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Thermodynamics of microbial growth and its

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ARTICLE in TRENDS IN BIOTECHNOLOGY DECEMBER 1994

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 483

Thermodynamics of microbial
growth and its implications for
process design
Sef J. Heijnen

Several different approaches have been used in an attempt to define and analyse
the thermodynamics of microbial growth in bioreactor culture. While
thermodynamic theory has been developed sufficiently to enable satisfactory
prediction of biomass and catabolic-product yield, prediction of non-catabolic-
product yield and growth kinetics has proven less successful. Further research in
this area is required to develop models that would be useful in process design
and optimization.

A wide variety of nutrients can support the growth of
microorganisms under a wide range of conditions
(pH, temperature). For process design, the key factors
that need to be considered are the biomass yield (Ysx)
and the maximal growth rate (/,m~x) on any particular
electron donor. These factors vary greatly: Ysx ranges
between 0.01-1.0 C-mol biomass per C-mol electron
donor, while /,m= ranges between 0.005 and 2 h <.
Generally applicable methods that can provide a pre-
liminary estimate of Ysx and/ohmaxfor any defined cul-
ture system are therefore clearly of interest for opti-
mizing process design.
One approach to characterizing microbial growth
in terms of measurable parameters is based on defin-
ing the thermodynamics of the system. This review
focuses on th e tJae'rmodynamic approach and specifi-
cally addresses the following points:
The definition of the growth system and its thermo-
dynamic analysis.
A critical evaluation of various approaches to pre-
dict Ysx.
The effects of heat production and energy dissi-
pation on growth.
Thermodynamic analysis of growth kinetics and the
effect of nonstandaM conditions.
Where appropriate, the predicted consequences for
fermentation strategies are outlined. For detailed cover-
age of the thermodynamics of growth, the reader is
referred to R e ~ 1-4.
j. j. Heijnen is at the Department of Biochemical Engineering, Delft
University of Technology, Kluyver Laboratory for Biotechnology,
Julianalaan 67, 2628 BC Delft, The Netherlands.
Definition of the growth system and its
thermodynamic analysis
Microbial growth can be interpreted as a 'black box'
(Fig. la), via which biomass is produced from an elec-
tron donor, a carbon (C) source, a nitrogen (N) source
and an electron acceptor. In addition, H 2 0 , CO2, H +
and other products are produced. A convenient way
of describing such 'black box' growth is in the form
of a macrochemical equation2,s which, by definition,
produces 1 C-mol ofbiomass (the amount ofbiomass
containing 1 mol carbon, equivalent to - 2 5 g dry
biomass). The stoichiometry of this equation, for any
particular system, can be derived by defining the sys-
tem input and the measured biomass yield using the
C, H, O, N and charge balance 5. These equations
express the fact that elements and charge can neither
be lost nor generated during a chemical reaction. An
example of such an equation is shown in Fig. lb,
for the growth of Pseudomonas oxalaticus on oxalate as
C source.
In order to analyse growth in terms of its thermo-
dynamics, values for the enthalpy (AH ) and Gibbs
energy of formation (AG 1) of the reactant under
standard biochemical conditions (i.e. 1 bar, 1 mol 1-1,
98 K and pH7) are usually used*. The thermodynamic
properties ofbiomass have been discussed extensively
by Battley2,6-11 and Mavrovouniotis 12, and reliable
estimates of the enthalpy and Gibbs energy for biomass
* The energies of formation (AHfand A Gfa), which carry the subscript
'f' to denote formation, are properties of chemical compounds (and
may be found in the 'Thermodynamic Tables'). These are distinct from
the changes in energy associated with the reaction (AH and A G I ) ,
which carry the subscript R, to denote reaction, and are calculated from
the reaction stoichiometry and energies of formation of the compounds
involved.

1994, Elsevier Science Ltd TIBTECHDECEMBER1994 (VOL 12)

