i r e d f o r s a t u r a t i o n o f
one s i t e , t h e n s = ( l / n ) c .
The m o d i f i e d r e a c t i o n mechanism must a l s o a c c o u n t f o r b i o m a s s
w a s t a g e (and t h e c o n c o m i t a n t l o s s o f c o n s u m p t i o n s i t e s ) t h r o u g h
r e s p i r a t i o n , e x c r e t i o n , d e a t h , e t c . B i o m a s s w a s t a g e i s assumed
to take p l a c e a t a r a t e p r o p o r t i o n a l t o t h e biomass c o n c e n t r a t i o n
o f t h e p o p u l a t i o n , i . e . , a t a mass f l o w r a t e wB = w B + wB . c n

F i n a l l y , s u b s t r a t e i s added t o a c o n t i n u o u s r e a c t o r a t a
mass f l o w r a t e d S , w h e r e S i s t h e c o n c e n t r a t i o n o f i n f l o w i n g
Q 0
s u b s t r a t e and d i s a d i l u t i o n r a t e e q u a l t o t h e r a t i o o f t h e
v o l u m e t r i c i n f l o w r a t e t o t h e volume o f f l u i d i n t h e r e a c t o r .
For a constant f l u i d volume, i n f l o w i n g s u b s t r a t e a l s o d i s p l a c e s
s u b s t r a t e , b i o m a s s , a n d m e t a b o l i t e a t mass f l o w r a t e s e q u a l ,
r e s p e c t i v e l y , t o d S , dB - d B + d B , a n d dM.
c n
A t t h i s p o i n t , a l l t h e n e c e s s a r y mass c o n c e n t r a t i o n s a n d
f l o w s h a v e b e e n d e f i n e d . The r e s u l t i n g r e a c t i o n mechanism i s
s c h e m a t i z e d i n F i g u r e 1. The s o l i d l i n e s r e p r e s e n t t h e mass
f l o w s , a n d t h e d a s h e d l i n e s show w h i c h c o n c e n t r a t i o n s i n f l u e n c e
t h e i r r a t e s . A y i e l d c o e f f i c i e n t Y must be i n t r o d u c e d t o make
t h e u n i t s o f S a n d M c o m p a t i b l e w i t h t h o s e o f B a n d Bn. E a c h c
u n i t o f S consumed i s assumed t o p r o d u c e Y u n i t s o f B. S i m i l a r
l y , each u n i t o f M excrete i unit
R a t e Laws
The r a t e o f c h a n g e o f e a c h c o n c e n t r a t i o n shown i n F i g u r e 1
may be o b t a i n e d b y summing a l l i t s mass i n f l o w s a n d o u t f l o w s :
•
dS/dt Ξ S = d ( S - S) - c S B / Y
0 c (6)
dB /dt = B
c c
- (c - s ) S B c + uB n - (w + d ) B c
(7)
dB / d t = sSB c - (u + w + d ) B n
(8)
η
dM/dt = M = e B / Y - dM.
n
(9)
Also, equations ( 7 ) a n d ( 8 ) may be a d d e d t o g e t h e r to yield:
dB/dt = Β = cSB c - (w + d ) B (10)
The r a t e s o f b i o m a s s p r o d u c t i o n a n d m e t a b o l i t e e x c r e t i o n
c a n be shown t o s a t u r a t e i n s u b s t r a t e c o n c e n t r a t i o n i f B i s c
assumed t o a d j u s t much more s l o w l y t o c h a n g e s i n S t h a n B d o e s . n
So, o n t h e t i m e s c a l e o f c h a n g e s i n B , B 2 0, a n d f r o m e q u a t i o n
c n
(8):
Β s SB /K C (Ha)
η
where Κ = (u + w + d ) / s (lib)
Now, f r o m e q u a t i o n s (5) and (11a):
B c Z B K / ( K + S) (12a)
B n ζ B S / ( K + S) (12b)

Figure 1. Reaction mechanism assumed to govern the rates of microbial biomass

production and metabolite excretion in batch (a = 0) and continuous (a > 0)
reactors. Symbols defined in text and in legend of symbols. B = B + B .
C n

Substrate Depletion. S u b s t i t u t i o n o f equation (12a) i n t o

(6) y i e l d s t h e f a m i l i a r c o n t i n u o u s - c u l t u r e e q u a t i o n for substrate
d e p l e t i o n (17,18):
S Ζ d ( S - S) - ^ ( B / Y j S / f K
Q + S) (13a)
where y = cK (13b)
m
The s e c o n d t e r m i n e q u a t i o n (13a) r e p r e s e n t s t h e r a t e o f s u b s t r a t e
consumption, o r n u t r i e n t uptake. I t s a t u r a t e s i n s u b s t r a t e con
c e n t r a t i o n a c c o r d i n g t o a Monod r a t e l a w where b o t h Κ a n d y / Y m
a r e known t o v a r y w i t h t e m p e r a t u r e a n d d i l u t i o n r a t e ( 1 1 - 2 1 ) .
Biomass P r o d u c t i o n . S i m i l a r l y , s u b s t i t u t i o n o f equation
(12a) i n t o ( 1 0 ) y i e l d s t h
l y used f o r continuous c u l t u r
Β/Β ζ y S/(Κ + S) - (w + d) (14)

m
The f i r s t t e r m i n e q u a t i o n ( 1 4 ) , r e p r e s e n t i n g t h e g r o s s r a t e o f
b i o m a s s p r o d u c t i o n , i s i d e n t i c a l w i t h t h e f u n c t i o n Monod ( 2 5 )
o r i g i n a l l y adopted " t o express c o n v e n i e n t l y t h e r e l a t i o n between
e x p o n e n t i a l growth r a t e and c o n c e n t r a t i o n o f an e s s e n t i a l n u t r i e n t .
Such a r e c t a n g u l a r h y p e r b o l i c f u n c t i o n h a s b e e n d e r i v e d many t i m e s
f r o m v a r i o u s r e a c t i o n mechanisms ( 2 6 - 3 0 ) , b u t none h a s a d d r e s s e d
the p r e s e n t case o f c o n t i n u o u s c u l t u r e systems where y and Κ m
have been o b s e r v e d t o v a r y w i t h temperature and d i l u t i o n r a t e .

Now, t h r o u g h e q u a t i o n s ( l i b ) a n d ( 1 3 b ) , t h e t e m p e r a t u r e v a r i a t i o n
o f y and Κ i s a t t r i b u t a b l e t o t h e m i c r o s c o p i c r a t e c o e f f i c i e n t s
m
c,s,u,w, and t h e i r v a r i a t i o n w i t h d i l u t i o n r a t e i s e x p l i c i t .
To e s t i m a t e t h e v a l u e s o f t h e m i c r o - c o e f f i c i e n t s c,s,u,w a s
f u n c t i o n s o f t e m p e r a t u r e , e q u a t i o n ( 1 4 ) c a n be r e w r i t t e n a s
Β/Β Ζ V ( S - S ) / ( S + K)
B T (15a)
where V. Ξ y - (w + d) (15b)
Β m
= (c - s) Κ + u (15c)
S T Ξ (w + d ) K / V B (15d)
E q u a t i o n (15a) r e p r e s e n t s a Monod r a t e l a w w i t h a t h r e s h o l d s u b
s t r a t e c o n c e n t r a t i o n S-j ( F i g u r e 2 ) . I t c a n be l i n e a r i z e d t o a
generalized Eadie-Augustinsson equation (31-33):
ν ζ V B - K(v/S) - V S ( 1 / S )
B T (16)

τ 1 1 1 1 1
GENERIC MONOD RATE - SATURATION LAW
WITH THRESHOLD SUBSTRATE CONCENTRATION
I ι ι ι ι ι ι I
Ο 10 20 30 40 50 60 70
S (ppm ELEMENT)
Figure 2. Properties of the rate law for net biomass production in batch (a = 0)
and continuous (d > 0) reactors. Symbols defined in text and in legend of
symbols.

where ν i s t h e s p e c i f i c n e t b i o m a s s p r o d u c t i o n r a t e B/B m e a s u r e d
i n the reactor a t p a r t i c u l a r values o f substrate concentration,
t e m p e r a t u r e , and d i l u t i o n r a t e . A l i n e a r r e g r e s s i o n o f ν v e r s u s
v/S and 1/S w i l l y i e l d v a l u e s f o r t h e m a c r o - c o e f f i c i e n t s V g , K,
and S- a s f u n c t i o n s o f t e m p e r a t u r e and d i l u t i o n r a t e . The v a l u e s
o f t h e m i c r o - c o e f f i c i e n t s a s f u n c t i o n s o f t e m p e r a t u r e c a n t h e n be
e s t i m a t e d f r o m e q u a t i o n s ( l i b ) , ( 1 5 c ) , a n d (15d) a s s u m i n g c = s .
Metabolite Excretion. S u b s t i t u t i o n o f e q u a t i o n (12b) i n t o

(9) y i e l d s a p u r e Monod r a t e l a w f o r m e t a b o l i t e e x c r e t i o n :
(M + dM)/(B/Y) Ζ V S / ( S + K) M (17a)
where V = e (17b)
m
Thermal Sensitivity of
Temperature i n f l u e n c e s t h e m a c r o - c o e f f i c i e n t s (K,y ,Vg,V ,

m m
S.j0 t h r o u g h t h e t e m p e r a t u r e d e p e n d e n c i e s o f t h e m i c r o - c o e f f i c i e n t s
( c , e , s , u , w ) . E a c h o f t h e m i c r o - c o e f f i c i e n t s i s assumed t o obey
an A r r h e n i u s temperature l a w o f t h e form ( 3 4 ) :
k = A exp ( - E / R T )
A K (18)
where k = a positively-valued micro

scopic rate c o e f f i c i e n t
A = a positively-valued coefficient
that i s p r a c t i c a l l y independent
of temperature
E^ Ξ a p o s i t i v e l y - v a l u e d apparent
l
a c t i v a t i o n energy ( c a l . mol )
R Ξ t h e e a s c o n s t a n t (1.987 c a l .
Y 1
mol- K- )
T R = degrees Kelvin
W i t h i n t h e t e m p e r a t u r e r a n g e 0 - 50°C, e q u a t i o n ( 1 8 ) may be
approximated as f o l l o w s ( 1 0 ) :
k ζ a exp (Τ/Θ ) α (19a)
α Ξ A exp (-1.843 χ 1 0 * 3
E^) (19b)
Τ = degrees Celsius
θ „ Ξ 1.481 χ 1 0 / E 5
A (19c)
α A

22. QUiNLAN Thermochemical Optimization All
This approximation considerably s i m p l i f i e s the i n v e s t i g a t i o n of

thermo-chemical r a t e - o p t i m i z a t i o n c o n d i t i o n s .
To i l l u s t r a t e some i n t e r e s t i n g a s p e c t s o f t h e t h e r m a l s e n
s i t i v i t y o f the m a c r o - e f f i c i e n t s , s p e c i f i c temperature depend
e n c i e s w e r e assumed f o r t h e f i v e m i c r o - c o e f f i c i e n t s . The assumed
d e p e n d e n c i e s a r e shown i n F i g u r e 3. The p a r a m e t e r v a l u e s w e r e
c h o s e n so i n b a t c h c u l t u r e (d = 0) a l l f i v e m a c r o - c o e f f i c i e n t s
w o u l d a l w a y s r i s e w i t h t e m p e r a t u r e ( F i g u r e s 4-6) and Κ w o u l d
r i s e more r a p i d l y w i t h t e m p e r a t u r e t h a n Vg and i n the tempera
t u r e r a n g e 20-30°C. T h i s t h e r m a l s e n s i t i v i t y p a t t e r n i s o f t e n
seen i n b a t c h c u l t u r e (e.g., 11-14).
H a l f - S a t u r a t i o n C o e f f i c i e n t K. For batch c u l t u r e , e q u a t i o n
( l i b ) reduces t o :
K|
I f s r i s e s more r a p i d l y w i t h t e m p e r a t u r e t h a n b o t h n u m e r a t o r t e r m s ,
then the b a t c h - c u l t u r e h a l f - s a t u r a t i o n c o e f f i c i e n t should always
d e c r e a s e as t e m p e r a t u r e i n c r e a s e s . I f s r i s e s more r a p i d l y t h a n
t h e n u m e r a t o r t e r m d o m i n a n t a t l o w t e m p e r a t u r e s b u t more s l o w l y
t h a n t h e t e r m d o m i n a n t a t h i g h t e m p e r a t u r e s , i t s h o u l d show a
t e m p e r a t u r e minimum. I f s r i s e s more s l o w l y t h a n b o t h n u m e r a t o r
t e r m s ( F i g u r e 3 ) , i t s h o u l d a l w a y s r i s e w i t h t e m p e r a t u r e ( F i g u r e 4,
d = 0)
The t h e r m a l s e n s i t i v i t i e s o f t h e b a t c h - c u l t u r e h a l f - s a t u r a t i o n
c o e f f i c i e n t s f o r s e v e r a l m i c r o b i a l p r o c e s s e s (11-14) h a v e b e e n
observed to resemble the l i n e a r p o r t i o n o f the d = 0 curve i n
F i g u r e 4. However, a few d e v i a t i o n s f r o m t h i s p a t t e r n h a v e b e e n
s e e n where t h e h a l f - s a t u r a t i o n c o e f f i c i e n t s showed e i t h e r a
n e g a t i v e s l o p e o r a maximum ( 1 3 ) . O n l y t h e t h e r m a l maximum
i s i n c o n s i s t e n t w i t h e q u a t i o n s (18) - ( 2 0 ) .
For continuous c u l t u r e ,
K
|d > ο - K
|d = ο + ( 1 / s ) d ( 2 1 )
According to equation (21), the continuous-culture h a l f - s a t u r a t i o n

c o e f f i c i e n t equals the b a t c h - c u l t u r e h a l f - s a t u r a t i o n c o e f f i c i e n t
augmented by a t e r m t h a t i n c r e a s e s l i n e a r l y w i t h d i l u t i o n r a t e .
However, t h e m a g n i t u d e o f t h i s t e r m s h o u l d d e c r e a s e e x p o n e n t i a l l y
w i t h i n c r e a s i n g t e m p e r a t u r e b e c a u s e o f t h e f a c t o r 1/s. Thus, a t
l o w t e m p e r a t u r e s where t h e d i l u t i o n t e r m may d o m i n a t e t h e b a t c h -
c u l t u r e h a l f - s a t u r a t i o n c o e f f i c i e n t , the continuous-culture h a l f -
s a t u r a t i o n c o e f f i c i e n t may d e c l i n e w i t h i n c r e a s i n g t e m p e r a t u r e .
I t s h o u l d grow w i t h t e m p e r a t u r e o n l y when t h e b a t c h - c u l t u r e h a l f -
s a t u r a t i o n c o e f f i c i e n t has a p o s i t i v e s l o p e and d o m i n a t e s t h e
d i l u t i o n term. I n t h i s c a s e , i t may h a v e a t h e r m a l minimum
( F i g u r e 4, d > 0 ) . M o r e o v e r , f o r t h e r a n g e o f d i l u t i o n r a t e s
where t h e l o c u s o f m i n i m a ( d a s h e d l i n e , F i g u r e 4) p a r a l l e l s t h e

Figure 3. Temperature dependencies assumed for the five microscopic rate coeffi-
cients defined in Figure 1. The microcoefficients c and s have units (ppm element
Khr' , e, u, and w have units Khr'K
1

QUINLAN Thermochemical Optimization 473
1 1 1 1 1 1 1—
VARIATION OF
HALF - SATURATION COE FFICIENT
WITH
TEMPERATURE AND DILUTION RATE
4
~d « 100 Khr -ι /
LU /
ΙΟ « 5 0
1
/
Lu
_J
LU
«25/
ε -Ο "
CL /
CL /
/
/
BATCH
/ d «Ο
u • w*d
w « 0.5 exp(T/5.l)
u « 10 exp(T/l6.9)
s « 5 0 exp (T/20)
I I I I L
10 20 30 40
TTC)
Figure 4. Theoretical thermal sensitivity of the half-saturation coefficient in batch
(a = 0) and continuous (a > 0) culture with the parameter values specified in
Figure 3. Dashed line, locus of thermal minima ( O).

