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
C-mol biomass produced and a convenient black box de-
scription of microbial growth, a general equation for the cal-
culation of the yield of biomass on electron donor has been
obtained. This black box model defines four formal electron
donating reactions for biomass, carbon source, electron
donor, and electron acceptor. The proposed description
leads to a simple equation which gives the biomass yield on
electron donor for chemotrophic growth systems under
carbon and energy limitation for which biomass is the only
anabolic product. The variables involved are the degrees of
reduction and the Gibbs energy characteristics of the four
compounds, and the required Gibbs energy dissipation per
C-mol produced of biomass. It appears that biomass yields
on electron donor for auto- and heterotrophic growth under
aerobic, denitrifying, and fermentative conditions can be
estimated with 10-15% error in a range of Y&-values of
0.01-0.80 C-mol/(C)-molelectron donor. Also, simple regu-
larities in the Gibbs energy and enthalpy of organic sub-
strates are found. Furthermore, simple relations are derived
to calculate the thermodynamic maximal biomass yield,
conditions required for growth to occur, heat production,
biomass yield on electron acceptor, and anaerobic product
yield. Finally a new definition of thermodynamic efficiency
is derived. 0 1992 John Wiley & Sons, Inc.
Key words: auto- and heterotrophic growth biomass yield
Gibbs energy dissipation heat production thermodynamic
efficiency second law of thermodynamics
INTRODUCTION
For industrial and environmental applications, the che-
motrophic microbial growth systems are of major impor-
tance. One of the key parameters in such processes is
the yield of biomass on the electron donating substrate
YDx(biomass (X)and electron donor (D)). Moreover,
an estimate of YDxis often required in situations where
the microorganism is unknown or poorly known with
respect to its biochemical properties. However, the
C-source, the N-source, electron donor, and electron
acceptor are almost always known. This information is
generally called a "black box" de~cription.~ Thus, it
would be very useful to provide a method which allows
an estimation of YDxin such black box situations.
In a previous article," a critical evaluation was pre-
sented of several published methods. It was concluded
* To whom all correspondence should be addressed.
Biotechnology and Bioengineering, Vol. 40, Pp. 1139-1154 (1992)
0 1992 John Wiley & Sons, Inc.
that none of these was satisfying in terms of the follow-
ing minimum set of requirements:
General applicability to all chemotrophic systems.
A clear limit of the used parameter based on the sec-
ond law of thermodynamics.
Only black box information (and no biochemical de-
tails) required to calculate YDx.
Absence of intrinsic problems.
However, it was shown4 that the parameter D;l/rAx,
which is the Gibbs energy dissipation per C-mol pro-
duced biomass in kJ/C-mol, does indeed satisfy the
above-mentioned requirements.
A large number of microbial growth systems, for
which the biomass yield YDxis known, has been evalu-
ated with respect to their Gibbs energy dissipation. For
all these systems growth occurred at higher growth rates
under C-limitation, and biomass was the only anabolic
product. Hence YDx-values used correspond to condi-
tions of maximal growth yield. The growth systems used
covered organic and inorganic C-sources, fermentation,
O2 and NO3- as electron acceptors and electron donors
with and without reversed electron transfer (RET).
A simple correlation for the Gibbs energy dissipation
per C-mol produced biomass was obtained (Fig. 1). It
appeared that DsO1/rAxis mainly determined by the na-
ture of the C-source, especially by its carbon chain
length (C) and degree of reduction ( y D ) . The value of
D;'/rAXseems only little influenced by the electron ac-
ceptor used. Also it appeared that for inorganic electron
donors, where RET is required, D;'/rAxis very high.
Table I contains the found dissipation values which
characterize microbial growth for different C-sources.
For C-sources that are not listed, Eqs. (la) and (lb)
provide a best first estimate4 for D;'/rAr:
-RET DsO1/rAx= 200 + 18 . (6 - C)'.8
+ exp[{(3.8 - yD)2}o.16(3.6 + 0.4C)I
* (la)
+RET D;l/rAx = 3500 (1b)
From Table I and Eqs. (la) and (lb) it appears that
DsO1/rAxvaries between 200 and 3500 k J/C-mol biomass
depending on C-source and Occurrence of RET. It has
been shown that these dissipation values can be used to
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.
