BIOTECHNOLOGY LETTERS

Vobune 15 No.5 (May 1993) pp.499-504

Received4th March

A COMPARATIVE STUDY OF GLUCOSE CONVERSION TO ETHANOL

by Zymumonas mobilis and a RECOMBINANT Escherichia cdl

Hugh G. Lawford* and Joyce D. Rousseau

Department of Biochemistry
University of Toronto
Toronto, Ontario, Canada ~5s 1A8
FAX (416) 978-8548

SUMMARY

Zymomonas mobilis and recombinant Escherichia coli B (pLOI297) were compared in

side-by-side bat& fermentations using a synthetic cellulose hydrolysate (glucose/salts)
medium with pH control at 6.0 and an inoculation cell density of 35-50 mg dry wt. cells/L
At a nominal glucose concentration of 696, both cultures achieved near maximal theoretical
ethanol yields; however, the 2. mobilis fermentation was complete at 13h compared to 33h
for the E.coli fermentation. With approx.1295 glucose, the 2. mobilis fermentation was
complete in 20h with a process yield of 0.49 g ethanol/g added glucose compared to the E.
coli fermentation which remained 20% incomplete after 6 days resulting in a process yield of
only 0.32 g/g. Nutrient supplementation (log tryptone/l) resulted in complete fermentation
of 12% glucose (pH 6.3) by the recombinant E. coli in 4 days, with a yield of 0.48 g/g.

INTRODUCTION

The growing demand for fermentation ethanol as an renewable, “environment friendly”,

alternative transportation fuel, means that the industry will soon be forced to expand beyond
its present starch and yeast-based technology (Wyman et al., 1990). Various bacterial
ethanologens are being investigated in relation to the production of ethanol from
lignocellulosic biomass (Tolan & Finn, 1987a and 1987b; Ingram et aZ., 1990; Lawford &
Rousseau, 1991a, 1992; Lacis & Lawford, 1989; Lynd, 1989). Zymomonas mobilis is
generally recognized as being superior to brewer’s yeast with respect to both glucose
conversion efficiency and productivity (Rogers er al., 1982; Baratti Bt Bu’L.ock, 1986;
Lawford, 1988), but both organisms are similarly disadvantaged with respect to the
fermentation of lignocellulosic feedstocks because of their inability to utilize the pentose
sugars that are present in hemicellulose, Whereas Escherichia coli is able to efficienctly
metabolize all the sugars present in lignocellulose, ethanol is only produced in very low yield
(Gottschalk, 1985). The ethanol production pathway of Z. mobilis (pdc and udh genes) has
been cloned and E. coli recombinants have been genetically engineered which combine and
exhibit the positive traits of both organisms such that the ethanol yield from both hexose and
pentose sugars closely approaches the theoretical maximum (Ingram et al., 1987; Ingram &
Conway, 1988; Alterthum & Ingram, 1988; Ohta ezal., 1990)

499
 In a study comparing the fermentation performance of yeast (S. cerevisiae) and 2.
mobilis, using acid-hydrolyzed softwood (pine) cellulose, the glucose-to-ethanol conversion
efficiency was reported to be respectively 94% and 96% of theox%ical maximum (Par&h et
al., 1989). Mart recently, in a study designed to promote the putative superior fermentation
characteristics of a recombinant E. coli strain KOl 1, Barbosa et al. (1992) reported that the
ethanol yield based on monomer sugars in a nutrient-supplemented, CaOH-treated, pine
cellulose acid hydrolysate exceeded 100% of the theoretical yield. Unfortunately, the vastly
diffenznt cell loadings used in these studies precludes any direct comparison with respect to
kinetic parameters.
The purpose of the present study was to compare the fermentation performance of two
recog&& high-perfom~ance bacterial ethanologens, namely 2. mobilis and a recombinant
E. coli l3, in side-by-side batch fermentations under identical environmental conditions with
respect to medium composition, pH and inoculation cell density. Since both these culture are
known to convert glucose to ethanol with very high efficiency, the technoeconomic parameter
of particular interest is the comparative productivity displayed by each biocatalyst.

