Heijnen Et Al-1992-Biotechnolo1gy and Bioengineering

In Search of a Thermodynamic Description
of Biomass Yields for the Chemotrophic

Growth of Microorganisms
J. J. Heijnen'* and J. P. van Dijken'
1
Department of Biochemical Engineering and of 2Microbiology and EnzymologF
Delft University of Technology, Julianalaan 6Z 2628 BC Delft, The Netherlands
Received June 12, 1991Mccepted October 18, 1991
Correlations for the prediction of biomass yields are valuable, and many proposals based on a number of parameters
(YATP,YA",, vo, V,, Gibbs energy efficiencies, and enthalpy
efficiencies) have been published. This article critically examines the properties of the proposed parameters with respect to the general applicability to chemotrophic growth
systems, a clear relation to the Second Law of Thermodynamics, the absence of intrinsic problems, and a requirement of only black box information. It appears that none of
the proposed parameters satisfies all these requirements.
Particularly, the various energetic efficiency parameters suffer from major intrinsic problems. However, this article will
show that the Gibbs energy dissipation per amount of produced biomass (kJ/C-mol) is a parameter which satisfies the requirements without having intrinsic problems. A
simple correlation is found which provides the Gibbs energy
dissipation/(=-mol biomass as a function of the nature of
the C-source (expressed as the carbon chain length and the
degree of reduction). This dissipation appears to be nearly
independent of the nature of the electron acceptor (e.g., Oz,
NO3-, fermentation). Hence, a single correlation can describe a very wide range of microbial growth systems. In
this respect, Gibbs energy dissipation is much more useful
than heat production/C-mol biomass, which is strongly
dependent on the electron acceptor used. Evidence is
presented that even a net heat-uptake can occur in certain
growth systems.
The correlation of Gibbs energy dissipation thus obtained shows that dissipation/C-mol biomass increases
for C-sources with smaller chain length (C, + C,), and
increases for both higher and lower degrees of reduction
than 4. It appears that the dissipation/C-mol biomass can
be regarded as a simple thermodynamic measure of the
amount of biochemical "work" required to convert the carbon source into biomass by the proper irreversible carboncarbon coupling and oxidation/reduction reactions. This is
supported by the good correlation between the theoretical ATP requirement for biomass formation on different
C-sources and the dissipation values (kJ/C-mot biomass)
found. The established correlation for the Gibbs energy dissipation allows the prediction of the chemotrophic biomass
yield on substrate with an error of 13% in the yield range
0.01 to 0.80 C-mol biomass/(C)-mol substrate for aerobic/
anaerobic/denitrifying growth systems.
Key words: biomass yield chemotrophic growth Gibbs
energy dissipation thermodynamic efficiencies energy
convertor
INTRODUCTION
Microbial growth occurs on a wide variety of compounds (Table I). For biotechnological processes of industrial interest, chemotrophic growth is most relevant.
Phototrophic growth i s rarely exploited at present. An
important parameter in biotechnological processes i s
Table I.
A sample list of microbial growth systems.
Electron donor
couple
Electron acceptor
couple
C-source
Organic
Organic
Organic
Oxalic acid/COz
Formic acid/COz
Glyoxalic acid/COz
Malic acid/C02
Citric acid/COz
Pyruvic acid/C02
Succinic acid/COz
Gluconic acid/COz
Formaldehyde/COz
Glucose/COz
Lactic acid/C02
Acetic acid/COz
Mannitol/COz
Glycerol/C02
2,3 Butanediol/acetoin
Et hanol/COz
Methanol/COz
n-Alkanes/COz
Methane/COz
Fumarate/succinate
Pyruvate/lactate
Acetaldehyde/ethanol
Acetoine/ butanediol
Oxalic acid
Formic acid
Glyoxalic acid
Malic acid
Citric acid
Pyruvic acid
Succinic acid
Gluconic acid
Formaldehyde
Glucose
Lactic acid
Acetic acid
Mannitol
Glycerol
2,3 Butanediol
Acetoine
Ethanol
Methanol
n-Alkanes
Methane
Inorganic
Inorganic
Inorganic
coz
co
* To whom all correspondence should be addressed.
Biotechnology and Bioengineering, Vol. 39,Pp. 833-858 (1992)

0 1992 John Wiley & Sons, Inc.
CCC 0006-3592/92/080833-026$04.00
the yield (YDx)

of biomass (X) on the available substrate
(electron donor, D). Yoxis defined as C-mol of biomass
produced per amount of electron donor consumed (in
C-mol for organic or in moles for inorganic donors).
Hence, YDxis in C-mol/(C)-mol. Because of its prime
importance, biomass yield for many different microbial
systems has been studied extensively, and it is currently
known for a wide variety of substrates that support microbial growth. Yields can vary widely, within the range
of 0.01 to 1.0 C-mol biomass/(C)-mol electron donor,
and depend strongly on the microorganism and its
growth substrates.
In practice, an estimate of the expected biomass yield
in a process is frequently required before the biochemical capabilities and properties of the microorganism(s)
are known. Considering the wide variety of potentially
useful microbial systems (Table I), this poses a difficult
problem. The solution lies in the selection of a parameter which can be quantified from an established simple
correlation, and which can be used to calculate YDx.
Such a parameter should, however, preferably comply
with a number of general requirements. First, it is required that the parameter can be generally applied to all
microbial growth systems. Second, it is well-known that
a theoretical upper limit also can be calculated for YDx
due to the Second Law of Thermodynamics. Thus, it
would be useful if the parameter itself has an upper or
lower limit which originates from the Second Law.
Third, one often faces the problem of providing an estimate of YDxfor a microbial system, for which the biochemistry is not well-known. Hence, one must be able
to use the parameter without having information about
the intracellular biochemical properties of the microorganism (e.g., electron transport chain or anabolic/
catabolic properties). The only information available,
known as black box information, would deal with the
carbon source, electron donor and acceptor, N-source,
and biomass composition. This information is generally
presented as a black box (Fig. 1, which indicates the
consumption/production of said chemicals), or as a
macrochemical equation which describes the material
balance for the production of 1 C-mole biomass. Figure 1 contains a simple example of a black box and
the macrochemical equation for the aerobic growth of
Pseudomonas oxalaticus on oxalate with a yield of
0.086 C-mol biomass/C-mol oxalate. Finally, such a
parameter should not suffer from intrinsic problems
(the nature of which will become apparent).
Because of its obvious importance, there have been
numerous attempts to establish biomass yield predictions (Table IIA). This article will present a critical
evaluation of these attempts in relation to the abovementioned requirements. The negative results of this
evaluation prompted us to develop a more satisfying
parameter, based on the Gibbs energy dissipation per
unit of biomass produced.
834
Figure 1. Black box description of microbial growth.
Table IIA.
Models for the prediction of biomass yields.
Correlation
based on
Metabolic
Black box
Black box
Black box
Black box
Metabolic, conservation
Metabolic, energy convertor
Black box
YATP
YAW
70
Y,
ATP
Available electrons
Oxygen
Carbon
Gibbs energy
Gibbs energy
Gibbs energy
Enthalpy
Table IIB.
yields.
Nature of
the model
Parameter
7BB
;Zc
7 H
Evaluation of the models for the prediction of biomass
1. Generally
applicable
2. Relation to
Second Law
3. Black box
4. Invariant to
frame of
reference of
Gibbsenergy
5 . Invariant to
choice of
input/output
process
-: Not relevant.
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 8, APRIL 5, 1992
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Yes
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No
Critical Evaluation of Proposed Methods for the

Prediction of Biomass Yields
trons per C-mol electron donor upon combustion). These

electrons generate the Gibbs energy needed for microbial
growth. The idea is that, assuming a constant Gibbs
In 1949, Monod proposed the concept of the yield of
energy
production per mole of electrons, the biomass
biomass on substrate.'l This yield was originally considyield
per
mole of available electrons might be constant.
ered to be a characteristic constant of a microorganThis
parameter
can be calculated for any microbial sysism for a specific substrate. Later, Herbert and Pirt
tem,
and
is
generally
applicable. YAvefollows directly
showed that the biomass yield was only constant at high
YDx,
using
only
black
box conservation relations
from
growth rates, and that the yield dropped at low growth
as
shown
by
Roels,28
and
is
therefore a true black box
rates because of maintenance and/or endogeneous proparameter.
The
obvious
limitation
is that YAveis not
c e ~ s e s .Following
~ ~ , ~ ~ these initial studies, maximal bioconstant
for
different
electron
donors
and acceptors.
mass yields have been determined experimentally for
For
example,
aerobic
and
anaerobic
growth
give a difmany microorganisms growing on a wide variety of
4
to
5
in
YAv,.
This
is
due
to the fact
ference
of
a
factor
substrates (for reviews, see refs. 14, 18, 20, and 23). In
that
the
Gibbs
energy
of
combustion
per
available
electhis article, only the so-called maximal biomass yield,
tron
depends
strongly
on
the
electron
donor/acceptor.
corrected for maintenance, designated by the symbol
Furthermore, there is no intrinsic Second Law-based
YDX[yield of biomass (X) on electron donor (D)], will be
limit
for YAve.
considered.
Minkevich
and EroshinZ3proposed the oxygen effiThe results of yield determinations have greatly stimuq,,
which
is defined as the ratio of amount of
ciency
lated the search for parameters to correlate biomass
electrons
conserved
in biomass over the amount of elecyields (Table IIA). A critical evaluation of these attrons
available
in
organic
substrate by aerobic combustempts will be provided here. This evaluation will be
tion to HC03-. Roels" has shown that q, follows from
qualitative. For the quantitative relationships between
Y D using
~
only black box conservation relations. Hence,
YDXand YATP,YAve, q,, K , TH, q C , qBB,or qECand a
q, is a true black box parameter. An obvious limitation
critical comparison of the established correlations to
is that q, can only be applied to aerobic systems and,
predict YATP,YAve, qo, K, TH, q C, q BB, and qEC,the
reader is referred to Reels:' We~terhoff:~S t o ~ t h a m e r , ~ ~therefore, lacks general applicability. Also, there is no
intrinsic limit based on the Second Law.
and battle^.^
Linton and Stephenson" proposed that the carbon
In this section, the less-known general aspects of
yield
of biomass on the C-source, Y,, could be used as a
these parameters will be elucidated in light of the reparameter
for growth. This is clearly a black box paquirements described above (see Table IIB for sumrameter
which
is identical to YDx for heterotrophic
mary). Initial attention will be given to chemical
growth.
However,
for autotrophic growth, this parameefficiencies and, subsequently, the Gibbs energy and
1.
Hence,
K is not a generally applicable
ter
is
always
enthalpy-based efficiencies will be dealt with.
parameter. Furthermore, there is also no intrinsic limit
based on the Second Law.
YATP,YA"~,~ 0 Yc,
Bauchop and Elsden6 proposed the concept of expressing the yield of biomass in terms of consumed ATP
(YA~pin grams of biomass dry weight/mol ATP). This
concept, which is, in principle, applicable to any microbial system, has subsequently been extended by
Stouthamer3' and others. The effects of maintenance
and different C- and N-sources has been taken into account, and the theoretical YATphas been shown to vary
between 2 and 30 g/mol. A major problem is, however,
that there is often a gap of about 50% between experimentally measured and the theoretically predicted YATp.
Moreover, for practical application, one needs detailed
biochemical knowledge about the ATP generated by the
specific microorganism to be able to calculate the
biomass yield, YDx. This concept can, therefore, not be
applied if one only has black box information. Furthermore, this parameter has no intrinsic limit based on the
Second Law.
Mayberry et al?' proposed the concept of biomass
in terms of grams of biomass per mole availyield (YAve)
able electrons (which is defined as the number of elec-
qBB
Roels proposed the use of the black box (BB) Gibbs