Chemo-heterotrophic,
Compounds Chemo-heterotrophic
Biomass CHi.80o.sNo.z
N-source NH~+
H+ H+
HC03- HCO3-
H20 HzO
C-source C16Hi206
Electron donor C6H1206
Oxidized donor HCO3-
Electron acceptor O2
Reduced acceptor H20
H', and H 2 0 are the main raw materials for biomass
synthesis. However, in addition to the raw materials, mi-
crobial systems need a source of Gibbs energy in order
to assemble the concentrated polymerized biomass from
the diluted monomeric raw materials. This Gibbs energy
is delivered by the electron transfer between electron
donor and the electron acceptor couples.
It is stressed that, although Table I1 contains 10 com-
pounds, it will appear in practice that certain com-
pounds are identical. Table I1 contains some examples to
illustrate this point, e.g., C-source and electron donor
are frequently the same. Furthermore, due to the ele-
mental and electric charge conservation relations,6most
microbial growth systems will only have two degrees of
freedom. This means that, in general, only two net con-
version rates (e.g., consumption of electron donor, rAD,
and production of biomass, T A X ) need be known in order
to calculate the net conversion rates of all the remaining
compounds listed in Table 11.
The list of relevant compounds is easily specified for
any microbial growth system. Of the 10 general com-
pounds 4 are nearly always the same (biomass, HC03-,
H', and H20). Only the N-source, C-source, electron
donor, and electron acceptor show a large diversity in
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
a reduced organic compound (heterotrophic growth) or
C 0 2 (autotrophic growth).
The electron donor couple contains the reduced or-
ganic or inorganic compound and its oxidized form (or-
ganic compound/HC03-, NH4'/N02-, H z / H C ,etc.).
The electron acceptor couple is, in systems with exter-
nal acceptor, the oxidized compound and its redu-
ced form ( 0 2 / H 2 0 ,SO:-/HS-, N03-/N2, etc.). For
microbial growth systems with an internal electron ac-
ceptor (fermentation), the definition of electron-donor/
acceptor couples is not so clear. Here the organic
substrate is partially oxidized to COz and some intracel-
lular compound X of primary metabolism. This oxi-
dized compound X is then also the internal electron
acceptor, which is subsequently reduced to the fermen-
HEIJNEN, VAN LOOSDRECHT, AND TIJHUIS: MODEL TO CALCULATE BIOMASS YIELDS
Examples
Chemo-autotrophic,
glucose/ethanol nitrification
CHi.sOo.sNo.z CHi.sOo.sNo.z
NH~+ NH~+
H+ H+
Hco3- Hco3-
H20 Hz0
C6H1206 Hco3-
C6H1206 NH~+
HCOs- NO2-
HCO3- 0 2
C&O HzO
tation product. Compound X is not excreted but re-
mains intracellular.
A normal procedure would now be to define the
donor couple as substrate/X and the acceptor couple
as X/fermentation product. From a practical point of
view, this is a complication because the nature of com-
pound X can vary considerably and is in general not
well known. However, due to the fact that in black box
systems the biomass is supposed to be in balanced (or
steady) state, there is no net production of the intracel-
lular compound X. Therefore any convenient compound
may be chosen as X, because X cancels out in the cal-
culations (no net production). The compound HC03- is
a convenient choice because this allows the use of the
same organic donor couples which occur in the external
electron acceptor situation (see Table I1 for examples).
A further aspect of fermentative growth is that, besides
the main catabolic product, there may occur multiple
other products. These situations can be simply handled
by suitable averaging (see Appendix 2). It is noted, how-
ever, that in order to apply the present method for YDx-
calculations in fermentative systems, one must know
the stoichiometry of the expected catabolic products
(Appendix 2).
DERIVATION OF THE RELATION BETWEEN
BIOMASS YIELD AND GIBBS ENERGY
DISSIPATION
As stated before, it is the intention of this article to find
a simple but general relationship between biomass yield
on electron donor (YDx)and the Gibbs energy dissi-
pation per C-mol biomass (D:'/rAX).In principle, this
relationship follows directly from the application of ele-
mental conservation relations and the Gibbs energy bal-
a n ~ e The
. ~ equations obtained are, however, complex
and different for different microbial systems. Therefore,
an alternative approach will be presented here. In this
description four redox half reactions will be proposed.
