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A Black Box Mathematical Model to

Calculate Auto- and Heterotrophic Biomass


Yields Based on Gibbs Energy Dissipation

J.J. Heijnen,* M.C. M. van Loosdrecht, and L. Tijhuis


Delft University of Technology, Department of Biochemical Engineering,
2628 BC Delft, The Netherlands
Received June 16, 19921Accepted June 23, 1992

On the basis of the estimated Gibbs energy dissipation per that none of these was satisfying in terms of the follow-
C-mol biomass produced and a convenient black box de- ing minimum set of requirements:
scription of microbial growth, a general equation for the cal- General applicability to all chemotrophic systems.
culation of the yield of biomass on electron donor has been
obtained. This black box model defines four formal electron A clear limit of the used parameter based on the sec-
donating reactions for biomass, carbon source, electron ond law of thermodynamics.
donor, and electron acceptor. The proposed description Only black box information (and no biochemical de-
leads to a simple equation which gives the biomass yield on tails) required to calculate YDx.
electron donor for chemotrophic growth systems under Absence of intrinsic problems.
carbon and energy limitation for which biomass is the only
anabolic product. The variables involved are the degrees of However, it was shown4 that the parameter D;l/rAx,
reduction and the Gibbs energy characteristics of the four which is the Gibbs energy dissipation per C-mol pro-
compounds, and the required Gibbs energy dissipation per duced biomass in kJ/C-mol, does indeed satisfy the
C-mol produced of biomass. It appears that biomass yields above-mentioned requirements.
on electron donor for auto- and heterotrophic growth under A large number of microbial growth systems, for
aerobic, denitrifying, and fermentative conditions can be
estimated with 10-15% error in a range of Y&-values of which the biomass yield YDxis known, has been evalu-
0.01-0.80 C-mol/(C)-molelectron donor. Also, simple regu- ated with respect to their Gibbs energy dissipation. For
larities in the Gibbs energy and enthalpy of organic sub- all these systems growth occurred at higher growth rates
strates are found. Furthermore, simple relations are derived under C-limitation, and biomass was the only anabolic
to calculate the thermodynamic maximal biomass yield, product. Hence YDx-values used correspond to condi-
conditions required for growth to occur, heat production,
biomass yield on electron acceptor, and anaerobic product tions of maximal growth yield. The growth systems used
yield. Finally a new definition of thermodynamic efficiency covered organic and inorganic C-sources, fermentation,
is derived. 0 1992 John Wiley & Sons, Inc. O2 and NO3- as electron acceptors and electron donors
Key words: auto- and heterotrophic growth biomass yield with and without reversed electron transfer (RET).
Gibbs energy dissipation heat production thermodynamic A simple correlation for the Gibbs energy dissipation
efficiency second law of thermodynamics
per C-mol produced biomass was obtained (Fig. 1). It
appeared that DsO1/rAxis mainly determined by the na-
INTRODUCTION ture of the C-source, especially by its carbon chain
length (C) and degree of reduction ( y D ) . The value of
For industrial and environmental applications, the che-
D;'/rAXseems only little influenced by the electron ac-
motrophic microbial growth systems are of major impor-
ceptor used. Also it appeared that for inorganic electron
tance. One of the key parameters in such processes is
donors, where RET is required, D;'/rAxis very high.
the yield of biomass on the electron donating substrate
Table I contains the found dissipation values which
YDx(biomass (X)and electron donor (D)). Moreover,
characterize microbial growth for different C-sources.
an estimate of YDxis often required in situations where
the microorganism is unknown or poorly known with For C-sources that are not listed, Eqs. (la) and (lb)
provide a best first estimate4 for D;'/rAr:
respect to its biochemical properties. However, the
C-source, the N-source, electron donor, and electron -RET DsO1/rAx= 200 + 18 . (6 - C)'.8
acceptor are almost always known. This information is
generally called a "black box" de~cription.~ Thus, it
+ exp[{(3.8 - yD)2}o.16(3.6 + 0.4C)I
* (la)
would be very useful to provide a method which allows +RET D;l/rAx = 3500 (1b)
an estimation of YDxin such black box situations.
In a previous article," a critical evaluation was pre- From Table I and Eqs. (la) and (lb) it appears that
sented of several published methods. It was concluded DsO1/rAxvaries between 200 and 3500 k J/C-mol biomass
depending on C-source and Occurrence of RET. It has
* To whom all correspondence should be addressed. been shown that these dissipation values can be used to

Biotechnology and Bioengineering, Vol. 40, Pp. 1139-1154 (1992)


0 1992 John Wiley & Sons, Inc. CCC 0006-3592/92/01001139-16
Number of carbon atom8 of carbon 8ource C- and energy limitation and under absence of anabolic
I 2 3 4 6 product formation. This calculation of YDxuses the
elemental conservation principles and the Gibbs energy
balance and is straightforward but cumbersome."
This article presents a convenient alternative calcula-
tion method which provides an analytical expression for
YDxas a function of
The value of D!l/rAxcalculated from Eqs. (la) and (lb)
or Table I.
The degree of reduction of the electron donor and
biomass.
, 1 5 1 I 1 5 1 I 1 5 1 I 1 5 1 I 3 5 1
The Gibbs energy characteristics of biomass, electron
Degree of reduction of carbon source donor, electron acceptor and, C-source.
Figure 1. Gibbs energy dissipation per C-mol biomass (kJ/C-mol)
for various C-sources with different degrees of reduction and car-
bon chain length4: (Reproduced with permission.) (+) aerobic + A BLACK BOX SYSTEM DEFINITION OF
denitrifying; (m) anaerobic. CHEMOTROPHIC GROWTH
provide a first estimate of YDxwith about 15% error in a Chemotrophic growth systems can be fully described in
range from 0.01 to 0.80 C-mol biomass/C-mol electron black box terms by the specification of the involved rele-
donor. It is stressed that this method of YDx-estimation vant compounds. It is generally sufficient to provide the
applies to microbial growth at high growth rate, under compounds in Table 11. The C-source, N-Source, HC03-,
Table I. Average Gibbs energy dissipation values (D:'/rAx)for growth on different C-
sources.a

D:'/rAx Standard
C-source Degree of Carbon chain Number of (kJ/C-mol deviation
compound reduction length observations biomass) (%)

c02 0 1 9 3494 23
C0zC 0 1 3 1061 16
Oxalate2- 1.o 2 2 1224 14
co 2.0 1 1 1105 -
Formate- 2.0 1 5 1107 25
Glyoxylate- 2.0 2 1 709 -
Tartrate2- 2.5 4 1 584 -
Malonatez- 2.67 3 1 751 -
Malate'- 3.0 4 2 380 13
itr rate'- 3.0 6 5 381 31
Pyruvate- 3.33 3 2 316 25
Succinate2- 3.5 4 5 422 17
Gluconate- 3.66 6 6 311 19
Formaldehyde 4 1 1 587 -
Acetate- 4 2 4 529 8
Lactate- 4 3 2 296 33
Di hydrox yacetone 4 3 1 257 -
Glucose 4 6 8 284 26
Glycerol 4.66 3 4 345 23
Mannitol 4.33 6 5 338 35
Propionate- 4.66 3 1 556 -
Et hyleneglycol 5.0 2 1 617 -
Acetoin 5.0 4 3 457 53
Butanediol 5.5 4 3 469 53
Aceton 5.33 3 1 813 -
Methanol 6 1 3 729 15
Ethanol 6 2 4 712 11
Propanol 6 3 2 725 9
n-Alkanes 6.13 6 1 662 -
Butane 6.5 4 1 1061 -
Methane 8 1 1 1011 -

a From ref. 4. Reproduced with permission.


For electron donor with reversed electron transfer.
' For electron donor without reversed electron transfer.

1140 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 10, DECEMBER 5, 1992
Table 11. General black box system definition of chemotrophic microbial growth.