energy efficiency, qBB,as a correlating parameter for
microbial growth processes. (Fig. 3.7 in ref. 28). qBBIs
defined as the ratio between the sum of the Gibbs energy associated with all consumed chemicals (input)
and the sum of the Gibbs energy associated with all
produced chemicals (output) (Fig. 1). The relation between qBBand Yox can be calculated from a black box
approach, using elemental conservation principles and
the macroscopic Gibbs energy balance.28
The attractive features of this type of approach are
that it can be applied to any microbial system, needs
only black box information, and has a maximal limit of
1 due to the Second Law of Thermodynamics. This
limit of qBB= 1 leads directly to the theoretical maximal limit in YDx by substitution of qBB= 1 in the equation between YDx and qBB.
An important aspect of energetic efficiencies is that
they, as well as being correlating parameters, are gener-
HEIJNEN AND VAN DIJKEN: THERMODYNAMICS OF MICROBIAL GROWTH
835
ally considered as a measure of thermodynamic process

performance. A high numerical value (e.g., vBB= 90%),
indicates that only 10% of the available Gibbs energy
has been lost, and hence indicates a good performance;
vBB= 10% is then considered bad. Moreover, one intuitively expects that qBBincreases as YDxincreases, and
vice versa. However, energetic efficiencies are troubled
with intrinsic problems, which makes such an intuitive
interpretation improper, as will be shown below.
To use the qBBconcept, one has to choose a certain
frame of reference within which the Gibbs energy of the
chemical compounds is defined. This choice is completely independent of the specific process studied, and
normally one uses the thermodynamic reference (zero
Gibbs energy for elements at standard temperature and
pressure). However, other choices are possible. For example, Roels has defined the combustion reference as
a particularly convenient choice.28Here, the Gibbs energy of 02,HC03-, H 2 0 , N-source, and H are defined as zero at standard temperature and pressure. This
leads to a Gibbs energy of organic compounds which is
equal to the combustion Gibbs energy (Appendix A,
Table A-I). It is obvious that a meaningful thermodynamic efficiency of a process should be independent
of the choice of the frame of reference of the Gibbs
energy of the chemical compounds. To test this, 7
has been calculated for the above-mentioned 2 frames
of reference for a set of aerobic and a set of anaerobic
growth data (taken from Rutgers3). Sample calculations
+
are presented in Appendix A. The results for 71 (thermodynamic reference) and 728 (combustion reference)
are listed in Table I11 and shown in Figure 2A (aerobic
growth) and Figure 2B (anaerobic growth) as a function
of the degree of reduction (yD)of the organic substrate/
electron donor. yD Is the fiumber of electrons liberated
upon oxidation of 1 C-mol of organic material to C02
or of 1 mol of inorganic material to its oxidized form.28
From Figures 2A and B and Table 111, the following
conclusions are obvious:
There is a large difference in the values of 17 1 and r/2BB
for the same microbial system.
For aerobic growth (Fig. 2A), a switch in the frame of
reference leads to a complete reversal of the efficiency
correlation and also in counterintuitive behavior. For
example, 7728 for oxalate is much lower than for ethanol. In addition, vfB correlates with YDx in an intu8 ~ a higher YDx.
itively expected way; a higher ~ 2 gives
However, for 71 the behavior is completely reversed,
now oxalate has a very high efficiency and ethanol the
lowest. Also, a higher YDx corresponds with a lower
71, which is quite counterintuitive.
For anaerobic growth (Fig. 2B) the obtained vBBcorrelation (for both reference frames) is completely different from that for aerobic growth. Apparently, different
correlations are found for different electron acceptors;
furthermore, a switch in reference frame no longer
gives such a dramatic change in behavior, as observed
in the aerobic case. However, now both frames of ref-
Table 111. Black box Gibbs energy efficiencies with thermodynamic frame of reference
(9:) and combustion frame of reference (7:)for aerobic and anaerobic growth.
7J
C-mol/C-mol
(-)
(-1
Pseudomonas oxalaticus (aerobic)

czo42OxaIatezFormateCHOzGlyoxylateCzH03TartratezC4H4062Malonate2C3Hz042citrate3CsH5073Succinate2c4 H 4 0 2 AcetateCzH302Fructose
C6 H 1 2 0 6
Glycerol
C3HaO3
Ethanol
CzH&
1
2
2
2.5
2.7
3.0
3.5
4
4
4.66
6.0
0.086
0.162
0.220
0.280
0.238
0.390
0.385
0.406
0.505
0.569
0.558
0.83
0.66
0.68
0.64
0.62
0.62
0.51
0.46
0.40
0.39
0.20
0.31
0.30
0.40
0.45
0.39
0.55
0.49
0.46
0.50
0.50
0.40
Klebsiella pneumoniae (anaerobic)

itr rate'CsH50:PyruvateC3H303GluconateC6H1107Fructose
C 6H 1 2 0 6
Glucose
C6H 1 2 0 6
Dihydrox yacetone
C3H603
Mannitol
C6Hi406
Glycerol
C3H803
3
3.33
3.66
4
4
4
4.33
4.66
0.073
0.083
0.121
0.173
0.176
0.150
0.154
0.093
0.95
0.93
0.90
0.87
0.84
0.83
0.86
0.86
0.96
0.95
0.93
0.93
0.92
0.92
0.94
0.95
Clostridium butyricum (anaerobic)

GluconateCsHi107Glucose
C6H1206
Mannitol
C6H1406
3.66
4
4.33
0.143
0.176
0.151
0.90
0.84
0.87
0.93
0.91
0.93
YDX
836
7J ,
YO
Substrate
Composition
060
/Y DX
060
Ps~udomnasoroloficus
YDX
0.40
Aerobic
0.20
0.00
1
0
YD
7)-
0.90 -
0.00
o.80
0.60
O''O1
0.50
0.50
0.40
0.40
0.30
0.30 -
0.20
020
0.10
\cL.
8
0.60
8
YD
0.90i
7)-
0.70
0.00
0.80 -
0.10
K/Cbsi.//o pneumonia*
Clorfridium bulyricum
Vrs
1
1
0.00
0
yo
Y D
(A)
(B)
Figure 2. Black box thermodynamic efficiency, T ' ~ ,and biomass yield on electron donor (YDx)
for microbial growth using a thermodynamic frame of reference, (qFB), or a combustion frame of reference, (T:'), for C-sources with different degrees of reduction.
(A) Aerobic growth (Pseudomonas oxala~icus)~';
(B) anaerobic growth (Klebsiella aerogenes and Clostridium butyricum).3'
erence do indicate that a higher YDxgives a lower qBB,

which is again counterintuitive.
From these results, it clearly follows that qBBis not a
meaningful process parameter because its value is very
sensitive to the chosen frame of reference. Also, counterintuitive behavior is observed. These intrinsic problems are general properties of any black box efficiency,
which is defined as a ratio of total Gibbs energy input
and total Gibbs energy output. Therefore, qBBis not
considered to be a meaningful parameter to correlate
biomass yields in the light of the requirements outlined
in the introduction.
Roels proposed a second definition of a Gibbs energy
efficiency, which is often also considered to be a black
box efficiency [eq. (3.67) in ref. 281. This parameter, in
contrast to the qEBdiscussed above, leads to a single
correlation which covers aerobic, denitrifying, and fermentative systems. From a correlative point of view, this
parameter is more successful than qBB,However, it is
easily shown that this parameter is not a black box
Gibbs energy definition, but that it resembles an energy
convertor efficiency of Gibbs energy. This proposal is

dealt with in a paragraph below on Gibbs energy efficiencies, qEC,based on the energy convertor concept of
microbial growth. Furthermore, Appendix C explains
the difference between a black box and an energy convertor efficiency, because these can easily be confused.
.I"
In 1960, Battley introduced the concept of Gibbs energy conservation efficiency qc to describe microbial
To calculate qc, two chemical reactions, the
conservative reaction and the nonconservative reaction, are defined.
The conservative reaction, also called the growth
equation, is in fact the macrochemical equation (Fig. l),
whereby proper multiplication the stoichimetric coefficient of substrate becomes -1. Hence, the conservative
equation represents the mass/elemental balance of the
conversion of 1 mol of substrate into biomass. From this
conservative reaction one can calculate AGc, the Gibbs
837
energy of the conservative reaction. Clearly, ( -AGc) is

then the Gibbs energy dissipated in this reaction.
The nonconservative reaction describes the process
which occurs when all the substrate of the conservative
reaction (being 1 mol) is converted according to the
catabolic pathway used by the microorganism. For example, for anaerobic growth of Saccharomyces cerevisiae on glucose, this is the conversion of 1 mol glucose
to ethanol and CO,; for its aerobic growth on ethanol,
this is the combustion of 1 mol ethanol to CO2 and
H 2 0 . This nonconservative reaction has a Gibbs energy
of AGNC. Hence, (-AGNc) is the maximum amount of
Gibbs energy which can be generated by the organism
by catabolizing all substrate.
In relation to the maximal available Gibbs energy
(-AGNC), the amount (-AGNC + AGc) represents then
a measure of the Gibbs energy, which has not been dissipated. Hence, '
7 = (-AGNC + AGc)/(-AGNc) is the
fraction of the maximal available Gibbs energy which
has not been dissipated, but can be considered as conserved into growth. Also, it is clear that in the hypothetical case of thermodynamic equilibrium AGc = 0,
and therewith qc has a maximal value of 1. Also, the qc
concept is generally applicable, and is not influenced by
different frames of reference of Gibbs energy for the
chemical compounds. Using this approach, Battley3 obtained a useful correlation for aerobic and anaerobic
7 and the maximal available Gibbs
growth between '
energy per C-mol of substrate. It is obvious that, in order
to apply qc, one must possess biochemical information
about the catabolic route, used by the organism, to
7 canestablish the nonconservative equation. Hence, '
not be considered as a black box parameter. Furthermore, an intrinsic problem appears to be that ( -AGNc)
represents the maximal available Gibbs energy if at1
substrate is catabolized.
In actuality, in heterotrophic growth, a part of the
substrate is always assimilated to biomass and only
a fraction of the substrate is catabolized to generate
7 appears not to represent
Gibbs energy. Therefore, '
the actual energy metbolism, but must be considered as
an operational definition. Nevertheless, from an empirical point of view, the 'q concept leads to an interesting
correlation.
rlEC
Kedem and Kaplan proposed the general concept of a

linear Gibbs energy convertor to describe nonequilib' ~the early 1980s this
rium processes in the late 1 9 6 0 ~In
concept was applied to describe microbial growth by
W e ~ t e r h o f f(Fig.
~ ~ 3).
Basically, the overall growth process, represented
by the measured macrochemical reaction, is split into
2 processes. Each of these processes can be described
by a chemical reaction, the sum of which is equal to the
already known macrochemical equation. The first process is a Gibbs energy-producing chemical reaction,
838
GIBBS ENERGY CONVERTOR DESCRIPTION OF MICROBIAL GROWTH

convertor
CATABOLISM
"Gibbs energy
"Gibbs energy
uptake"
(Y,
5.315 C,Or
2.657 0.
- 5.315 (0
= O.Ce6)
/
ANABOLISM
CATA0OLISU
ANABOLISM
1391 kJ
3 4 3 kJ
+ 10.63 HCO;
- 0.5 c p y
- 0.2 FH:
- 0 . 1 yo
- 0s K
ANABOLISM
0 5 C,O:'
- 02
NH,'
- 0
1 H,O
0 8 H'
1 CH,O,,N,,
0 8 0,
CATABOLISM
5 315 C,O:-
5 315 H,O
- 2 6 5 7 0, +10 63
HCO;
SUM MACROCHEMICAL EQUATION
5 815 C,O:-
- 0 2 NH,' + 1 CH,,O,,N,,
5 415 H,O - 0 8 H' - 1 8 5 7 0,