It is stressed that these reactions are defined from the
point of view of calculatory convenience and that there
is no relation whatsoever to the actual biochemical re-
1141
actions, which occur inside the microorganisms. The
convenience will be shown to reside in the fact that
The degrees of reduction7of biomass, C-source, elec-
tron donor, and electron acceptor are derived in a
simple way from these redox half reactions.
It will be possible to use, in an uncomplicated way, the
Gibbs energy characteristics of the C-source, biomass,
electron donor, and electron acceptor half reactions,
which can then be identified as the well-known redox
potentials of half reactions.
A direct calculation of the relation between YDx,
D!'/rAx, and the Gibbs energy characteristics is
possible.
DEFINITION OF A SET OF FORMAL REDOX
HALF REACTIONS
This proposed formalism using four redox half reac-
tions is mathematically completely equivalent to the
black box description using only the relevant compounds
of Table 11. A convenient set of four redox half reactions
is the oxidation of biomass (reaction 1) and carbon
source (reaction 2) to HC03-, H + , H 2 0 , N-source, and
electron donor (reaction 3) and electron acceptor (reac-
tion 4) to their respective oxidized and reduced forms:
Reaction 1 biomass:
-1 C-biomass + 1Hco3- + ( . . . ) H 2 0
+ (. . .)N-source + (. . .)H+ + yx(e-) = 0
Reaction 2 C-source/substrate:
-1 C-source + 1HC03- + (. . . ) H 2 0
+ (. . .)N-source + (. . .)H+ + ys(e-) = 0
Table 111. Degree of reduction yi and electron Gibbs energies and enthalpies (AG:, AG:',
AH:, kJ/e-mol) for electron donating redox half reactions.
Reaction 3 electron donor:
-1 electron donor + (. . .)oxidized donor
+ (...)N-source + ( . . . ) H 2 0 + (...)HC03-
+ (. . .)H- + yD(e-) = 0
Reaction 4 electron acceptor:
-1 electron acceptor + ( .. .)reduced acceptor
+ (. . .)N-source + ( . . . ) H 2 0 + ( . . .)HC03-
+ ( . . .)H+ + yA(e-) = 0
These four reactions should contain all listed relevant
chemicals of the growth system (Table 11). Appendix 1
shows some examples of such redox half reactions. Typi-
cally 1 C-mol of organic or 1 mol of inorganic compound
is oxidized. Products have positive and substrates have
negative stoichiometric coefficients. In each reaction a
number of electrons is produced. This number is called
the generalized degree of reduction for biomass (yx),
C-source (ys), electron donor (yD), and electron accep-
tor (yA). For organic compounds this degree of reduc-
tion coincides with the definition from R ~ e l s and
~ ~ *has
a simple relation to the alternative concepts like the
amount of redoxons' or available electrons.738 However,
the present description also includes the degree of re-
duction of arbitrary donor and acceptor couples, where
oxidation does involve other reactants (see, e.g., Appen-
dix 1, the NH4+/N02-electron donor redox half reac-
tion). As such the proposed definition of yi in reactions
1-4 has a more generalized meaning than the definition
from Roels7,*or Minkevich.'
Table I11 shows that the degree of reduction for C-
sources, biomass, and electron donors varies between

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-
tion is negative, which is logical, and varies between
0 and -8.
THE AG, AND AH, CONCEPT AND ENERGETIC
REGULARITIES
For the established redox half reactions it is possible to
calculate the Gibbs energy and enthalpy of reaction
(AGR and AHR) in the usual way. For biochemical sys-
tems one assumes as the standard condition 298 K,
pH 7, the aqueous standard state of ions and solutes,
and the gaseous standard state of slightly soluble gases
(02, Nz,CH4, etc.). Furthermore, for AGR calculations
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
includes the rather large heat effect of the HC03-(aq)
to C O A d transition. For the AG!' and AH!(') the val-
ues fr~m"Wilhoit,'~ Thauer et al.," Roels,'s8 and Rutgers
et al.9 were used. For biomass the values of -91 and
-67 kJ/C-mol were used as enthalpy and Gibbs energy
of f~rmation.'~~It is noted that there is a discrepancy for
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
here. The calculation of AG:' and AH: for some redox
half reactions is shown in Appendix 1.