Examples

Chemo-heterotrophic, Chemo-autotrophic,
Compounds Chemo-heterotrophic glucose/ethanol nitrification

Biomass CHi.80o.sNo.z CHi.sOo.sNo.z CHi.sOo.sNo.z


N-source NH~+ NH~+ NH~+
H+ H+ H+ H+
HC03- HCO3- Hco3- Hco3-
H20 HzO H20 Hz0
C-source C16Hi206 C6H1206 Hco3-
Electron donor C6H1206 C6H1206 NH~+
Oxidized donor HCO3- HCOs- NO2-
Electron acceptor O2 HCO3- 0 2
Reduced acceptor H20 C&O HzO

H', and H 2 0 are the main raw materials for biomass tation product. Compound X is not excreted but re-
synthesis. However, in addition to the raw materials, mi- mains intracellular.
crobial systems need a source of Gibbs energy in order A normal procedure would now be to define the
to assemble the concentrated polymerized biomass from donor couple as substrate/X and the acceptor couple
the diluted monomeric raw materials. This Gibbs energy as X/fermentation product. From a practical point of
is delivered by the electron transfer between electron view, this is a complication because the nature of com-
donor and the electron acceptor couples. pound X can vary considerably and is in general not
It is stressed that, although Table I1 contains 10 com- well known. However, due to the fact that in black box
pounds, it will appear in practice that certain com- systems the biomass is supposed to be in balanced (or
pounds are identical. Table I1 contains some examples to steady) state, there is no net production of the intracel-
illustrate this point, e.g., C-source and electron donor lular compound X. Therefore any convenient compound
are frequently the same. Furthermore, due to the ele- may be chosen as X, because X cancels out in the cal-
mental and electric charge conservation relations,6most culations (no net production). The compound HC03- is
microbial growth systems will only have two degrees of a convenient choice because this allows the use of the
freedom. This means that, in general, only two net con- same organic donor couples which occur in the external
version rates (e.g., consumption of electron donor, rAD, electron acceptor situation (see Table I1 for examples).
and production of biomass, T A X ) need be known in order A further aspect of fermentative growth is that, besides
to calculate the net conversion rates of all the remaining the main catabolic product, there may occur multiple
compounds listed in Table 11. other products. These situations can be simply handled
The list of relevant compounds is easily specified for by suitable averaging (see Appendix 2). It is noted, how-
any microbial growth system. Of the 10 general com- ever, that in order to apply the present method for YDx-
pounds 4 are nearly always the same (biomass, HC03-, calculations in fermentative systems, one must know
H', and H20). Only the N-source, C-source, electron the stoichiometry of the expected catabolic products
donor, and electron acceptor show a large diversity in (Appendix 2).
microbial growth systems. In general, these compounds
are easily identified. The N-source is often NH4+,N2,
NO3-, or an organic N-compound. The C-source is often DERIVATION OF THE RELATION BETWEEN
a reduced organic compound (heterotrophic growth) or BIOMASS YIELD AND GIBBS ENERGY
DISSIPATION
C 0 2 (autotrophic growth).
The electron donor couple contains the reduced or- As stated before, it is the intention of this article to find
ganic or inorganic compound and its oxidized form (or- a simple but general relationship between biomass yield
ganic compound/HC03-, NH4'/N02-, H z / H C ,etc.). on electron donor (YDx)and the Gibbs energy dissi-
The electron acceptor couple is, in systems with exter- pation per C-mol biomass (D:'/rAX).In principle, this
nal acceptor, the oxidized compound and its redu- relationship follows directly from the application of ele-
ced form ( 0 2 / H 2 0 ,SO:-/HS-, N03-/N2, etc.). For mental conservation relations and the Gibbs energy bal-
microbial growth systems with an internal electron ac- a n ~ e The
. ~ equations obtained are, however, complex
ceptor (fermentation), the definition of electron-donor/ and different for different microbial systems. Therefore,
acceptor couples is not so clear. Here the organic an alternative approach will be presented here. In this
substrate is partially oxidized to COz and some intracel- description four redox half reactions will be proposed.
lular compound X of primary metabolism. This oxi- It is stressed that these reactions are defined from the
dized compound X is then also the internal electron point of view of calculatory convenience and that there
acceptor, which is subsequently reduced to the fermen- is no relation whatsoever to the actual biochemical re-

HEIJNEN, VAN LOOSDRECHT, AND TIJHUIS: MODEL TO CALCULATE BIOMASS YIELDS 1141
actions, which occur inside the microorganisms. The Reaction 3 electron donor:
convenience will be shown to reside in the fact that
-1 electron donor + (. . .)oxidized donor
The degrees of reduction7of biomass, C-source, elec-
tron donor, and electron acceptor are derived in a + (...)N-source + ( . . . ) H 2 0 + (...)HC03-
simple way from these redox half reactions. + (. . .)H- + yD(e-) = 0
It will be possible to use, in an uncomplicated way, the
Gibbs energy characteristics of the C-source, biomass, Reaction 4 electron acceptor:
electron donor, and electron acceptor half reactions,
which can then be identified as the well-known redox
-1 electron acceptor + ( .. .)reduced acceptor
potentials of half reactions. + (. . .)N-source + ( . . . ) H 2 0 + ( . . .)HC03-
A direct calculation of the relation between YDx, + ( . . .)H+ + yA(e-) = 0
D!'/rAx, and the Gibbs energy characteristics is
These four reactions should contain all listed relevant
possible.
chemicals of the growth system (Table 11). Appendix 1
shows some examples of such redox half reactions. Typi-
DEFINITION OF A SET OF FORMAL REDOX cally 1 C-mol of organic or 1 mol of inorganic compound
HALF REACTIONS is oxidized. Products have positive and substrates have
This proposed formalism using four redox half reac- negative stoichiometric coefficients. In each reaction a
tions is mathematically completely equivalent to the number of electrons is produced. This number is called
black box description using only the relevant compounds the generalized degree of reduction for biomass (yx),
of Table 11. A convenient set of four redox half reactions C-source (ys), electron donor (yD), and electron accep-
is the oxidation of biomass (reaction 1) and carbon tor (yA). For organic compounds this degree of reduc-
source (reaction 2) to HC03-, H + , H 2 0 , N-source, and tion coincides with the definition from R ~ e l s and
~ ~ *has
electron donor (reaction 3) and electron acceptor (reac- a simple relation to the alternative concepts like the
tion 4) to their respective oxidized and reduced forms: amount of redoxons' or available electrons.738 However,
the present description also includes the degree of re-
Reaction 1 biomass: duction of arbitrary donor and acceptor couples, where
-1 C-biomass + 1Hco3- + ( . . . ) H 2 0 oxidation does involve other reactants (see, e.g., Appen-
dix 1, the NH4+/N02-electron donor redox half reac-
+ (. . .)N-source + (. . .)H+ + yx(e-) = 0 tion). As such the proposed definition of yi in reactions
Reaction 2 C-source/substrate: 1-4 has a more generalized meaning than the definition
from Roels7,*or Minkevich.'
-1 C-source + 1HC03- + (. . . ) H 2 0 Table I11 shows that the degree of reduction for C-
+ (. . .)N-source + (. . .)H+ + ys(e-) = 0 sources, biomass, and electron donors varies between
Table 111. Degree of reduction yi and electron Gibbs energies and enthalpies (AG:, AG:',
AH:, kJ/e-mol) for electron donating redox half reactions.

Degree of AG:, AG:', AH,^,^


Redox half reaction reduction, yi kJ/e-mol k J/e-mol kJ/e-mol

Biomass
Biomass/HC03-/N H 4f +4.2 -13.8 +33.8 -26.1
Biomass/HCO3-/NOs- +5.8 -33.2 +15.04 -44.2
Biomass/HCOs-/N2 +4.8 - 15.0 +33.2 -26.3
Organic electron donor
Oxalate/HC03- +1 +13 +52.30 -20
Formate/HCOs- +2 +7.5 +47.80 -15.50
Glyoxylate/HCO3- +2 +1.0 +51.30 NA
Tartrate/HCOs- +2.5 -8.6 +39.70 NA
Malonate/HCOs- +2.67 -15.8 +29.70 NA
Fumarate/HCOs- +3.0 -12.8 +34.30 -31.60
Malate/HCOs- +3.0 -13.2 +33.50 -32.20
Citrate/HCO~- +3.0 -14.2 +32.40 -33.90
Pyruvate/HC03- +3.33 -13.7 +34.20 -23.60
Succinate/HCOJ- +3.50 -17.0 +28.49 -36.30
Gluconate/HCO3- +3.67 -11.1 +39.30 NA
Formaldehyde/HCOs- +4.0 -4.6 +45.38 -0.10
Acetate/HCOs- +4.0 -18.1 +26.86 -33.50
Lactate/HCOs- +4.0 -15.0 +31.70 -28.90

enthalpy based on COz(g) and NH4+ as reactant in the redox half reaction for the car-
bonaceous compounds. NA, not available.