+ 10 63 HCO;
Figure 3. Gibbs energy convertor description of microbial growth
commonly identified as catabolism. The second process

is the chemical reaction which produces the biomass,
commonly called anabolism. This second process normally requires Gibbs energy. Now the ratio of the required Gibbs energy in anabolism to the produced Gibbs
energy in catabolism is a measure of the Gibbs energy
conservation, and is called the thermodynamic coupling
efficiency qECof the convertor (EC from energy convertor). Figure 3 shows an example to illustrate this concept. From the Second Law, it follows directly that qEC
is maximally 1. This concept can be generally applied,
and the value of ''7 is not influenced by different
choices of frames of reference of Gibbs energy of the
chemical compounds, because qECis a ratio of the
Gibbs energies of reaction of the defined anabolic and
catabolic processes.
Furthermore, it has been shown, that an exact value
of qECcan be derived from a supposed optimization
criterion according to which the biomass energy convertor
For example, a maximal rate of biomass formation at optimal efficiency gives 7" = 0.24.
Such efficiencies have subsequently been calculated for
many aerobic growth systems,43suggesting that growth
has perhaps evolved toward maximal growth rates at
optimal qEC.The independence of''7 from the frame
of reference, in combination with the possible link to
an optimal criterion, makes the parameter vECa more
interesting candidate than qBBor qc. However, close

inspection reveals a basic problem in the use of qEC.
This resides in the requirement to specify the chemical
reactions of catabolism (providing the Gibbs energy input into the convertor) and anabolism (which contains
the conserved Gibbs energy and represents the convertor output of Gibbs energy). As already stated, this boils
down to a split of the macrochemical reaction. Making
such a split presents 2 problems.
It is assumed that only black box information is available, and not specific biochemical knowledge about
the microbial system. Hence, only generally available
biochemical knowledge can be used. Within this
framework, many splits are still possible. Thus, it is of
importance to find out whether qECis sensitive toward
different splits.
The 2 processes resulting from a proposed split are
not independent. Their sum must equal the measured
macrochemical equation. Hence, a proposed catabolic
(or anabolic) reaction implicitly defines the corresponding anabolic (or catabolic) reaction. In the literature, different proposals for the catabolic (or anabolic)
process have been made on the basis of generally
available biochemical arguments. However, the corresponding implicitly defined anabolic (or catabolic)
process also has to be questioned from a general biochemical viewpoint. Hence, it is of obvious interest to
study both the defined catabolic and anabolic processes from a general biochemical point of view, for
the various proposed qECdefinitions.
To answer the first point, 3 different splits for aerobic

and anaerobic growth data sets have been made. It is
stressed that these splits are only for illustrative purposes to test the qECsensitivity toward different splits.
Appendix B shows the chosen splits and gives the calculations. The results are shown in Table IV, Figure 4A
(aerobic growth), and Figure 4B (anaerobic growth). It
is clear that qECis very sensitive to the chosen split.
Thus, it is important to take into account as much as
possible the generally available biochemical arguments
for a particular split in order to obtain a meaningful
thermodynamic efficiency qEC.
It is interesting to pay some attention to the various
proposed splits associated with the definitions given by
a number of proponents of qEC.
Roels implicitly proposed a qECdefinition (Appendix D) which gave a useful correlation of aerobic, anaerobic, and denitrifying growth.28A general anabolic
definition was proposed, which is equivalent to the
statement that biomass formation occurs from COzwith
production of Oz. The virtue of this definition is that
there is always Gibbs energy uptake in this process,
which assures that 0 < qEC< 1. From a biochemical
point of view, it is clear that this anabolic process is very
unrealistic for heterotrophic growth systems (especially
the fermentative and the denitrifying systems). It is
now illuminating to see which implicitly defined catabolic processes arise from Roels' anabolic definition
(Appendix D). For aerobic growth, one finds that the
catabolic reaction is the aerobic combustion of the or-
Table IV. Gibbs energy convertor efficiencies vECwith 3 different definitions of input
and output processes for aerobic growth and anaerobic g r ~ w t h . ~ '
:"
:"
11
C-mol/C-mol
11
(-)
11
(-)
Pseudomonas oxalaticus (aerobic)

1
OxaIate2Formate2
Glyoxylate2
Tartrate*2.5
Malonate2.7
citrate33.0
3.5
Succinate2Acetate4
Fructose
4
Glycerol
4.66
Ethanol
6.0
0.086
0.162
0.220
0.280
0.238
0.390
0.385
0.406
0.505
0.569
0.558
0.246
0.169
0.233
0.233
0.200
0.268
0.164
0.085
-0.003
-0.170
-0.350
-0.078
-0.055
-0.111
-0.042
+0.025
+0.017
+0.044
+0.053
-0.057
-0.045
-0.020
0.31
0.30
0.40
0.45
0.39
0.55
0.49
0.46
0.50
0.50
0.40
Klebsiella pneumoniae (anaerobic)

citrate33
Pyruvate3.33
Gluconate3.66
Fructose
4
Glucose
4
Dihydroxyacetone
4
Mannitol
4.33
Glycerol
4.66
0.073
0.083
0.121
0.173
0.176
0.150
0.154
0.093
-0.014
-0.028
-0.086
-0.144
-0.125
-0.126
-0.094
-0.065
+0.035
-0.002
- 0.079
-0.144
-0.125
-0.123
-0.094
-0.069
-0.157
-0.120
-0.119
-0.144
-0.122
-0.111
-0.115
-0.110
Clostridium butyricum (anaerobic)

3.66
GluconateGlucose
4
Mannitol
4.33
0.143
0.176
0.151
-0.100
-0.125
-0.094
-0.092
-0.125
-0.094
-0.139
-0.122
-0.115
YDX
Substrate
YD
(-1
839
yoxo601
0 60
YDX
Pseudomonos oxolOt,CuS
Aerobic
0 40
Klrbsrdla pmumonia8
Clostndum bulyricum
Anaerobic
0 40
0 20
0.20
'
0.00
1
7)"
. .
0 50
yo
0001
0.4 1
0.40
0.30
o'201-
0.20
0.10
0 lo
0.00 -
0.00
-0.10
-0 20
-020
-0.30
-0.30
-0 10
yo
(A)
yo
(B)
Figure 4. Gibbs energy convertor efficiency, T ~and

~ biomass
,
yield on electron donor, ( Y D x )using
,
three different definitions of input
and output (qFc,qFc, qFc) for C-sources with different degrees of reduction. (A) Aerobic growth (P.~xalaticus)~';
(B) anaerobic growth
( K . aerogenes and C. b~tyricurn).~'
ganic substrate, which is biochemically quite acceptable.

strates than biomass. This then gives the possibility of
This shows that an unrealistic anabolic reaction somenegative values of qEc.The implicitly defined catabolic
times can lead to an acceptable catabolic reaction. Howreaction for aerobic growth is the combustion of the
ever, the corresponding catabolic process for anaerobic
substrate with 02,which is biochemically quite valid.
growth is unusual, being the partial oxidation, with 02,
For anaerobic growth, it has been pointed out that
of the substrate to fermentation products. In concluthis anabolic definition is unreali~tic,~~
and it has been
sion, it appears that qECas defined by Roels, although
proposed to replace O2with H2. However, for fermentaproviding a satisfying empirical correlation of microbial
tion processes without H 2 this proposal is again unsatisgrowth, cannot be considered as a valid measure of
factory. In addition, the qEc correlation obtained for
thermodynamic process performance.
anaerobic growth totally differed from aerobic
Westerhoff and Van Dam43 proposed, for aerobic
The question then arises whether one can formulate
growth, an anabolic process (Appendix E) in which bioan anabolic reaction equation which is based on biomass formation occurs from the organic substrate with
chemical evidence. Such equations have indeed been
production of O2 (for substrates more oxidized than
proposed.8 It is easily shown, however, that the Gibbs
biomass) or consumption of O2 (for substrates more reenergy of these anabolic reactions is invariably close to
duced than biomass). The O2 production is, for heterozero, a fact which was already recognized by Battley' in
trophic growth, clearly unrealistic from a biochemical
his studies of qc. He proposed a similar anabolic reacpoint of view. Also the above-mentioned hypothesis that
tion where biomass is produced from organic substrate,
growth has evolved to a maximal rate at optimal qEC C02, and N-source, without involvement of 02.This
seems to be questionable, because the experimentally
leads to partly C 0 2 fixation for substrates more reduced
found qEC= 0.24 is based on this biochemically unrealthan biomass and C 0 2production for substrates less reistic split.
duced than biomass. In fact, this proposal of anabolism
In addition, a substantial Gibbs energy production is
is identical to the anabolism used in split 2 (Appenobtained in the anabolic process for more reduced subis differdix B). Figure 4A and B clearly show that
840
rent for aerobic and anaerobic growth. Also qECappears to be close to zero for this anabolic proposal
where biochemistry is taken into account as much as
possible. This value of qEc= 0 does not correspond to
any optimization criterion.43
Before closing this section on Gibbs energy-based efficiency proposals, it is interesting to remark that, for
aerobic heterotrophic growth, qBB(with combustion reference), qEC(Roels), and qc from Battley all give identical results. It appears that totally different concepts may
lead to exactly the same efficiency.
In conclusion, it appears that qECcannot be applied
as a biochemically meaningful measure of thermodynamic process performance, given only black box information. Extensive biochemical knowledge is required
for a useful application, and major intrinsic problems
are present.
Enthalpy Efficiencies qH
Enthalpy efficiencies have been defined in analogous

terms as the previously discussed Gibbs energy efficiencies. Minkevich and EroshinZ3and Roels28proposed the
use of the black box efficiency using the combustion
reference for enthalpy values. Battley3 has proposed the
use of the heat efficiency derived from the conservation
concept of microbial growth. As with the Gibbs energy
efficiencies, these parameters are generally applicable.
However, they lack a theoretical upper limit based on
the Second Law. The Second Law does allow for heatuptake during growth, which would result in an enthalpy efficiency > 1. More serious is, however, that
the intrinsic problems which have been pointed out for
the various Gibbs energy efficiencies also apply to the
analogous enthalpy efficiencies.
Summarizing, it can be concluded that none of the parameters discussed above provide a satisfactory framework for the correlation of microbial growth yields of
chemotrophic systems (Table IIB). Their range of application is too limited (yC and v,),most have no relation
to the Second Law of Thermodynamics (YATp, K, q,,
enthalpy efficiencies, YAVe),
some require biochemical
information (YATp, qc, vEC),and intrinsic problems occur due to frames of reference (vBB),making a split
(vEC),and assuming catabolism of all substrate (77'). In
the next section, another parameter, which can be used
to achieve a biomass yield correlation, will be presented.
This parameter fulfills the requirements mentioned in
the introduction.
Gibbs Energy Dissipation Per C-Mol Biomass
Produced as a Predictive Parameter for
Chemotrophic Growth
It has been suggested by Roels that the Gibbs energy