It will now prove to be useful to define two new quan-
tities [eqs. (2) and (3)] based on the energies AG: and
AH&of the redox half reaction i:
-AH:,
AH; =- (3)
Yi
Because -AG:f gives the liberated Gibbs energy in an
electron donating redox half reaction relative to the hy-
drogen reference (where the Gibbs energy of the elec-
tron per definition is zero), AG:' represents a measure
of the Gibbs energy level of an electron in a redox half
reaction, relative to the H2 reference.
The same holds for AH:, which provides the enthalpy
level of the electrons in the redox half reaction. It is
noted that AG:' is directly related to the well-known
redox potential of electrons in redox half reactions:
AG:' = -F X Ei, (4)
where F is the Faraday constant (96.5 kJ/V e-mol). From
calculations of AGjf and AH:, for redox half reactions
as in Appendix 1 and using Eqs. (2) and (3), the values
of AG:' and AH: were obtained for many redox half
reactions and tabulated (Table 111).
Further energetic parameters of interest for complete
redox reactions can be calculated directly from AG:'-
and AH:-values. These are the available difference of
Gibbs energy [Eq. (5a)l and enthalpy [Eq. (5b)l per
electron between electron donor and electron acceptor
couples, or the combustion Gibbs energy [Eq. (5c)l and
combustion enthalpy [Eq. (5d)l for a complete donor/ac-
cepter reaction (calculated per C-mole for organic donor
or per mole for inorganic donor):
AG: = AG$ - AG:q)r (54
AH:^ = - AH:A (5b)
AG:L= 70 AG:; (5c)
A H L ~= AH:^ (54
Using the values of AGf'and AH: of Table I11 some ob-
servations can be made:
1. AG:' and AH: for all organic compounds are rela-
tively constant (32 28 kJ/e-mol and (-28) 2 5 kJ/
e-mol, respectively).
2. AG:' and AH: for biomass (NH4+or N2as N-sources)
are not very different from the average values for or-
ganic compounds. It is noted that the N-source in-
fluences the values somewhat. It is known that AGfO'
for biomass is not well known. Recently3 a value of
-45 kJ/C-mol was proposed. This only changes AG:'
1144 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 10, DECEMBER 5, 1992
from +33.8 to +26.6 kJ/C-mol. Hence for organic
donors a very important energetic regularity is that
AG:; = AG:h
AH:^ =
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
clear from AG:'-values.
4. From the AG:'-values of Table I11 it is immediately
clear which redox half reaction functions as donor
(the higher AG:'-value) and as acceptor (the lower
AG :'-value), because the electrons move from high
to low energy level (second law of thermodynamics).
Also AG: [Eq. (5a)l gives directly the energetic qual-
ity of the donor/acceptor combination. For organic
donors and 02/N03 as acceptors AG:; is typically
about 110 kJ/e-mol, for fermentations the value is
much lower (2 to 20 kJ/e-mol).
5. For oxygen as electron acceptor and organic com-
pounds as electron donor and using the average
AG:'- and AH:-values for organic compounds men-
tioned above, Eqs. (5a) and (5b) show rather constant
values for AG: and AH:, of 110.8 and 115 kJ/e-mol,
which holds for all organic compounds. This is in
close agreement with earlier values2 of 108.7 and
110.9 kJ/e-mol. Also it appears that for aerobic
growth AH:v = AG:;. It is stressed that this is cer-
tainly not so for anaerobic growth. Because 1 mol O2
accepts four electrons, it follows directly that for
organic compounds the available combustion Gibbs
energy and enthalpy are 435 and 460 kJ/mol 02, which
is also well known?,','
In conclusion, it appears that the formalism of redox
half reactions leads directly to convenient definitions of
degrees of reduction and useful values of AGi1 and
AH:i. Furthermore, these values immediately reveal
useful regularities in the energetics of organic and inor-
ganic compounds, the electron donor and acceptor
couple can immediately be identified, and the need for
reversed electron transfer can be noticed directly.
AN ANALYTICAL RELATION BETWEEN Yox
and Dg'/rAx
It is now very simple to calculate the dissipation of the
growth process if one assumes that the four described
redox half reactions run with a rate rl to r4. The Gibbs
energy dissipation under biochemical standard con-
ditions (D!')is then obtained by multiplying each
rate (r;) with the released Gibbs energy (y;AGeiaccording
to Eq. (2)) and summing up, leading to
D;' = rl(yxAG:!) + rz(rsAGl') + b(yDAG:h)
+ rd(yAAG:!) (6)
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;;
(Table 111).
the electron acceptor, because this influences AG;;
(Table 111).