1142 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 10, DECEMBER 5, 1992
Table 111. (continued)

Degree of AG:, AG:', AH:,"


Redox half reaction reduction, 'yi k J/e-mol kJ/e-mol kJ/e-mol
Glucose/HCOs- +4.0 -10.2 +39.80 -25.75
Mannitol/HCOs- +4.33 -10.3 +38.90 NA
Glycerol/HCOs- +4.67 -10.8 +37.80 -24.30
Propionate/HCOs- +4.67 -18.6 +26.99 -33.80
Et hyleneglycol/HC03- +5.0 -9.9 +38.50 NA
Acetoin/HCOs- +5.0 -15.2 +33.10 NA
Butyrate/HC03- +5.0 -20.3 +27.07 -33.30
Propanediol/HC03- +5.33 -14.3 +33.60 NA
Acetone/HCOs- +5.33 -18.8 +28.70 -30.90
Butanediol/HCOs- +5.50 -16.1 +31.36 NA
Methanol/HC03- +6.0 -10.7 +36.17 -22.98
Ethanol/HC03- +6.0 -16.1 +30.50 -28.90
Propanol/HC03- +6.0 -17.5 +29.60 -32.50
n-Alkane/HC03- +6.13 -19.9 +26.60 NA
Propane/HCOs- +6.66 -20.1 +25.90 -31.90
Ethane/HC03- +7.0 -20.3 +25.40 -31.40
Methane/HCOs- +8.0 -22.0 +22.98 -31.50
Inorganic electron donor
H 2(1 bar)/H + +2 0 +40.3 0
H2(lO-' bar)/HC +2 -5.6 +34.4 0
H2(10-3bar)/Hf +2 -8.3 +31.7 0
H z ( ~ Obar)/Hf
-~ +2 -11.2 +28.8 0
CO/HCOs- +2 - 12.5 +47.5 -1.5
HS-/SO;- +8 -24.0 +21.0 -43.9
szo:-/so42- +8 -26.8 +23.3 -27.5
so/so;- +6 -34.0 +19.3 -55.2
N H4+/N02- +6 -86.2 -32.8 -99.7
N02-/NOs- +2 -81.6 -41.6 -92.7
FeZf/FeSf +1 -74.3 -74.3 -46.8
H20/02 +2 -118.7 -78.8 -143.0
Electron acceptors
H+/H2 -2 0 +40.3 0
HC03-/ethanol -6 -16.1 +30.50 -28.9
HCO,-/acetate -4 -18.1 +26.86 -33.5
HCOs-/CHd -8 -22.0 +22.98 -31.5
SO;-/HS- -8 -24.0 +21.0 -43.9
Acetaldehyde/ethanol -2 -20.9 +19.1 -14.8
Pyruvate/lactate -2 -21.6 + 18.4 -45.0
SO?-/HS- -6 - 15.5 +11.2 -41.9
Fumarate/succinate -2 -43.1 -3.1 NA
NOs-/Nz -5 -120 -72.0 -130.1
Fe3'/Fe2+ -1 -74.3 -74.3 -46.8
02/H20 -4 -118.7 -78.8 - 143.0
N20-/N2 -3 -145.7 -92.3 -145.9
N20/N2 -2 -170.1 -130.7 -183.8

a enthalpy based on COz(g) and NH4+as reactant in the redox half reaction for the car-

bonaceous compounds. NA, not available.

0 and +8. For an electron acceptor the degree of reduc- and the gaseous standard state of slightly soluble gases
tion is negative, which is logical, and varies between (02, Nz,CH4, etc.). Furthermore, for AGR calculations
0 and -8. HC03- is used as the carbon dioxide form (at pH 7 the
predominant form). For AHR calculations C02(g) has
been used because the measured heat production always
THE AG, AND AH, CONCEPT AND ENERGETIC
REGULARITIES includes the rather large heat effect of the HC03-(aq)
to C O A d transition. For the AG!' and AH!(') the val-
For the established redox half reactions it is possible to ues fr~m"Wilhoit,'~ Thauer et al.," Roels,'s8 and Rutgers
calculate the Gibbs energy and enthalpy of reaction et al.9 were used. For biomass the values of -91 and
(AGR and AHR) in the usual way. For biochemical sys- -67 kJ/C-mol were used as enthalpy and Gibbs energy
tems one assumes as the standard condition 298 K, of f~rmation.'~~It is noted that there is a discrepancy for
pH 7, the aqueous standard state of ions and solutes, AG? of formate between Thauer et al." (-351 kJ/mol)

HEIJNEN, VAN LOOSDRECHT, AND TIJHUIS: MODEL TO CALCULATE BIOMASS YIELDS 1143
and Wilhoit14(-335 kJ/mol). The latter value was used from +33.8 to +26.6 kJ/C-mol. Hence for organic
here. The calculation of AG:' and AH: for some redox donors a very important energetic regularity is that
half reactions is shown in Appendix 1.
AG:; = AG:h
It will now prove to be useful to define two new quan-
tities [eqs. (2) and (3)] based on the energies AG: and AH:^ =
AH&of the redox half reaction i:
3. Inorganic electron donors have AG:'-values which
are much lower than for biomass. The need for RET
(reversed electron transport, lifting up the low-energy
donor electrons to the biomass level) is immediately
-AH:, clear from AG:'-values.
AH; =- (3)
Yi 4. From the AG:'-values of Table I11 it is immediately
clear which redox half reaction functions as donor
Because -AG:f gives the liberated Gibbs energy in an
(the higher AG:'-value) and as acceptor (the lower
electron donating redox half reaction relative to the hy-
AG :'-value), because the electrons move from high
drogen reference (where the Gibbs energy of the elec-
to low energy level (second law of thermodynamics).
tron per definition is zero), AG:' represents a measure
Also AG: [Eq. (5a)l gives directly the energetic qual-
of the Gibbs energy level of an electron in a redox half
ity of the donor/acceptor combination. For organic
reaction, relative to the H2 reference.
donors and 02/N03 as acceptors AG:; is typically
The same holds for AH:, which provides the enthalpy
about 110 kJ/e-mol, for fermentations the value is
level of the electrons in the redox half reaction. It is
much lower (2 to 20 kJ/e-mol).
noted that AG:' is directly related to the well-known
5. For oxygen as electron acceptor and organic com-
redox potential of electrons in redox half reactions:
pounds as electron donor and using the average
AG:' = -F X Ei, (4) AG:'- and AH:-values for organic compounds men-
tioned above, Eqs. (5a) and (5b) show rather constant
where F is the Faraday constant (96.5 kJ/V e-mol). From values for AG: and AH:, of 110.8 and 115 kJ/e-mol,
calculations of AGjf and AH:, for redox half reactions which holds for all organic compounds. This is in
as in Appendix 1 and using Eqs. (2) and (3), the values close agreement with earlier values2 of 108.7 and
of AG:' and AH: were obtained for many redox half 110.9 kJ/e-mol. Also it appears that for aerobic
reactions and tabulated (Table 111). growth AH:v = AG:;. It is stressed that this is cer-
Further energetic parameters of interest for complete tainly not so for anaerobic growth. Because 1 mol O2
redox reactions can be calculated directly from AG:'- accepts four electrons, it follows directly that for
and AH:-values. These are the available difference of organic compounds the available combustion Gibbs
Gibbs energy [Eq. (5a)l and enthalpy [Eq. (5b)l per energy and enthalpy are 435 and 460 kJ/mol 02, which
electron between electron donor and electron acceptor is also well known?,','
couples, or the combustion Gibbs energy [Eq. (5c)l and
combustion enthalpy [Eq. (5d)l for a complete donor/ac- In conclusion, it appears that the formalism of redox
cepter reaction (calculated per C-mole for organic donor half reactions leads directly to convenient definitions of
or per mole for inorganic donor): degrees of reduction and useful values of AGi1 and
AH:i. Furthermore, these values immediately reveal
AG: = AG$ - AG:q)r (54 useful regularities in the energetics of organic and inor-
AH:^ = - AH:A (5b) ganic compounds, the electron donor and acceptor
couple can immediately be identified, and the need for
AG:L= 70 AG:; (5c) reversed electron transfer can be noticed directly.
A H L ~= AH:^ (54
AN ANALYTICAL RELATION BETWEEN Yox
Using the values of AGf'and AH: of Table I11 some ob- and Dg'/rAx
servations can be made:
It is now very simple to calculate the dissipation of the
1. AG:' and AH: for all organic compounds are rela- growth process if one assumes that the four described
tively constant (32 28 kJ/e-mol and (-28) 2 5 kJ/ redox half reactions run with a rate rl to r4. The Gibbs
e-mol, respectively). energy dissipation under biochemical standard con-
2. AG:' and AH: for biomass (NH4+or N2as N-sources) ditions (D!')is then obtained by multiplying each
are not very different from the average values for or- rate (r;) with the released Gibbs energy (y;AGeiaccording
ganic compounds. It is noted that the N-source in- to Eq. (2)) and summing up, leading to
fluences the values somewhat. It is known that AGfO'
for biomass is not well known. Recently3 a value of D;' = rl(yxAG:!) + rz(rsAGl') + b(yDAG:h)
-45 kJ/C-mol was proposed. This only changes AG:' + rd(yAAG:!) (6)