d - 0 curve, the temperature t h a t minimizes the h a l f - s a t u r a t i o n

c o e f f i c i e n t r i s e s l i n e a r l y w i t h the l o g a r i t h m o f t h e d i l u t i o n
rate.
In c o n t i n u o u s c u l t u r e , t h e h a l f - s a t u r a t i o n c o e f f i c i e n t has
b e e n o b s e r v e d t o r e s p o n d t o r i s i n g t e m p e r a t u r e s by d e c l i n i n g
m o n o t o n i c a l l y (15-17) and by s h o w i n g a minimum ( 1 8 , 1 9 ) . B o t h
c a s e s a r e c o n s i s t e n t w i t h t h e d > 0 c u r v e s i n F i g u r e 4.
In c o n t r a s t , experiments p r o b i n g i t s response t o r i s i n g
d i l u t i o n r a t e s h a v e n ' t shown c l e a r - c u t t r e n d s b e c a u s e o f e x t r e m e
l y l a r g e measurement u n c e r t a i n t i e s ( 2 0 , 2 1 ) . I n one i n s t a n c e , no
t r e n d c o u l d be d e t e c t e d ( 2 0 ) . I n a n o t h e r , t h e mean v a l u e s showed
a maximum, b u t l i n e a r and s a t u r a t i o n c u r v e s c o u l d a l s o be drawn
through the + o n e - s t a n d a r d - d e v i a t i o n u n c e r t a i n t y ranges f o r each
mean v a l u e ( 2 1 ) . B e t t e r d a t a a r e c l e a r l y needed t o t e s t t h e
l i n e a r d e p e n d e n c e on d i l u t i o predicted b equation ( l i b )
and 2 1 ) .
M a x i m u m - S p e c i f i c - G r o w t h - R a t e C o e f f i c i e n t V^. In batch c u l t u r e ,
e q u a t i o n s ( l i b ) and (13b) c o m b i n e t o y i e l d :
w
«|d = 0 = c u / s + c w / s ( 2 2 )
The b a t c h - c u l t u r e m a x i m u m - s p e c i f i c - g r o w t h - r a t e c o e f f i c i e n t s h o u l d
f a l l w i t h i n c r e a s i n g t e m p e r a t u r e i f s r i s e s more r a p i d l y t h a n b o t h
c u and cw. I t s h o u l d show a minimum i f s r i s e s more r a p i d l y t h a n
the n u m e r a t o r o f t h e t e r m d o m i n a n t a t l o w t e m p e r a t u r e s b u t l e s s
r a p i d l y than the o t h e r term. I t should always i n c r e a s e i f s r i s e s
l e s s r a p i d l y t h a n t h e n u m e r a t o r s o f b o t h t e r m s . When c = s , i t
a l s o s h o u l d a l w a y s i n c r e a s e ( F i g u r e 5, d = 0 ) .
The d a t a (11-14) a v a i l a b l e t o t e s t t h e p r e d i c t e d t h e r m a l s e n
s i t i v i t y of the b a t c h - c u l t u r e maximum-specific-growth-rate co
e f f i c i e n t were o b t a i n e d from s u b s t r a t e consumption e x p e r i m e n t s .
From e q u a t i o n ( 1 3 a ) , t h e m a x i m u m - v e l o c i t y c o e f f i c i e n t f o r s u b
s t r a t e c o n s u m p t i o n i s V^/Y. D a t a p r e s e n t e d i n t h i s form must be
m u l t i p l i e d by a y i e l d c o e f f i c i e n t Y t o c o n v e r t t o y « I f the m
y i e l d c o e f f i c i e n t i s assumed t o be p r a c t i c a l l y c o n s t a n t , t h e n
y / Y s h o u l d show t h e same t h e r m a l s e n s i t i v i t y p a t t e r n as y .
m m
The t h e r m a l s e n s i t i v i t y o f y / Y i n b a t c h c u l t u r e i s w e l l d e s c r i b e d
m
by t h e l i n e a r p o r t i o n o f t h e d = 0 c u r v e i n F i g u r e 5, and s o i s
t h a t o f y as l o n g as Y i s n e a r l y c o n s t a n t .
m
In continuous c u l t u r e , the maximum-specific-growth-rate co

e f f i c i e n t i s a c c o r d i n g t o e q u a t i o n s ( l i b ) , ( 1 3 b ) , and ( 2 2 ) :
\|d > ο • "Jd = ο + ( c / 8 ) d ( 2 3 )
The c o n t i n u o u s - c u l t u r e y s h o u l d i n c r e a s e l i n e a r l y w i t h d i l u t i o n
m
r a t e , and t h e i n f l u e n c e o f t h e d i l u t i o n t e r m s h o u l d grow w i t h
r i s i n g t e m p e r a t u r e i f s r i s e s more s l o w l y t h a n c . I f c = s , t h e
c o n t i n u o u s - c u l t u r e y should a s y m p t o t i c a l l y approach the d i l u t i o n
m

0 10 20 30 40
T(°C)
Figure 5. Theoretical thermal sensitivity of the maximum specific growth-rate

coefficient in batch (d = 0) and continuous (d > 0) culture with the parameter
values specified in Figure 3.

r a t e a s t e m p e r a t u r e f a l l s and t h e b a t c h c u l t u r e p a s t e m p e r a t u r e
m
r i s e s ( F i g u r e 5, d > 0 ) .
The l i n e a r p o r t i o n s o f t h e d > 0 c u r v e s i n F i g u r e 5 r e s e m b l e
the t h e r m a l s e n s i t i v i t i e s o b s e r v e d f o r y (17,18) and y / Y
m (15,16,
m
19) i n c o n t i n u o u s c u l t u r e . M o r e o v e r , i n agreement w i t h e q u a t i o n
(23) y / Y has b e e n o b s e r v e d t o i n c r e a s e l i n e a r l y w i t h d i l u t i o n
m
r a t e ( 2 0 ) . I t has a l s o b e e n s e e n t o d e c r e a s e s l i g h t l y w i t h i n
c r e a s i n g d i l u t i o n r a t e (21), which d i s a g r e e s w i t h equation (23).
M a x i m u m - V e l o c i t y C o e f f i c i e n t s Vft V^.
t According to equations
( l i b ) and ( 1 5 c ) , t h e m a x i m u m - v e l o c i t y c o e f f i c i e n t f o r b i o m a s s p r o
d u c t i o n depends on t h e m i c r o s c o p i c r a t e c o e f f i c i e n t s a s f o l l o w s :
V B = (1/s) [ ( c - s) w + cu] + [ ( c - s ) / s ] d (24)
In g e n e r a l , depending e s p e c i a l l
t h i s m a x i m u m - v e l o c i t y c o e f f i c i e n t may v a r y i n a c o m p l i c a t e d f a s h i o n
w i t h b o t h t e m p e r a t u r e and d i l u t i o n r a t e . However, a s c a p p r o a c h e s
s i n v a l u e , the i n f l u e n c e o f a l l the m i c r o - c o e f f i c i e n t s but u
v a n i s h e s , and i n t h e l i m i t o f c = s ,
V B = u (25)
In t h i s s p e c i a l case o n l y , the maximum-velocity c o e f f i c i e n t f o r

b i o m a s s p r o d u c t i o n w o u l d show t h e same e x p o n e n t i a l t e m p e r a t u r e
dependence i n c o n t i n u o u s c u l t u r e as i t does i n b a t c h c u l t u r e .
The m a x i m u m - v e l o c i t y c o e f f i c i e n t f o r m e t a b o l i t e e x c r e t i o n
a s g i v e n by e q u a t i o n (17b) i s i d e n t i c a l l y e q u a l t o e. As a
r e s u l t , i t should g e n e r a l l y r i s e e x p o n e n t i a l l y w i t h temperature
and be i n s e n s i t i v e t o d i l u t i o n r a t e . Thus, the maximum-velocity
c o e f f i c i e n t f o r metabolite excretion i n batch culture should
a l s o be i d e n t i c a l t o t h a t s e e n i n c o n t i n u o u s c u l t u r e .
No t h e r m a l s e n s i t i v i t y d a t a c o u l d be f o u n d f o r Vg o r
So, t h e v a l i d i t y o f e q u a t i o n s ( 1 7 b ) , ( 2 4 ) , and (25) a w a i t s t e s t
ing.
T h r e s h o l d C o e f f i c i e n t S T * F o r b a t c h c u l t u r e and t h e s p e c i a l
case c = s, the t h e r m a l s e n s i t i v i t y o f t h e t h r e s h o l d c o e f f i c i e n t
i s d e t e r m i n e d a s f o l l o w s by e q u a t i o n s ( 1 5 d ) , ( 2 0 ) , and ( 2 5 ) :
S |
T d m 0 = (w/s) + (w/u) (w/s) (26)
A t t e m p e r a t u r e s where u d o m i n a t e s w, t h e b a t c h - c u l t u r e t h r e s h o l d
c o e f f i c i e n t should f a l l e x p o n e n t i a l l y w i t h temperature i f s r i s e s
more r a p i d l y w i t h t e m p e r a t u r e t h a n w. A t t e m p e r a t u r e s where w
d o m i n a t e s u, i t s h o u l d a l s o f a l l e x p o n e n t i a l l y w i t h r i s i n g tem
2
p e r a t u r e i f s u r i s e s more r a p i d l y t h a n w . I f b o t h terms d e
c r e a s e as t e m p e r a t u r e r i s e s , i t s h o u l d a l w a y s d i m i n i s h as tem
perature r i s e s . I f the term dominant a t low temperatures f a l l s

w i t h r i s i n g temperature and t h e o t h e r term i n c r e a s e s , i t s h o u l d

h a v e a t h e r m a l minimum. I f b o t h t e r m s r i s e w i t h t e m p e r a t u r e , i t
s h o u l d a l w a y s r i s e w i t h t e m p e r a t u r e ( F i g u r e 6, d = 0 ) .
None o f t h e b a t c h - c u l t u r e t h e r m a l s e n s i t i v i t y e x p e r i m e n t s
r e p o r t e d any v a l u e s f o r S^, s o t h e v a l i d i t y o f e q u a t i o n (26)
a n d t h e d = 0 c u r v e i n F i g u r e 6 c a n ' t be j u d g e d now.
For c o n t i n u o u s c u l t u r e and t h e s p e c i a l c a s e c = s , e q u a t i o n s
( l i b ) , ( 1 5 d ) , and (26) d e t e r m i n e t h e t h e r m a l s e n s i t i v i t y o f t h e
t h r e s h o l d c o e f f i c i e n t as f o l l o w s :
2
S | T d > 0 - S |T d m 0 + (1/s) [ 1 + 2 (w/u)] d + ( l / s u ) d (27)
According to equation (27), the continuous-culture threshold co

e f f i c i e n t i s a q u a d r a t i c f u n c t i o n o f d i l u t i o n r a t e when c = s ;
however, t h e magnitude o
t i a l l y w i t h r i s i n g temperatur
l i n e a r t e r m w i l l v a n i s h t o o i f s u r i s e s more r a p i d l y w i t h tem
p e r a t u r e than w does. The c o n t i n u o u s - c u l t u r e t h r e s h o l d c o
e f f i c i e n t s h o u l d i n c r e a s e when t h e b a t c h c u l t u r e t h r e s h o l d
c o e f f i c i e n t r i s e s w i t h temperature and dominates t h e d i l u t i o n
terms. I t s h o u l d have a t h e r m a l minimum i f a d e c l i n i n g t e r m
d o m i n a t e s a t l o w t e m p e r a t u r e and a n i n c r e a s i n g t e r m d o m i n a t e s
a t h i g h t e m p e r a t u r e ( F i g u r e 6, d > 0)· The t e m p e r a t u r e a t
w h i c h t h i s minimum o c c u r s r i s e s a l m o s t l i n e a r l y w i t h t h e l o g
a r i t h m o f t h e d i l u t i o n r a t e as l o n g as t h e l o c u s o f minima
( d a s h e d l i n e , F i g u r e 6) p a r a l l e l s t h e d = 0 c u r v e .
In continuous r e a c t o r s , the t h r e s h o l d c o e f f i c i e n t i s i d e n t i
c a l w i t h the steady s t a t e substrate c o n c e n t r a t i o n (17,18). For
i n c r e a s i n g t e m p e r a t u r e s , i t has b e e n o b s e r v e d t o f a l l (17,18)
and show a minimum ( 1 8 ) . T h e s e r e s p o n s e s a r e c o n s i s t e n t w i t h
the d > 0 c u r v e s shown i n F i g u r e 6. I t h a s a l s o b e e n o b s e r v e d
to i n c r e a s e n o n l i n e a r l y w i t h d i l u t i o n r a t e u n t i l w a s h o u t o c c u r r e d
(17,18). I n b o t h i n s t a n c e s , t h e s l o p e a t low d i l u t i o n r a t e s de
c r e a s e d w i t h r i s i n g t e m p e r a t u r e . The o b s e r v e d r e s p o n s e s t o i n
creasing d i l u t i o n rate are also consistent with equation (27).
A t t h i s p o i n t , i t w o u l d a p p e a r t h a t one c a n a c c o u n t f o r most
of t h e observed e f f e c t s o f temperature and d i l u t i o n r a t e on t h e
m a c r o - c o e f f i c i e n t s by s i m p l y a s s u m i n g t e m p e r a t u r e d e p e n d e n c i e s
s u c h a s t h o s e shown i n F i g u r e 3 f o r t h e m i c r o - c o e f f i c i e n t s i n
e q u a t i o n s (20) - ( 2 7 ) . These e q u a t i o n s w i t h t h e parameter v a l u e s
s p e c i f i e d i n F i g u r e 3 w i l l now be u s e d t o a n a l y z e t h e t h e r m a l
s e n s i t i v i t y o f t h e n e t b i o m a s s p r o d u c t i o n and m e t a b o l i t e e x c r e t i o n
rates.
Thermal S e n s i t i v i t y o f Process Rates
R a t e I s o t h e r m s . D e p e n d i n g on s u b s t r a t e c o n c e n t r a t i o n , t h r e e
t h e r m a l s e n s i t i v i t y p a t t e r n s may be s e e n i n b o t h b a t c h a n d c o n
t i n u o u s r e a c t o r s when t h e n e t b i o m a s s p r o d u c t i o n r a t e ( a s s p e c i f i e d
by e q u a t i o n s ( l i b ) and ( 1 5 ) and F i g u r e s 3, 4, and 6) i s p l o t t e d

1 1 1 1 1 1 1
VARIATION OF
T H R E S H O L D S U B S T R A T E CONCENTRATION
WITH
_ T E M P E R A T U R E AND DILUTION R A T E _
T(°C)
Figure 6. Theoretical thermal sensitivity of the threshold coefficient in batch (à
= 0) and continuous (à > 0) culture with the parameter values specified in Figure
3. Dashed line, locus of thermal minima (O).