dissipation, which occurs per unit of biomass produced,
is perhaps constant for many C-sources, and probably
independent of the type of electron acceptor involved.28
If 0,"'
is designated as the Gibbs energy dissipated
(kJ/m3 h) and biomass production (C-mol/m3h) is represented by TAX, then Dsol/rAxprovides the Gibbs energy
which must be dissipated by the microbial system in
order to produce 1 C-mol of biomass from the available
C-source, electron donor, and electron acceptor. If
D;'/rAx is considered with respect to the requirements
mentioned in the introduction it appears that:
The dissipation of Gibbs energy can be calculated for
any microbial growth system.
The Second Law of Thermodynamics requires that
Dsol/rAxis positive, hence there is a theoretical lower
limit: ~ s o l / r A L
~ 0.
The value of D;'/rAxcan be calculated from black box
information alone. RoelsZ8has shown that there is a
In fact,
unique relationship between YDxand Dsol/rAx.
Dso'/rAxis equal, but of opposite sign, to the reaction
Gibbs energy of the macrochemical reaction (Fig. 1,
Appendix F).
Df'/rAxdoes not suffer from the intrinsic problems
which were found for qBBand qEc.It is clear (See Appendix F) that Dsol/rAxis independent of the chosen
frame of reference. Furthermore, there is no need to
provide a split in input and output processes for its
calculation; D;l/rAx can be calculated directly from
black box information alone as represented in the
macrochemical equation (Appendix F).
It is clear that the parameter Dsol/rAx
possesses all the
properties required to function as a thermodynamically
based, black box, predictive parameter for the calculation of biomass yields.
It must be remembered that the actual concentrations
of the reactants must be considered in order to calculate
meaningful values of Gibbs energy dissipation. This information is often not available. As a compromise, the
Gibbs energy at pH 7, and otherwise standard conditions (1 mol/L or 1 atm and 25"C), can be calculated.
This choice is indicated by the superscript "01". Deviations from the standard conditions generally exert a
limited effect. However, in some special microbial systems [e.g., anaerobic cultures with lo-' to lo-' atm Hz,
or systems with a low pH (e.g., pH l),or low values of
YDx],
significant effects can occur which necessitate the
use of the actual concentration^.^^ A set of published
data for which chemotrophic microbial growth yield
(electron donor limited) has been measured in batch or
continuous culture has been collected. In these experiments, well-defined mineral media were used and unknown product formation was excluded by showing that
the carbon and redox balances were satisfied. Reliable
macrochemical equations could thus be calculated, giving reliable and meaningful values of D:'/rAx.A sample
calculation of D,ol/rAx
is shown in Appendix F.
The microbial systems used cover a wide range of
conditions (Table VA-H) which encompass:
A large variety of microorganisms.
Chemoheterotrophic (Table VA-G) and chemoautotrophic (Table 5H) growth.
841
Table VA. Biomass yield and Gibbs energy dissipation for the aerobic growth of Pseudomonas oxalaticus on different organic substrate^.^'
YO
C-mol/C-mol
D,0/rAx
k J/C-mol biomass
1
2
2
2.5
2.7
3.0
3.5
4
4
4.66
6.0
0.086
0.162
0.220
0.280
0.238
0.390
0.385
0.406
0.505
0.569
0.558
1048
1089
709
584
757
383
504
567
470
473
702
YDX
Substrate
Composition
OxaIate2FormateGlyoxylateTartrate2Malonate2-
itr rate'Succinate2AcetateFructose
Glycerol
Ethanol
Table VB. Biomass yields and Gibbs energy dissipation for aerobic growth of Candida
utilus on different organic s~bstrates.~
YO
C-mol/C-mol
D?lrAX
k J/C-mol biomass
3.0
3.33
3.50
3.666
4.0
4.0
4.0
4.666
5.00
5.50
6.0
0.411
0.434
0.448
0.559
0.595
0.490
0.455
0.692
0.424
0.446
0.617
340
396
366
296
327
497
455
316
845
890
592
YOX
Substrate
Composition
CitratePyruvateSuccinate2GluconateGlucose
Xylose
AcetateGlycerol
Acetoin
2-3 Butanedic
Ethanol
Table VC. Biomass yield and Gibbs energy dissipation for aerobic growth of Paracoccus
denitrificans.39s43
Substrate
Composition
YO
C-mol/C-mol
D ,/TAX
k J/C-mol biomass
FormateMalate2Succinate2GluconateMannitol
Methanol
C02HC4H40:C4H40.tC6H1107C6H1406
CH40
2
3
3.5
3.666
4.333
6.0
0.12
0.42
0.48
0.51
0.62
0.54
1636
333
311
371
345
809
YDX
Table VD. Biomass yield and Gibbs energy dissipation for aerobic growth of Thiobacillus
acidophilus.27
Substrate
Composition
YO
C-mol/(C)-mol
D?/rAx
k J/C-mol biomass
FormateL-MalateZPyruvate Glucose
Glycerol
COzHC4H40:C3H303c 6H 1 2 0 6
C3H803
2
3.0
3.33
4
4.66
0.10
0.25
0.32
0.40
0.55
2058
880
704
717
512
YDX
Different C-sources, either more reduced or more oxidized than biomass (with degree of reduction yDfrom
0 -+8) and C-sources with highly different carbon
chain lengths (from C1 to C6).
Different electron acceptors such as O2(Table VA-E),
NO3- (Table VF) and fermentation (Table VG).
842
- A number of microbial systems where growth yield
data were measured for the same microorganism growing on a wide variety of electron donors ( = C-source).
From the available biomass yield data, the values of
were calculated (see
Gibbs energy dissipation (DP/rAx)
Appendix 6 for the procedure). The results are provided
Table VE. Biomass yield and Gibbs energy dissipation for aerobic growth of various heterotrophic microorganisms on various organic substrate^.^^'^.'^
YO
C-mol/C-mol
D?/rAx
kJ/C-mol biomass
1
2
3
3
3.5
3.666
4
4
4
4
4.333
4.666
4.666
5.33
6
6
6
6.13
6.5
8
0.07
0.18
0.375
0.365
0.400
0.51
0.61
0.5 1
0.41
0.47
0.56
0.67
0.480
0.445
0.53
0.54
0.575
0.57
0.445
0.55
1399
933
429
442
467
371
308
394
557
587
433
335
556
813
765
809
658
662
1061
1011
YDX
Substrate
Composition
OxaIate2FormateMalate2citrate3Succinate2GluconateGlucose
LactateAcetateFormaldehyde
Mannitol
Glycerol
Propionate
Acetone
Ethanol
Methanol
Propanoi
n-Alkanes
Butane
Methane
Table VF. Biomass yield and Gibbs energy dissipation for denitrifying growth of microorganisms on organic substrates using NO3-/N2 as a ~ c e p t o r . ~ '
YDX
Microorganism
Campylobacter
sputum
Paracoccus
denitrificans
P. denitrificans
C. sputum
P. denitrificans
Compound
Composition
YD
C-mol/C-mol
DPl/rAx
kJ/C-mol
biomass
Formate-
C02H-
0.166
999
Succinate2GluconateLactateMannitol
C4H402C6H1107C3Hs03C6H1406
3.50
3.666
4
4.333
0.387
0.505
0.274
0.506
466
358
1064
500
in Table 5A-H and Figures 5A-G. If one studies the

values of D:'/rAXfor the various microbial systems, a
very simple correlation becomes evident (Fig. 6A). It
appears that DsO1/rAx:
depends strongly on the degree of reduction and the
carbon chain length of the carbon source.
is only weakly dependent on the electron acceptor.
for chemoautotrophic systems with an electron donor
where reversed electron transport occurs, the required
dissipation is much higher than for chemoautotrophic
systems where reversed electron transport is absent.
Within both groups, the dissipation is influenced little
by the nature of the electron donor.
The effect of the degree of reduction of the C-source is
clearly observed in Figures 5A-G. Figure 6A contains
all data. The data in these figures cover a very wide
spectrum of C-sources, microorganisms, and electron
acceptors. Nevertheless, the same pattern arises. D:l/rAx
is minimal around yD = 3.5 to 4.5, and increases for
more reduced or more oxidized substrates. Moreover,
most Gibbs energy dissipation data for the different
microorganisms are fairly close. Typically, D:'/rAx is
around 200 to 350 kJ/C-mol for yD = 3.5 to 4.5, and

around 900 kJ/C-mol for YD = 0 to 2 and YD = 6 to 8.
Systematic deviation from the typical average behavior
of microorganisms can, however, also be observed. For
example Thiobacillus acidophilus (Table VD) has a
much higher dissipation than most aerobic microorganisms. One reason might be the low pH of 3.5, which
might increase dissipation. Another example is the anaerobic bacterium Butyribacterium methylotrophicum,
which has a significantly lower dissipation than most
fermentative microorganisms. The reasons for this are
not known.
The effect of the carbon chain length of the C-source,
as a function of its degree of reduction, is shown in
Figure 6A (based on the data of Table VA-H, without
T. acidophilus and B. methylotrophicum). For carbon
sources with the same number of C-atoms, one observes
the above-mentioned typical effect of its degree of reduction. Dissipation is minimal for the degree of reduction around 4 and increases on both sides. Table VI
contains the average dissipation values for a number of
C-sources.
843
Table VG. Biomass yield and Gibbs energy dissipation for anaerobic growth of defined heterotrophic microorganisms on organic
substrates.
Ref.
31
31
31
31
31
31
31
31
31
31
31
9,40
1
45
45
1
1
1
1
33
33
33
33
33
33
33
33
33
34
34
34
35
Microorganism
Substratel
electron Donor
Klebsiella pneumoniae
itr rate'-
Clostridium butyricum
PyruvateGluconateFructose
Glucose
Dihydrox y-acetone
Mannitol
Glycerol
Gluconate
Glucose
Mannitol
HZ
atm)
FormateAcetateMethanol
Hz/COz
Methanobacterium A Z
M. formicicum
M. soehngenii
Methanosarcina barkeri
Butyribacterium methylofrophicum
Pelobacter propionicus
P. carbinolicus
C. magnum
Saccharomyces cerevisiae
YD
C-mol/(C)-mol
D?/rAx
kJ/C-mol
3
3.33
3.666
4
4
4
4.33
4.666
3.666
4
4.33
2
2
4
6
2
2
4
6
4
5
5.5
6
6
5
0.073
0.083
0.121
0.173
0.176
0.150
0.154
0.093
0.143
0.176
0.151
0.019
0.053
0.024
0.13
0.056
0.11
0.250
0.30
0.085
0.08
0.063
0.028
0.019
0.073
185
236
237
210
236
257
191
254
219
229
222
822
880
539
570
440
350
110
584
197
358
390
785
792
617
5
5.5
3
4
5
5.5
4
0.070
0.036
0.03
0.32
0.08
0.072
0.14
259
244
55 1
139
364
353
255
YDX
Composition
co
Glucose
Methanol
Lactate
Acetoin
Butanediol
Ethanol
Propanol
Ethylene
Glycol
Acetoin
Butanediol
citrate3Glucose
Acetoin
Butanediol
Glucose
Table VH. Biomass yield and Gibbs energy dissipation for chemoautotrophic growth.
Ref.
Microorganism
Acceptor
No reversed electron transport systems

24
Methanobacterium arborophilus
34
Alcaligenes eutrophus
22
Carboxydotrophic bact.
9,40
M. A Z
Reversed electron transport systems
30
Thiosphaera pantotropha
30
Thiobacillus neapolitanus
11
Thiobacillus ferrooxidans
27
Thiobacillus acidophilus
11
Thwbacillus ferrooxidans
39
Thiobacillus denitrificans
12
Thiobacillus ferrom'dans
25
Nitrosomonas europaea
25
Nitrobacter sp.
It appears that the carbon sources containing more

C-atoms (for the same degree of reduction) require less
Gibbs energy dissipation. For example, consider the
carbon sources with degree of reduction 4 (formaldehyde, acetate, lactate, dihydroxy-acetone, glucose, see
844
Donor
(at4
YO
YDX
(-1
C-mol/mol
D?lrAx
k J/C-mol
biomass
0.015
0.13
0.16
0.019
1076
1267
1105
840
0.16
0.16
0.22
0.23
0.41
0.30
0.010
0.06
0.017
4627
4627
3237
3076
2761
2186
2927
4117
3892
~~(10-~)
Hz(lo-')
co
~~(10-~)
szoszs20:-
s20:s20sz-
s402HSFe2+(pH 1.6)
NH4+
N02-
8
8
8
8
14
8
1
6
2
Table VI). It appears that DP1/rAxis halved when the

chain length of the carbon source increases from 1 to 3.
An analogous trend is observed for compounds with
degrees of reduction 2 to 2.5 (CO/formate, glyoxylate,
tartrate), 2.7 to 3 (malonate, malate, citrate), and 5 to
5.5 (ethyleneglycol, acetoin). It also appears that the effect of carbon chain length is most significant between
1 to 4 carbon atoms. There is no large effect if one considers 4 to 6 C-atoms.
The effect of a change in electron acceptor is rather

limited, as can be observed if one compares 02,NO3-,
and fermentation systems (Fig. 5A-E, G, and F). As a
first approximation, it appears that the Gibbs energy
1 - 0 0
0.00
Utolug
Candad-
aerobic
0.00'
'
"
'
'
'
'
'
'
2000
1600
s
a
1200
?i
-B
Boo
is
A00
c?
800
400
d
0
degree of
degree of
reduction
.oo
reduction
ThiobaCllluS
aCldODhllUB
aerobic
I
I
_
0.50
0
0
0
0
O.=O
0.00
2000
0.00
1600
sm
D
1200
u)
v,
'0
1
!i
-B
800
!i
400
(3
0
degree of
(C)
reduction
degree of
reduction
(D)
Figure 5. Biomass yield, Y D X and