1144 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 10, DECEMBER 5, 1992
The rates of reaction ri follow from the net conversion formation for D!'/rAX[Table I, Eq. (l)] and AGei-and
rate rAi by component balancing6based on the reaction yi-values (Table 111).
stoichiometry. From the four defined redox half reac- the direct calculation of biomass yield on electron ac-
tions one can write directly ceptor (YAx),yield of electron acceptor on donor (Ym),
and heat production per C-mole biomass (Q/rAx)from
rl = -TAX (74 Gibbs energy dissipation.
r2 = -rAs (7b) thermodynamic limits to AG,,, YDX,YAX,and YDA.
the relation between D:l/rAx and alternative biomass
yield correlator~.~
a new black box thermodynamic efficiency proposal.
Furthermore, the yield of biomass on electron donor
and of C-source on biomass [Eqs. (8) and (9)] can be A SIMPLE RELATION TO CALCULATE BIOMASS
introduced: YIELDS FROM D:'/rAx
Equation (12) can function as a basis for a first estimate
of biomass yields on a particular electron donor. This is
shown clearly if one rewrites Eq. (12) as
(9) Yx
YDX-
YLJ
Now obviously it is required that the generated electrons
-
-
AG:; - AG:i
in the four redox half reactions are conserved, leading to
Do' 1
- AGfqfr) + -s- X - + (AG:; - AG:;)]
yxrl + ysr2 + y ~ r 3+ Y A r 4 = 0
Combination of Eqs. (loa) and (7a)-(7d) by elimination
( W (AG:;
[ TAX 'yx

(13)
of r l to r 4 leads to Eq. (lob), which can be recognized as
the well-known balance of degree of reduction': Equation (13), particularly, shows how YDxdepends on
(see also Fig. 2)
3/xrAx + YsrAs + Y D r A D + Y A r A A = 0
(lob) the N-source, because this changes yx and AG: (see
Combination of Eqs. (6), (7), (8), (9), and (lob) leads to Table 111).
the desired relation between YDXand D ! l / r A x : the C-source, because this changes D!'/rAx [Table
Eq. (1)l.

-
- * (AG:; - AG:.)
D!l/rAx + yx(AG,; - AG:.) - Yxsys(AG:: - AG:.)

Equations (lob) and (11) are the equations of central


interest. These relations can be simplified considerably
for large classes of microbial growth.
In many microbial systems of practical interest there
is not a separate C-source because the electron donor is
both carbon and energy source (heterotrophic growth).
This means that reaction 2 can be removed [r2 = -rAs =
0; Yxs= 0, see Eq. (9)].
For autotrophic growth with inorganic donors C 0 2 is
the C-source, for which ys = 0. Hence for heterotrophic
and autotrophic growth one can simplify Eqs. (lob) and
(11) by setting Yxs and ys = 0, which yields 0.00
~ ~~ '
YxrAx + 3/DrAD + 3/ArAA = 0 (10c)
0 40
A@; = AG:
80
- AGZ
120 160
(kJle-mol)

Influence of N-source: Y.
Influence of C-source: Dy/ru
Influence electron Donor: Yo and AG:
Equations (12) and (1Oc) can now be used to illustrate Influence electron Acceptor: AGZ
some relevant aspects of microbial growth: Figure 2. Effect of N-source ( y x ) , C-source ( L l ~ ' / r ~electron
~),
the direct calculation of YDx-values for arbitrary mi- donor ( y ~ AG;;),
, and electron acceptor (AG;i) on YDXaccording
crobial growth systems based on available general in- to Bq. (14).

HEIJNEN, VAN LOOSDRECHT, AND TIJHUIS: MODEL TO CALCULATE BIOMASS YIELDS 1145
the electron donor, because this changes yD and AG;; the yield of electron acceptor on electron donor, YDA
(Table 111). [Eq. (16)] (this is, e.g., the yield of product on substrate
the electron acceptor, because this influences AG;; in anaerobic fermentations or the yield of O2 on sub-
(Table 111). strate in aerobic growth):
In general, the term AG,: - AG,; is only about 5-10%
of the denominator value, because AG!; = AG!; as
mentioned above. This remains even true for RET sys-
tems, like nitrification (where AG,' % AG:;), because
then D!'/rAxis very large [3500 kJ/C-mol; see Eq. (lb)].
Hence, one can write as a good approximation the sim-
pler equation (14), where AG:: has disappeared as a If it is further assumed that only autotrophic and het-
variable and where Eq. (5a) has been used: erotrophic growth is considered, equations (1Oc) and
(12) can be used in combination with the definitions
[Eqs. (15) and (16)] to obtain

Obviously YDX increases (i) with ' y ~ ;(ii) with a larger


value of AG::, which is the available Gibbs energy per
transferred electron; (iii) due to decreased dissipation
D!'/rAx;and (iv) if yx decreases (see Fig. 2).
It is now possible to estimate YDxfor all of the micro-
Equations (17a) and (18a) give YAxand YDAdirectly as a
bial growth systems described in reference 4 where the function of D!'/rAx.These equations can be simplified
best estimate of D!'/rAx(Table I) or the correlation considerably by invoking the found energetic regular-
Eq. (1) is used for the various carbon sources. Further- ity (AG:; = AG;;) for organic substrates. This gives
more, yx is assumed to be 4.2 and AG,: = +33.8 kJ/ Eqs. (17b) and (18b), where Eq. (5a) has also been used:
mol (NH4+as N-source). Table I11 contains all the nec-
essary AG:' data for these growth systems. It is now
also of interest to use the exact equation (13) or the ap-
proximate equation (14) to calculate the estimated value
of YDx.Figures 3A-D show the resulting comparison of
measured and estimated YDxvalues. The error in Fig-
ures 3A-D is calculated as the average absolute value Figure 4A shows that the biomass yield on electron ac-
of the ratio of (measured minus predicted YDx)to mea- ceptor YAx[Eq. (17b)I increases if AG:: increases, if the
sured YDx.First it is noted that Eq. (13) or (14) shows dissipation decreases, and if the degree of reduction of
virtually no difference (compare Figs. 3A and B or 3C the acceptor ( - y A ) increases.
and D). Clearly the use of Table I leads to better predic- Figure 4B shows that the yield of acceptor on donor
tions than Eq. (1) (compare Fig. 3A to 3C and 3B to YDA[Eq. (18b)l approaches the stoichiometry of the pure
3D). The reason is that Table I contains the best average acceptor/donor redox reaction (yD/(-yA) for high values
dissipation data while Eq. (l), which is the best approxi- of D!'/rAx.This is to be expected because, for the limit
mation to these data, gives less accurate value^.^ Inspec- D!' = 03, there is no biomass formation [YDx= 0; see
tion of the most serious deviations shows that especially Eq. (14)]. For anaerobic fermentations, the reduced ac-
the substrates butanediol, citrate, formate, and acetoin ceptor represents the fermentation product; hence YDA is
show large errors in YDxestimations. Table I shows that the anaerobic product yield on electron donor. From
for these substrates large variations in the found average Eq. (18b) and Figure 4B it immediately appears that
dissipation occur. Clearly it can be concluded that increased dissipation and a low value of AG:: leads to
Eq. (14), in combination with available information on maximal anaerobic product yield, YDA . Furthermore,
D!'/rAx[Table I, Eq. (l)], enables the estimation of YDx- it clearly follows that YDA > 1 in situations where Y D >
values for auto- and heterotrophic growth over a range ( -yA). This means that for anaerobic processes with re-
of 0.01-0.80 C-mol/(C)-mol donor with an error duced substrates (e.g., methanol, yD = 6) and more oxi-
of 10-20%. dized products (e.g., acetate, -yA = 4) HCO3- fixation
must occur.
EQUATIONS TO CALCULATE OTHER YIELDS AND The heat production Q (in kJ/m3 h) is of obvious im-
HEAT PRODUCTION DIRECTLY FROM GIBBS portance in microbial growth systems. Clearly, using the
ENERGY DISSIPATION first law of thermodynamics, the heat production can be
In addition to the yield of biomass on eletron donor, calculated from an equation equivalent to Eq. (6) by re-
other yield values are often required, e.g., the yield placing D!' with Q and by replacing AG:' with AHi. By
of biomass on electron acceptor, YAx [Eq. (15)], and the same line of reasoning which leads to Eq. (12), one