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
variable and where Eq. (5a) has been used:
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-
bial growth systems described in reference 4 where the
best estimate of D!'/rAx(Table I) or the correlation
Eq. (1) is used for the various carbon sources. Further-
more, yx is assumed to be 4.2 and AG,: = +33.8 kJ/
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
of the ratio of (measured minus predicted YDx)to mea-
sured YDx.First it is noted that Eq. (13) or (14) shows
virtually no difference (compare Figs. 3A and B or 3C
and D). Clearly the use of Table I leads to better predic-
tions than Eq. (1) (compare Fig. 3A to 3C and 3B to
3D). The reason is that Table I contains the best average
dissipation data while Eq. (l), which is the best approxi-
mation to these data, gives less accurate value^.^ Inspec-
tion of the most serious deviations shows that especially
the substrates butanediol, citrate, formate, and acetoin
show large errors in YDxestimations. Table I shows that
for these substrates large variations in the found average
dissipation occur. Clearly it can be concluded that
Eq. (14), in combination with available information on
D!'/rAx[Table I, Eq. (l)], enables the estimation of YDx-
values for auto- and heterotrophic growth over a range
of 0.01-0.80 C-mol/(C)-mol donor with an error
of 10-20%.
EQUATIONS TO CALCULATE OTHER YIELDS AND
HEAT PRODUCTION DIRECTLY FROM GIBBS
ENERGY DISSIPATION
In addition to the yield of biomass on eletron donor,
other yield values are often required, e.g., the yield
of biomass on electron acceptor, YAx [Eq. (15)], and
1146 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 10, DECEMBER 5, 1992
the yield of electron acceptor on electron donor, YDA
[Eq. (16)] (this is, e.g., the yield of product on substrate
in anaerobic fermentations or the yield of O2 on sub-
strate in aerobic growth):
If it is further assumed that only autotrophic and het-
erotrophic growth is considered, equations (1Oc) and
(12) can be used in combination with the definitions
[Eqs. (15) and (16)] to obtain
Equations (17a) and (18a) give YAxand YDAdirectly as a
function of D!'/rAx.These equations can be simplified
considerably by invoking the found energetic regular-
ity (AG:; = AG;;) for organic substrates. This gives
Eqs. (17b) and (18b), where Eq. (5a) has also been used:
Figure 4A shows that the biomass yield on electron ac-
ceptor YAx[Eq. (17b)I increases if AG:: increases, if the
dissipation decreases, and if the degree of reduction of
the acceptor ( - y A ) increases.
Figure 4B shows that the yield of acceptor on donor
YDA[Eq. (18b)l approaches the stoichiometry of the pure
acceptor/donor redox reaction (yD/(-yA) for high values
of D!'/rAx.This is to be expected because, for the limit
D!' = 03, there is no biomass formation [YDx= 0; see
Eq. (14)]. For anaerobic fermentations, the reduced ac-
ceptor represents the fermentation product; hence YDA is
the anaerobic product yield on electron donor. From
Eq. (18b) and Figure 4B it immediately appears that
increased dissipation and a low value of AG:: leads to
maximal anaerobic product yield, YDA . Furthermore,
it clearly follows that YDA > 1 in situations where Y D >
( -yA). This means that for anaerobic processes with re-
duced substrates (e.g., methanol, yD = 6) and more oxi-
dized products (e.g., acetate, -yA = 4) HCO3- fixation
must occur.
The heat production Q (in kJ/m3 h) is of obvious im-
portance in microbial growth systems. Clearly, using the
first law of thermodynamics, the heat production can be
calculated from an equation equivalent to Eq. (6) by re-
placing D!' with Q and by replacing AG:' with AHi. By
the same line of reasoning which leads to Eq. (12), one
 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):
- y,(AH,O! - AH$)
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
(20) produced biomass is mainly determined by the enthalpy

HEIJNEN, VAN LOOSDRECHT, AND TIJHUIS: MODEL TO CALCULATE BIOMASS YIELDS 1147

I /,-(kJ/C-ml
C02 is the donor and C02/ethanol the formal acceptor.