22. QUiNLAN Thermochemical Optimization 479
v e r s u s s u b s t r a t e c o n c e n t r a t i o n w i t h t e m p e r a t u r e as a p a r a m e t e r
( F i g u r e 7 ) . A t l o w s u b s t r a t e c o n c e n t r a t i o n s (S < 1 . 2 ) , t h e r a t e
d i m i n i s h e s a s t e m p e r a t u r e r i s e s and h e n c e shows a n e g a t i v e t h e r m a l
s e n s i t i v i t y ; w h e r e a s , a t h i g h s u b s t r a t e c o n c e n t r a t i o n s (S > 4·4),
t h e r a t e i n c r e a s e s w i t h t e m p e r a t u r e and t h u s has a p o s i t i v e t h e r m a l
sensitivity. A t i n t e r m e d i a t e s u b s t r a t e c o n c e n t r a t i o n s (1.2 < S <
4 . 4 ) , t h e r a t e f i r s t r i s e s and t h e n f a l l s w i t h i n c r e a s i n g tem
perature. I n o t h e r words, a t moderate s u b s t r a t e c o n c e n t r a t i o n s ,
the thermal s e n s i t i v i t y s w i t c h e s s i g n from p o s i t i v e to n e g a t i v e ,
w h i c h means t h e r a t e goes t h r o u g h a maximum a s t e m p e r a t u r e r i s e s .
M o r e o v e r , t h e maximum r a t e i s d e t e r m i n e d by a h i g h e r t e m p e r a t u r e
i s o t h e r m a s s u b s t r a t e c o n c e n t r a t i o n i n c r e a s e s . As a c o n s e q u e n c e ,
t h e optimum t e m p e r a t u r e s h o u l d r i s e when s u b s t r a t e c o n c e n t r a t i o n
increases.
The r a t e i s o t h e r m s f o m e t a b o l i t excretio (a s p e c i f i e d b
e q u a t i o n s ( l i b ) and (17
thermal s e n s i t i v i t y pattern
isotherms. T h u s , t h e r a t e o f m e t a b o l i t e e x c r e t i o n may a l s o h a v e
an optimum t e m p e r a t u r e t h a t s h i f t s t o h i g h e r v a l u e s a s s u b s t r a t e
concentration r i s e s .
Rate I s o c o n c e n t r a t e s . The i n c r e a s e o f optimum t e m p e r a t u r e

w i t h i n c r e a s i n g s u b s t r a t e c o n c e n t r a t i o n i s p l a i n l y s e e n by p l o t t i n g
r a t e v e r s u s t e m p e r a t u r e w i t h s u b s t r a t e c o n c e n t r a t i o n as a p a r a
m e t e r ( F i g u r e s 8 and 9 ) . The f a m i l i e s o f r a t e i s o c o n c e n t r a t e s
f o r n e t b i o m a s s p r o d u c t i o n and m e t a b o l i t e e x c r e t i o n a l s o a p p e a r
qualitatively similar. However, t h e maxima o f c o r r e s p o n d i n g i s o
concentrates occur at q u i t e d i f f e r e n t temperatures. For example,
when S = 1 ppm, t h e optimum t e m p e r a t u r e f o r t h e n e t b i o m a s s p r o
d u c t i o n r a t e w o u l d be a b o u t 16°C ( F i g u r e 8) and t h a t f o r t h e
m e t a b o l i t e e x c r e t i o n r a t e w o u l d be a b o u t 28°C ( F i g u r e 9 ) . Note
t h a t i f the o p e r a t i n g t e m p e r a t u r e o f t h e r e a c t o r were s e t a t t h e
optimum t e m p e r a t u r e f o r t h e m e t a b o l i t e e x c r e t i o n r a t e , t h e n e t
b i o m a s s p r o d u c t i o n r a t e w o u l d be s e v e r e l y s u b o p t i m a l ( F i g u r e 8 ) .
But i f i t were s e t a t t h e optimum t e m p e r a t u r e f o r t h e n e t b i o m a s s
p r o d u c t i o n r a t e , t h e m e t a b o l i t e e x c r e t i o n r a t e w o u l d be o n l y
s l i g h t l y suboptimal (Figure 9).
S h i f t o f Optimum T e m p e r a t u r e . F i g u r e 10 shows how t h e tem

p e r a t u r e t h a t maximizes the r a t e o f each process v a r i e s w i t h b o t h
s u b s t r a t e c o n c e n t r a t i o n and d i l u t i o n r a t e . I n t h i s regard, the
t h e r m a l s e n s i t i v i t i e s o f t h e two p r o c e s s e s d i f f e r s u b s t a n t i a l l y .
The optimum o p e r a t i n g t e m p e r a t u r e f o r t h e n e t b i o m a s s p r o
d u c t i o n r a t e i s n e a r l y i n s e n s i t i v e t o d i l u t i o n r a t e , but i n
creases almost l i n e a r l y w i t h the l o g a r i t h m of s u b s t r a t e con
c e n t r a t i o n when t h i s c o n c e n t r a t i o n e x c e e d s t h e minimum v a l u e
of the t h r e s h o l d c o e f f i c i e n t ( F i g u r e 6 ) .
I n c o n t r a s t , t h e optimum o p e r a t i n g t e m p e r a t u r e f o r t h e
m e t a b o l i t e e x c r e t i o n r a t e i s s t r o n g l y a f f e c t e d by b o t h d i l u t i o n
r a t e and s u b s t r a t e c o n c e n t r a t i o n . As s u b s t r a t e c o n c e n t r a t i o n

1 1 1 1 1 Γ
NET BIOMASS PRODUCTION
S (ppm ELEMENT)
Figure 7. Predicted influence of substrate concentration on the rate of net biomass

production, with temperature as a parameter. Parameter values correspond to
Figures 3-6. Note shift of optimum temperature to higher values with increasing
substrate concentration.

τ 1 1 1 τ—
NET BIOMASS PRODUCTION
THEORETICAL RATE ISOCONCENTRATES
Τ CO
Figure 8. Predicted influence of temperature on the rate of net biomass production

with substrate concentration as a parameter. Parameter values correspond to
Figures 3-7. Dashed line, locus of thermal rate maxima (O). Note shift of optimum
temperature to higher values as substrate concentration rises.

— ι 1 1 1 1 1 1
METABOLITE EXCRETION
T H E O R E T I C A L RATE ISOCONCENTRATES
CONTINUOUS CULTURE ! d = 2 5 Khr" 1
0.5
M • dM
(B/Y)
(Khr )1
0.1
0.05
S s
0 . 0 2 5 ppm
ELEMENT!
0.02
0.01
Figure 9. Predicted influence of temperature on the rate of metabolite excretion

with substrate concentration as a parameter. Solid lines, curves generated from
Equations lib and 17 with the parameter values given in Figures 3 and 4. Dashed
line, locus of thermal rate maxima (O).

QUINLAN Thermochemical Optimization 483
Figure 10. Predicted influence of substrate concentration and dilution rate on the
temperature that maximizes the rates of net biomass production and metabolite
excretion in batch (a = 0) and continuous (a > 0) culture.

becomes v e r y l o w , t h e optimum t e m p e r a t u r e a s y m p t o t i c a l l y a p p r o a c h
es a minimum v a l u e c h a r a c t e r i s t i c o f e a c h d i l u t i o n r a t e . The
minimum optimum t e m p e r a t u r e i s t h e t e m p e r a t u r e a t w h i c h t h e s l o p e
o f t h e h a l f - s a t u r a t i o n c o e f f i c i e n t ( F i g u r e 4) e q u a l s t h a t o f t h e
maximum-velocity c o e f f i c i e n t , namely, the m i c r o s c o p i c e x c r e t i o n
r a t e c o e f f i c i e n t e. As s u b s t r a t e c o n c e n t r a t i o n i n c r e a s e s , t h e
i n f l u e n c e o f t h e d i l u t i o n r a t e p r o g r e s s i v e l y d i m i n i s h e s , and t h e
optimum t e m p e r a t u r e c u r v e s c o n v e r g e . A t h i g h s u b s t r a t e c o n
c e n t r a t i o n s , t h e optimum t e m p e r a t u r e i n c r e a s e s a l m o s t l i n e a r l y
w i t h the logarithm of substrate concentration.
Thermo-Chemical O p t i m i z a t i o n o f P r o c e s s Rates
A t t h i s p o i n t , t h e g o a l o f t h i s p a p e r has b e e n a c h i e v e d .
The two f a m i l i e s o f c u r v e s shown i n F i g u r e 10 c o n s t i t u t e t h e r m o
chemical rate-optimizatio
ture, substrate concentration
o f t h e s e t h r e e v a r i a b l e s a r e f i x e d , e a c h f a m i l y s p e c i f i e s what
v a l u e t h e t h i r d must h a v e t o m a x i m i z e i t s r a t e . The s e p a r a t i o n
o f t h e two f a m i l i e s means t h a t b o t h r a t e s c a n n o t be s i m u l t a n e
o u s l y maximized. As a r e s u l t , an o p t i m i z a t i o n s t r a t e g y may be
n e e d e d , s u c h a s o p e r a t i n g a t t h e optimum t e m p e r a t u r e o f t h e
p r o c e s s whose r a t e i s most s e n s i t i v e t o t e m p e r a t u r e .
Summary and C o n c l u s i o n s
T h i s p a p e r showed how c h a n g e s i n s u b s t r a t e c o n c e n t r a t i o n
and d i l u t i o n r a t e may s h i f t t h e optimum t e m p e r a t u r e o f m i c r o b i a l
p r o c e s s e s whose r a t e s s a t u r a t e i n s u b s t r a t e c o n c e n t r a t i o n a c c o r d
i n g t o Monod r a t e l a w s . The optimum t e m p e r a t u r e s h i f t was a t t r i b u
t e d t o t h e e f f e c t s o f t e m p e r a t u r e and d i l u t i o n r a t e on t h e m a c r o -
c o e f f i c i e n t s i n t h e Monod r a t e l a w s . E q u a t i o n s d e s c r i b i n g t h e s e
e f f e c t s were d e r i v e d from a s u i t a b l y m o d i f i e d M i c h a e l i s - M e n t e n
r e a c t i o n mechanism. W i t h o n l y a few e x c e p t i o n s , t h e s e e q u a t i o n s
a g r e e d w i t h p u b l i s h e d d a t a when A r r h e n i u s t e m p e r a t u r e d e p e n d e n c i e s
were assumed f o r t h e m i c r o - c o e f f i c i e n t s i n t h e r e a c t i o n mechanism.
As a c o n s e q u e n c e , t h e s e e q u a t i o n s and t h e optimum t e m p e r a t u r e
c u r v e s may be c o n s i d e r e d a s p l a u s i b l e , e x p e r i m e n t a l l y t e s t a b l e
hypotheses. They d e s e r v e c a r e f u l q u a n t i t a t i v e t e s t i n g b e c a u s e
t h e y p r o m i s e t o u n i f y h e r e t o f o r e f r a g m e n t a r y and s e e m i n g l y c o n
t r a d i c t o r y b a t c h and c o n t i n u o u s c u l t u r e d a t a . S u c h u n i f i c a t i o n
i s needed t o d e v e l o p a r a t i o n a l m e t h o d o l o g y f o r o p t i m i z i n g t h e
d e s i g n and o p e r a t i o n o f b i o l o g i c a l r e a c t o r s i n w h i c h s u b s t r a t e
c o n c e n t r a t i o n , t e m p e r a t u r e , and d i l u t i o n r a t e a r e e c o n o m i c a l l y
important v a r i a b l e s .

Legend o f Symbols
A = pre-exponential f a c t o r i n Arrhenius temperature law;

same u n i t s a s k.
Β Ξ t o t a l b i o m a s s c o n c e n t r a t i o n i n r e a c t o r ; e q u a l s sum o f
B p l u s B ; mg d r y w e i g h t p e r l i t e r .
c n
B c = biomass c o n c e n t r a t i o n a s s o c i a t e d w i t h s u b s t r a t e con
s u m p t i o n s i t e s c a p a b l e o f c o n s u m i n g s u b s t r a t e ; mg
dry weight per l i t e r .
B n = biomass c o n c e n t r a t i o n a s s o c i a t e d w i t h s u b s t r a t e con
sumption s i t e s n o t c a p a b l e o f consuming s u b s t r a t e ;
mg d r y w e i g h t p e liter
C Ξ t o t a l concentratio consumptio
i n m i c r o b i a l p o p u l a t i o n ; e q u a l s sum o f C p l u s C^;
c
number o f s i t e s p e r l i t e r .
C c = c o n c e n t r a t i o n o f s u b s t r a t e consumption s i t e s capable
o f consuming s u b s t r a t e ; number o f s i t e s p e r l i t e r .
C n = c o n c e n t r a t i o n o f s u b s t r a t e consumption s i t e s n o t
c a p a b l e o f c o n s u m i n g s u b s t r a t e ; number o f s i t e s
per l i t e r .
Ε = c o n c e n t r a t i o n o f f r e e enzyme.
= apparent a c t i v a t i o n energy; cal./mol.
ES = concentration o f enzyme-substrate complex.
Κ = half-saturation coefficient; same u n i t s a s S.
KJJJ Ξ the Michaelis-Menten half-saturation coefficient.
Μ Ξ concentration of metabolite of interest; same u n i t s a s S.
Ν Ξ c o n c e n t r a t i o n o f m i c r o o r g a n i s m s i n r e a c t o r ; number o f
organisms per l i t e r .
Ρ = concentration of product(s).
1 1
R Ξ g a s c o n s t a n t = 1.987 c a l . m o l " Κ" .
S = s u b s t r a t e c o n c e n t r a t i o n i n r e a c t o r ; ppm o f a n e l e m e n t .
S Q = i n l e t s u b s t r a t e c o n c e n t r a t i o n ; same u n i t s a s S.
C o n t i n u e d on n e x t page

threshold c o e f f i c i e n t ; steady-state substrate concentra

t i o n t h a t must be e x c e e d e d f o r a p o s i t i v e n e t b i o m a s s
p r o d u c t i o n r a t e ; same u n i t s a s S.
t e m p e r a t u r e ; °C.
t e m p e r a t u r e ; °K.
maximum p o s s i b l e s p e c i f i c r a t e o f n e t b i o m a s s p r o d u c t i o n
1 1
i n the reactor; time" (e.g., K h r " ) .
maximum p o s s i b l e r a t e o f g r o s s m e t a b o l i t e formation;
1 1
time" (e.g., K h r " ) .
y i e l d c o e f f i c i e n t ; ppm d r y w e i g h t p r o d u c e d p e r ppm
e l e m e n t consumed
microscopic substrate-consumption rate coefficient;

1 1
(ppm e l e m e n t ) " time" .
d i l u t i o n r a t e (flow/volume); r a t e a t which s u b s t r a t e ,
b i o m a s s , and m e t a b o l i t e d i s p l a c e d f r o m c u l t u r e v e s s e l ;
1
time" .
1
microscopic metabolite-excretion rate c o e f f i c i e n t ; time" .
g e n e r i c m i c r o s c o p i c r a t e c o e f f i c i e n t , e.g., c,f,s,u,w.
microscopic rate coefficient f o r f o r m a t i o n o f enzyme-

s u b s t r a t e complex.
microscopic rate coefficient f o r d i s a s s o c i a t i o n of

enzyme-substrate complex.
microscopic rate coefficient f o r formation of product.
number o f s u b s t r a t e m o l e c u l e s needed t o f i l l a substrate

consumption s i t e .
microscopic substrate-saturation rate coefficient;

1 1
(ppm e l e m e n t ) " time" .
m i c r o s c o p i c u n s a t u r a t i o n r a t e c o e f f i c i e n t ; t i m e *.
1
m i c r o s c o p i c wastage r a t e c o e f f i c i e n t ; time" .
value of generic microscopic rate c o e f f i c i e n t a t 0°C;

same u n i t s a s k.