1146 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 10, DECEMBER 5, 1992
1 1

0.1
!k
w
0.1
E
a
t

0.0 1 0.01
0.01 0.1 1 0.0 1 0.1 1

Y, meawed Y, meawed

(A) (B)

1 1

U
c

0.1
E
c 0.1
E
?

Error = 19%

0.0 1
0.01 0.1 1 0.0 1 0.1 1

Y, measued

(D)
Figure 3. Comparison between measured and estimated values of YDx (data from ref. 4): (A) YDXcalculated with Eq. (13)
using Dj'/rAx from Table I; (B) Yox calculated with Eq. (14) using D:'/rAX from Table I; (C) YDXcalculated with Eq. (13) using
D:'/rAx from Eq. (1); (D) YDxcalculatedwith Eq. (14) usingD:'/rA, from Eq. (1). (+) Aerobic + denitrifying; (D) anaerobic.

can then arrive at Eq. (19), which holds for auto- and Due to the fact that for organic substrates and NH4+or
heterotrophic growth: Nz as N-source, the energetic regularities hold (AH:' =
and AG:' = AGfh), Eq. (20) can be simplified
considerably into

The relation between heat production and Gibbs energy


dissipation is found by elimination of YDxfrom Eqs. (19)
and (12) and using Eqs. (5a) and (5b): It is easily shown that the differences between the heat
production calculated from Eq. (21) are, within a few
percent, equal to the exact Eq. (20). Equation (21)
shows directly that the heat production per C-mole of
- y,(AH,O! - AH$) (20) produced biomass is mainly determined by the enthalpy

HEIJNEN, VAN LOOSDRECHT, AND TIJHUIS: MODEL TO CALCULATE BIOMASS YIELDS 1147
C02 is the donor and C02/ethanol the formal acceptor.
I /,-(kJ/C-ml biomass) Hence from Table I11 one obtains
AHL = -25.75 - (-28.9) = +3.15 kJ/e-mol and
AG:: = 39.8 - 30.5 = +9.3 kJ/e-mol
Equation (21) shows then that Q/rAx = (3.15/9.3) x
284 = 96 kJ/C-mol biomass, which is indeed a normal
value.l2

THERMODYNAMIC LIMITS TO AGav, YDX,Ymx, YDA


It is easily shown that for all microbial growth sys-
tems the denominator of Eq. (12) is always positive.
Since DSol/rAx > 200 kJ/C-mol biomass4and, in general,
AG;; > AG;;. Therefore, in order to achieve a positive
value of YDx,it is necessary that AG,D > AGeA (or
0 40 80 120 160 AGav> 0). This provides a clear necessary limit to AGav
AGE
(kJ/e-moll for all growth systems.
(A) In principle, AGei must be calculated from actual con-
centrations. Because of lack of data one normally as-
sumes biochemical standard conditions. If AG;; is large
(>20 kJ/e-mol), the concentration effects are minor and
W
assumption of biochemical standard conditions is ap-
propriate. However, especially for systems with low
values for AG::, ( 4 0 kJ/e-mol) these concentration ef-
fects can be very substantial (Appendix 3). A very well
known example" is the anaerobic conversion of ethanol
into 1 acetate and 2 HZ, where ethanol/acetate is the
donor couple and H 2 / H f is the acceptor couple. It is
easily shown that AG, is only positive if the H2pressure
is below about atm.
The condition AGav> 0, in combination with
Table 111, directly shows whether envisaged growth sys-
tems are thermodynamicallypossible, whereas the value
of AGavindicates directly whether concentration effects
might be important.
0.00
0
u 40 00 120 160
The thermodynamic maximal limits to various yields
(YDx, YAX, YAD)are obtained by applying the second law
AGE (kJ/e-mol)
limit (D!'/rAx = 0) to the exact equations [(12), (17a),
(B) and (lsa)] for these yields. Very incorrect yield lim-
Figure 4. Different microbial yields as a function of AG:: and its are obtained if the approximative equations [(14),
D!l/rAx (using -yx = 4.2): (A) &x, biomass yield on electron accep- (17b), and (18b)], which were obtained by applying the
tor (C-mol biomass/mol acceptor), Eq. (17b); (B) Ym,yield of ac- energetic regularity AG;; = AGzh to the exact equa-
ceptor on donor (C-mol/C-mol), Eq. (18b).
tions [(12), (17a), and (lsa)], are combined with D!'/
and Gibbs energy properties of the energy generating rAx= 0. This point was also stressed recently by Sandler
donor/acceptor couple (AH:v and AG::) and the neces- and Orbey."
sary Gibbs energy dissipation. For aerobic growth on It is now convenient to define the parameter z , which
organic substrates it is known" that AH:v = AG:: (see is a measure of the deviation of said energetic regularity:
also energetic regularities), and from Eq. (21) one can AG,' - AG,;
conclude that for such growth systems the heat produc- z = (22)
AG,":, - AG:;
tion is virtually equal to di~sipation.~
Equation (21) also
shows that heat production can be much smaller or For z = 1, AG;; = AG$, which is the energetic regu-
larger than the Gibbs energy dissipation depending on larity. For z < 1, it appears that AG;; > AG;;, which
the ratio of AHL and AG::. As an example one could indicates that the donor electrons are at a higher Gibbs
calculate the heat production for the anaerobic ethanol energy level than biomass electrons (energy enriched
fermentation on glucose. Table I shows that D!'/rAxfor regime). For z > 1 one obtains that AG$ < AG;:,
glucose is 284 kJ/C-mol biomass. Furthermore glucose/ which indicates that the donor electrons must be lifted

1148 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 10, DECEMBER 5, 1992
up to the biomass electron Gibbs energy level (energy
deficient regime).
Now using Eqs. (22) and (12), (17a), and (18a), one
obtains Eqs. (23a)-(23c) for the thermodynamic limits,
called Y&, YA$, Y&:
I \ I .,
aerobic growth
denitrifying growth
anaerobic growth

Y&X-- z - 1
-- 0 1 2 3 4
Z AGO:,-AGO,:
Z-
AGrD-AG:L
Figures 5A-C show the dependence of the thermody-
namic maximal yield values on the parameter z and the
various degrees of reduction. Also, in Figure 5A the cal-
culated z-values for the microbial growth systems used
in reference 4 are shown. Clearly z is always positive,
which shows that apparently AG; < AGf; for all growth
systems. This provides a useful limit for electron accep-
tors; a redox couple with AG, > AG,, obviously cannot
function as an acceptor in microbial growth systems.
Further z has a value in the range 0.4-3, which clearly
indicates the deviations from the energetic regularity.
However, Figure 5A also shows that for growth systems
with a strong electron acceptor (02, NO3-) z is always
close to 1 (range 0.85-1.10). Obviously for such systems
the value of z = 1 is an admissible approximation,
0 1 2 3 4
which leads to the particularly simple statement that AGO:,-AGtA
Y& = yD/y,, which was already found earlier.597*8 z=
AG;D-AGO,:
However, Eqs. (23a)-(23c) plus Eq. (22) provide the
exact thermodynamic limits. Some remarks can now be (B)
made on these thermodynamic limits.
For z = 1 one finds that Y& = yD/y,, while Y;: = 0
and YA$ = 03. This situation of z close to 1 often occurs
for aerobic or denitrifying growth and for some an-
y:&)
aerobic growth systems. Clearly this theoretical limit
case means zero consumption of electron acceptor 1
(Y,& = m). Also for very reduced substrates (yD > y x ) ,
it follows that Y& > 1. This means that C 0 2 fixation is
thermodynamically possible, where the electrons of the 0
reduced substrates are used for C 0 2 reduction to bio-
mass. The acetate fermentation from methanol serves as
an example. -1
The situation z < 1 means that AG;; > AG;' and 0 1 2 3 4
that therefore the substrate electrons have more Gibbs AGP,:-AGO,:
energy than the biomass electrons. Figures 5A-C show
z-
AG",b-AG::
that Y& X ( y x / y D )can now become much larger than (C)
1, while Y,& becomes negative! This means that in this
Figure 5. Thermodynamic maximal yield values as a function of
theoretical limit case an electron acceptor is produced. parameter z : (A) maximal biomass yield on donor Y&; (B) maxi-
For aerobic growth this would mean a theoretical but mal biomass yield on acceptor Y&; (C) maximal yield of acceptor
thermodynamically allowed" production of 0 2 .The on donor YG.
case z < 1 occurs mainly for the typical energy-enriched
substrates (Table I11 like glucose, mannitol, gluconate, energy input to be lifted up to the Gibbs energy level
glycerol, pyruvate, and formate, all of which are also of the biomass electrons. Therefore, even under this
good substrates for anaerobic growth. Forz > 1, AG; < thermodynamic limit situation theoretically a minimal
AGf', and therefore the donor electrons, need a certain amount of Gibbs energy must be produced and fully