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

0 40 80 120
AGE
(kJ/e-moll
(A)
W
0.00
0
u 40 00 120
AGE (kJ/e-mol)
(B)
Figure 4. Different microbial yields as a function of AG:: and
D!l/rAx (using -yx = 4.2): (A) &x, biomass yield on electron accep-
tor (C-mol biomass/mol acceptor), Eq. (17b); (B) Ym,yield of ac-
ceptor on donor (C-mol/C-mol), Eq. (18b).
and Gibbs energy properties of the energy generating
donor/acceptor couple (AH:v and AG::) and the neces-
sary Gibbs energy dissipation. For aerobic growth on
organic substrates it is known" that AH:v = AG:: (see
also energetic regularities), and from Eq. (21) one can
conclude that for such growth systems the heat produc-
tion is virtually equal to di~sipation.~
Equation (21) also
shows that heat production can be much smaller or
larger than the Gibbs energy dissipation depending on
the ratio of AHL and AG::. As an example one could
calculate the heat production for the anaerobic ethanol
fermentation on glucose. Table I shows that D!'/rAxfor
glucose is 284 kJ/C-mol biomass. Furthermore glucose/
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
160 AGav> 0). This provides a clear necessary limit to AGav
for all growth systems.
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
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.
160
The thermodynamic maximal limits to various yields
(YDx, YAX, YAD)are obtained by applying the second law
limit (D!'/rAx = 0) to the exact equations [(12), (17a),
and (lsa)] for these yields. Very incorrect yield lim-
its are obtained if the approximative equations [(14),
(17b), and (18b)], which were obtained by applying the
energetic regularity AG;; = AGzh to the exact equa-
tions [(12), (17a), and (lsa)], are combined with D!'/
rAx= 0. This point was also stressed recently by Sandler
and Orbey."
It is now convenient to define the parameter z , which
is a measure of the deviation of said energetic regularity:
AG,' - AG,;
z = (22)
AG,":, - AG:;
For z = 1, AG;; = AG$, which is the energetic regu-
larity. For z < 1, it appears that AG;; > AG;;, which
indicates that the donor electrons are at a higher Gibbs
energy level than biomass electrons (energy enriched
regime). For z > 1 one obtains that AG$ < AG;:,
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-
sipation occurs in this theoretical limit case). So a cer-
tain number of electron donors and acceptors are
combined to provide said Gibbs energy. Hence (see also
Figs. 5A and 5B), not all donor electrons go into biomass
and Ygx X (yx/yD)< 1, while there is also consump-
tion of electron acceptors ([Y& x (yX/(-yA))]-l > 0).
Typical examples of such energy deficient electron
donors are alcohols and acids (Table 111), all of which
are typical fermentation products. Recently Sandler and
Orbey lo have also discussed the implication of energy
enriched and energy-deficient electron donors on ther-
modynamic limits at great length using a different cal-
culatory approach.
RELATIONS BETWEEN D:'/rAxAND OTHER
BIOMASS YIELD CORRELATING PARAMETERS
In literature a number of alternative parameters have
been proposed to correlate biomass yields. Although it
was shown4that all these parameters suffer from major
intrinsic problems, it is still useful to show the relation
of Dsol/rAxwith these earlier attempts. Appendix 4 shows
these relations for the biomass yield on available elec-
trons and various thermodynamic efficiencies. (Battley,
Roels, Westerhoff) as a function of D!'/rAx,AG,', AG,&
that for aerobic growth systems T B a t = 7~~~ = 7th.
Equation (A.2) shows that 7 B a t = 7 t h for all growth sys-
tems with an external electron acceptor and for fermen-
tative growth with only a single product. Equation (25)
therefore appears to be a suitable generalization of these
earlier proposals. Battley has advanced the hypothesis
that 7 t h is linearly related to the available Gibbs energy
per C-mole or per mole of substrate. In general terms
this available Gibbs energy per C-mole is equal to X
AG::. Figure 6A shows the correlation between 7 t h and
yD X AG:: for the aerobic, denitrifying, and anaerobic
growth systems compiled in reference 4. It appears that
the aerobic and denitrifying systems are situated at
70 X AG:: values of about 150 kJ/C-mol and more. This
is due to the fact that for aerobiddentirifying systems
AG;: = llSkJ/e-mol and varies from 1 to 8. The an-
aerobic growth systems are situated at yD X AGtJ-values
below 100 kJ/C-mol. Figure 6A shows that 7 t h appears
to increase more or less linearly with yDAG:' in the
range 0-400 kJ/C-mol, which is in accordance with
Battley's original proposal.' The decrease in 7 t h above
yDAG:: = 500 kJ/C-mol is not according to Battley's
proposal. An alternative hypothesis has been put for-
ward by Roels' that 7 t h is constant in the lower range

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
limited in this regime, while 7 t h decreases for higher
yDAG::-values due to carbon limitation and energy sur-
plus. It is clear from Figure 6A that this hypothesis is
also not entirely correct.