3 = a v e r a g e b i o m a s s p e r m i c r o o r g a n i s m ; mg d r y w e i g h t p e r
organism.
Ύ = a v e r a g e number o f s u b s t r a t e c o n s u m p t i o n s i t e s p e r
o r g a n i s m ; number o f s i t e s p e r o r g a n i s m .
PJJJ = 1
maximum s p e c i f i c g r o w t h r a t e c o e f f i c i e n t ; t i m e " " .
θ = r e t a r d a t i o n t e m p e r a t u r e ; °C.
Acknowledgments
The r e s e a r c h r e p o r t e d i n t h i s p a p e r was s u p p o r t e d i n p a r t
by t h e D e p a r t m e n t o f M e c h a n i c a
a g r a n t f r o m t h e Therm
The f i g u r e s w e r e drawn b y Michèle H a l v e r s o n , a n d t h e t e x t

was p r e p a r e d f o r p u b l i c a t i o n by Anna M. P i c c o l o . The a u t h o r
greatly appreciates their efforts i nher behalf.
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R E C E I V E D July 7, 1 9 8 2

23
Kinetics of Yeast Growth and Metabolism
in Beer Fermentation
IVAN MARC and JEAN-MARC ENGASSER

Institut National Polytechnique de Lorraine, Laboratoire des Sciences du Génie
Chimque, CNRS-ENSIC, Nancy 54042 France
MANFRED M O L L and BRUNO DUTEURTRE
Centre de Recherche TEPRAL, Champigneulles 54250 France
An experimental and t h e o r e t i c a l k i n e t i c study o f

yeast growth and
uptake, ethanol,
during beer fermentation is presented. C e l l u l a r growth,
and consequently amino a c i d s uptake and some metabo-
lites production, stops a f t e r about 100 hours o f f e r -
mentation. Yeast f l o c c u l a t i o n , which s t a r t s when gluco-
se is totally removed from the medium, then becomes a
predominant phenomenon. Fermentable sugars are sequen-
tially taken up, maltose and m a l t o t r i o s e being repres-
sed by glucose. Ethanol and CO production r a t e remain
2
e s s e n t i a l l y p r o p o r t i o n a l to the t o t a l r a t e o f sugar
uptake both during the growth and f l o c c u l a t i o n phase.
The increase with temperature o f s u b s t r a t e uptake,
yeast growth and f l o c c u l a t i o n , and metabolite produc-
t i o n r a t e is simply described by Arrhenius type o f
relationships.
The conversion by yeast o f sugars and amino a c i d s i n t o aro-

matic compounds represents a c e n t r a l process i n brewing. A q u a n t i -
t a t i v e understanding o f the m i c r o b i a l events t a k i n g p l a c e during
the t r a n s f o r m a t i o n o f wort i n t o beer i s e s s e n t i a l when c o n s i d e r i n g
the automatic c o n t r o l o f t h i s fermentation.
A f i r s t k i n e t i c modeling o f yeast growth, sugar and amino
a c i d uptake and aromatic m e t a b o l i t e p r o d u c t i o n was p r e v i o u s l y
proposed (1,2). On the b a s i s o f recent experimental r e s u l t s we
propose a new model which takes i n t o account the e v o l u t i o n o f the
yeast v i a b i l i t y during the f e r m e n t a t i o n , and d e s c r i b e s i n g r e a t e r
d e t a i l the f l o c c u l a t i o n phase and the p r o d u c t i o n o f CO2. The k i n e -
t i c study i s s p e c i a l l y aimed a t e s t a b l i s h i n g r e l a t i o n s p h i p s bet-
ween the wort i n i t i a l composition, the f i n a l beer q u a l i t y and
v a r i o u s process parameters, such as temperature, pressure and i n o -
cculum s i z e .
0097-6156/83/0207Ό489$06.00/0

EXPERIMENTAL
The experimental p a r t of t h i s work was performed at the

TEPRAL Beer Research Center i n Champigneulles, France. Beer pro
d u c t i o n was c a r r i e d out e i t h e r i n l a b o r a t o r y t u b u l a r fermentors,
1,80 m h e i g h t and 4,5 cm inner diameter, c o n t a i n i n g 2 l i t e r s of
medium, or i n 1000 l i t e r s c y l i n d r o - c o n i c a l p i l o t p l a n t fermentors
without mechanical a g i t a t i o n . Standart i n d u s t r i a l wort and yeast
Saccharomyces uvarum 0019 were used. The i n i t i a l d i s s o l v e d oxygen
c o n c e n t r a t i o n i n the wort was f i x e d at 8 ppm. The temperature was
maintained constant at e i t h e r 10° C or 14° C during the whole f e r
mentation. The overhead pressure was 1 atm w i t h the l a b o r a t o r y
fermentor and 2,2 atm w i t h the p i l o t p l a n t .
Samples of 80 cm^ and 4 l i t e r s were p e r i o d i c a l l y withdrawn
from the l a b o r a t o r y and p i l o t v e s s e l r e s p e c t i v e l y Yeast
t r a t i o n was determined e i t h e
ter counter. The v a r i o u sugars, glucose, , ,
charose, m a l t o t r i o s e were analyzed by high pressure l i q u i d chroma
tography. The c o n c e n t r a t i o n o f the twenty amino a c i d s were d e t e r
mined by i o n exchange chromatography. Ethanol was assayed by micro-
c a l o r i m e t r y and other m e t a b o l i t e s by gas chromatography.(3)
THE KINETIC MODEL
In c o n v e n t i o n a l fermentation models the c e n t r a l equation

u s u a l l y expresses c e l l u l a r growth as a f u n c t i o n o f s u b s t r a t e , pro
duct and c e l l u l a r c o n c e n t r a t i o n i n the medium. Product formation i s
then r e l a t e d to c e l l growth or c e l l u l a r c o n c e n t r a t i o n , whereas
s u b s t r a t e uptake r a t e may i n c l u d e s e v e r a l c o n t r i b u t i o n s from c e l l u
l a r growth, maintenance and product formation. Moreover, these mo
d e l s most o f t e n are based on constant y i e l d s .
In view of the experimental r e s u l t s (4) i t i s c l e a r t h a t the
c o n v e n t i o n a l approach cannot be used to model beer fermentation.
F i r s t , the fermentation medium c o n t a i n s s e v e r a l sugars which are
a s s i m i l a t e d at d i f f e r e n t r a t e s . The uptake of each sugar may be l i
mited by i t s own c o n c e n t r a t i o n and i n h i b i t e d by the presence of
others. Second, the i n v e s t i g a t e d beer fermentation from a k i n e t i c
p o i n t of view i s complicated by yeast f l o c c u l a t i o n which becomes
predominant during the second stage of the process, and which i s
a f f e c t e d by one of the fermentable sugars, namely glucose. T h i r d ,
the v a r i o u s y i e l d s between fermentable sugars uptake, yeast growth,
ethanol and CÛ2 formation are not constant during the course of the
fermentation. F i n a l l y , the r a t e o f aromas production i s r e l a t e d to
e i t h e r yeast growth r a t e or sugars uptake r a t e .
In order to d e s c r i b e the i n f l u e n c e of the i n i t i a l wort compo
s i t i o n on the fermentation process the proposed model i s based on
separate r a t e equations f o r the v a r i o u s sugars uptake. Production
of y e a s t , ethanol and carbon d i o x i d e i s then r e l a t e d to the t o t a l
uptake of fermentable sugars w i t h v a r i a b l e y i e l d s . The r a t e of
amino a c i d s uptake and other m e t a b o l i t e s production i s expressed
as a f u n c t i o n of growth r a t e or sugars uptake r a t e w i t h constant
or v a r i a b l e y i e l d s .

23. MARC ET AL. Kinetics in Beer Fermentation 491
Y e a s t g r o w t h and f l o c c u l a t i o n . D i s t i n c t i o n i s made between

t h e t o t a l y e a s t p r o d u c e d d u r i n g t h e g r o w t h p h a s e and t h e s u s p e n d e d
y e a s t c o n c e n t r a t i o n i n t h e f e r m e n t a t i o n medium. The i n c r e a s e i n
t o t a l yeast c o n c e n t r a t i o n i n the fermentor i s d i r e c t l y r e l a t e d t o
the t o t a l r a t e o f fermentable sugar consumption.
d(X ) T
d(TFS)
= - R (1)
dt X/S dt
(Xy) being the t o t a l yeast concentration
(TFS) t h e t o t a l fermentable sugar c o n c e n t r a t i o n
R the yeast t o sugar y i e l d
X/S
The e x p e r i m e n t a l l y o b s e r v e d d e c r e a s e o f R x / s d u r i n g t h e f e r
mentation has p r e v i o u s l
c e l l u l a r sterol concentratio
a b s e n c e o f o x y g e n . We f o u n d i t most c o n v e n i e n t t o m a t h e m a t i c a l l y
r e l a t e t h e d e c r e a s e o f Rx/s t o t h e i n c r e a s e o f t h e e t h a n o l c o n c e n
t r a t i o n i n t h e medium, ( E t h ) , a s :
R = Y
(2)
X/S X/S 4
(Eth)
1 +
with Y ^ g t h e i n i t i a l yeast t o sugar yield

the ethanol i n h i b i t i o n constant.
Y e a s t f l o c c u l a t i o n , an e s s e n t i a l phenomenon i n b e e r f e r m e n t a
t i o n , i s i n f l u e n c e d by t h e medium c o m p o s i t i o n , e s p e c i a l l y by t h e
g l u c o s e c o n c e n t r a t i o n , and i s d e l a y e d by t h e m i x i n g e f f e c t o f CO2
p r o d u c t i o n . The t i m e v a r i a t i o n o f t h e s u s p e n d e d y e a s t c o n c e n t r a
t i o n i s t h u s t a k e n a s t h e d i f f e r e n c e between t h e g r o w t h and f l o c
c u l a t i o n r a t e as :
d(X ) T
dt dt
- k
CO,
(x )
s
(3)
K
G1
w i t h ( X ) t h e suspended y e a s t c o n c e n t r a t i o n
5
k f
the yeast f l o c c u l a t i o n constant
(Gl) the glucose c o n c e n t r a t i o n
K
the i n h i b i t i o n constant o f f l o c c u l a t i o n by g l u c o s e
G1
k t h e m i x i n g c o n s t a n t by CO^
m
d
t h e r a t e o f CO^ d e s o r p t i o n
r
co 2

Sugars consumption. Saccharose i s f i r s t hydrolysed i n t o g l u

cose and f r u c t o s e a t the c e l l s u r f a c e , then the four fermentable
sugars, glucose, f r u c t o s e , maltose and m a l t o t r i o s e are a s s i m i l a t e d
at d i f f e r e n t r a t e s depending on the medium composition. Glucose i s
taken up the f a s t e s t as the maltose, m a l t o t r i o s e and f r u c t o s e i n t r a
c e l l u l a r t r a n s p o r t s are i n h i b i t e d by glucose. Assuming that the
s u b s t r a t e s are mainly consumed by the suspended y e a s t s , the f o l l o
wing r a t e expressions are proposed t o describe the time v a r i a t i o n
of fermentable sugars :
d(Gl) _ (Gl) , γ χ 180 d(Sac) m

V U ; K J
dt " " G1 K G 1 + (Gl) S " 342 d t
d(Mal) _ (Mal) 1 , χ , n
V
dt " " Mal K + (Mal M a l
K
G1
d(Sac)
dt " * sac k (
S a c ) (
V ( 6 )
d(Mlt) . (Mit) 1 , γ χ m
"dt - - Mit v
KMit
Z T W- I V , , J W
:
1—+ (cry (
v ( 7 )
1
K K
G1
d(Fr) _ (Fr) 1 , . 180 d(Sac) Y
~dF~~ " * F r K
V
r + (Fr) ~ (GÎT V " 342 "dt ( W
Fr 1 + -pr-
K
G1
with ( G l ) , (Mal), (Sac), ( M i t ) , ( F r ) the glucose, maltose, saccha

rose, m a l t o t r i o s e and f r u c t o s e c o n c e n t r a t i o n .
n e m a x m a r a e
^ G l ' ^Mal* ^ M l t ' V r ^ ^ ^ ^ °^ glucose, maltose, malto
t r i o s e and f r u c t o s e consumption.
f
i i i " u c o s e
^Gl' ^ G l ' ^Gl 9 ^ i n h i b i t i o n constants
k K K K K k i n e t i c c o n s t a n t s
s a c ' G1' M a l ' M l t ' F r -
The time v a r i a t i o n o f the t o t a l fermentable sugar concentra
t i o n i s c a l c u l a t e d by adding the p r e v i o u s l y defined r a t e s o f v a r i a
t i o n o f the c o n c e n t r a t i o n o f the i n d i v i d u a l sugars.
Amino a c i d s consumption. Two r a t e expressions were used. For

threonine, s e r i n e , methionine, i s o l e u c i n e , l e u c i n e , l y s i n e , the
r a t e o f consumption i s p r o p o r t i o n a l t o the r a t e o f yeast growth,
but can be l i m i t e d by the amino a c i d c o n c e n t r a t i o n

(AA) being the amino a c i d c o n c e n t r a t i o n i n the medium

the maximal r a t e of amino a c i d consumption
a k i n e t i c constant.
For other amino a c i d s , such as a s p a r t i c a c i d , glutamic a c i d ,
a l a n i n e , v a l i n e , t y r o s i n e , p h e n y l a l a n i n e , h i s t i d i n e , a r g i n i n e , an
a d d i t i o n n a i i n h i b i t o r y e f f e c t of threonine demonstrated by expe
riments w i t h threonine enriched wort, i s considered
d(AA) (AA) 1 d (
V n n )
dt AA Κ Δ + (AA) ~ TThrT dt v J
Thr
w i t h K.^ the i n h i b i t i o n constant by threonine

Ethanol p r o d u c t i o n
r e l a t e d to the t o t a l r a t e of fermentable sugar consumption
Φ* - - Vs <">
The ethanol to sugar y i e l d , R£/s, was found to s l i g h t l y i n
crease during the fermentation as a r e s u l t of the decrease of the
growth y i e l d . Based on a carbon balance of the sugar conversion
i n t o yeast, ethanol and CO2, the v a r i a t i o n of R^/s can be r e l a t e d
to the p r e v i o u s l y expressed v a r i a t i o n of Rx/s as
1 , °> 4 3 ( Y
x/s - R
x/s }
(12)
R = Y
E/S E/S +
0,52 Y E / s + 0,27 Y C Q 2 / S
a s n e
w i t h Yçyg and Yfjg^/s ^ i n i t i a l ethanol to sugar and CO2 to
sugar y i e l d , r e s p e c t i v e l y .
Other metabolite p r o d u c t i o n . The r a t e of production of aro

matic coumpounds - higher a l c o h o l s , e s t e r s , e t h e r s , a c i d s - i s
assumed p r o p o r t i o n a l to the r a t e of yeast growth. Some, l i k e d i k e -
tones, are r e a s s i m i l a t e d or c h e m i c a l l y transformed a f t e r e x c r e t i o n ,
so t h a t the f o l l o w i n g general equation was used :
Μ . γ ÎÎV . k ( M ) ( 1 3 )
Y k W
dt " M/X dt M
with (M) the metabolite c o n c e n t r a t i o n

Y a r o o r
M/X P P t i o n a l i t y constant between metabolite and yeast
production
k^ a f i r s t order t r a n s f o r m a t i o n constant.