HEIJNEN, VAN LOOSDRECHT, AND TIJHUIS: MODEL TO CALCULATE BIOMASS YIELDS 1149
coupled to said lifting up of the donor electrons (no dis- that for aerobic growth systems T B a t = 7~~~ = 7th.
sipation occurs in this theoretical limit case). So a cer- Equation (A.2) shows that 7 B a t = 7 t h for all growth sys-
tain number of electron donors and acceptors are tems with an external electron acceptor and for fermen-
combined to provide said Gibbs energy. Hence (see also tative growth with only a single product. Equation (25)
Figs. 5A and 5B), not all donor electrons go into biomass therefore appears to be a suitable generalization of these
and Ygx X (yx/yD)< 1, while there is also consump- earlier proposals. Battley has advanced the hypothesis
tion of electron acceptors ([Y& x (yX/(-yA))]-l > 0). that 7 t h is linearly related to the available Gibbs energy
Typical examples of such energy deficient electron per C-mole or per mole of substrate. In general terms
donors are alcohols and acids (Table 111), all of which this available Gibbs energy per C-mole is equal to X
are typical fermentation products. Recently Sandler and AG::. Figure 6A shows the correlation between 7 t h and
Orbey lo have also discussed the implication of energy yD X AG:: for the aerobic, denitrifying, and anaerobic
enriched and energy-deficient electron donors on ther- growth systems compiled in reference 4. It appears that
modynamic limits at great length using a different cal- the aerobic and denitrifying systems are situated at
culatory approach. 70 X AG:: values of about 150 kJ/C-mol and more. This
is due to the fact that for aerobiddentirifying systems
RELATIONS BETWEEN D:'/rAxAND OTHER
AG;: = llSkJ/e-mol and varies from 1 to 8. The an-
BIOMASS YIELD CORRELATING PARAMETERS aerobic growth systems are situated at yD X AGtJ-values
below 100 kJ/C-mol. Figure 6A shows that 7 t h appears
In literature a number of alternative parameters have to increase more or less linearly with yDAG:' in the
been proposed to correlate biomass yields. Although it range 0-400 kJ/C-mol, which is in accordance with
was shown4that all these parameters suffer from major Battley's original proposal.' The decrease in 7 t h above
intrinsic problems, it is still useful to show the relation yDAG:: = 500 kJ/C-mol is not according to Battley's
of Dsol/rAxwith these earlier attempts. Appendix 4 shows proposal. An alternative hypothesis has been put for-
these relations for the biomass yield on available elec- ward by Roels' that 7 t h is constant in the lower range
trons and various thermodynamic efficiencies. (Battley,
Roels, Westerhoff) as a function of D!'/rAx,AG,', AG,&

1 1
AG,!, yx, and YD. 1.0 I

0.8
A NEW BLACK BOX THERMODYNAMIC
EFFICIENCY PROPOSAL c
0.6
c
It has been shown4that thermodynamic efficiency defi- 0.4
nitions (which are based on the black box and the Gibbs
energy convertor concept) are all subject to serious in- 0.2
trinsic problems. However, the present description offers
0.0
a very simple and straightforward possibility to define a
0 200 400 600 800 1000
thermodynamic efficiency which is not subject to these
problems. It is proposed to define this efficiency as the
y,AG,: [kJ/C-moll
ratio of the actual and thermodynamic maximal yield of
biomass on electron donor [Eq. (24)]: (A)

0.25

Using Eqs. (12) (23a), and (22), one obtains


0.20
.
c
0.1 5
.. I

For YDxas a function of 7th one obtains 0.05


I
0.00
0 20 40 60 80 100
From Eq. (25) it is clear that 7 t h = 1 for D;'/rAx = 0
and that 7 t h > 0 because in general AG,; > AG:; (see y,AG,: [kJ/C-moll
above). Hence 0 < 7 t h < 1. (B)
If one compares the definition for 7 t h with earlier
Figure 6. Efficiency 7 t h as a function of the available Gibbs energy
proposals, one obtains the interesting result that 7 t h is content of the electron donor (yDAG::) for heterotrophic growth
closely related to 7 B a t [Eq. (A.2) in Appendix 41 and [drawn line is Eq. (27)]: (A) all data from Fig. 1; (B) blow-up of part
vRoe[Eq. (A.4) in Appendix 41. Equation (A.4) shows of part A . (e)Aerobic + denitrifying; (m) anaerobic.

1150 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 10, DECEMBER 5, 1992
of yDAG:,! (400 kJ/C-mol) because growth is energy correlating parameter to calculate biomass yields for
limited in this regime, while 7 t h decreases for higher hetero- and autotrophic growth systems under carbon
yDAG::-values due to carbon limitation and energy sur- and energy limitations where biomass is the only an-
plus. It is clear from Figure 6A that this hypothesis is abolic product. To facilitate the calculation of YDxfrom
also not entirely correct. D;l/rAxfor such growth systems, a black box description
The behavior of 7 t h seems best "explained" [see has been proposed based on the formal definition of
Eq. (25)] by the notion that dissipation is minimal four redox half reactions for biomass, C-source, electron
around = 4 and rises for higher and lower yD-values, donor, and electron acceptor. From these redox half re-
leading to a maximal Tth-value around YD = 4. actions one obtains directly the degree of reduction (yi),
In principle, the correlation for 7 t h in Figure 6A electron Gibbs energy (AGei), and electron enthalpy
could be used in combination with Eq. (26) to estimate (AH,.) for said compounds. The values of AGeiand AHei
YDX. This correlation can be represented by the poly- reveal directly important energetic regularities for or-
nome equation ganic compounds. Based on this black box description,
simple relations are found which relate D;l/rAxto YDx,
7th = 2.12 x i0-3yDAG:,! - 2.2 x 10-6(yDAG:,!)2
YAx, YAD,and Q/rAx.Based on available values for
- 2.4 X 1010(yDAG::)3 (27) D ; l / r ~[Table
~ I, Eq. (l)], YDx can be estimated using
Using this correlation in conjunction with Eq. (26), it is said relation with 10-15% accuracy over a range of
possible to compare the estimated YDx-valueswith the 0.01-0.80 C-mol biomass/C-mol donor. Furthermore,
measured YDx-values. relations are obtained to calculate the thermodynamic
The results are shown in Figure 7. Clearly the method maximal values of YDX, YAx, and YDA. Also, useful limit-
based on the 7 t h correlation is inferior to the results ing values are obtained (AGav> 0 and AG; < AG:).
based on the dissipation correlation, as shown in Fig- Finally it is shown that a thermodynamic efficiency 7 t h
ures 3A-D. In particular, the prediction of anaerobic can be defined and a 7 t h correlation is obtained. How-
biomass yields is much worse. This is due to the large ever, prediction of YDxis much better if based on
scatter of the anaerobic data around the 7 t h correlation D;l/rAxas a correlating parameter than on qth.Also the
(Fig. 6B). Therefore the present approach using the dis- D;l/rAxcorrelation is very well understandable in bio-
sipation correlation [Table I, Eq. (l)] and Eq. (12) is chemical terms: contrary to the 7th correlation. There-
preferred. Moreover the dissipation approach is pre- fore, Dil/rAxis the preferred correlating parameter to
ferred for its good agreement with general biochemical estimate biomass yields.
knowledge!
APPENDIX 1
CONCLUSION
Setting Up the Redox Half Reactions and
It has been shown that the amount of Gibbs energy dis- Calculation of the Degree of Reduction and of the
sipated per C-mole of produced biomass is a suitable Gibbs Energy and Enthalpy of Electrons in the
Redox Half Reaction

1
In the thermodynamic description of microbial growth one can
A conveniently use a set of four redox half reactions for (1) biomass,
(2) C-source, (3) electron donor, and (4) electron acceptor. The
reaction format is that, in addition to the two main reactants, H',
H20, HCOj-, and the N-source of the growth system are used to
obtain a complete formulation of the redox half reaction. The pro-
cedure is best illustrated with some examples.