MATERIALS AND MEfHODS

Organisms Zymomona~ mobilis ATCC 29191 is a neotype strain and was obtained from
the American Type Culture Collection (RocKlle, MD). Escherichia cofi B (ATCC 11303
canying pLGI297) (Alter&m & Ingram, 1989) was a gift from L. 0. Ingram (University
of Florida, Gain&lie, FL, USA).
Fermentation medium The synthetic glucose mineral salts medium contained 1.5 g/L
yeast extract (Difco) and ammonium chloride (1.6 g/L) as sources of assimilable nitxogen.
Thiamine was added at O.OSmg/L. The composition with respect to all other inorganic
elements and vitamins was as previously described (Lawford & Ruggiero, 1990). Certain
experiments +th E. coZi employed a complex LB medium, the composition of which was as
previously described by Lawfoxd and Rousseau (1992).
Equipment and Fermentation Conditions Batch fermentations were conducted
anaerobically in 2L stirred-tank biorectors (MultiGen= F2000 from New Brunswick
Scientific Co., Edison, NJ) containing about ISL medium. Agitation was minimal at about
150 RPM. The temperature was 30’C and the pH was controlled automatically at the
specified value by addition of 2N KOH. Inoculation was made from an overnight static flask
cultme. The iuitial cell density was in the range 35-50 mg dry wt. cells per litnz (equivalent to
anODat55OnmofaboutO.l too.15
Analytical Procedures Growth was measured turbidometrically at 55Onm (lcm lightpath)
and culture dry weight was measured by microfrltration (0.45~), washing and drying the
filter to constant weight under an i&a-red heatlamp. Compositional analyses of fermentation
media and cell-free spent media were determined using an HPLC equipped with an
HPX-87H column and a RI monitor with computer-interfaced controller/integrator (Bio-Rad
Labs) as described previously (Lawford & Rousseau, 1992).
Calculation of Operational Parameters The average volumetric productivity ( ) was
determined by dividing the foal ethanol concentration by the time (h) required to 9 hieve
complete glucose utilization. The maximum volumetric productivity (Q -) was estimated as
the maximum slope in plots of ethanol concentration versus elaspsed f&mentation time. The
‘pmcess”ethano1 yield (Y ) was determined as the mass of ethanol produced divided by the
mass of glucose added% the medium. In determining glucose-to-ethanol conversion
efficiency (I theoretical max.), the theoretical maximum value for Ytis is assumed to be 0.51

500
 Glucose Utilization (pH 6)

0 20 40 60 60 100 120 140

7
Ethanol Production

7d recombinant /

0 20 40 60 80 100 120 140

Time thl

FIGURE 1 Fermentation Profiles for 2. mobilis and Recombinant E. coli:

[A] Glucose. Symbols: A and A, 2. mobilis and E. coli (p~OI297) as per lines 4 and
3 of Table 1 respectively; W,r E. coli in LB-supplemented medium as per line 1 in Table 2.
[B] Bwoduction. Symbols: 0 and 0, E. coli (pLOI297) and E. coli (KOl 1) as per lines 2
and 3 of Table 2 respectively; other symbols as in (A).

501
 TABLE 1
Comparative Fermentation Parameters for Z mobilis and
Recombinant E. co/i (pLOI297) In a Synthetic Ceilulose Hydrolysate
(Glucose/salts) Medium at pH 6.0
PRODUCTS PRODUCTIVITY YIELD
[Glucose] [Biomass] [EtOH] av.QP QPmax Yp,‘ Conver.
Organism an- gDwh sn gPhnp de Effi. (s)
E. co/i 6 (p~o1297) 66.7 2.4 30.7 0.93 1.10 0.46 90
z. Il7ObiiiS (29191) 61.5 2.1 30.8 2.40 4.78 0.50 98
E. co/i B(pumn) 134.0 43.1 0.30 0.50 0.41' 80
z. mObiliS (29191) 123.5 2.5 60.5 3.03 4.80 0.49 96
l Sii ghmse utilii was incompkIe aftfz lSOh, the ‘process’ Yield (based on sugar added to the medium)
is 0.32 g &OH/g gl~se.

TABLE 2
Comparative Fermentation Parameters for Recombinant E. co/i
in Nutrient-supplemented Glucose Media

PRODUCTS PRODUCTIVITY YIELD

[Glucose] [Biomass] (EtOH] av.Q cl ma
P
YP/s Conver.
Organism pn gDwh tin- t&- BIB Effic. (Q)

Lurla broth w
E. co/i B(pmmn)
This work [expt. B13 ] 6.3 123.0 2.7 59.0 0.61 0.92 0.48 94
E. co/i B (PLDIZW)~ 7.0 120.0 3.6 58.0 0.48 1.40' 0.48 94
E. co/i B (K011)2 6.0 100.0 3.8 53.0 0.74 1.40 0.53 104
E. co/i B (~011)
pinewood cellulose 6.6 63.1 (-IRS) 34.0 0.71 1.33 0.61f 119
acid hydrolysate3
Note: The hostculture is E. coli B (ATCC 11303) - in strain pLOI297. the ‘PET operon is plasmid encoded,
whereas for strain KOll. it is chromosomally integrated (ref. Ohta ti al.. 1991a)
1. Altmthum and Ingram (1989) [data taken from Fii. 1A]
2 Guimaaea u al. (1992) [data taken from Fig. 2A]
3. Barbosa et ul. (1992) [Fig. lC] @yptone-supplanenti. CaOH-treated. pinewood cellulose hydrolysate;
TRS was 82.4% glucose. whae TRS = total reducing sugars
f high yield may result from catabolii of cellobiose and/or complex exogenous nutrients (Barbosa et al.. 1992)
* In all these fermentation trials (refs, 1-3). the inoculation cell density was approx. 10 times (330 mg dry wet. ceb/f.,)
that used in the present investigation