C02 p r o d u c t i o n . The r a t e of carbon d i o x i d e production i s

taken p r o p o r t i o n a l to the t o t a l r a t e of sugar consumption. Produ
ced CO2 i s e i t h e r d i s s o l v e d , or transformed i n t o bicarbonate, or
i s desorbed i n t o the gas phase uhen the d i s s o l v e d c o n c e n t r a t i o n
exceeds the s a t u r a t i o n c o n c e n t r a t i o n . The d e s o r p t i o n of CO2 i s en
hanced by the n a t u r a l convection r e s u l t i n g from the r i s i n g of CO2
bubbles. I t i s thus assumed t h a t the d e s o r p t i o n r a t e i s propor
t i o n a l to the r a t e of CO2 p r o d u c t i o n , i . e . approximately to the
r a t e of fermentable sugar uptake. Consequently the r a t e of v a r i a
t i o n o f d i s s o l v e d CO2 i s w r i t t e n as :
d ( C t
W ρ
= — Κ
diTFS)
d(TFS) qd
< HH CU U
^ °3") ;
Π ) ( wld(TFS) ( .
dt
c o y s ~6t~ " St + k
d[ ( C 0
2d - ) ( C 0
2 )
j^t^ ( }
w i t h (CO^çj) the d i s s o l v e
(CO2)* the s a t u r a t i o n CO2 concentration
(HCO^") the bicarbonate concentration
k^ the d e s o r p t i o n constant
w a s
The CO2 to sugar y i e l d , Rc02/S> observed to s l i g h t l y i n c r e a s e
d u r i n g the fermentation. As p r e v i o u s l y f o r the v a r i a b l e ethanol to
c a n
sugar y i e l d , the value of Rc02/S be r e l a t e d to the i n i t i a l CO2
anc
to sugar y i e l d , Yc02/S * the v a r i a b l e yeast to sugar y i e l d as
R
C0 /S = Y
C02/S
1 + °> 4 3 (Y
x/s - X/S R }
(15)
2
0,52 Y E / 5 + 0,27 Y C 0 2 / S
Bicarbonate i s considered i n e q u i l i b r i u m w i t h d i s s o l v e d CO2, w i t h

the pH decreasing from 5 to 4.2 during the fermentation. Desorbed
CO2 leaves the fermentor when the overhead pressure reaches the
maximal tank pressure. Under these c o n d i t i o n s
ν =
-^[ ν- ν*]^- (ϋ ε (16)
Θ η
^CÛ2 ^ * 9 the CO2 flow r a t e l e a v i n g the fermentor.
The r e s u l t i n g s e t of d i f f e r e n t i a l equations i s n u m e r i c a l l y
i n t e g r a t e d by s t a n d a r t Runge Kutta procedure. The d i f f e r e n t para
meters of the model are determined t o o b t a i n the best agreement
between the theory and the experimental r e s u l t s .
RESULTS AND DISCUSSION

Experiments were f i r s t performed i n l a b o r a t o r y tube fermentors
to study the yeast growth phase. The time change o f t o t a l fermen
t a b l e sugar c o n c e n t r a t i o n , t o t a l and suspended yeast c o n c e n t r a t i o n
during beer fermentation i s shown i n Figure 1. As seen, d e s p i t e a

high l e v e l o f fermentable sugars, yeast growth c o n s i d e r a b l y slows

down and f l o c c u l a t i o n becomes a predominant phenomenon a f t e r 80
hours of fermentation. When the i n i t i a l wort i s enriched i n g l u
cose, one observes i n F i g u r e 2, t h a t yeast growth r a t e i s not
i n f l u e n c e d , but f l o c c u l a t i o n i s delayed u n t i l glucose consumption
i s completed, which confirms p r e v i o u s l y reported dependence of
f l o c c u l a t i o n on glucose c o n c e n t r a t i o n ( 5 ) .
Figure 3 shows the e x p e r i m e n t a l l y determined and the c a l c u l a
ted time v a r i a t i o n of suspended yeast c o n c e n t r a t i o n i n p i l o t p l a n t
fermentor at two d i f f e r e n t temperatures. As seen, decreasing the
temperature decreases the i n i t i a l growth r a t e , delays the onset of
f l o c c u l a t i o n and decreases the r a t e o f f l o c c u l a t i o n . A s a t i s f a c t o
ry modeling i s provided by equations 1 to 3 using a yeast to sugar
y i e l d decreasing from 0,065 to 0,018 w i t h i n c r e a s i n g ethanol con
c e n t r a t i o n , and an Arrheniu typ f temperatur dependenc f th
maximal r a t e s of sugar consumptio
The e x p e r i m e n t a l l y
time of the f i v e fermentable sugars at 14° C i n the p i l o t p l a n t
fermentor are represented i n F i g u r e 4. As expected, glucose, sac
charose, f r u c t o s e , maltose and m a l t o t r i o s e are removed at d i f f e
r e n t r a t e s from the medium. Glucose, which i s p a r t l y produced by
the saccharose h y d r o l y s i s , i s taken up the f a s t e s t , the i n i t i a l
a s s i m i l a t i o n of maltose and m a l t o t r i o s e being repressed by the
presence of glucose.
Figure 5, which represents the experimental and t h e o r e t i c a l
r e s u l t s obtained f o r four of the twenty considered amino a c i d s ,
shows t h a t amino a c i d s present i n the wort are consumed at very
d i f f e r e n t r a t e s . P r o l i n e i s p r a c t i c a l l y not taken up, whereas
threonine i s t o t a l l y consumed a f t e r 70 hours of fermentation.
Others, l i k e l e u c i n e and v a l i n e , are p a r t i a l l y a s s i m i l a t e d during
the yeast growth phase. I t has been v e r i f i e d t h a t amino a c i d s are
not l i m i t i n g at the end of the growth phase.
Ethanol p r o d u c t i o n , which i s shown i n F i g u r e 6 at 10° C and
14° C, i s adequately r e l a t e d to the fermentable sugars consumption
by equations 11 and 12, w i t h an ethanol t o sugar y i e l d i n c r e a s i n g
s l i g h t l y from 0,46 to 0,485. The time v a r i a t i o n of two o f the
other i n v e s t i g a t e d m e t a b o l i t e s , a c e t o l a c t a t e and phenyl e t h a n o l ,
as e x p e r i m e n t a l l y observed and c a l c u l a t e d from equation 13, i s
represented i n Figure 7.
F i n a l l y Figure 8 shows the measured change w i t h time of d i s
s o l v e d CO2 c o n c e n t r a t i o n , fermentor overhead p r e s s u r e , and CO2
o u t l e t fow r a t e at 14° C i n the p i l o t p l a n t . As demonstrated by
the good agreement between the experimental p o i n t s and the c a l c u
l a t e d curves, equations 14 to 16 provide an adequate modeling of
CO2 p r o d u c t i o n , d i s s o l u t i o n and d e s o r p t i o n .
CONCLUSIONS
The experimental and t h e o r e t i c a l r e s u l t s of t h i s study are

expected to improve the q u a n t i t a t i v e understanding of beer fermen-

TIME, h
Figure 1. Time variation of total yeast, suspended yeast, and total fermentable
sugar concentrations in a laboratory scale fermentation. Key: O , total yeast; ·,
suspended yeast; +, total fermentable sugar.
Figure 2. Time variation of glucose and

suspended yeast concentration with stand-
ard and glucose-enriched (+20 g/L)
wort.
Figure 3. Experimental and theoretical

yeast growth and flocculation kinetics at 300
14°C (%) and 10°C (O).

MARC E T AL. Kinetics in Beer Fermentation 497
Figure 5. Experimental and theoretical kinetics of uptake of amino acids at 14°C.

Key: A , threonine; • , valine; |, leucine; O , proline; ·, total amino acids.

Figure 6. Experimental and theoretical kinetics of ethanol production f | and •,)

and total fermentable sugar consumption ( · and O) at 14°C and 10°C. Open
symbols, 10°C; solid symbols, 14°C.
100 200 300

TIME, h
100 200 300

TIME, h
Figure 7. Experimental and theoretical time variation of acetolactate and phenyl-

ethanol concentration at 10°C (O) and 14°C (Φ).

23. MARC E TAL. Kinetics in Beer Fermentation 499
τ Γ τ Γ
ο^ 8h
g Ι" € + * ^ ι ο γ ϊ : ·. ·
^ 4h/ saturation^
ο
100 200 300 Figure 8. Experimental and theoretical

time variation of dissolved C0 , overhead
2
TIME, h pressure, and C0 exitflowrate at 14°C.

2

t a t i o n . An e s s e n t i a l f i n d i n g o f b o t h l a b o r a t o r y and p i l o t p l a n t
experiments i s the decrease i n c e l l u l a r growth a c t i v i t y d u r i n g the
c o u r s e o f the f e r m e n t a t i o n , the growth phase ending a f t e r about
100 h o u r s o f f e r m e n t a t i o n . T h i s f a l l i n c e l l u l a r v i a b i l i t y i s n o t
c a u s e d by s u g a r o r amino a c i d s l i m i t a t i o n s . I t may p a r t l y r e s u l t
f r o m e t h a n o l a c c u m u l a t i o n i n t h e medium, o r as s u g g e s t e d by o t h e r
s t u d i e s ( 6 ) , from l i m i t a t i o n s i n i n t r a c e l l u l a r s t e r o l s which are
no l o n g e r s y n t h e s i z e d u n d e r a n a e r o b i c c o n d i t i o n s . A c c o r d i n g t o
our r e s u l t s , the r a t e o f sugar uptake i s d i r e c t l y p r o p o r t i o n a l t o
t h e s u s p e n d e d y e a s t c o n c e n t r a t i o n , t h e r a t e o f amino a c i d s u p t a k e
and o f some a r o m a t i c m e t a b o l i t e s p r o d u c t i o n i s d i r e c t l y r e l a t e d
t o t h e y e a s t g r o w t h r a t e , w h e r e a s e t h a n o l and C02 p r o d u c t i o n i s
e s s e n t i a l l y p r o p o r t i o n a l to the t o t a l r a t e o f sugar consumption.
As d e m o n s t r a t e d by t h e good agreement between e x p e r i m e n t s
and t h e o r y , t h e t i m e v a r i a t i o f th d i f f e r e n t specie i th
f e r m e n t a t i o n medium c a
s i m p l e model b a s e d on y e a s physica processe
o f y e a s t f l o c c u l a t i o n , CO2 d e s o r p t i o n and CO2 m i x i n g . E s s e n t i a l
i n t h e model i s t h e i n t r o d u c t i o n o f a y e a s t t o s u g a r y i e l d d e p e n
d i n g on t h e m e a s u r a b l e e t h a n o l c o n c e n t r a t i o n t o r e p r e s e n t t h e l o s s
of c e l l u l a r v i a b i l i t y .
F u r t h e r e x p e r i m e n t a l and t h e o r e t i c a l i n v e s t i g a t i o n s a r e b e i n g
done t o s t u d y i n g r e a t e r d e t a i l t h e p o s s i b l e i n f l u e n c e o f o t h e r
key p r o c e s s p a r a m e t e r s , p i t c h i n g r a t e , p r e s s u r e , w o r t a e r a t i o n , on
t h e f e r m e n t a t i o n p r o c e s s and t h e f i n a l b e e r q u a l i t y .
LITERATURE CITED
1. Tepper, P. ; Marc, I. ; Engasser, J.M. ; M o l l , M. ; Duteurtre,

B. i n Current Developments i n Yeast Research (G. Stewart and
I. Russel, eds) 1981, p. 129, Pergamon Press, Toronto
2. Engasser, J.M. ; Marc, I. ; M o l l , M. ; Duteurtre, B.
Proc. 18 th Eur. Brew. Conv. 1981, p. 579
3. Lehuede, J.M. ; Flayeux, R. ; M o l l , M. ; 9 th Technicon I n t e r -
n a t i o n a l Symposium, P a r i s , 1979, p. 1
4. Marc, I. , Thesis Nancy, 1982
5. Amri, M.A. ; Bonaly, R. ; Duteurtre, B. ; M o l l , M.
Eur. J . Appl. M i c r o b i o l . 1979, 7, 227-234
6. Haukeli, A.D. ; L i e , S.
Proc. 17 th Eur. Brew. Conv. 1979, p. 35.

24
Growth Inhibition Kinetics for the Acetone—
Butanol Fermentation
1
JEANINE M. COSTA and ANTONIO R. MOREIRA
Colorado State University, Department of Agricultural and Chemical Engineering,
Fort Collins, CO 80523
The i n h i b i t o r y e f f e c t of each fermentation

product on the cell
of product formatio
butanol fermentation with C l o s t r i d i u m acetobutylicum
ATCC 824. I n h i b i t i o n of cell growth was s t u d i e d by
c h a l l e n g i n g c u l t u r e s with v a r y i n g concentrations of
each product. There was a t h r e s h o l d c o n c e n t r a t i o n
which must be reached before growth i n h i b i t i o n
occurred. This c o n c e n t r a t i o n was found to vary with
each i n h i b i t o r . Above the t h r e s h o l d c o n c e n t r a t i o n ,
there was a l i n e a r decrease of the growth r a t e with
an increase in product c o n c e n t r a t i o n .
Due t o t h e r i s i n g c o s t s a s s o c i a t e d w i t h p r o d u c i n g c h e m i c a l s
f r o m f o s s i l f u e l s , a l c o h o l f e r m e n t a t i o n s b a s e d on r e n e w a b l e
r e s o u r c e s a r e b e c o m i n g more and more a t t r a c t i v e a s an a l t e r n a t i v e
r o u t e f o r o b t a i n i n g commodity c h e m i c a l s . However, a p r o b l e m
common t o many o f t h e s e f e r m e n t a t i o n s i s t h e r e l a t i v e l y l o w c o n -
c e n t r a t i o n of the d e s i r e d product i n the f i n a l fermented b r o t h .
B a c t e r i a l ethanol fermentations stop a t a l c o h o l l e v e l s i n the
r a n g e o f 5-8 w e i g h t p e r c e n t C l ) . A more d r a m a t i c t o x i c e f f e c t i s
o b s e r v e d i n t h e a c e t o n e - b u t a n o l f e r m e n t a t i o n . The t o t a l s o l v e n t s
c o n c e n t r a t i o n i s 2-3 w e i g h t p e r c e n t a t t h e p o i n t where s o l v e n t
l e v e l s a r e t o x i c t o t h e p r o d u c i n g C l o s t r i d i u m (2). Butanol,
b u t y r i c a c i d , and a c e t i c a c i d a r e t h e m o s t t o x i c p r o d u c t s o f t h e
acetone-butanol fermentation. I t has b e e n p r e v i o u s l y shown (3)
t h a t a t b u t a n o l c o n c e n t r a t i o n s i n t h e r a n g e o f 0.10-0.15 M, 50%
i n h i b i t i o n i s o b s e r v e d s i m u l t a n e o u s l y f o r t h e maximum s p e c i f i c
g r o w t h r a t e , t h e n u t r i e n t u p t a k e r a t e , and t h e membrane-bound
ATPase a c t i v i t y . This end-product t o x i c i t y r e s u l t s i n l a r g e
1
Current address: International Flavors and Fragrances, Inc., Union Beach, N J 07735
0097-6156/83/0207-0501$06.00/0

energy e x p e n d i t u r e s f o r p r o d u c t r e c o v e r y as w e l l as i n l a r g e
c a p i t a l i n v e s t m e n t s due t o t h e l a r g e s i z e e q u i p m e n t r e q u i r e d t o
r e a c h an e c o n o m i c a l l y a t t r a c t i v e s c a l e o f p r o d u c t i o n . Conse
q u e n t l y , k i n e t i c d a t a i s needed t o d e v e l o p a b a s i c u n d e r s t a n d i n g
of t h e s e f e r m e n t a t i o n p r o c e s s e s and t o p e r m i t an o p t i m a l d e s i g n
f o r t h e f e r m e n t o r s y s t e m s t o be u s e d f o r t h e p r o d u c t i o n o f t h e s e
chemicals. T h i s paper addresses t h e k i n e t i c s o f end-product
i n h i b i t i o n o f c e l l growth i n t h e acetone-butanol f e r m e n t a t i o n .
M a t e r i a l s a n d Methods
M i c r o o r g a n i s m and C u l t u r e C o n d i t i o n s . C l o s t r i d i u m a c e t o -
b u t y l i c u m s t r a i n ATCC 824 was u s e d i n t h i s s t u d y . Maintenance
c u l t u r e s were grown i n c o r n mash medium f o r 72 h o u r s a t 37 C a n d
then r e f r i g e r a t e d a t 4 t 5 C Th h mediu c o n s i s t e d f
5% (w/v) c o r n m e a l w i t
anaerobic conditions. Th appropriat
volume o f d i s t i l l e d w a t e r f o r one h o u r . The mash was t h e n d i s
t r i b u t e d i n t o s c r e w c a p t u b e s (16 χ 125 mm) a n d a u t o c l a v e d f o r 15
m i n u t e s a t 121 C.
The m i c r o o r g a n i s m was r o u t i n e l y t r a n s f e r r e d a n a e r o b i c a l l y i n
s c r e w c a p t u b e s w i t h 10 m l o f medium c o m p r i s e d o f 5 0 % ( v / v ) t h i o -
g l y c o l l a t e 135C medium ( D i f c o L a b o r a t o r i e s , D e t r o i t , M i c h i g a n ) a n d
50% ( v / v ) s o l u b l e medium c o n t a i n i n g t h e f o l l o w i n g components i n
g/1: K H P 0 , 0.75; Κ 2 Η Ρ Ο 4 , 0.75; MgS04, 0.20; M n S 0 . H 0 , 0.01;
2 4 4 2
FeS04.7H20, 0.01; N a C I , 1.00; c y s t e i n e , 0.50; y e a s t e x t r a c t , 5.00;