,
Gibbs energy dissipation as a function of the degree of reduction of the C-source for different carbon sources, electron acceptors, and microorganisms. (A) P. oxahticus, aerobic, heterotrophic; (B) Candida utilis, aerobic, heterotrophic; (C) Paracoccus denitrificans, aerobic, heterotrophic; (D) Thiobaciffus acidophilus, aerobic, heterotrophic;
(E) chemoheterotrophic microorganisms, aerobic; (F) chemoheterotrophic microorganisms, denitrifying; (G) Chemoheterotrophic microorganisms, fermentative. (+) Aerobic; (+) denitrifying; (B)anaerobic systems.
845
2000
2000
.=.
1600
6
m
1200
-B
8
d
400
800
+ +
+
400
3
d
0
0
degree of
degree of reduction
reduction
(F)
(E)
2000
degree of
reduction
(G)
Figure 5. (continued)
dissipation is independent of the external electron acceptor 0 2 , NO3-, or fermentation. There appears to
be, however, a tendency that microbial systems which
do not use an electron transport chain (fermentative
growth) have less Gibbs energy dissipation than systerns which do. The difference appears to be about 50
846
to 150 kJ/(C-mol biomass) (compare Fig. 5G w i t h

Fig. 5A-F).
The effect of a change in electron acceptor can be
illustrated nicely with data from Von Stockar and
B h - 0 who
~ ~ ~measured substrate yield of yeast under different regimes of oxygen supply and ethanol production
Number of carbon atoms of carbon source

4
YD
am
OD
0 " " '
Degree of reduction of carbon source

(A)
I'
Error
1,
30%
'
400
800
1200
Actual dissipation (kJ/C-mol
1600
biomass)
(B)
Figure 6. (A) The effect of carbon source (carbon chain length and degree of reduction) on the
required Gibbs energy dissipation per C-mol biomass produced. Solid line is the estimation according to eq. (1).(*) Aerobic; (+) denitrifying; (D)anaerobic systems. (B) Comparison between actual
and estimated [eq. (l)]dissipation data (from Table VA-H). (*) Aerobic; (+) denitrifying; (D)anaerobic systems.
(Table VII). It can be seen that the biomass yield YDx

changed strongly, but that D:'/rAxremained fairly constant. Furthermore, it should be noted that the measured heat production per C-mol produced biomass was
not nearly as constant; there was a decrease by a factor
3.5 if the electron acceptor changed from aerobic to fermentat ion.
In relation to heat production per C-mol biomass for

different electron acceptors, another interesting feature
can be shown. It is known that the total Gibbs energy
dissipation in chemical reaction systems is due to the
sum of heat-related and chemical entropy-related Gibbs
energy dissipation. The total Gibbs energy dissipation
must be positive (Second Law), but there are no restric-
847
Table VI.
Average Gibbs energy dissipation values (Dpl/rAx)for C-sources.

Degree of
reduction
Carbon
chain
length
No. of
observations
kJ/C-mol
biomass
Cola
COzh
OxaIatez-
0
0
1.o
1
1
2
9
3
2
3494
1061
1224
co
2.0
2.0
2.0
2.5
1
1
2
4
1
5
1
1
1105
1107
709
584
itr rate'-
2.67
3.0
3.0
3
4
6
1
2
5
757
380
381
PyruvateSuccinate2Gluconate-
3.33
3.5
3.66
3
4
6
2
5
6
316
422
311
Formaldehyde
AcetateLactateDi hydrox yacetone
Glucose
4
4
4
4
4
1
2
3
3
6
1
4
2
1
8
587
529
296
257
284
Glycerol
Mannitol
Propionate
4.66
4.33
4.66
3
6
3
4
5
1
345
338
556
Et hyleneglycol
Acetoin
Butanediol
Acetone
5.O
5.0
5.5
5.33
2
4
4
3
1
3
3
1
617
457
469
813
Methanol
Ethanol
Propanol
n-Alkanes
Butane
Methane
6
6
6
6.13
6.5
8
1
2
3
6
4
1
3
4
2
1
1
1
729
712
725
662
1061
1011
C-source
compound
FormateGlyoxylateTartratezMaionateMalate*-
SD
(%)
For electron donor with reversed electron transfer.

For electron donor without reversed electron transfer.
tions on heat- or chemical entropy-related Gibbs energy

dissipation; each can be positive or negative. Table VIII
shows some data for aerobic and anaerobic growth with
glucose, H2, or acetate as electron donor, which use
glucose, C 0 2 , and acetate as C-source. It can be seen
that the total Gibbs energy dissipation, D:l/rAx,remains
very similar for the same C-source, with different elecTable VIJ. Biomass yield, heat production, and Gibbs energy dissipation for growth of yeast under aerobic, partly anaerobic, and
anaerobic condition^?^
Biomass
yield
C-mol/C-mol
Ethanol
yield
C-mol/C-mol
Measured
heat production
kJ/C-mol
biomass
0.57
0.52
0.40
0.23
0.19
0.14
0
0.082
0.228
0.440
0.512
0.566
339
313
250
160
114
95
848
DplrAX
Gibbs energy
dissipation
(DsU1/rAx)
k J/C-mol
biomass
332
307
306
312
230
255
tron acceptors. The relative contribution of heat- and

chemical-related Gibbs energy dissipation is, however,
quite different. During aerobic growth on glucose, there
is a small chemical entropy consumption, but nearly
all dissipation is due to heat. In anaerobic growth on
glucose, the main dissipation comes from chemical entropy production which is due to the degradation of a
large molecule (glucose) into small fragments (ethanol
and Con).
Because the total dissipation is more or less constant,
this results in much less heat production under anaerobic conditions. With aerobic growth on H2,there is a significant chemical entropy consumption, because small
molecules (H2,C 0 2 )are converted into larger molecules
(biomass). This entropy consumption must be paid for
by extra dissipation from heat production. This effect is
even more pronounced under anaerobic conditions
(4H2 COz combine to give CH4 2Hz0), resulting
in a very large entropy consumption and, therefore, a
very large heat production. In contrast to glucose, the
use of H 2 as an electron donor gives a heat production
Table VIII. Gibbs energy dissipation and heat production for aerobic and anaerobic growth on glucose, H2, and acetate, and the relative
contribution of heat- and chemical entropy-related dissipation.
Dissipation due to
C-mol/(C)-mol
donor
Total
dissipation
kJ/C-mol
biomass
0.57
332
+339
-7
0.14
270
+95
+175
0.13
1265
+ 1686
-421
0.015
1035
+3923
-2888
0.406
562
+593
-31
0.024
597
-90
YDx
Microbial
system
Ref.
35
Saccharomyces cerevisiae
35
S. cerevisiae
34
H Zbacterium
24
Methanobacterium arborophilus
31
Pseudomonas oxalaticus
45
Methanobacterium soehngenii
Conditions
Glucose/02
aerobic
Glucose/
anaerobic
Hz + COz/Oz
aerobic
Hz + COz/CO2
anaerobic
Acetate/Oz
aerobic
Acetate/CH4
anaerobic
per C-mol biomass under anaerobic conditions, which is

much higher than under aerobic conditions. Finally, for
microbial growth on acetate under aerobic conditions,
nearly all of the dissipation comes from heat production, and there is only a small amount of chemical
entropy consumption. During anaerobic growth on
acetate (which is converted to CH4 and C02/HC03-),
there is a calculated net hear uptake, because the conversion of acetate into gaseous C 0 2 and CH4 produces
so much entropy.
These results clearly show the inadequacy of heat production as a correlating parameter for biomass yields,
because large changes are associated with a change in
electron acceptor. Furthermore, it is quite interesting to
see that microbial growth can possibly be accompanied
by heat uptake.
Different organic electron donors have the same effect as different organic C-sources. During autotrophic
growth, it appears that there is a fairly constant value
of DP'/rAxof about 1000 kJ/C-mol for different electron
donors when reversed electron transfer is not required
(H2, CO, Table VH). This value is in line with the
extrapolated values for a C-source (COz) where y = 0
(Fig. 6A). However, when reversed electron transport
is involved, a significantly increased value of DP1/rAx
of about 3000 to 4000 kJ/C-mol biomass is found
(Table VH). The nature of the electron donor (Fe2+,
NOz-, NH4+,S2032-, etc.) exerts no systematic influence. It is remarkable that, for photoautotrophic growth
of Chlorella vulgaris, a Gibbs energy dissipation of
3575 kJ/C-mol biomass occurs.15 This coincides with
the value for chemoautotrophic systems, which use
reversed electron transport.
DISCUSSION
It is evident that one can use DP'/rAxas a correlating

parameter to estimate microbial growth yields. As a first
heat
k J/C-rnol
biomass
Chemical
entropy
k J/C-mol
biomass
+687
approximation, one can conclude that Dj)'/rAxis mainly

determined by the nature of the C-source and whether
reversed electron transport is required.
Table VI contains the average values of DS'/rAxwhich
are found for a large number of C-sources.
Furthermore, a correlation has been found [eq. (l)]
which describes the data of Table VA-H for electron
donors when no reversed electron transport occurs.
D:'/rAX = 200
+ 18(6 - c)'"
+ ExpC((3.8 - Y ~ ) ' } " *. ~(3.6

~ + 0.4C)I
(1)
For electron donors that necessitate reversed electron

transport one finds a constant value of DF/rAxof about
3500 kJ/C-mol. In eq. (l),C is the number of C-atoms
and ys is the degree of reduction of the substrate
C-source.
Figure 6A shows how the correlation eq. (1)compares
to the data. Figure 6B shows the comparison of actual
and calculated [eq. (l)]dissipation values. It can be conx
for an arbicluded that eq. (1) gives the D f l / r A value
trary C-source with 30% error. Hence, eq. (1) can be
used for nonlisted C-sources to calculate a first estimate
of D:l/rAX.It is now interesting to compare calculated
and measured biomass yield values. YDxcan be calcufor any particular microbial system
lated from DP1/rAx
by a simple procedure (Appendix F). It should be kept
in mind that the relation between YDx and D:l/rAX is
do lead
nonlinear to such an extent that errors in DP1/rAx
to smaller errors in Yox. Said calculation (YDxfrom
D:'/rAx)is, however, lengthy (Appendix F), and therefore, a simple mathematical relation has been derived
which gives YDxas a function of the Gibbs energy characteristics of C-source, electron donor, electron acceptor, and biomass (J. J. Heijnen, manuscript submitted).
Figure 7A shows the result when the values of D:'/rAx
from Table VI are used to calculate the YDxvalues of
the microbial systems of Table VA-H. Figure 7B shows
HEIJNEN AND VAN DIJKEN: THERMODYNAMICS
OF MICROBIAL GROWTH
849
0.1
Y,
0.0 1
0.0 1
measwed
0.1
Y,
(A)
measwed
(B)
Figure 7. Comparison between actual (Table VA-H) and estimated YDXdata from D y / r A xvalues. (+) Aerobic; ( + ) denitrifying;
(m) anaerobic systems. (A) Average dissipation data from Table VI. (B) Calculated dissipation data from eq. (1).
the result when D,"'/rAx