The behavior of 7 t h seems best "explained" [see
Eq. (25)] by the notion that dissipation is minimal
around = 4 and rises for higher and lower yD-values,
leading to a maximal Tth-value around YD = 4.
In principle, the correlation for 7 t h in Figure 6A
could be used in combination with Eq. (26) to estimate
YDX. This correlation can be represented by the poly-
nome equation
7th = 2.12 x i0-3yDAG:,! - 2.2 x 10-6(yDAG:,!)2
- 2.4 X 1010(yDAG::)3 (27)
Using this correlation in conjunction with Eq. (26), it is
possible to compare the estimated YDx-valueswith the
measured YDx-values.
The results are shown in Figure 7. Clearly the method
based on the 7 t h correlation is inferior to the results
based on the dissipation correlation, as shown in Fig-
ures 3A-D. In particular, the prediction of anaerobic
biomass yields is much worse. This is due to the large
scatter of the anaerobic data around the 7 t h correlation
(Fig. 6B). Therefore the present approach using the dis-
sipation correlation [Table I, Eq. (l)] and Eq. (12) is
preferred. Moreover the dissipation approach is pre-
ferred for its good agreement with general biochemical
knowledge!
CONCLUSION
It has been shown that the amount of Gibbs energy dis-
sipated per C-mole of produced biomass is a suitable
1
A conveniently use a set of four redox half reactions for (1) biomass,
0.01
0.01 0.1 1
Y, measwed
Figure 7. Comparison of measured and estimated YDx-values
using Eq. (26) and the gth-correlation[Eq. (27)]: (+) aerobic +
denitrifying; (m) anaerobic.
HEIJNEN, VAN LOOSDRECHT, AND TIJHUIS: MODEL TO CALCULATE BIOMASS YIELDS
correlating parameter to calculate biomass yields for
hetero- and autotrophic growth systems under carbon
and energy limitations where biomass is the only an-
abolic product. To facilitate the calculation of YDxfrom
D;l/rAxfor such growth systems, a black box description
has been proposed based on the formal definition of
four redox half reactions for biomass, C-source, electron
donor, and electron acceptor. From these redox half re-
actions one obtains directly the degree of reduction (yi),
electron Gibbs energy (AGei), and electron enthalpy
(AH,.) for said compounds. The values of AGeiand AHei
reveal directly important energetic regularities for or-
ganic compounds. Based on this black box description,
simple relations are found which relate D;l/rAxto YDx,
YAx, YAD,and Q/rAx.Based on available values for
D ; l / r ~[Table
~ I, Eq. (l)], YDx can be estimated using
said relation with 10-15% accuracy over a range of
0.01-0.80 C-mol biomass/C-mol donor. Furthermore,
relations are obtained to calculate the thermodynamic
maximal values of YDX, YAx, and YDA. Also, useful limit-
ing values are obtained (AGav> 0 and AG; < AG:).
Finally it is shown that a thermodynamic efficiency 7 t h
can be defined and a 7 t h correlation is obtained. How-
ever, prediction of YDxis much better if based on
D;l/rAxas a correlating parameter than on qth.Also the
D;l/rAxcorrelation is very well understandable in bio-
chemical terms: contrary to the 7th correlation. There-
fore, Dil/rAxis the preferred correlating parameter to
estimate biomass yields.
APPENDIX 1
Setting Up the Redox Half Reactions and
Calculation of the Degree of Reduction and of the
Gibbs Energy and Enthalpy of Electrons in the
Redox Half Reaction
In the thermodynamic description of microbial growth one can
(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
reactants except the electron:
AGL = +0.2 X (-80) + 1 X (-588) - 2.5 X (-238)
-1X (-67) = +58 kJ
AGE = +5 X (-40) + 0.2 X (-80) + 1 X (-588)
- 2.5 X (-238) -1X (-67) = -142 kJ
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)

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