g l u c o s e , 60; a s p a r a g i n e . H 2 0 , 2.00; ( N H 4 ) S 0 4 , 2.00.
2
G r o w t h C h a l l e n g e S t u d i e s . The e f f e c t o f t h e f e r m e n t a t i o n
p r o d u c t s o n t h e g r o w t h r a t e o f C I . a c e t o b u t y l i c u m was d e t e r m i n e d
by t h e f o l l o w i n g p r o c e d u r e . A 24-hour o l d c u l t u r e was u s e d a s a
5% i n o c u l u m t o f l a s k s c o n t a i n i n g 200 m l o f 2% (w/v) g l u c o s e
s o l u b l e medium. A f t e r 10-12 h o u r s , t h e s e c e l l s w e r e u s e d a s a 2 0 %
(v/v) i n o c u l u m f o r f l a s k s c o n t a i n i n g s o l u b l e m e d i a w i t h 2% (w/v)
g l u c o s e . A f t e r a l a g p h a s e o f 30-45 m i n u t e s , t h e c u l t u r e s were
c h a l l e n g e d w i t h v a r i o u s c o n c e n t r a t i o n s o f e t h a n o l , b u t a n o l , and
acetone. G r o w t h was m o n i t o r e d b y h o u r l y measurements o f o p t i c a l
d e n s i t y a t 560 nm.
The p r o c e d u r e f o r d e t e r m i n a t i o n o f g r o w t h r a t e s i n t h e
p r e s e n c e o f a c e t i c a n d b u t y r i c a c i d was m o d i f i e d a s f o l l o w s . The
i n o c u l u m was p r e p a r e d b y u s i n g c e l l s f r o m a c o r n t u b e a s a 1 0 %
(v/v) i n o c u l u m f o r t h i o g l y c o l l a t e / s o l u b l e medium c o n t a i n i n g 20 g/1
MES b u f f e r ( 2 - [ N - m o r p h o l i n o ] e t h a n e s u l f o n i c a c i d ) , (Sigma Chemi
c a l Co., S t . L o u i s , MO). A f t e r 10-12 h o u r s , t h e s e c e l l s w e r e
used as a 20% (v/v) inoculum f o r f l a s k s c o n t a i n i n g s o l u b l e media
(20 g/1 g l u c o s e + 20 g/1 MES b u f f e r ) . A f t e r a l a g p h a s e o f 30-45
m i n u t e s , t h e c e l l s were c h a l l e n g e d w i t h v a r i o u s c o n c e n t r a t i o n s o f
a c e t i c and b u t y r i c a c i d .

24. COSTA AND MOREiRA Growth Inhibition Kinetics 503
pH C o n t r o l l e d F e r m e n t a t i o n . A 7 - l i t e r New B r u n s w i c k M i c r o -
f e r m f e r m e n t o r (New B r u n s w i c k s c i e n t i f i c , E d i s o n , NJ) w i t h a
w o r k i n g volume o f 4 l i t e r s was u s e d i n t h i s s t u d y . The a g i t a t i o n
s p e e d was m a i n t a i n e d a t 2 0 0 rpm d u r i n g t h e f e r m e n t a t i o n . The pH
o f t h e f e r m e n t a t i o n s t a r t e d a t 6.2 and was a l l o w e d t o f a l l t o pH
5.0. The pH was t h e r e a f t e r m a i n t a i n e d a t 5.0 by t h e a d d i t i o n o f
1 Ν NaOH on a demand b a s i s . The t e m p e r a t u r e was c o n t r o l l e d a t
37 C
Analysis. The c e l l d e n s i t y was m e a s u r e d a t 560 nm u s i n g a

B a u s c h and Lomb S p e c t r o n i c 2 0 s p e c t r o p h o t o m e t e r .
F e r m e n t a t i o n s a m p l e s were c o l l e c t e d and c e n t r i f u g e d i m m e d i
a t e l y u n d e r r e f r i g e r a t i o n a t 15,000 rpm f o r 1 0 m i n u t e s . The
s u p e r n a t a n t s were s t o r e d i n p l a s t i c v i a l s and i m m e d i a t e l y f r o z e n
for subsequent a n a l y s i s
d i s t i l l e d w a t e r . The c e l l
w a t e r and p l a c e d i n t a r e d c o n t a i n e r s . The c e l l d r y w e i g h t was
d e t e r m i n e d a f t e r d r y i n g f o r 24 h o u r s a t 80 C.
The c o n c e n t r a t i o n o f p r o d u c t s i n t h e s u p e r n a t a n t s was
d e t e r m i n e d on a V a r i a n m o d e l 2400 gas c h r o m a t o g r a p h e q u i p p e d w i t h
a flame i o n i z a t i o n d e t e c t o r . The c h r o m a t o g r a p h had i n s t a l l e d a
6 f t χ 1/8 i n s t a i n l e s s s t e e l t e f l o n l i n e d c o l u m n p a c k e d w i t h
C h r o m o s o r b W-AW coated w i t h 1 0 % AT-1000. The t e m p e r a t u r e o f t h e
c o l u m n was programmed f r o m 1 0 0 C t o 180 C a t a r a t e o f 2 0 C/min.
H e l i u m was u s e d as t h e c a r r i e r gas a t a f l o w r a t e o f 30 m l / m i n .
The f l a m e i o n i z a t i o n d e t e c t o r t e m p e r a t u r e was 230 C. A 10% (w/w)
n - p r o p a n o l s o l u t i o n c o n t a i n i n g 4% (w/w) H 2 S O 4 was u s e d a s an
i n t e r n a l standard.
The g l u c o s e c o n c e n t r a t i o n o f t h e f e r m e n t a t i o n s a m p l e s was
m e a s u r e d by t h e DNSA ( d i n i t r o s a l i c y l i c a c i d ) method (4) u s i n g
g l u c o s e as a s t a n d a r d . Samples were d i l u t e d t o c o n t a i n l e s s t h a n
1 g/1 g l u c o s e .
Product C h a l l e n g e d Growth S t u d i e s . To s t u d y t h e i n h i b i t o r y
f a c t o r s of the acetone-butanol fermentation, the growth r a t e s of
CI. acetobutylicum i n the presence of each fermentation product
were d e t e r m i n e d . The end p r o d u c t s u s e d i n t h i s s t u d y i n c l u d e d
e t h a n o l , b u t a n o l , a c e t o n e , a c e t i c a c i d , and b u t y r i c a c i d . From
the slopes of the l e a s t squares r e g r e s s i o n l i n e s of o p t i c a l
d e n s i t y v s . t i m e d a t a , t h e maximum s p e c i f i c g r o w t h r a t e s i n t h e
p r e s e n c e o f v a r y i n g c o n c e n t r a t i o n s o f e a c h i n h i b i t o r ( y ) were
m
d e t e r m i n e d . The r e s u l t s f o r e a c h f e r m e n t a t i o n p r o d u c t a r e shown
in Figures 1 - 3. T h e r e a p p e a r s t o be a t h r e s h o l d c o n c e n t r a
t i o n w h i c h must be r e a c h e d b e f o r e g r o w t h i n h i b i t i o n o c c u r s . This
c o n c e n t r a t i o n was f o u n d t o v a r y w i t h e a c h i n h i b i t o r s t u d i e d .
Above t h e t h r e s h o l d c o n c e n t r a t i o n , t h e g r o w t h i n h i b i t i o n c a n be
d e s c r i b e d by a l i n e a r r e l a t i o n s h i p o f t h e f o r m :

.ο
Σ!
2
•s-s
δο
* ο
s;
c α
i «
t°
•8 6
8*
ed
Ο
_· s:
U S
si
S "S
•s I
â -S
Ο «ο
§·§
s-S
sΡ
§«
•S ?
s
.00

24. COSTA AND MOREiRA Growth Inhibition Kinetics 505
.ο
s:
* §
%<
1&
u *
° ?
s:
M
•S s:
Si
3
00

BUTYRIC ACID CONCENTRATION (9/1)
Figure 3. Maximum specific growth rate of CI. acetobutylicum when challenged

with various concentrations of butyric acid.

24. COSTA AND MOREIRA Growth Inhibition Kinetics 507
μ 1
= μ [1 - (Ρ - Ρ ) ] forΡ > Ρ C
m m κ ο = ο
Ρ
C o n c e n t r a t i o n s c a u s i n g a 5 0 % r e d u c t i o n i n g r o w t h r a t e were a l s o
determined. A summary o f t h e i n h i b i t i o n d a t a o b t a i n e d i s g i v e n
i n Table I .
Table I. Summary o f G r o w t h I n h i b i t i o n D a t a
Product K p (M) Ρ (M) Cone, a t w h i c h g r o w t h was

ο —
i n h i b i t e d by 50%
M 2/1
Butyric Acid 0.07
Butanol 0.15 0.06 0.15 11.0
Acetic Acid 0.19 0.05 0.13 8.0
Acetone 0.98 0.26 0.88 43.5
Ethanol 1.01 0.26 1.10 51.0
The i n h i b i t i o n c o n s t a n t Kp f o r e a c h p r o d u c t was c a l c u l a t e d
from t h e s l o p e o f the l e a s t squares r e g r e s s i o n l i n e o f y ^ / V ^ v s .
Ρ data. The i n h i b i t i o n c o n s t a n t s f o r e t h a n o l a n d a c e t o n e a r e
approximately t e n times g r e a t e r than t h a t f o r b u t a n o l , a c e t i c
a c i d , and b u t y r i c a c i d . T h i s i s i n d i c a t i v e o f t h e r e l a t i v e l y low
t o x i c i t y o f a c e t o n e a n d e t h a n o l a s compared w i t h t h e o t h e r f e r
mentation products.
F o r e a c h f e r m e n t a t i o n p r o d u c t , t h e P v a l u e was p l o t t e d
Q
a g a i n s t t h e Kp v a l u e a s shown i n F i g u r e 4. The t h r e s h o l d c o n
c e n t r a t i o n was f o u n d t o i n c r e a s e l i n e a r l y w i t h a n i n c r e a s i n g Kp
value. The e q u a t i o n o f t h i s l i n e was d e t e r m i n e d t o b e :
Κ = 3.94 Ρ - 0.03 (2)

Ρ ο
by a l e a s t s q u a r e s r e g r e s s i o n a n a l y s i s . The c o r r e l a t i o n c o e f f i
c i e n t was f o u n d t o be 0.997.
S i m i l a r growth c h a l l e n g e e x p e r i m e n t s were p e r f o r m e d u s i n g
t e r t - b u t y l a l c o h o l and n-hexanol. Although t e r t - b u t y l a l c o h o l
and n - h e x a n o l a r e n o t p r o d u c t s o f t h i s f e r m e n t a t i o n , t h e Kp a n d
P v a l u e s o b t a i n e d f o r t h e s e two a l c o h o l s w e r e f o u n d t o l i e on
0
t h e s t r a i g h t l i n e shown i n F i g u r e 4. L i n d e n e t a l . (3) h a v e
shown t h a t t h e e n d p r o d u c t t o x i c i t y i n t h e a c e t o n e - b u t a n o l f e r
m e n t a t i o n o c c u r s b y a l t e r i n g membrane f u n c t i o n a l i t y . The l i n e a r
r e l a t i o n s h i p b e t w e e n Kp a n d P may i n d i c a t e t h a t t h e i n h i b i t i o n
Q
o f e a c h o f t h e s e v a r i o u s compounds o c c u r s b y t h e same mechanism.

The r e s u l t s o f a 39-hour b a t c h f e r m e n t a t i o n a r e shown i n
F i g u r e 5. The l e v e l s o f a c e t o n e a n d e t h a n o l t y p i c a l l y o b s e r v e d
d u r i n g a f e r m e n t a t i o n (5 g/1 a n d 1.5 g/1 r e s p e c t i v e l y ) w e r e

Figure 4. Relationship between K p and ?0 from the product inhibition studies.

24. COSTA A N D MOREiRA Growth Inhibition Kinetics 509
f o u n d t o be n o n i n h i b i t o r y t o c e l l g r o w t h . The h i g h e s t c o n c e n t r a
t i o n s o f b u t y r i c a c i d and a c e t i c a c i d w h i c h h a v e b e e n o b s e r v e d
d u r i n g f e r m e n t a t i o n a r e i n t h e r a n g e o f 3.5 t o 4.0 g/1. These
l e v e l s o f a c i d s a p p r o a c h t h o s e c a u s i n g 50% i n h i b i t i o n o f g r o w t h .
T y p i c a l butanol concentrations observed d u r i n g a fermentation are
11-12 g/1. Butanol i s the only product t h a t reaches l e v e l s
a c t u a l l y c a u s i n g 50% g r o w t h i n h i b i t i o n .
R e c e n t r e s u l t s r e p o r t e d by L e u n g and Wang (5) show a c e t i c
and b u t y r i c a c i d c o n c e n t r a t i o n s c a u s i n g 50% i n h i b i t i o n o f g r o w t h ,
w h i c h a r e about t w i c e t h e l e v e l s o b t a i n e d i n t h i s work. The
a p p a r e n t d i s a g r e e m e n t b e t w e e n t h e two s e t s o f d a t a c o u l d be due
t o t h e d i f f e r e n t methods u s e d i n e a c h c a s e t o s t a b i l i z e t h e pH o f
t h e f e r m e n t a t i o n m e d i a d u r i n g an a c i d c h a l l e n g e .
The m e t h o d o l o g y u s e d i n t h i s s t u d y i n v o l v e d t h e use o f a
b i o l o g i c a l b u f f e r , MES ( 2 - [ N - m o r p h o l i n o ] e t h a n e s u l f o n i a c i d )
s t a b i l i z e t h e pH. Cell
t a i n i n g MES b u f f e r and t h
d i f f e r e n c e i n g r o w t h r a t e s . T h i s i n d i c a t e s t h a t MES has no
e f f e c t on t h e g r o w t h r a t e o f t h e o r g a n i s m .
The m e t h o d o l o g y t o s t a b i l i z e t h e pH o f t h e f e r m e n t a t i o n
m e d i a r e p o r t e d by L e u n g and Wang (5) i n v o l v e d t h e p r e p a r a t i o n o f
200 g/1 s o l u t i o n s o f a c e t a t e and b u t y r a t e by t i t r a t i o n w i t h NaOH.
I n t h i s manner, t h e c e l l s were e x p o s e d t o Na+ c o n c e n t r a t i o n s i n
t h e r a n g e o f 2-7 g/1. The h i g h c o n c e n t r a t i o n s o f Na+ may h a v e an
o s m o t i c o r membrane s t a b i l i z i n g e f f e c t w h i c h may a c c o u n t f o r t h e
h i g h e r t o l e r a n c e t o a c i d s . However, i n s p i t e o f t h e s e d i s c r e p a n
c i e s , t h i s i n v e s t i g a t i o n supports the c o n c l u s i o n t h a t the con
c e n t r a t i o n o f b u t a n o l i s an i m p o r t a n t p a r a m e t e r i n t h e a c e t o n e -
butanol fermentation.
Attempts t o model the c e l l growth curve d u r i n g a b a t c h f e r
m e n t a t i o n by c o n s i d e r i n g t h e i n h i b i t i o n due t o e a c h p r o d u c t
s e p a r a t e l y r e s u l t e d i n much h i g h e r g r o w t h r a t e s t h a n were a c t u a l l y
observed. Two f a c t o r s c o u l d c o n t r i b u t e t o t h i s d i s c r e p a n c y . On
t h e one h a n d , t h e p r o d u c t i n h i b i t i o n e x p e r i m e n t s were c a r r i e d o u t
u s i n g e a c h f e r m e n t a t i o n p r o d u c t s e p a r a t e l y . However, i n an a c t u a l
fermentation the products are present together. T h i r t e e n hours
i n t o t h e f e r m e n t a t i o n , as shown i n F i g u r e 5 , t h e c o n c e n t r a t i o n o f
e a c h p r o d u c t i s b e l o w t h e t h r e s h o l d c o n c e n t r a t i o n when g r o w t h
i n h i b i t i o n was d e t e r m i n e d t o o c c u r . T h i s i n d i c a t e s t h a t no g r o w t h
i n h i b i t i o n s h o u l d be o b s e r v e d . However, t h e c e l l g r o w t h r a t e a t
t h i s t i m e was d e t e r m i n e d t o be o n e - h a l f o f t h e maximum s p e c i f i c
c e l l g r o w t h r a t e . T h i s i n d i c a t e s t h a t some s y n e r g i s m o c c u r s
among t h e s e v e r a l f e r m e n t a t i o n p r o d u c t s and i t i s p r o b a b l y t h e
t o t a l concentration of products which i s important i n determining
t h e t o x i c e f f e c t on g r o w t h r a t e .
The o t h e r f a c t o r c o u l d be t h e d i f f e r e n c e i n t o x i c e f f e c t s o f
p r o d u c e d s o l v e n t s as o p p o s e d t o a d d e d s o l v e n t s . Novak e t a l . (6)
have shown t h a t e t h a n o l p r o d u c e d d u r i n g a b a t c h f e r m e n t a t i o n o f
S a c c h a r o m y c e s c e r e v i s i a e i s more i n h i b i t o r y t h a n e t h a n o l added t o
the fermentation. Therefore, i t i s p o s s i b l e t h a t the i n h i b i t i o n