is calculated from eq. (1). It can
be seen that YDXcan be predicted with 13% error over a
YDxrange from 0.01 to 0.8 C-mol biomass per (C)-mol
donor if the dissipation values of Table VI are used, and
with 19% error if eq. (1) is used.
On the basis of these results, it appears that chemotrophic microbial growth can be characterized and predicted by the simple black box parameter D;l/rAx.
T h e effect of C-source, electron donor, and electron
acceptor on Dpl/rAxcan be summarized as follows. It appears that D:l/rAx is mainly dependent on the C-source
used. Typically, the degree of reduction (0 to 8) and
the C-chain length (1 to 6) exert a major influence. Values of D , " l / r A x range between 150 and 3500 kJ/C-mol
biomass. Electron acceptor (02,NO3-, fermentation)
has little or no effect on D : l / r A x . Organic electron
donors, which function as C-source (chemoheterotrophic
growth), have the same effect as organic C-sources.
With inorganic donors, where CO, is the C-source
(chemoautotrophic growth), the effect of the electron
donor depends on the occurrence of reversed electron
transport. If this does not occur (e.g., H2, CO as elecis about 1000 kJ/C-mol
tron donor) the value of Df1/rAX
biomass, and seems to be independent of the electron
donor. When reversed electron transport does occur
(e.g., Fez', N H 4 + ,NO2-, SZO3*-,etc.), the value of
D P ' / r A x is about 3500 kJ/C-mol biomass and, again, appears to be independent of the electron donor.
One might wonder about the origins of the observed
correlation (Fig. 6A). In the opinion of the authors, the
850
correlation is the result of the fact that biochemical processes are similar in all microorganisms. Depending on
the available C-source, a microbial system must carry
out many or few biochemical reactions to arrive at the
correct redox level and carbon chain length of the building blocks for biomass synthesis. A microbial system
which has C 0 2 as its C-source must carry out more reduction reactions and carbon-carbon coupling reactions
than a microbial system which uses glucose. Glucose is
much closer to the redox level of biomass and the typical biomass building blocks which contains about 4 to
5 C-atoms. Thus, much more reaction steps, and hence
much more dissipation of Gibbs energy, are involved in
C 0 2 assimilation than in glucose utilization. Analogous
reasoning applies to substrates such as formaldehyde or
methanol.
Similarly, the presence of an electron transport chain
(02,
NO3-) results in somewhat more dissipation per
C-mol biomass compared with its absence (fermentation). Thus, the extra facility of electron transport
phosphorylation requires further chemical reactions, resulting in the additional Gibbs energy dissipation of
about 50 to 150 kJ/C-mol. In the same way, the consequence of reversed electron transfer is apparently an
additional dissipation of about 2500 k J/C-mol biomass,
as compared to autotrophic growth without reversed
electron transfer. Reversed electron transfer seems a
very costly process.
The Gibbs energy dissipated per C-mol produced
biomass thus appears to be a straightforward measure
Table IX. Comparison between the theoretical ATP requirement for biomass synthesis on
different carbon s o ~ r c e s , and
~ ~ ~found
~ " values of Gibbs energy dissipation (see Table VI).
Carbon source
YATP
theoretical
biomass production
o n ATP
g/mol ATP
Theoretical
ATP need
for biomass
synthesis
mol ATP/C-mol biomass
Found
Gibbs energy
dissipation
kJ/C-mol biomass
Glucose
Malate
Acetate
Ethanol
COZ a
C02
28.8
15.4
10
10
6.6
2.5
0.85
1.69
2.46
2.46
3.73
9.85
280
380
530
710
1060
3500
No reversed electron transport.

Reversed electron transport.
of the amount of work required and spent to synthesize

biomass from a given C-source, electron donor, and
electron acceptor. As such, this parameter should resemble 1/YATp, which provides the theoretically needed
amount of ATP to synthesize biomass from a specific
C-source.
Table IX and Figure 8 show a comparison of the theoretical ATP requirement for biomass synthesis in moles
ATP/C-mol b i o m a ~ s ' and
~ , ~the
~ Gibbs energy dissipation values (in kJ/C-mol biomass) found for different
C-sources. There appears to be a very good correlation,
indicating that the parameter D:'/rAx can be considered
the thermodynamic equivalent of the biochemicallybased parameter 1/YATp.
Finally, some words of caution. The derived relationship [Fig. 6A, eq. (l),Table VI], which gives D,"'/rAxfor
various C-sources, will give only a first approximation
3500
2.5
3000
2500
of the biomass yield. It is bound to be of limited accuracy because the actual biochemistry involved has not
been taken into account. Of course, the absence of the
need for biochemical details is the attractive feature of
this approach, but it also limits the accuracy of yield
predictions.
This point is illustrated by the system where microorganisms convert sugar anaerobically to ethanol. Using
the present approach described here, one would estimate a biomass yield of about 0.13. This is correct for
S. cerevisiae, but wrong for Zymomonas mobilis, where
YDx = 0.06. The difference between the two microorganisms is biochemical. S. cerevisiae employs the glycolysis route, which gives 2 mol ATP/mol glucose, while
Zymomonas mobilis uses the Entner-Doudoroff pathway, which generates only 1 ATP/mol glucose.
A second illustrative example is the aerobic growth of
microorganisms on formate. Biochemically, two different types of microorganisms are known. The autotrophs
(such as P. oxalaticus or Paracoccus denitrijicans) oxidize formate to COz, and subsequently assimilate the
COz to biomass. Typically, these microorganisms have
a biomass yield of about 0.14 C-mol/C-mol, and a Gibbs
energy dissipation of about 1300 kJ/C-mol.
The heterotrophs (such as Pseudomonas sp. 1 or 135)
can assimilate formate directly to biomass without first
oxidizing it to COz. The yield obtained with heterotrophs is about 0.23 C-mol/C-molZ9and the Gibbs energy dissipation is about 600 kJ/C-mol biomass. Using
the approach described here, a yield of about 0.16 for
both formate systems would have been calculated.
'ooov
,;
,
500
15.4
0
0
l/YA,p
(mol ATP/C-mol
10
biomass)
Figure 8. Comparison between ~/YATPand found dissipation values. Data from Table IX.
CONCLUSION
It has been shown that all published parameters to establish a prediction for biomass yield are subject to
limitations and problems. Notably, the Gibbs energy efficiencies suffer from intrinsic problems. However, a
simple (and biochemically understandable) prediction
can be based on the Gibbs energy dissipation per C-mol
biomass produced. It is shown that, for a wide variety of
851
microbial growth systems, in which YDxvaries between

0.01 and 0.80, this method provides an estimated biomass yield with an error of about 13%.
The authors thank Prof. K. van Dam, Prof. U. von Stockar,
and Dr. H.V. Westerhoff for fruitful discussions, and
Dr. L. Robertson for correction of the text.
yield of biomass on electron donor (per mol or per C-mol

for carbon compounds) [C-mol/(c)-moll
yield of biomass on ATP (g/mol ATP)
yield of biomass on available electrons (g/mol electron)
carbon efficiency (-)
oxygen efficiency (-)
enthalpy efficiency (-)
Gibbs energy efficiency from black box description (-)
Gibbs energy efficiency from the conservation description (-)
Gibbs energy efficiency for Gibbs energy convertor description (-)
Gibbs energy dissipation in microbial growth systems
(kJ/m3 h)
net production rate of biomass (C-mol/m3 h)
degree of reduction of electron donor (-)
degree of reduction of substrate (-)
reaction Gibbs energy (kJ)
reaction Gibbs energy conservative reaction (kJ)
reaction Gibbs energy nonconservative reaction (kJ)
APPENDIX A: CALCULATION OF qBB

For microbial growth systems, one can formulate the black box description if the electron acceptor, the N-source, and the yield of
biomass on the electron donor is known. This description can be
presented in various ways, but it is easiest to calculate the macrochemical reaction equation. In this equation, biomass is formed
from the C-source, the N-source, and electron acceptor. The stoichiometric coefficients follow from:
use of the elemental and electric charge conservation principles.
setting the stoichiometric coefficient of biomass to + l .
using the available yield value of biomass on C-source/electron
donor.
qBBCan now be calculated from the macrochemical equation. The
procedure will be illustrated for the aerobic growth of Pseudomonas
oxulaticus and the anaerobic growth of Klebsiellu uerogenes on a
variety of organic carbon sources.
Aerobic Growth of Pseudomonas oxalaticus on

Oxalate as C-source
Given a yield of 0.086 C-mol biomass/C-mol substrate, and using
the fact that NH4+ is the N-source and 0 2 the electron acceptor,
while H2O and HC03- are produced, one can establish the following macrochemical equation as the black box description (including
the reaction Gibbs energy AG g). For biomass, the composition
quoted by Roels is used.28 The stoichiometric coefficients follow
from charge, C, H, 0, and N conservation.
- 0.2NH4'
- 0.8H'
- 1.85702
+ lCHI.gOo.~N0.z+ 10.63HC03-
=0
- 5.42Hz0
AG:' = -1048 k J
The compounds with a negative stoichiometric coefficient are obviously consumed, while the others are produced. The conservation
relations are obeyed in this equation. qBBCan now be calculated
for two frames of reference.
852
Based on the values of Table Al, qFBcan be calculated as

qFB
input Gibbs energy thermodyn. ref. frame

output Gibbs energy thermodyn. ref. frame
- 5.815(-676)
+ 0.2(-80) + 0.8(-40) + 1.85 * (0) + 5.42(-238)

1 * (-67) + 10.63 * (-588)
- 5269
= -= 0.834
-6317
NOMENCLATURE
-5.815CzO42-
Thermodynamic Reference q F0
Combustion Reference
q p=
output Gibbs energy combustion ref. frame

input Gibbs energy combustion ref. frame
5.815(+262)
1 * 474.6 + 10.63 * 0
+ 0.2(0) + 0.8(0) + 1.857 * 0 + 5.42 * 0
474.6
= 0.311
1523.5
It should be noted that for both frames of reference, the Gibbs

energy of the macrochemical reaction is identical, being -1048 kJ
(-6317 + 5269 = -1048 or 475 - 1523 = -1048). This is, of
course, logical, because a difference of Gibbs energy values is not
influenced by a change in reference frame (contrary to a quotient
of Gibbs energy values).
Aerobic Growth of R oxalaticus on Ethanol as

C-source
From a yield of 0.558 C-mol biomass/(=-mol substrate, the following
macrochemical equation can be calculated.
- 1.6402 - 0.2NH4' + 1CHl.sOo.sNo.z

+ 0.79HC03- + 0.99H' + 1.296H20 = 0 AC:
-0.896C2H60
= -702kJ.
It can be calculated that (using values of Table A-I).
qp
0.896 * (-182) + 1.64 * (0) + 0.2(-80)

0.99(-40)
1.296 * -238)
1 * (-67) + 0.79(-588)
= 0.203
-880.8
1 I
qp=
,
.
I
1 * (474.6) + 0.79 * (0) 0.99 * (0) 1.296 * (0)

0.896 * (1314) + 1.64 * (0) + 0.2 * (0)
474.6
- 0.403
1177.3
Here again, both frames of reference provide the same reaction

Gibbs energy of -702 kJ.
Anaerobic Growth of Klebsiella aerogenes

on Citrate
The following macrochemical equation has been provided by
Rutgers3'
- 4.6820 - 0.2NH4' + 1CHI.aOo.sNo.z

+ 4.096C2H3O2- + 0.192C4H4042- + 0.068CH02+ 3.657HCO3- + 0.63H2 + 1.56Ht = 0
-2.283C6HsO;-
The Gibbs energy AG;' = -185 kJ.

This macrochemical equation shows that elemental and charge
conservation is obeyed, and that YOX= 1/6 * 2.283 = 0.073; C-mol
biomass/C-mol substrate and that citrate is fermented to acetate,
succinate, formate, and H z . For q?", it can be calculated (using the
Gibbs energy values of the thermodynamic frame of reference in
Table A-I.
Gibbs energy values for two different frames of reference (pH 7).
Compound
Composition
Degree
of
reduction
Thermodynamic
reference
(PH 7)
k J/mol
-67
-238
-588
-80
-40
0
4.2
0
0
0
0
-4
Biomass
Hz0
Hco3NH4
H+
0 2
OxaIate2Carbon monoxide
FormateGlyoxylateTartrate2-
1
2
2
2
2.5
-674
-137
-335
-458
-1010
Malonate2Fumarate2Malatecitrate3Pyruvate-
2.66
3.0
3.0
3.0
3.33
-700
-602
-845
-1170
-475
Succinate2GluconateFormaldehyde
AcetateDi hydroxyacetone
3.50
3.66
4
4
4
- 688
-1154
-130
-372
-450
LactateGlucose
Mannitol
Glycerol
Propionate-
4
4
4.33
4.66
4.66
Ethylene glycol
Acetoin
ButyratePropanediol
Butanediol
Methanol
Ethanol
Propanol
n-Alkanes
Propane
Ethane
Methane
Hz
Combustion
reference
(PH7)
kJ/mol
kJ/(C-)mol
+474.6
0
0
0
0
0
+474.6
0
0
0
0
0
+ 262
+ 131
+254
+253
+260
+296
+254
+253
+520
+1184
+867
+ 1356
+ 1352
+2004
+1131
+ 1504
+289
+339
+338
+334
+377
+2580
+498
+844
+1437
+376
+430
+498
+422
+479
-519
-918
-944
-489
-361
+ 1326
+442
+2856
+3066
+ 1638
+1481
+476
+511
+546
+493
5.0
5.0
5.0
5.33
5.50
-323
- 280
-378
-327
- 322
+1172
+2236
+2096
+1797
+2432
+586
+559
+524
+599
+608
6
6
6
6.13
6.66
7.0
8.0
2
- 175
+692
+1314
1950
+9720
+2100
1464
+816
+238
+692
+ 657
+ 650
+648
+ 700
+ 732
+816
+ 238
- 182
- 176
+60
-24
-32
-51
0
le A-I) that
llp
1 * (-67)
-3780
--=
-
-3965
r)2BB one
2.283 * (-1170) + 4.6 * (-238) + 0.2(-80)

+ 4.096(-372) + 0.192(-688) + 0.068(-335) + 3.657(-588) + 1.56(-40)
0.95
obtains:
ll;
=
-
{l * (474.6) + 4.096
* (844) + 0.192(1504) + 0.068(253) + 0.63(238)}

2.283(2004)
+4390
- 0.960
+4575
853
It should be noted that one can calculate the same Gibbs energy of
the macrochemical reaction for both reference systems (= -185 kJ).

on Gluconate
The following macrochemical equation has been p r ~ v i d e d . ~ '
+ 1CHl.aOo.sNo.z
+ 1.876CzH302- + 0.0661C4H4042- + 0.504C~H60
+ 2.256HCO3- + 2.O91H2 + 3.083H'
-1.3773C6HiiO7- - 2.149H20 - 0.2NH4'
=0
The Gibbs energy of this reaction AG;' = -237 kJ.