502
 A pH of 6.0 is near optimal for growth and ethanol production by genetically engineered
Escherichia coli B (Beall et al., 1991; Ohta et al., 199la). For Zymomonas mobilis, pH 6.0
is near optimal for growth (Lawford et al., 1988); however, because maintenance
metabolism is increased at lower PI-I, pH 6.0 is not optimal with respect to the rate of ethanol
production (Lawford & Ruggiero, 1990) and a pH value of 5.0 is most often used in
Zymomonas fermentations (Rogers et al., 1982). Therefore, by choosing to control the pH at
6.0, the recombinant E. coli culture receives an advantage in terms of productivity.
Using a 6% (W/V) glucose defined salts medium with pH controlled at 6.0, Z. mobilis
(ATCC 29191) and recombinant E. coli B (pLOI297) (Alterthum & Ingram. 1989) produced
ethanol at a conversion efficiency which was 98% and 90% (max. theoretical) respectively
(Table 1). However, the volumetric pmductivity of the Z. mobilis fermentation was 2.6 times
that of the recombinant E. coli fermentation with times for complete fermentation being 13h
and 33h respectively (I’able 1).
When the glucose concentration was doubled (range 12-13s w/v), there was a more
pronounced difference in fermentation performance displayed by these two bacterial
ethanologens (Fig. 1, Table 1). Whereas the Zymomonas culture maintained high
performance characteristics with respect to both product yield and productivity, the
recombinant E. coli displayed a markedly lower fermentation performance (Table 1). At the
higher glucose concentration, about 20% of the sugar remained unfermented by the E. coli
culture after 6 days (Fig. 1), resulting in a process yield (based on sugar added) of only 0.32
g/g (equivalent to a conversion efficiency of 63%) (Table 1). Nutrient supplementation
(log/L tryptone) resulted in an improved performance by the recombinant E. coli with
complete fermentation in 4 days at a conversion efficiency of 94% (max. theoretical) (Fig. 1,
Table 2). For comparison purposes, data taken from Alterthum and Ingram (1989) relating to
the production of ethanol by E. coli B (pLOI297) using a nutrient-rich medium (Luria broth),
containing 12% glucose, is plotted in Figure 1B and presented in Table 2. Also included, for
comparison purposes, is data taken from Guimaraes et al. (1992) in connection with their
investigation of a closely related genetic construct, namely strainKOl1 (Ohta et at., 1991b),
in which, for purposes of culture stability, the pdc and adh genes from Zymomonas were
chromosomally integrated into E. coli B (Fig. 1B and Table 2). Figure 1B illustrates that the
fermentation performance characteristics of recombinant E. coli B (pLO1297) and strain
KOll, in a nutrient-rich medium, are very similar. The faster rates of ethanol production
(Fig. 2) observed in the experiments of Alterthun & Ingram (1989) and Guimaraes et al.
(1992), are due to the 10 times inoculation cell density employed (see Table 2). Recently,
Barbosa et al. (1992) proposed using recombinant E. coli strain KOll for fuel ethanol
production from softwood (pine) acid hydrolysate. The results of their investigation relating
specifically to the fermentation of the acid hydrolyzed cellulose fraction (detoxified using
CaOH) are presented in Table 2. The fact that the observed product yield (0.61 g EtOH/g
glucose) exceeded the theortical maximum may be due to the catabolism of cellobiose and/or
the tryptone used as a nutritional supplement (Barbosa et al., 1992). The inoculation cell
density used by Barbosa et al. (1992) was also approx. 10 times that used in the present
investigation.
Using a similar fermentation feedstock (acid hydrolyzed pinewood cellulose), Parekh et
al. (1989) compared the fermentation performance of S. cerevisiae and Z. mobilis and found
that both organisms exhibited high conversion efficiencies; however, the productivity of the
bacterial fermentation was 2.6 times higher than the yeast fermentation. Unfortunately,
because of the vastly different inoculation densities employed by Pa&h et al. (1989). the
results of these investigations using different ethanologens cannot be compared directly in
terms of productivity.
The newly developed and patented ethanologenic recombinants of Esherichia coli have
recently been the focus of considerable attention in terms of their potential for the production
of fuel ethanol from biomass and wastes (Ingram et al., 1991; Lawford & Rousseau, 199 1
and 1993; Beall & Ingram, 1992). Nevertheless, the results of the present study, together
witb observations reported in the literature, suggest that the fermentation performance
characteristks of Zymovnonusare not inferior to those of the recombinant E. coli cults and
that, in f&t, further R&D with 2. mobilis seemsjustified particularly in terms of the potential
fa implwzd productivity.
Acknowledgements
This research was supported by an aperating grant from the Natural Sciencesand Engineering ResearchCouncil
of Canada. We are grateful to Professor Lonnie Ingram (University of Florida) for the patented recombinant
Escherichia coli B (pLOI297)

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