s sa
δ·
ο
-Ci
ε §
JD . -s:
s «ο
ci
>>«*§
1*1
Η<
Ο Ο
£ ,3
^ ©Ο
«ο
la
"Ι
s:
§δ

24. C O S T A A N D MOREiRA Growth Inhibition Kinetics 511
p a r a m e t e r s d e t e r m i n e d i n t h i s s t u d y , based on added s o l v e n t s ,
m i g h t be l e s s s e v e r e t h a n t h e a c t u a l v a l u e s due t o t h e s o l v e n t s
produced during the fermentation.
Conclusions
From t h e s e s t u d i e s o f g r o w t h i n h i b i t i o n a n d f e r m e n t a t i o n
k i n e t i c s i n the acetone-butanol fermentation, the f o l l o w i n g
c o n c l u s i o n s may be made:
1. A t l e v e l s normally observed during fermentation

a. B u t a n o l , a c e t i c a c i d , and b u t y r i c a c i d a r e i n h i b i t o r y t o
c e l l growth.
b. A c e t o n e a n d e t h a n o l show no i n h i b i t i o n e f f e c t s .
2. F o r each f e r m e n t a t i o
t i o n below which n growt
a l i n e a r decrease i n growth r a t e i s observed w i t h an i n c r e a s e
i n product concentration.
3. There i s a l i n e a r r e l a t i o n s h i p between t h e t h r e s h o l d concen

t r a t i o n ( P ) a n d t h e i n h i b i t i o n c o n s t a n t (Kp) f o r e a c h
Q
fermentation product.
4. P r e l i m i n a r y o b s e r v a t i o n s seem t o i n d i c a t e t h a t t h e g r o w t h
i n h i b i t i o n c a u s e d by s o l v e n t s p r o d u c e d d u r i n g f e r m e n t a t i o n
i s d i f f e r e n t from t h e i n h i b i t i o n caused by e x t e r n a l l y added
solvents.
Legend o f Symbols
Κ i n h i b i t i o n constant (M)
Ρ —
Ρ p r o d u c t c o n c e n t r a t i o n (M)
P^ threshold product concentration (M)
μ maximum s p e c i f i c g r o w t h r a t e (hr~l)
m
μί maximum s p e c i f i c g r o w t h r a t e i n p r e s e n c e o f
1
i n h i b i t o r (hr" )
Acknowledgments
P a r t i a l s u p p o r t f o r t h i s r e s e a r c h was p r o v i d e d b y t h e U.S.
D e p a r t m e n t o f E n e r g y u n d e r S u b c o n t r a c t No. ΧΚ-φ-9059-l. The
a u t h o r s a l s o w i s h t o a c k n o w l e d g e D r . James C. L i n d e n f o r h e l p f u l
discussions.
Literature Cited
1. Reed, G.; Peppier, H. J . "Yeast Technology;" A v i P u b l i s h i n g

Co.: Westport, 1973; 185.

2. S t e e l , R. "Biochemical Engineering: U n i t Processes in

Fermentation;" Macmillan: New York, 1958; 125.
3. Linden, J . C.; Ulmer, D. C.; Moreira, A. R. "A Mechanism f o r

A l i p h a t i c Alcohol-Induced T o x i c i t y in C l o s t r i d i u m acetobuty-
licum, " presented a t the 182nd N a t i o n a l Meeting American
Chemical S o c i e t y , D i v i s i o n o f M i c r o b i a l and Biochemical Tech-
nology New York, New York. 1981.
4. M i l l e r , G. C. A n a l . Chem. 1959, 31, 426.
5. Leung, J. C. Y.; Wang, D. I . C. Proc. 2nd World Congress o f

Chemical Engineering. 1981, 1, 348.
6. Novak, M.; Strehaiano P.; Moreno M.; Goma G Biotechnol

Bioeng., 1981, 23
R E C E I V E D August 2, 1982

INDEX
A Biocatalysts—See Immobilized cell

catalysts
Acetone-butanol fermentation, growth
Biochemical engineering—See
inhibition kinetics
Biotechnology
experimental methods 502, 503
Biomass production
growth challenge experiments ... 503-508
rate equations 468-469/
p H controlled 509,510/
thermochemical optimization ... 463-488
Activator mechanisms of gene
regulation 10
Aerobic growth without product
Biotechnology
formation, thermodynamic
functional hierarchies 355-357/
theory 304-311
history 3
Agrobacterium tumejaciens, as
structural hierarchies 355-357/
example of demand theory 22
Bubble columns
Airlift columns—See Reactor equip
mass transfer 345, 346
ment performance, systems with
performance as bioreactor 345, 346
stationary internals
Butanol-acetone fermentation—See
Allosteric inhibitors 74
Acetone-butanol fermentation
Amensalism
mixed cultures 209
scheme for 211/ C
Anaerobic growth, thermodynamic Candida tropicalis, competition in
theory 314 mixed cultures 208
Antagonism in mixed cultures 209 Carbon source management in
Antibiotics optimizing fermentations 180-198
optimizing fermentation processes 53-67 Catalysts—See Immobilized cell
production, penicillin 65 catalysts
Antiterminator mechanisms of Cell control system, cybernetic
gene regulation 11/ perspective 165-176
Cell cycle, models 137
Β Cell division cycle, Saccharomyces
cerevisiae 151, 152/
Bacillus brevis Cell mass production, optimization 180-186
optimization of fermentation Cell, as control system 165-176
processes 53-67 Cellulase production by solid
in vivo inactivation of enzymes in . 55 state fermentation 421-442
Bacillus cereus, competition in mixed Cellulose, hydrolysis of, kinetic
cultures 208 analysis of enzyme reaction 37
Bacteria, enteric, as example of Chaetomium cellulolyticum, growth
demand theory 15 on corn stover 429-432
Baker's yeast—See Saccharomyces Chemiosmotic coupling 324, 325/
cerevisiae See also Ion pumps
"Bang-bang" policy 168 Chemiosmotic hypothesis of
Beer fermentation oxidative phosphorylation 324
comparison of kinetic theory Chemosensory movement properties—
and experiment 494-500 See Chemotaxis
kinetic analysis 490-494 Chemotactic responses of various
kinetics of yeast growth and species 268,269/
metabolism 489-500 Chemotaxis 265, 266
515

Chemotaxis— Ε
Continued
and competition in mixed cultures 276/ Eccrinolysis in mixed cultures 214
Chromatography, size exclusion, Ecology, and demand theory of
procedure 445-449 gene regulation 15
Clostridium acetobutylicim, acetone- Ecosystems, effects of cell motility
butanol fermentation 501-512 on populations 265-293
Clumping, mass transfer in viscous Efficiency
systems with microbial network energetic, of ion pumps 326
structure 351 ion pumps 323, 326
Colpoda steinii, feeding behavior thermodynamic, for non-
in mixed cultures 215, 216, 219 equilibrium 300-304
Commensalism, in mixed cultures 210-212 Electrodes, enzyme 46, 48, 50/
Competition in mixed cultures 205-209 Electrogenic pumps—See Ion pumps
effect of cell motility 286-289 Enteric bacteria
mathematical models 206-209 demand theory 15
Continuous culture gene regulation 15, 18/-21/
Hansenula polymorpha 187, 188/ and demand theory of gene
single-cell protein productio
Continuous reactors
rate laws 466, 468 Enzymatic hydrolysis of starch 443-461
reaction mechanism 464-467/ effect of starch recrystallization 457, 458/
Control effect of temperature 448
See also Regulation Enzyme activity
of reactor in reactor design 370-373/ mechanisms of regulation 73/
Cooper-Helmstetter model 137, 138, 140 physiological patterns affecting 71,72/
Cornell model 100-102/, 114-121/ Enzyme amount
predictions 116-121/ mechanisms of regulation 77-83
Crowding in mixed cultures 222, 223 physiological mechanisms of
Cybernetic approach, diauxic growth 164 regulation 75
Cybernetic perspective physiological patterns of
cell control system 165-176 regulation 76, 77
microbial growth 161-178 Enzyme electrodes 46, 48, 50/
Cytometry, flow 135, 139/ Enzyme inactivation in vivo 55, 57
Enzvme systems, kinetics 27-52
Henri equation 27-32
D
Enzyme-catalyzed reactions, kinetics
Demand theory immobilized enzymes 38
Agrobacterium tumefaciens as particulate substrates 32-37
example 22 soluble substrates 27-32
enteric bacteria as example 15 Enzymes
gene regulation 3, 7 See also specific enzymes
development 13 degradation of insoluble substrates,
and physiology 16/ mass transfer 345
Depolymerization of cell wall, kinetic immobilized cells as catalysts—See
analysis of enzyme reaction 35 also Immobilized cells
Design—See Reactor design preparation and reaction
Diauxic growth performance 377-392
dissolved oxygen as indicator 171/ comparison of kinetics, with
example of cybernetic approach .... 164 soluble enzymes 40/, 42/, 43
Dictoystelium discoideum kinetics 38-48, 343, 345
feeding behavior in mixed cultures 215 production, strategies for
predator for Escherichia coli 254 optimization 189-191
Didinum nasutum, feeding behavior production of heparinase by
in mixed cultures 214,215 Flavobacterium heparinum 191-192/
D N A synthesis in bacteria 140 strategies for optimization 191-192/

INDEX 517
Enzymes—Continued Gene regulation

production of maltase by in enteric bacteria 15, 18/-21/
Saccharomyces italicus 189-191 demand theory 3, 7
production optimization 186-192 Gene regulation mechanisms
reducing in vivo inactivation 53-67 activator 10/
Escherichia coli antiterminator 11/
immobilized in epoxide beads, as negative 14/, 80
immobilized cell catalyst 384-388/ positive 14/, 80
mathematical models 98, 99 proterminator 12/
transient response 123 repressor 9/
mechanisms of enzyme regulation 80, 82 Generalist microorganism,
mechanisms of regulation of Thiobacillus A 2
enzyme activity 74 as example 232, 234/, 235/
model of single-cell kinetics 138-143 Generalists in microbial population
in mixed cultures 209 interactions 229-251
and parasitism in mixed cultures 221 Generalists and specialists—See
physiological patterns in enzyme Specialists and generalists
regulation 76, 77 Glucose oxidation, as example of
population, mathematical growt
model 138-14
as prey for Dictyostelium factors affecting inactivation 57-61
discoideum 254 inactivation in vivo 55, 56/, 59, 60
regulation of enzyme activity in 73/ mechanism 62, 63/
structural genes 84 kinetics of inactivation 57, 58/
Growth inhibition—See Kinetics,
growth inhibition
F Growth of microorganisms in solid
state fermentation of lignocel-
Feeding luloses for protein and cellulase
See also Prédation production 421-442
in mixed cultures 214-220 Growth in multiple substrate
mathematical model 219 systems 166-176
Fermentation—See Solid state Growth patterns of Phycomyces
fermentation sporangiophores 403-420
Fermentation processes, measurement 406-407
optimization 179-198 Growth
through control of in vivo inactiva- mathematical models of
tion of biosynthetic enzymes 53-67 single cells 93-133
Flavobacterium heparinum and product formation 179-198
optimization of production 191, 192/ microbial, cybernetic
Flow cytometry perspective 161-178
cell division of Schizosaccharomyces and product formation 179-198
pombe 144, 146, 147/ organisms with rigid
and frequency function 137, 139/ exoskeletons 415-417
measurement and analysis 155
Flow in reactors, effect on interparticle H
mass transfer rates 339-342
Frequency function, experimental Halobium, measurement of potential
changes in membrane 329
determination 137, 139/
Hansenula polymorpha
Fungal growth, mechanism in solid
continuous culture 187, 188/
state fermentation 426-437
single-cell protein source 187, 188/
strategy for optimization of
growth 187, 188/
G Henri equation, enzyme conversion
Gene control models 3-25 of soluble substrates 27-32

Heparinase, optimization of Ion pumps—Continued

production 191-192/ measurement of potential changes
Heat transfer in reactor design 368, 369 by light absorption 329
History Isoleucine, pathway for formation 85/, 86/
biotechnology 3 Isosteric inhibitors 74
enzyme kinetics 28
mathematical models of Κ
cell population 94-96
solid state fermentation 421-423 Kinetic(s)
Hydrodynamics in reactor design 358-359 in biological reactor design 335-354
Hydrolysis of cellulose, kinetic of enzyme systems 27-52
analysis of enzyme reaction 37 Henri equation 27-32
Hydrolysis of particulate substrate, expeditiousness of ion pumps 323-331
kinetic analysis of enzyme growth inhibition, acetone-
reaction 32 butanol fermentation 501-512
Hydrolysis of starches by amylase, experimental methods 502, 503
kinetic analysis of enzyme growth challenge experiments 503-508
reaction 36 p H controlled 509,510/
Hyphomicrobiwn, and Pseudomonas
interactions in mixed culture
of yeast growth and metabolism
in beer fermentation 489-500
I Kinetic analysis
biological systems by power-
Immobilized cell catalysts law formalism 4-7
deactivation 390, 391 derivation 5-7
effectiveness and Thiele evidence 6
modulus 383-386 complex biological systems 4
effectiveness, cleavage of peni enzyme reactions
cillin G by Escherichia hydrolysis of cellulose 37
coli 384-388/ hydrolysis of particulate
operational stability 390, 391 substrate 32
preparation entrapment hydrolysis of starch by amylase . 36
in polymeric carriers by iono- general methods 3-25
tropic gelation of poly- Klebsiella aerogenes
electrolytes 379/, 380, 381/ physiological patterns of
in polymeric carriers by polycon- enzyme regulation 76, 77
densation of epoxy as prey for Tetrahymena
resins 380-382/ pyrijormis 254
Immobilized enzymes
enzyme electrode as example 46, 48, 50/
kinetics 38-48, 343, 345
comparison with soluble Lactobacillus plantarum, commensal
enzymes 39-43 interaction in mixed cultures 212
pancreatic lipase 43 Lignocelluloses, fermentation—See
optimization 386 Solid state fermentation
Immobilized cell catalysts, prepa
ration and reaction M
performance 377-392
Inhibition of product—See Maltase, optimization of production 189-191
Acetone-butanol fermentation Mass transfer
Interfacial phenomena, effect in See also Transport, Transfer
reactor design 338-340/ in reactor design 359-365
Interparticle mass transfer rates 342 with reaction 360, 361/
Intraparticle bioreaction rates 342 effect of viscosity 365-367/
Ion pumps gas phase phenomena 326
coupling models 327 interfacial coefficients 360
energy transducers 323-331 intraparticle 362