This equation shows that gluconate is fermented to acetate, succinate, ethanol, and Hz, with a biomass yield of 1/(6 * 1.3773) =
0.121 C-mol biomass/C-mol substrate.
For 7FB,one obtains (using the thermodynamic reference frame
Gibbs energy values of Table A-I):
7;B
{l
Output Processes
+ CHix0o.sNo.z
- 0.2NH4' - 0.1H20 - 0.8H'
1. -1C20:-
+ 0.802 = 0
AGF = +343 kJ
1.3773(-1154) + 2.149(-238) + 0.2(-80)

+ 0.0661(-688) + 0.504(-182) + 2.256(-588) + 2.091(0) + 3.083(-40)}
* (-67) + 1.876(-372)
-2117 kJ
-2354 kJ
thermore, charged species are involved. This means, having 5 conservation relations (4 elemental, 1 electric charge), that this reaction should contain at least a total of 6 compounds. Biomass, H 2 0 ,
N-source and H' must he present. This leaves 2 more compounds
which must he specified from the 3 additional compounds (Csource, HC03-, 0 2 ) . Thus, there are 3 simple options for the
2 remaining compounds:
C-source and 0 2 ;
C-source and Hco3-;
HCO3- and 02.
These three options then lead to the following three output processes, all of which produce 1 C-mol biomass (anabolism).
- 0.90
For 728' it follows (from the combustion reference frame values of

Table A-I)
7p = {l * (474.6) + 1.876(844) + 0.661(1504) + 0.504(1314) + 2.256(0) + 2.091(238) + 3.083(0)}

1.3773(2580) + 2.149(0)
---=+3317
+3554
+ 0.2(0)
0.93
It should be noted that, for both reference frames, the same amount
of Gibbs energy is liberated in the macrochemical reaction, being
237 kJ (-2117 - (-2354) = 237 or 3554 - 3317 = 237 kJ).
Thus, in general, provided that one knows the biomass yield and
the complete product formation, the macrochemical equation can
be drawn up and the values of ':7
and 7;' can be calculated. For
the microbial systems shown in Table Ill, this gives the 7" values
of Figure 2A and B.
APPENDIX 6: CALCULATION OF qEc

Given the biomass yield on the substrate and complete information
for the amounts of excreted products, it is always possible to establish the macrochemical reaction equation (see also Appendix A).
In order to use the Gibbs energy convertor concept, the input
(catabolic) and output (anabolic) processes must be defined by separating the macrochemical reaction into two reactions.
There are, in principle, an infinite number of ways of dividing
the macrochemical reaction and different vECvalues are obtained
for different divisions. This will be illustrated for two of the microbial growth systems which were dealt with in Appendix A.
2. -2.1C1042- - 0.2NH4'
1.7Hz0 - 0.8H'
+ 3.2HCO33. -HCO3-
0.2NH4' - 0.8H'
As shown in Appendix A, the macrochemical reaction and the process Gibbs energy are as follows:
-5.815C2042- - 0.2NH4' - 0.8H' - 1.85702 - 5.415H20
+ I.OCH1xOusNoz + 10.63HCO3- = 0
AGk = -1048 kJ
854
AG;' = +474.6 kJ
The input processes (catabolism) are obtained by subtraction of the

output process from the macrochemical reaction. This leads to the
following 3 options for the inputprocesses (catabolism).
1. -5.315C2042- - 2.65702 - 5.315H20
+ 10.63HC03-
=0
AGf = -1391 kJ
2. -3.71.5C2042-
1.85702 - 3.715HzO
+ 7.43HCO3-
=0
AG;' = -972 kJ
3. -5.815C202- - 2.90702 - 5.815H20
+ 11.63HCO3- = 0
AG;' = -1522 kJ
For the 3 values of q", there follows

7pc = +343/(1391) = 0.246;
':77
= -80.6/(972) = 0.083;
= 474.6/(1522) = 0.312
It is stressed t h y , for these 3 options, the sum of input and output

processes always equals the black box macrochemical equation.

on Citrate
The macrochemical reaction was provided by Rutgers3'
-2.283C6Hs07'-
Three alternative anabolic reactions can now be defined as

examples to represent 3 alternative output processes. The anabolic
reaction produces biomass, hence, 4 elements are involved. Fur-
AG;' = -80.6 kJ
+ CH~sOosNoz+ 1.0502
+ 0.4H20 = 0
'
:
7
Aerobic Growth of Pseudomonas oxalaticus on

Oxalate as C-Source
=0
+ CH~xOosNuz
4.6H20
0.2NH4'
+ CHix00sNoz
+ 4.096C2H302- + 0.192C4H40:- + 0.068CH02+ 3.657HC03- + 0.63H2 + 1.5hH' = 0 AG;' = -185
kJ
In analogy to the aerobic case treated above, one can define various
output reactions. Using similar reasoning as before, 3 simple proposals are obtained. These 3 reactions all have biomass, N-source,
H20, and H' as reactants, but contain in addition
C-source, H 2
* C-source, HC03HCO3-, H2
as reactants to complete the output process definition where
1 C-mol biomass is produced (anabolism).
This leads then to the following 3 output process definitions
1. -0.1667C6Hs073-
0.2NH.4' - 0.3H' - 0.60H2
+ CHl.8Oo.sNIj.z+ 0.666H20 = 0
AGF = -2.5 kJ
2. -0.233C6H5073- - 0.2NH4' - 0.1H' - 0.066H20
+ CH1,gOasNo.z+ 0.4HCO3-
AGE' = +6.7 kJ
+ CHl.8Oo.sNo.z
+ 2.5H20 = 0 AGE = -25.2
3. -HCOs- - 0.2NH4' - 0.8H' - 2.1Hz
kJ
The input reaction is obtained by subtraction of the output reaction

from the macrochemical reaction. For the 3 obtained input reactions, the following reaction Gibbs energy can be calculated:
mally, one uses the thermodynamic frame of reference (chemical

elements have zero Gibbs energy). Because qBBis a ratio of Gibbs
energy sums, the effect of a different frame of reference is obvious
(Appendix A).
The processes of Gibbs energy generation or consumption form
the basis of the Gibbs energy convertor efficiency qEC.
qEc Is defined as the ratio of the Gibbs energies of reaction of
2 processes. The denominator contains the Gibbs energy generated
by the specified catabolic process. The numerator contains the
Gibbs energy taken up in the specified growth process (anabolism).
It is most important to stress the fact that the sum of both specified
processes must be equal to the macrochemical equation. Appendix B contains illustrative examples. qECIs a ratio of Gibbs energies of reaction (which are reference independent) of defined
processes, but now qECis dependent on the chosen split into the
defined processes of anabolism and catabolism (Appendix B).
It is noted that the sometimes claimed" black box character" of
the Gibbs energy convertor (Westerhoff and Van Dam43)is based
on the fact that one does not need to specify the mechanisms which
couple catabolism to anabolism. Hence, the coupling mechanism is
regarded as a black box. However, the needed specification of
catabolism and anabolism processes certainly makes this approach
no longer a black box.
1. AGE' = -185 - ( - 2 . 5 ) = -182.5 kJ

2. AGE' = -185
(+6.7) = -192 kJ
3. AGE' = -185
(-25.2) = -160 kJ
These 3 definitions of input/output reactions then give the following vEc values:
7':
= -2.5/(182.5) = -0.014;
7
':
= 6.7/(192) = +0.035;
qfc = -25.2/(160) = -0.157
Using these simple procedures, one can calculate qECvalues for

a microbial system with known biomass yield and product yields
after the input and output processes have been defined.
These examples only show 3 particular choices of the definition.
In principle, one can generate any other combination by simple,
arbitrary selections (whose sum must equal the macrochemical
reaction).
This will lead, for the same microbial system, to an infinite number of possible qEcvalues.
For the microbial systems of Table IV, one can calculate the 3
q-values for aerobic and anaerobic situations. The result is given in
Figure 4A and B. It should be clear that a biochemically meaningful qEc can only be obtained if the split is based on extensive biochemical information (see also Appendices D and E).
APPENDIX C: WHAT IS THE DIFFERENCE

BETWEEN qBBand qEC?
A black box Gibbs energy efficiency qBB(ref. 28, Fig. 3.7) is the
ratio between the sum of the Gibbs energy associated with all consumed chemicals and the sum of Gibbs energy associated with all
produced chemicals. If the black box is represented by the macrochemical equation, then this definition leads to a simple calculation. The Gibbs energy of the chemical compounds with a negative
stoichiometric coefficient (consumed chemicals) is summed up,
and the same is done for the compounds with positive coefficients
(produced chemicals). qBBIs the ratio of these sums. Appendix A
shows examples of this calculation. qBBIs thus a clear operational
definition, without any consideration about Gibbs energy, which is
made available for growth, or Gibbs energy which is conserved in
growth-such mechanisms are not considered. However, to calculate these sums of Gibbs energy, one has to assume a frame of reference of Gibbs energy for the chemical compounds. This choice is,
in principle, completely independent from the process studied. Nor-
APPENDIX D: qEcDEFINITION FROM ROELS

Roels has defined a general thermodynamic efficiency as follows
[ref. 28, eq. (3.67)]. The numerator represents the Gibbs energy
conserved in biomass. This Gibbs energy is calculated by definition
as the Gibbs energy of combustion of biomass with 0 2 . This definition is supposed to hold for all growth systems (aerobic, anaerobic
denitrifying, etc.).
The denominator represents the Gibbs energy that is available
for growth. It is calculated as the Gibbs energy of combustion of all
the substrate, which must be diminished with the combustion
Gibbs energy associated with organic products other than biomass.
Alternatively, it appears that this definition can also be considered as an energy convertor concept. In convertor terminology,
Roels has defined a general anabolic process where the biomass is
produced from HC03- with 0 2 production (which is, of course, the
reversed combustion). The catabolic process definition should
then follow from subtraction of the anabolic process from the
macrochemical reaction. The following examples will show that
both procedures are identical and lead to the same result. Consider
aerobic growth of yeast on glucose with YDX= 0.55 C-mol/C-mol.
The macrochemical equation runs as:
-0.33C6Hl206
0.9302 - 0.2NH4'
+ O.4HzO + 1.18H'
+ 0.98HC03- + CHiaOosNoz = 0
According to Roels, the numerator of the efficiency definition represents the combustion Gibbs energy of the biomass, which is
+474.6 kJ. The denominator should contain the combustion Gibbs
energy of all the substrate, being 0.33 * 2856 = 942 kJ. There
are no organic products other than biomass. Hence, one obtains
q K = 474.6/942 = 0.50. Alternatively one can formulate, according to the energy convertor concept, the anabolic process
-HC03-
0.2NH4' - 0.8H'
+ C H I B O O S N U+ Z1.0502
+ 0.4H20 = 0
AG"' = +474.6 kJ
Subtraction of this equation from the macrochemical equation

leads to the implicitly defined catabolic process
-0.33C6H1206 - 1.9802
+ 1.98HCO3- 4-
1.98H' = 0
AG"' = -942 kJ
Hence, from 942 kJ released from catabolism only 474.6 kJ are

conserved leading to qEC= 0.50. It is clear that both procedures
lead to the same result.
855
Now consider the anaerobic growth of yeast on glucose, where

only ethanol is produced, with YDX= 0.14 C-molfC-mol. Th e
macrochemical equation can then be calculated as
-1.1904CsH1206
- 0.2NH4' - 1.63H20 + 1CHl8005N0.2