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Glossary

- Degree of reduction of a chemical compound, y is a stoichiometric property of a chemical compound that describes
its electron content (mol electrons C-mol compound<: for example, for CO2, 3, = O; for glucose, ~, = 4; for CH4, -y = 8).
~/'/e or z~Ge - Enthalpy or Gibbs energy of formation of a chemical compound expressed per mole of electrons present
in the compound (kJ mol electrons-i).
"Y~/'/e or ~,AGe - Enthalpy or Gibbs energy of formation of a chemical compound expressed in kJ per mole of carbon
(kJ C-mol-1).
~Gex - Gibbs energy of formation per electron in biomass (kJ mol electrons in biomassq).
~GeD - Gibbs energy of formation per electron for the electron donor (kJ mol electrons in electron donor-Z).
z~GeA - Gibbs energy of formation per electron for the electron acceptor (kJ mol electrons in electron acceptorq).
Ysx - Yield of biomass on substrate or electron donor (C-mol biomass C-mol donor-Z).
YDA- Yield of electron acceptor on electron donor (C-mol acceptor C-mol donor-i).
Dsl/r x - The dissipation of Gibbs energy needed to produce 1 C-mol of biomass from a given C source (kJ C-mol
biomassq).
ASR, ~/'/R - The entropy (J moll Kq) and enthalpy (kJ moll) of a reaction. They can be calculated from reaction
stoichiometry and tabulated entropies and enthalpies of formation for the chemical compounds involved.
Black-box thermodynamic efficiency - The ratio of the total Gibbs energy of formation associated with the chemi-
cal compounds produced to the total Gibbs energy of formation of the chemicals consumed.
Energy-convertor efficiency - The ratio of the Gibbs energy of reaction of a defined output process to the Gibbs
energy of reaction of a defined input process.
Conservation efficiency- The ratio of the Gibbs energy of reaction of the macrochemical equation to the Gibbs energy
of reaction of the catabolic reaction in which all the substrate from the macrochemical equation is combusted.

a For the example shown in Fig. lb, with a measured

C-source Biomass
value of Ysx = 0.086 C-mol biomass C-mol oxalat~ 1,
IL one can calculate, using tabulated values of Gibbs
N-source C02 energy of formation, that AGRI = -1048kJ and
Electron donor H20 IL
AHR = -939 kJ. This means that the formation of
Electron acceptor Products 1 C-mol ofbiomass from oxalate requires dissipation
}t
of 1048 kJ of Gibbs energy and produces 939 kJ heat.
In this article, the parameter (-A GRm) of the macro-
b Ysx=o.086 C-mol biomass C-mol oxalate -1 chemical equation is defined as Dsl/rx, measured in
kJ C-mol biomass-1.
Although this calculation is straightforward, Heijnen
+10.63 HCOa-
-0.2 NH4+ }' et al. 13 showed that the macrochemical equation can be

l I simplified still further by introducing a reference system

-5.415 H20

-1.857 O2 i -0.8 H+
Corresponding macrochemical equation
-5.815 0 2 0 4 2 - -0.2 NH4+ -1.857 02 -0.8 H
-5.415 H20 +1.0 CH,.800.sNo.2 +10.63 HCO a- = 0
Figure 1
(a) Black-boxdescription of microbial growth. (b) Aerobic growth of
Pseudomonas oxalaticus on oxalate described in terms of a macro-
chemical equation. The yield value, Ysx,means that (0.086)-I =
11.63 C-mol oxalate, or 5.815 mol oxalate are used to produce 1
C-tool biomass. The other stoichiometric values (-0.2, -1.857,
10.63, -5.415 and -0.8) follow from the C, H, O, N electric-charge-
conservation equations. (Redrawnfrom Ref. 5.)
are now available. It is therefore relatively straight-
forward to calculate the heat produced (AHR0) and the
Gibbs energy dissipation (AGR1) for the production
of 1 C-mol biomass using the macrochemical equation,
which incorporates an empirically determined value
for Ysx (biomass yield).
that combines the assignment of values for the enthalpy
and Gibbs energy of formation with a definition of the
'degree of reduction' of the chemical reactants (Box 1).
(The concept of'degree of reduction' has already been
used extensively to simplify stoichiometric calculations
by R o d s 1, for example.) In the reference system, each
chemical compound is characterized by its electron
content [which is equal to the degree of reduction, and
is defined as Ti (units: electrons C-mol-1)] and the
standard enthalpy, or Gibbs energy of formation per
mole of electrons (AHe and AGei, respectively). The
enthalpy or Gibbs energy of formation for a chemical
compound in this reference system is then equal to
(yiAHe) and (yiAGel). In this reference system, Yi,
AHe and AGem of H +, H C O 3 (or C02) , the N source
and H 2 0 have, by definition, values of zero, and this
forms the basis for the simplified equations. It should
be noted that this proposed reference system is a gen-
eralization of the 'aerobic-combustion reference' pro-
posed by Roels 1. In the new reference system, the
energedcs of growth become more obvious:

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The enthalpy of formation per electron (AHe) is
virtually constant for all organic (including biomass)
materials13; AHe = -28 kJ e-mol-L For O2, AHe =
-143kJe-mol -i. This means that the oxidation of
organic matter releases -[(-143)-(-28)] = 115 kJ of
heat per transferred electron. This is equivalent to
4.115 = 460kJ heat released per mol 02 consumed,
which is a well-known constant (see Refs 1,4).
The Gibbs energy of formation per electron for
organic matter (including biomass) is also nearly con-
stand 3, AGe = +32 kJ e-mol <. This means that there
is hardly any difference in Gibbs energy between the
organic electron donor and the biomass. AG R (and
also AHR) of the anabolic process is therefore close to
zero (an observation known as the 'Battley hypoth-
esis', and stressed again recently by Battley2,6-il).
Battley's observation, that the values of AG R, A H R,
and ASR are close to zero for anabolic reactions, results
from the fact that anabolism involves the conversion
of low-molecular-weight compounds into biomass,
without the transfer of electrons in catabolic reactions.
The AG R value referred to by Battley relates to this
anabolic reaction only, and is much smaller than the
AG associated with the consumption of ATP that is
also required for biomass production. If ATP is also
taken into account, the resulting overall values when
calculating AG, AS and AH for anabolic reactions are
very large. The constants of enthalpy and Gibbs
energy of formation per electron for organic com-
pounds were recently refined and modifications have
been proposed TM.
As the values for enthalpy and Gibbs energy of reac-
tion in anabolism during growth on organic com-
pounds are always close to zero, it is obvious that heat
production and Gibbs energy dissipation will only be
associated with catabolism2,7,15.
As the AG e ofbiomass and organic donor are nearly
equal (AGex = AGED), a simple equation that couples
Y~x to the Gibbs energy dissipation per C-mol pro-
duced biomass (Dsl/r x in kJ C-mol biomass<) may be
derived (Box 2) 13. This equation shows that Y~x
decreases at higher dissipation values, is lower for elec-
tron donors with fewer electrons (TD is then smaller),
and is a hyperbolic function of A GeD-A GeA, the Gibbs
energy released in the catabolic reaction per electron
present in the electron donor. AGeD-AGeA applies
only to the catabolic reaction (electron donor-ac-
ceptor reaction). For aerobic growth on a variety of
organic compounds, this value is nearly constant at
~110 kJ e-mol <. For anaerobic growth, however, this
value is variable (2-25kJe-mol <) (Ref. 13). The
derived equation for Y~x (Box 2) shows how Y~x
depends directly on the value of AGeD-AGeA of the
donor-acceptor couple. The greater the energy
released in catabolism, the higher the value of Ysx.
The Y~xequation also gives us the thermodynamic
theoretical maximal biomass yield, which is reached
at equilibrium, where D ? l / r x = 0. Ysx is equal to

Box 1. Stoichiometric calculations with the concept of degree of reduction

(1) Consider the example in Fig. l b and suppose that the stoichiometric coefficient 0 2 (coefficient O) is not known.
The values of 1, (degree of reduction) of the chemicals are:
Oxalate 1, = 1 C-tool -t = 2 moll
02 1, = - 4
Biomass 1, = 4.2
NH4+, H+, H20, HCO 3- 1, = 0
We can now write the balance of degree of reduction:
Oxalate electrons + 02 electrons + biomass electrons = 0
(-5.815x2) + (-4xO) + 4.2 =0 (Eqnl)
This means that 0 = -1.857
(2) Consider a 'short' macrochemical equation involving only donor, acceptor and biomass, because 3, for all other
compounds is zero
1 1
- Ysx C-mol donor - ~ acceptor + 1 C- mol biomass = 0 (Eqn 2)

The degree of reduction of the donor is 1,D,for the acceptor is 1,A and for biomass 1,x = 4.2. Using these values, we can
now write the balance of degree of reduction:

~o - +?A +4.2=0
(Eqn 3)

1
Solving for leads to the relationship between YAx andYsx :
YAx

YAx - 1,X -
(Eqn 4)

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Box 2. Thermodynamic analysis of the macrochemical equation

Macrochemical equation

A short macrochemical equation (omitting H20, C02, N source and H +, for which the degree of reduction 7 and
1
AGe is zero) runs as (using the relationship derived in Box 1, which relates ~ to the stoichiometric coefficient
of acceptor 1 ) .
YAx"
-1 d o n o r - [ T D - ~ , D ] [ 1 ] acceptor +1C-molbiomass =0
Ysx LY~x JL-~/Ad
(Eqn 1)
Gibbs energy balance over the macrochemical equation using the Gibbs energy of formation for:

Electron donor = TDAGeD01; electron acceptor = TAAGeA01; biomass = TxAGex1 = TxAGeD01

O= Gibbsenergy of Gibbs energy of Biomass

donor + acceptor + Gibbsenergy + Dissipation
[ - -
i i [ 1 I - - 1

= + Dl/rx
S (Eqn 2)

~IATD]
SolvingforYsxandYDA(=[YSX~AA )givestheequatinsthatshwhwYsxandYDAdependnDsz/rx:
01 01
~'D(AGeD-AGeA ) ^ ,
rsx=, ----~----
01 01 01
,b-moJ biomass C- mol electron donor
(Eqn 3)