INDEX 519
Mass transfer in reactor Metabolite production—

design—Continued Continued
volumetric coefficient 363, 364/ strategies for optimization 193-196
Mathematical models Microbial growth
of beer fermentation kinetics 490-494 cybernetic perspective 161-178
comparison with experiment 494-500 and product formation,
cell motility 265 optimizing 179-198
Microbial populations 135-158
of cell population
Microbial regulatory mechanisms 71-92
Cornell model 123, 125/
Micrococcus lysodeikticus
description and history 94-96
comparison of immobilized and
of commensalism in mixed
soluble enzymes in lysis 42
cultures 212
kinetic analysis of enzymatic lysis . 35
of competition in mixed
Microorganisms as energy
cultures 206-209
transducer 297/
of competition of Thiobacillus
Mixed cultures
A 2 , T. neapolitanus and
competition of chemotactic
Spirillum G 7 243/-245/
strains 276/
comparison with experimen
Cornell single-cell model
effect of motility on compe-
examples of ideal model 96-99 tition 286-289
predictions 116-121/ interactions 201-227
transient response 118, 122-125 interactions of specialists and
rationale 93,94,96 generalists 229-251
metabolism 135-158 predator-prey dynamics 253-254
of Escherichia coli 98, 99 Thiobacillus A 2 and Spirillum Gl 237
effect of cell motility on popu- Thiobacillus A 2 with T. neapoli-
lation 277-289 tanus, Spirillum G7, or
of feeding behavior in mixed both 230-249
cultures 219 Mixing in reactors
of growth of single cells 93-133 See also Reactor equipment per-
of interactions of mixed formance, mechanically
cultures 201-227 stirred tanks
of population growth, Escherichia effect on interparticle mass
coli 138-143 transfer rates 342
Models
predator-prey dynamics 253-254
See also Mathematical models
Mechanisms of gene regulation
cell cycle 137
activator 10/ gene function 3-25
antiterminator 11/ stochastic, of Phycomyces
proterminator 12/ sporangiophores 407-415
repressor 9/ Molecular size distribution of
Medium conservation equations 135 starch during enzymatic
Metabolic models, single-cell 135-158 hydrolysis 443-461
Metabolite excretion rates Motility
equations 470 chemotactic responses of various
thermochemical optimization 463-488 species 268, 269/
effect in mixed cultures 265-293
Metabolite flow
experimental observations 270-276/
enzymic mechanisms of
mathematical model 277-289
regulation 73/
peritrichously flagellated
physiological patterns of bacteria 266,267/
regulation 72 Multiple substrate systems,
Metabolite production growth 166-176
optimization 186-192 Mutation
penicillin by Pénicillium Escherichia coli strains as immo-
chrysogenum 193-196 bilized cell catalysts 386, 388/

Mutation—Continued Penicillin production—

and parasitism in mixed cultures .. 221 Continued
Saccharomyces italicus 189, 191/ strategies for optimization 193-196
scheme for producing strain of Phosphorylation, chemiosmotic
Serratia marcescens 87-90 hypothesis of oxidative 324, 325/
as tool for biochemical engineer 83-90 Phycomyces sporangiophores
Mutualism in mixed cultures 213-214 growth patterns 403-420
sensory apparatus 403-420
Physiology and demand theory 16/
Ν
Polyporus versicolor, destruction
Negative mechanisms of gene of wood 428-429
expression regulation 13 Population(s)
dissolved oxygen in Pseudo
monas ovalis colonies 395-401
Ο interactions in mixed culture 201-227
Optimal strategy in micro classification 202
organisms 161-178 of mixed specialists and
Optimization generalists 229-251
immobilized cell catalyst, Aceto
bacter simplex 386
microbial growth and product metabolic models 135-158
formation 178-198 Population balance
of cell mass 186-192 equations 135, 136, 146
of enzymes 186-192 Poria monticola, destruction of
of metabolites 186-192 wood 428
thermochemical, of microbial Positive mechanisms of gene
biomass-production and expression regulation 13
metabolite-excretion rates 463-488 Power-law formalism, for kinetic
and in vivo inactivation of bio- analysis of biological systems .... 4-7
synthetic enzymes 53-67 derivation of 5-7
Optimum temperature for biological evidence for 6
processes, relation to substrate Prédation dynamics, microbial 253-264
concentration 463-488 Predator-prey dynamics
Osmotic processes—See Chemios Dictyostelium discoideum as
motic coupling predator for Escherichia
Oxidation, glucose, immobilized coli 255-263/
enzyme reaction 45,47/ mathematical model 253-254
Oxygen transfer in mold pellets 343, 344/ Process synthesis at reactor level,
Oxygen, dissolved in reactor design 367
contours in Pseudomonas ovalis Propionibacterium shermanii, com-
colonies 395-401 mensal interaction in mixed
as indicator in diauxic growth 171/ cultures 212
Protein amount, physiological pat-
terns of regulation 76, 77
Protein formation, mechanisms of
Ρ regulation 77-83
Pancreatic lipase, comparison of Protein
kinetics of immobilized and microbial, reactor design 355
soluble enzymes 43 single-cell
Paramecium and Didinum nasutum continuous culture 185-187
in mixed cultures 214,215 Hansenula polymorpha 187, 188/
Parasitism in mixed cultures 221 strategies for optimizing
Penicillin G , cleavage by Escherichia production 185-187
coli, immobilized cell from lignocelluloses by solid
catalyst 384-388/ state fermentation 421-442
Penicillin production Proterminator mechanisms of
computer control 193-196 gene regulation 12/

INDEX 521
Protocooperation in mixed S
cultures 213-214 Saccharomyces cerevisiae
Proton pumps—See Ion pumps cell division cycle 151, 152/
Pseudomonas and Hyphomicrobiwn, fed-batch fermentation 181-184
interactions in mixed cultures 214 computer control 181-184
Pseudomonas ovalis colonies, dis- strategy for fermenter
solved oxygen contours 395-401 operation 181-184
mathematical model of growth
R kinetics 150-154
Reactor design Saccharomyces italicus
See also Reactor design fundamentals mutation 189,191/
constraints 335, 336 optimization of maltase
effect of flow 339-342 production 189, 191/
effect of interfacial phe- Scale-up in reactor design 370, 374
nomena 338, 340/ Schizosaccharomyces pombe
effect of interparticle transfer cell cycle 145/
rates 339-342 flow cytometer measure-
kinetics and transport phe- ments 144, 146, 147/
nomena 335-35
effect of mixing 342 kinetics 142,144-150
rate controlling steps 336-338 trajectory tracking kinetics 149/, 150
stagnant reactor 339 Serratia marcescens
systems approach 355-358 mutation for production of
Reactor design fundamentals 335-376 isoleucine 87/
heat transfer 368, 369 production of isoleucine 84-86/
hydrodynamics 358-359 Sewage treatment 250
mass transfer 359-365 See also Thiobacillus A 2 , T.
process synthesis at reactor level 369 neapolitanus, Spirillum G 7
reactor control 369-373/ Single-cell kinetics, steady-state
scale-up 370 models 138
Reactor equipment performance Single-cell metabolic models 135-158
bubble columns 345, 346 Single-cell protein
mechanically stirred tanks 347-351 continuous culture 185-187
systems with stationary internals 346 optimizing production 185-187
Reaction mechanism source, Hansenula polymorpha 187, 188/
modified Michaelis-Menten 464-467/ Size distribution of starch
for process whose rates determination by size exclusion
saturate in substrate chromatography 445-449
concentration 464-467/ during enzymatic hydrolysis ... 443-461
Reaction performance, immobilized Size exclusion chromatography,
cells as catalysts—See Immo- procedure 445-449
bilized cells Size of starch
Reaction rate laws effect of recrystallization 457, 458/
development, for processes whose effect of temperature 448
rates saturate in substrate Solid state fermentation
concentration 466, 468-477 of grains 423
thermal sensitivity 470-477 of lignocellulose 421-442
Reaction rates, intraparticle 342-345 choice of microorganisms 426
Reactors mechanism of fungal growth 426-437
effect of start-up conditions on physiological aspects 437,438
growth of Schizosac- pretreatment of lignocelluloses 424-426
charomyces pombe 146, 147/ requirements 437, 438
microbial, population balance Spirillum G7 as example 232, 234/, 235/
equations 135, 136 Thiobacillus neapolitanus as
Regulatory mechanisms, microbial 71-92 example 232, 234/, 235/
Repressor mechanisms of gene Specialists and generalists
regulation 9/ growth rate 230,231

Specialists and Generalists—Continued Thermodynamics—

model bacteria 233/ Continued
Specialists in microbial population thermodynamic constraints 299
interactions 229-251 balance equations 298
Spirillum G 7 Thermodynamic constraints 299
See also Thiobacillus A 2 Thermodynamic efficiency 300-304
as example of specialist 232, 234/, 235/ of growth, constant efficiency
metabolism 233/ hypothesis 316
specific growth rates 236/ Thiobacillus A 2
SSF—See Solid state fermentation specific growth rates 236/
Starch, effect of recrystallization competition with both T. neapoli-
on molecular size 457, 458/ tanus and Spirillum Gl
Starch, effect of temperature on 232, 237-241/, 247, 249/
molecular size 448 mathematical model 242-245/
Starch, hydrolysis of as example of generalist .232, 234/, 235/
as example of kinetic analysis metabolism 233/
of enzyme reaction 36 Thiobacillus neapolitanus
physical model 459, 460/ See also Thiobacillus A 2
Starch, molecular size distributio
during enzymatic hydrolysi
Steady-state models of single-cell specific growth rates 236/
kinetics 138 Time-series analysis 418,419
Strategies applied to Phycomyces blakeslee-
optimizing microbial growth and anus sporangiophores 403-420
product formation 179-198 Trajectory tracking kinetics, in
survival, specialist and generalist Schizosaccharomyces pombe 149/, 150
populations 250 Transfer rates in reactors
Symbiosis in mixed cultures 222 dependence on flow and mixing 339-342
Systems approach to reactor design— interparticle 339-342
See Reactor design Transfer, mass
in airlift columns 346
in bubble column reactors 345, 346
Τ in mechanically stirred reactors 347-351
Tetrahymena pyriformis Transfer, oxygen
feeding behavior in mixed in mold pellets 343-344/
cultures 215-221 in Pseudomonas ovalis colonies 395-401
predator for Klebisiella aerogenes .. 254 Transient experiments, test of
Thermal sensitivity of process rates, mathematical models 143/
relation of substrate con Transient response in single cell,
centration 477-484 mathematical model 118, 122-125/
Thermal sensitivity of reaction rate Transport phenomena, in biological
laws 470 reactor design 354-354
Thermochemical optimization—See
Optimization, thermochemical U
Thermodynamic(s) Urease electrode 46
macroscopic, and growth and
product formation of micro
organisms 295-322
of microorganisms 296 Valine, pathway for formation 85/, 86/
nonequilibrium, of ion pumps 329
nonequilibrium, for living Y
organisms 295-322 Yeast growth and metabolism in beer
aerobic growth without fermentation
product formation 304-311 comparison with experiment 494-500
growth with electron acceptors kinetics 489-500
other than oxygen 312-313 theory 489-494


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In Foundations of Biochemical Engineering; Blanch, H., et al.;

Harve W Blanch EDITOR

Based on the 1982 Winter Symposium

of the ACS Division of Industrial

and Engineering Chemistry jointly

sponsored by the Division of Microbial

and Biochemical Technology,

January 17-20, 1982

ACS SYMPOSIUM SERIES 207

AMERICAN CHEMICAL SOCIETY

In Foundations of Biochemical Engineering; Blanch, H., et al.;

In Foundations of Biochemical Engineering; Blanch, H., et al.;

David L. Allara Robert Ory

Robert Baker Geoffrey D. Parfitt

Donald D. Dollberg Theodore Provder

Brian M . Harney Charles N. Satterfield

W. Jeffrey Howe Dennis Schuetzle

Herbert D. Kaesz Davis L. Temple, Jr.

Marvin Margoshes Charles S. Tuesday

Donald E. Moreland C. Grant Willson

In Foundations of Biochemical Engineering; Blanch, H., et al.;

In Foundations of Biochemical Engineering; Blanch, H., et al.;

THIS V O L U M E IS BASED on the 1982 Winter Symposium. Following recent

• the interdisciplinary nature of biotechnology. We thus made every

In Foundations of Biochemical Engineering; Blanch, H., et al.;

In Foundations of Biochemical Engineering; Blanch, H., et al.;

In Foundations of Biochemical Engineering; Blanch, H., et al.;

Models of Gene Function

Alternative molecular designs for gene control

The primary purpose o f t h i s symposium i s t o have b i o l o g i s t s

In Foundations of Biochemical Engineering; Blanch, H., et al.;

Egyptian tombs (1)· Of course, t h i s was a very e m p i r i c a l a r t t h a t

In Foundations of Biochemical Engineering; Blanch, H., et al.;

found i n t e c h n o l o g i c a l systems are very d i f f e r e n t from those found

D e r i v a t i o n o f t h e B a s i c Equations. The b a s i c equations are

In Foundations of Biochemical Engineering; Blanch, H., et al.;

The behavior of complex b i o l o g i c a l systems i s d e t e r m i n e d by

Exact Representation. Although the power-law f o r m a l i s m was

Justification. The power-law formalism i s j u s t i f i e d by f o u r

In Foundations of Biochemical Engineering; Blanch, H., et al.;

deduced with t h i s formalism and t e s t e d a g a i n s t experimental

Demand Theory o f Gene Regulation

The c e n t r a l theme t h a t I would l i k e t o emphasize i n t h i s

In Foundations of Biochemical Engineering; Blanch, H., et al.;

Molecular Design o f Gene C o n t r o l

By the c o n c l u s i o n o f t h i s paper you w i l l come t o a p p r e c i a t e t h e

A l t e r n a t i v e M o l e c u l a r Designs. Let us begin w i t h the

In Foundations of Biochemical Engineering; Blanch, H., et al.;

SG| SG 2 |Q| SGi SG 2

• OPERON- RNA transcript ^

Ï1 IPIOJ SG| I S62 \T\j { |R J jf SGi SG 2

• OPERON- RNA transcript ^

In Foundations of Biochemical Engineering; Blanch, H., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

SGi SG 2 1 Π » Π ( SGi I SG 2 1τ|$

In Foundations of Biochemical Engineering; Blanch, H., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Figure 3. Antiterminator control of transcription termination. A regulatory gene, R, codes for an

In Foundations of Biochemical Engineering; Blanch, H., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Figure 4. Proterminator control of transcription termination. A regulatory gene, R, codes for a

In Foundations of Biochemical Engineering; Blanch, H., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1983.