+ 2.0308C~H60+ 2.081HC03- + 2.288'
= 0.
According to Roels, the numerator still represents the aerobic

combustion Gibbs energy, being 474.6 kJ. The denominator is the
combustion Gibbs energy of the glucose diminished with the combustion Gibbs energy of the produced ethanol, leading to 1.1904 *
2856 - 2.0308 * 1314 = +723 kJ. This gives T~~ = 474.6f723 =
0.65, which is quite analogous to the aerobic growth. Alternatively,
one can formulate the anabolic process as above. The catabolic
process is found by subtracting this anabolic process from the
macrochemical reaction leading to the catabolic process.
- 2.03HzO - 1.0502 + 2.0308CzH60

+ 3.081HCO3- + 3.08H' = 0 AG;' = -723
-1.1904CsH1206
kJ
It is again noted that this way of calculation leads exactly to the

same result (vEC= 474.6f723 = 0.65). It if further noted that this
catabolic reaction can also be found by writing out the appropriate
oxidation reactions of glucose and ethanol with subsequent subtraction. Hence, the combustion of 1.1904 mol glucose or 2.0308 mol
ethanol (from the macrochemical equation) runs as:
-1.1904ChH1206 - 7.142402
-2.0308CzH60
6.092402
+ 7.1424HCO3- + 7.14248'
=0
+ 4.0616HCO3- + 4.0616H'
+ 2.0308HzO = 0
It is easily seen that subtraction of these equations leads to the

above found catabolic process.
Using this approach, Roels28has shown that a satisfying empirical correlation for efficiency is obtained, which describes aerobic/
anaerobicidenitrifying growth. Moreover, it is noted that, for
aerobic heterotrophic growth, this T~~ definition leads to exactly
the same value as the qBBdefinition with the combustion reference
(compare
in Table 111 with ofc in Table I V for aerobic growth
of P. oxalaticus). This might be a reason why this approach of Roels
is often confused with the black box definition which he has also
given.28Also, it can be shown that, for aerobic growth, Roels' proposal is equal to Battley's proposal of qc.
However, if we consider this energy convertor from a general biochemical point of view, then it is clear that the proposed anabolism
is unrealistic. Heterotrophic growth uses seldom COz fixation and
0 2 production is not observed, certainly not under anaerobic conditions. The resulting catabolic reaction is acceptable for aerobic
growth. A partial aerobic oxidation of glucose to ethanol is definitively incorrect as a catabolic reaction for alcohol fermentation.
In general, the procedure of Roels leads to the involvement of 0 2
in the catabolic reaction of fermentative, dentrifying or other (non0 2 electron acceptor) systems. This is biochemically unrealistic.
So, in conclusion, Roels' proposal has the virtue of a simple correlation, but the intrinsic problem that this efficiency cannot be
considered as a realistic measure of thermodynamic process performance. This because the proposed split is biochemically unrealistic.
negative qEcvalues. The implicitly defined catabolic process is the

combustion of the remaining substrate, which is not used for
biomass formation. Using this approach, a value of T~~ = 0.24 was
found. However, the proposed anabolic reaction is highly unrealistic, 0 2 is seldom involved in anabolism and never produced in
chemotrophic growth as pointed out by Battley.' The resulting
catabolic reaction is biochemically acceptable. In conclusion, it appears that the aerobic T~~ value cannot be considered as a realistic
measure of thermodynamic performance of growth, because the
proposed split lacks biochemical reality.
For anaerobic processes, a different anabolic definition has been
proposed.31 0 2 has been replaced by H I in the anabolic reaction
(see split no. 1 for anaerobic growth in Appendix B). However, for
the many anaerobic processes where Hz is not a reactant this proposal is also very unrealistic.
Finally, it is mentioned that, contrary to Reels:' the approach of
Westerhoff and Van Dam leads to differrent correlations for anaerobic and aerobic growth systems. In addition, the obtained efficiencies cannot be regarded as realistic measures of thermodynamic
process performance due to the biochemically unrealistic split
which is used.
APPENDIX F: CALCULATION OF 0,"'f A x FROM Yox

AND THE CALCULATION OF Yox FR M D,"'/fA,
The dissipation per C-mol produced biomass is calculated most

simply by first establishing the macrochemical equation as introduced in Appendices A and B.
The macrochemical equation follows directly from a measured
YDXvalue. Suppose that one studies the aerobic growth of a microorganism on oxalate. One has checked the absence of products
or other substrates, and the N-source is NH4+.Thus, one can formally write the macrochemical equation as
-1
* 0.086
Cz02-
+ aNH4+ + bH' + c02 + dH2O

+ 1CH18005NU.2+ eHC03-
By definition, the macrochemical equation produces 1 C-mol of

biomass. The yield on substrate is 0.086, indicating that for the
production of 1 C-mol biomass there is needed a consumption of
1/0.086 C-mol oxalate or 1f0.086 * If2 = 5.815 mol oxalate. The
minus sign is because oxalate is consumed. Hence, there are 5
unknown stoichiometric coefficients (a, b,c,d, e), which can be calculated from the 5 (C, H, 0,N, and electric charge) conservation
equations.
C
-5.815 * 2 + 1 + e = 0
4a + b + 2d + 1.8 + e = 0
H
-5.815 * 4 + 2c + d + 0.5 + 3e = 0
0
a + 0.2 = 0
N
Charge -5.815 * (-2) + a + b + e * (-1) = 0
Solving these equations leads to a = -0.2; b = -0.8; c = -1.857;
d = -5.42; e = +10.63.
This gives the macrochemical equation given in Appendix A
(P. oxalaticus on oxalate). Now it is easy, using tabulated values of
AG$ of the various chemicals, to calculate AGE'. This leads to
+ 1 * (-67) - 5.42 * (-238)
APPENDIX E: qECDEFINITION FROM

WESTERHOFF AND VAN DAM43
AG;' = 10.63 * (-588)
For aerobic growth, Westerhoff and Van Dam have proposed an

anabolic process where biomass is produced from substrate and 0 2
production (substrates more oxidized than biomass) or 0 2 consumption (substrates less oxidized than biomass). An example is given in
Appendix B, split 1 for growth of P. oxalaticus on oxalate where 0 2
production is clearly seen. It is easy shown that, for more reduced
substrates, one obtains O 2 consumption in the anabolic reaction.
This leads to a release of Gibbs energy in anabolism and finally to
Now, clearly, this means that for the formation of 1 C-mol biomass,
1048 k J of Gibbs energy is dissipated. Hence, D f l / r A x= 1048.
A second question is how to calculate YDXif the C-source,
N-source, electron donor, and electron acceptor are known, using
the correlation eq. (1). Suppose a microorganism grows anaerobically on methanol and HCO3- and produces acetate. The C-source
is methanol, and from eq. (1) one then obtains (c = 1, y3 = 6) for
DQl/rAx= 698 kJfC-mol.
856
- 0.8 * (-40) - 0.2(-80) - 5.815(-676) = -1048 kJ.
The growth system contains biomass, NH4+, acetate, methanol,

HCOs-, H', and HzO as chemicals of interest. Hence, one can
write the macrochemical equation as
fCHsOH
+ u N H ~ ' + bH+ + CCZHJOZ-+ dHzO

+ lCH1.800.5N0.2+ eHCO3-
We know from the estimated dissipation per C-mol biomass that

the reaction Gibbs energy is -698 kJ. Now, one can calculate
straightforwardly the 6 coefficients a
f from the element and
charge conservation relations and the Gibbs energy balance.
C
H
0
N
Charge
Gibbs energy
f + 2c+ 1 + e = O
4f + 4a + b + 3c + 2d + 1.8 + e = 0
f + 2c + d + 0.5 + 3e = 0
a + 0.2 = 0
a+b-c-e=O
-175f - 80a - 406 - 372c - 238d - 67
- 588e + 698 = 0
Solving these equations leads to the following values: a = -0.2;

b = 2.866; c = 8.898; d = 12.964; e = -6.232; f = -12.564.
This then leads to YDX= 1/12.564 = 0.080 C-mol biomass/C-mol
methanol.
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In Search of a Thermodynamic Description

of Biomass Yields for the Chemotrophic

A sample list of microbial growth systems.

* To whom all correspondence should be addressed.

Biotechnology and Bioengineering, Vol. 39,Pp. 833-858 (1992)

the yield (YDx)

Figure 1. Black box description of microbial growth.

Models for the prediction of biomass yields.

Evaluation of the models for the prediction of biomass

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 8, APRIL 5, 1992

Critical Evaluation of Proposed Methods for the

trons per C-mol electron donor upon combustion). These

Roels proposed the use of the black box (BB) Gibbs

HEIJNEN AND VAN DIJKEN: THERMODYNAMICS OF MICROBIAL GROWTH

ally considered as a measure of thermodynamic process

Pseudomonas oxalaticus (aerobic)

Klebsiella pneumoniae (anaerobic)

Clostridium butyricum (anaerobic)

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 8, APRIL 5, 1992

erence do indicate that a higher YDxgives a lower qBB,

convertor efficiency of Gibbs energy. This proposal is

HEIJNEN AND VAN DIJKEN: THERMODYNAMICS OF MICROBIAL GROWTH

energy of the conservative reaction. Clearly, ( -AGc) is

Kedem and Kaplan proposed the general concept of a

GIBBS ENERGY CONVERTOR DESCRIPTION OF MICROBIAL GROWTH

SUM MACROCHEMICAL EQUATION

5 415 H,O - 0 8 H' - 1 8 5 7 0,

Figure 3. Gibbs energy convertor description of microbial growth

commonly identified as catabolism. The second process

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 8, APRIL 5, 1992

interesting candidate than qBBor qc. However, close

To answer the first point, 3 different splits for aerobic

Pseudomonas oxalaticus (aerobic)

Klebsiella pneumoniae (anaerobic)

Clostridium butyricum (anaerobic)

HEIJNEN AND VAN DIJKEN: THERMODYNAMICS OF MICROBIAL GROWTH

Figure 4. Gibbs energy convertor efficiency, T ~and

ganic substrate, which is biochemically quite acceptable.

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 8, APRIL 5, 1992

Enthalpy efficiencies have been defined in analogous

It has been suggested by Roels that the Gibbs energy

HEIJNEN AND VAN DIJKEN: THERMODYNAMICS OF MICROBIAL GROWTH

- A number of microbial systems where growth yield

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 8, APRIL 5, 1992

in Table 5A-H and Figures 5A-G. If one studies the

around 200 to 350 kJ/C-mol for yD = 3.5 to 4.5, and

HEIJNEN AND VAN DIJKEN: THERMODYNAMICS OF MICROBIAL GROWTH

No reversed electron transport systems

It appears that the carbon sources containing more

Table VI). It appears that DP1/rAxis halved when the

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 8, APRIL 5, 1992

The effect of a change in electron acceptor is rather

Figure 5. Biomass yield, Y D X and

to 150 kJ/(C-mol biomass) (compare Fig. 5G w i t h

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 39, NO. 8, APRIL 5, 1992

Number of carbon atoms of carbon source

0 " " '

Degree of reduction of carbon source

Actual dissipation (kJ/C-mol

(Table VII). It can be seen that the biomass yield YDx

In relation to heat production per C-mol biomass for

HEIJNEN AND VAN DIJKEN: THERMODYNAMICS OF MICROBIAL GROWTH

Average Gibbs energy dissipation values (Dpl/rAx)for C-sources.

For electron donor with reversed electron transfer.