Biomass Redox Half Reaction to HC03-


(NH,' as N-Source)
-CH1.800.5N0.2 - 2.5H2O + HCOs- + 0.2NH.4'
+ 5H' + 4.2e- = 0
The degree of reduction of biomass can be found immediately from
the 4.2e- produced. Hence yz = 4.2.
The values of AG; and AG;' follow by taking into account all
0.01 reactants except the electron:
0.01 0.1 1
AGL = +0.2 X (-80) + 1 X (-588) - 2.5 X (-238)
Y, measwed
-1X (-67) = +58 kJ
Figure 7. Comparison of measured and estimated YDx-values
using Eq. (26) and the gth-correlation[Eq. (27)]: (+) aerobic + AGE = +5 X (-40) + 0.2 X (-80) + 1 X (-588)
denitrifying; (m) anaerobic. - 2.5 X (-238) -1X (-67) = -142 kJ

HEIJNEN, VAN LOOSDRECHT, AND TIJHUIS: MODEL TO CALCULATE BIOMASS YIELDS 1151
This leads [using Eqs. (2) and (3)] to AGg = -13.8 and AG% = a fermentation by Klebsiella aerogenes using 2.283 mol citrate/C-mol
+33.8 kJ/e-mol. For AH! one finds, from the biomass redox half biomass (Appendix 1, ref. 4):
reaction with C02(g) as product and NH,++(aq)as N-source, 4.096 C2H302- (acetate)
0.192 C4H40:- (succinate)
AH! = -0.2(-80) - 394 - 1.5 X (-286) 0.068 CHO2- (formate)
- 1 X (-91) = +109.8 kJ 0.63 H Z
The number of electrons involved follows as
This gives AH: = -26.1 kJ/e-mol. Acetate: 4.096 X 2 X 4 = 32.76
Succinate: 0.192 X 4 X 3.5 = 2.688
Formate: 0.068 X 1 X 2 = 0.136
Glucose (C-Source) Redox Half Reaction to HC03- HI: 0.63 X 2 = - 1.26
The redox half reactions are defined per C-mole of substrate. 36.85
Hence we can write Here use has been made of the degree of reduction (Table III),
remembering that this is the number of electrons per C-mole for
-&H1206 - 2 H 2 0 + 1Hco3- + 5 H + + 4e- = 0 carbon compounds. The average acceptor electron Gibbs energy
is now found (see Table 111 for separate AC,-values) by using the
The degree of reduction 'ys is then read to be +4. The value of AG& appropriate AG,-value of each compound in conjunction with their
and AGE follows as electron number (as calculated above) and averaging over all elec-
trons (36.85):
1
AG& = +1(-588) - 2(-238) - - X (-918)
6
= +41 kJ AG;; = (32.76 X 26.9 + 2.668 X 28.6 + 0.136
X 47.8 + 1.26 X 40)/(36.85) = +27.54 kJ/e-mol
1
AGE = +5(-40) +1(-588) -2(-238) - -(-918) = -159 kJ
6
APPENDIX 3
This leads to AG;$ = -10.2 kJ/e-mol and AG: = +39.75 kJ/e-mol;
for AH! of the glucose redox half reaction to C02(g) one finds
Effect of Nonstandard Concentrations on
+lo3 kJ, leading to AH: = -25.75 kJ/e-mol (see also Table 111).
-
(AG& AGeA)Values
Normally one utilizes the biochemical standard condition to cal-
NH4+Redox Half Reaction to NO2- (Donor) culate AG;:-values. However, especially for systems with small
AG,D - AG,A values, the incorporation of the actual concentrations
-NH4+ - 2 H 2 0 + NOz- + 8H' + 6e- =0
is generally required to obtain meaningful AG,-values which are
The degree of reduction of HN4+ in this redox half reaction to NOz- needed in Eq. (12). For microbial growth systems with large (20 to
is seen to be +6. For the reaction Gibbs energy one obtains 150 kJ/e-mol) values of AG,, the use of actual concentrations only
AGiD = +517 kJ and AC& = +197 kJ. results in minor corrections compared to the biochemical standard
This leads to AG,oD= -86.2 kJ/e-mol and AG;; = -32.8 kJ/e-mol. conditions. However, in the range 0-20 kJ/e-mol, one can observe
For AH! one obtains +598 kJ, giving AH~D = -99.7 kJ/e-mol. significant effects. The influence of concentration on AG, follows
directly from the related redox half reaction (Appendix 1) using the
well-known relation between AGfi and concentration C,:
O2 Redox Half Reaction to H20 (Acceptor) AGf, = AGfq + RT In C ,
-02 + 2H20 - 4H' - 4e- = 0 In microbial systems, the actual electron donor concentrations are
often much lower than 1M or 1 bar (e.g., 10-2-10-4M or bar), and
The degree of reduction of 0 2 is seen to be -4. For the reaction
pH can deviate substantially from pH 7. Table IV shows some ef-
Gibbs energy one obtains A G ~ A = +2 X (-237.4) = -474.8 kJ and
fects of concentration, partial pressure, and pH on the value of AG,
AG;; = +2 X (-237.4) - 4 X (-40) = -314.8 kJ.
for several compounds.
This leads to AGL = -118.7 kJ/e-mol and AG; = -78.8 kJ/e-mol.
For AH! one obtains +572 kJ, giving AHk = -143 kJ/e-mol. Table IV. Effect of concentration/partial pressure" and pH on AGe
An alternative method is to calculate AG,A using tabulated redox (k J/e-mol) .
potentials [Eq. (4)]. For 0 2 one finds Eo' = +0.82 V; hence AG; =
-(+0.82) . 96.5 = -79 kJ/e-mol. This is, within round-off errors, PH 7 PH 2
the same as found before (-78.8 kJ/e-mol).
Compound 1 lo-' 1 10-~

Glucose (as) +39.8 +39.3 +38.8 +4.1 +3.6 +3.1


APPENDIX 2 Methanol (aq) +36.0 +34.1 +32.2 +2.7 +0.9 -1.0
Citrate3- (aq) +32.4 +31.8 +31.2 -0.9 -1.5 -2.1
Calculation of the Gibbs Energy of Acceptor Formate- (aq) +47.8 +42.1 +36.4 +10.8 +5.2 -0.4
Electrons in Multiproduct Fermentations € 3 2 (g) +40 +34.4 +28.8 +11.4 +5.8 +0.2
0 2 (g) -78.7 -81.5 -84.4 -107.3 -104.5 -101.7
Fermentative microbial growth systems often have more than one
product. For example, ethanol fermentation on glucose only pro- It appears that especially small molecules with a low degree of re-
duces ethanol, and hence, the formal electron acceptor couple can duction are significantly affected by nonstandard concentration or
be taken as COz/ethanol and AGd can be readily found. pressure on Ace,. This means that one should be especially cautious
In fermentations which contain more products, a suitable average in the calculation of dissipation of Gibbs energy for such com-
based on the various products is needed. This average is easily pounds (H2, formate). Also, lower concentrations lead to lower
found as the electron-based average using the tabulated degrees of AG,-values, which is to be expected.
reduction and AG,-values of the separate compounds (Table 111). a Ratio of actual to standard concentration or pressure: 1,
For example, one finds the following molar amounts of products in

1152 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 10, DECEMBER 5, 1992
APPENDIX 4 R ~ e l s ' .has
~ defined his thermodynamic efficiency for biomass
formation as
Relations Between Biomass Yield Correlators aerobic combustion Gibbs energy of biomass
and D !'/rAx TRoe =
+
dissipation aerobic combustion Gibbs energy of biomass
In literature a number of biomass yield correlators have been For the aerobic combustion Gibbs energy of biomass one can write
proposed. Examples are YAWand the Gibbs energy efficiencies as rAx X yx X (AG;; - AG:;,). This leads to
proposed by Battley, Roels, or Westerhoff (see ref. 4 for a review).
'
Battley has defined a thermodynamic efficiency of biomass for-
mation as
maximal dissipation - actual dissipation Westerhoff l 3 has defined his efficiency for aerobic systems as the
'))Bat =
maximal dissipation ratio of anabolic consumed and catabolic produced Gibbs energy.
Westerhoff has defined a catabolic process which leads to the fol-
The actual dissipation is D;'; the maximal dissipation is found by lowing catabolic energy production:
converting all the substrate involved in a proper catabolic process
which generates useful energy. For example, for aerobic growth this AGc = (TAD - rAx) X X
' y ~ (AG;; - AG&)
process is oxidative combustion, whereas for the alcoholic fermen- The anabolic consumed Gibbs energy follows from the Gibbs en-
tation of glucose this process is the conversion of glucose into ergy balance
ethanol/COz. This chosen catabolic process can be characterized
by a certain available Gibbs energy per electron, (AGE)B (with the AGa = D.? - AGc
index B meaning from Battley). For simple growth systems, like Now the efficiency is defined as the ratio of consumed Gibbs energy
aerobic growth or denitrifying growth without product formation or in anabolism (AGA) to the released catabolic energy (-ACc). This
anaerobic growth with only a single product (which is then necessar- leads to
ily the energy generating product), one may write (AG:')B = AG;:.
Here AG:; is the value of AG;; - AG:, calculated in the usual way.
However, for anaerobic growth systems with multiple products, of
which some are not assumed to be involved in energy generation,
(AG:t)B # AG:,!. For such systems AG:,! follows from AGZh - Division of nominator and denominator with rAxand elimination of
AGS, where AG;; is calculated according to Appendix 2. The term rAx/(-rAo) = YDXwith Eq. (12) leads to
(AG:')B is calculated from the selected energy generating product.
Battley' provides an example of anaerobic growth on glucose
(a - Y D ) (AG:! - AG%J + YD(AG;; - AG;;)
72' =
with ethanol and glycerol production. Ethanol is the product of the D!l/rAr + (7.r - Y D ) (AG;; - AG%,) + yD(AGE - AG:;)
energy generating process; glycerol is a byproduct. (A.6)
For glucose AG:D = +39.80 and for ethanol AG;; = +30.50 kJ/
Clearly becomes negative for 7~ > -yx, because AG;; AG;;.
i=
e-mol. Hence (AC:'), = 39.8 - 30.5 = 9.3 kJ/e-mol.
The biomass yield on available electrons, YAve,is easy to calcu-
Ethanol and glycerol are produced in a molar ratio of 0.332 mol
late. The available electrons are equal to 7~ X ( - T A D ) and the
glycerol to 1.0 mol ethanol. According to Appendix 2 and Table 111,
biomass production (in gram units) is equal to 24.8 X T A X . Hence
this gives
1X 2 X 6 X 30.5 + 0.332 X 3 X 4.666 X 37.80
AG:; =
2 X6 + 0.332 X 3 X 4.666
Using Eq. (12) to eliminate YDX,one obtains
= +32.54 kJ/e-mol
Hence one obtains AG:,! = +39.80 - 32.54 = 7.26 kJ/e-mol, which
differs significantly from (AC:,!), = 9.3 kJ/e-mol.
According to Battley, the maximal dissipation is equal to Because for organic substrates AGE = AG% is a very good approxi-
mation and using AG:,! = (AG;; - AG;i), Eq. (A.8) can be ap-
(-TAD) ?'D (Act,!)B
proximated with
and we obtain from the above-mentioned definition

Dividing numerator and denominator by rAx, taking into account NOMENCLATURE


that (rAx/-rAD) = YDX,
and using Eq. (12) to eliminate YDXleads to
the final relation Gibbs energy dissipation (kJ/m3 h)
heat production (kJ/m3 h)
DF/rA,(1 - AG%/(AG:J)B) -t yx(AGE - AG;;) yield of compound j on compound i (C-mol j/C-mol i)
Teal =
Djl/rAx + rx(AG,O: - AG;;) (A4
number of C-atoms in C-source
degree of reduction of compound i
For simple growth systems as mentioned above Act,! = (AG:,!)B and
net rate of conversion of compound i (mol/m3 h)
Eq. (A.2) can be simplified into
rate of reaction i (mol/m' h)
concentrat ion (mol/L)
Gibbs energy per mole of electrons in half reaction i
(k J/e-mol)
It is of interest to note that this early proposal is equal to the enthalpy per mole of electrons in half reaction i
presently proposed 7 t h definition [Eq. (25)] for the simple growth (k J/e-mol)
system. Gibbs energy of formation of compound i (kJ/mol)

HEIJNEN, VAN LOOSDRECHT, AND TIJHUIS: MODEL TO CALCULATE BIOMASS YIELDS 1153
AGR~ Gibbs energy of reaction in half reaction i (kJ) 2. Erickson, L. E., Patell, S.A. 1982. CRC Crit. Z Rev. Biomed.
AGcomb Gibbs energy of combustion of electron donor (k J/C-mol) Eng. 8: 311.
AHR, enthalpy of reaction in half reaction i (kJ) 3. Gross, R., Stephanopoulos, G. 1983. Statistical mechanical
AHcomb enthalpy of combustion of electron donor (kJ/C-mol) estimation of free energy of formation of E-coli biomass for
available Gibbs energy per electron between donor/ use with macroscopic bioreactor balances. Biotechnol. Bioeng.
acceptor (kJ/e-mol) 25: 2149-2163.
available enthalpy per electron between donor/acceptor 4. Heijnen, J. J., Van Dijken, J.A. 1992. In search of a thermo-
(k J/e-mol) dynamic description of biomass yields for the chemotrophic
thermodynamic maximal biomass yield on donor growth of micro-organisms. Biotechnol. Bioeng. 39: 833-858.
(C-mol/C-mol) 5. Minkevich, I. G. 1983. Mass-energy balance for microbial prod-
thermodynamic maximal biomass yield on electron uct synthesis- biochemical and cultural aspects. Biotechnol.
acceptor (C-mol/mol) Bioeng. 25: 1267-1293.
thermodynamic maximal yield of acceptor on donor 6. Noorman, H., Heijnen, J. J., Luyben, K.Ch.A. M. 1991. Lin-
(C-mol/C-mol donor) ear relations in microbial reaction systems, a general overview
yield on available electrons (g/e-mol) of their origin, form and use. Biotechnol. Bioeng. 38: 603-618.
thermodynamic Gibbs energy efficiency according to 7. Roels, J.A. 1980. Application of macroscopic principles to
Battley microbial metabolism. Biotechnol. Bioeng. 22: 2457-2514.
thermodynamic Gibbs energy efficiency according to 8. Roels, J. A. 1983. Energetics and kinetics in biotechnology, 1st
Roels edition. Elsevier Biomedical Press, Amsterdam.
thermodynamic Gibbs energy efficiency according to 9. Rutgers, M., Van der Gulden, H. M.L., Van Dam, K. 1989.
Westerhoff Thermodynamic efficiency of bacterial growth calculated from
thermodynamic efficiency growth yield of Pseudomonas oxalaticus 0x1 in the chemostat.
Redox potential (biochemical standard) of redox half Biochim. Biophys. Acta 973: 302-307.
reaction i (V) 10. Sandler, S.I., Orbey, H. 1991. On the thermodynamics of
Faraday constant (kJ/V e-mol) microbial growth processes. Biotechnol. Bioeng. 38: 697-718.
Gas constant (kJ/mol K) 11. Thauer, R. K., Jungermann, K., Decker, K. 1977. Energy con-
absolute temperature (K) servation in chemotrophic anaerobic bacteria. Bacteriol. Rev.
pressure (atm) 41: 100-180.
12. Von Stockar, U., Marison, I.W. 1989. The use of calorimetry
Subscripts i = in biotechnology. pp. 93-136 In: A. Fiechter (ed.), Advances
X biomass in biochemical engineering/biotechnology, vol. 40. Springer-
S substrate/C-source Verlag, Berlin and Heidelberg.
D electron donor 13. Westerhoff, H.V., Van Dam, K. 1987. Mosaic non-equilibrium
A electron acceptor thermodynamics and the control of biological free energy trans-
duction. Elsevier, Amsterdam.
Superscripts 14. Wilhoit, R. C. 1969. Thermodynamical properties of biochemi-
0 standard condition cal substances. In: M. D. Brown (ed.), Biochemical micro-
01 biochemical standard condition calorimetry. Academic, New York.
th thermodynamic limit condition

References
1. Battley, E. H. 1960. Growth-reaction equations for Saccharomy-
ces cerevisiae. Physiol. Plant. 13: 192-203.

1154 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 10, DECEMBER 5, 1992