YDA =
ro/(-rA)(Dsl/'x ) mol acceptor C- tool donor -1
+ (,Gel-,Go ') (Eqn 4)

TD/Tx, which is the ratio of the degree of reduction
of electron donor (TD) and biomass (Tx)- This rela-
tionship has been noted previously by tLoels 1, and a
refined equation has recently been derived using the
A G e frame of reference 13.
The ratio between heat production and Gibbs-
energy dissipation during growth is equal to the ratio
of heat production and Gibbs-energy dissipation of
the catabolic process 13. Using the A G e frame of refer-
ence, it is obvious that heat production and Gibbs-
energy dissipation are nearly equal during aerobic
growth. However, this is not the case for anaerobic
growth, where there are major differences between
heat production and Gibbs-energy dissipation (see
below).
It is easy to derive an equation to determine prod-
uct yield for a particular electron donor under anaer-
obic conditions (Box 2) 13. This yield YDA (C-tool
acceptor/C-tool donor <) increases with increasing
energy dissipation (D~1 rx) and decreasing values of
(AGeD-AGeA). This is quite logical because it implies
lower values of Y~x, and it is obvious that a lower
biomass yield leads to a higher product yield. Recog-
nition of this relationship enables selection of the
optimal electron donor and process conditions, and
thus increases the yield of catabolic products.
The derived relationships in Box 2 are generally
applicable to any donor-acceptor-N source combi-
nation. The equations in Box 2 show that the only
parameter needed to predict the biomass yield Y~x or
product yield YDA is D s l / r X, because for a given
growth system Tx, ")/D, AGeD and AGeA are known.
Correlations to estimate D s l / r x have recently been
obtained 5,13 (see Box 3).
Thermodynamic approaches to predicting
biomass yield
A critical evaluation of the published methods of
predicting Y~has recently been performed s,1s,16. The
criteria used for the evaluation were:
(1) General applicability to all chemotrophic growth
systems.
(2) Conforming to the second law of thermo-
dynamics.
(3) The requirement for black-box information only
(such as that depicted in Fig. 1), and no need for
detailed knowledge of the reaction pathway.
(4) The absence of intrinsic problems in the approach.

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Box 3. Correlations to calculate the dissipation of Gibbs energy for growth

Total dissipation is the sum of growth-related and maintenance-related components:

DsOl/rx = (DsOl/rx)gr + mE~IX

, , ,--~ , , (Eqn 1)
Total dissipation Growth Maintenance

The maintenance-related component of the Gibbs-energy dissipation is temperature dependent according to an

Arrhenius relationship:

rr~ : 4.5 x e x p[-69000

[~ [~-f1 - ~ 1 ) ] kJ C- mol < h_ 1 (Eqn 2)

The growth-related component of the Gibbs-energy dissipation depends on the C source (C, 70 for heterotrophic
growth/autotrophic growth [-reversed electron transport (RET)] and is a constant for autotrophic growth (+RET),
Heterotrophic growth/autotrophic growth (-RET):

(Dsm/rx)g r = 200 + 18(6-C) 1.8 + exp [((3.8 - 702).16 x (3.6 + 0.4C)] kJ C-mo1-1 (Eqn 3)

Autotrophic growth (+RET):

(DsOl/rx)gr = 3500 kJ C-mo1-1 (Eqn 4)

C = Number of C-atoms of C source

"/s = Degree of reduction of C source
T = Absolute temperature (K)
R = Gas constant (= 8.314 J mo1-1 K-1)

The most disturbing conclusion reached by this sur-
vey is that none o f the proposed methods satisfies all
o f these simple criteria s,ls. To illustrate some o f the
problems, let us consider attempts to correlate
thermodynamic efficiency with biomass yield.
This approach has been pursued by B a t t l e y 2, Roels 1,
and Westerhoffand Van Dam 3, all of whom, however,
proposed different definitions of efficiency. A problem
with all definitions of efficiency is that one arbitrary
assumption is always made in formulating the deft-
nition 16. For example, 1Loels's 'black box' definition
o f efficiency would vary if a different frame of refer-
ence for Gibbs energy of formation were chosen.
Recently, the feasibility of using a umque reference
for all growth systems has been discussed16,17; how-
ever, it appears that it is impossible to define such a
unique reference.
Westerhoff and Van Dam's 'energy convertor' ef-
ficiency3 is based on a definition of anabolism that is
biochemically unrealistic because 0 2 can be produced
in heterotrophic growth 16. Their conclusion 18 that
growth can be described as an optimized Gibbs-
energy convertor for maximal growth rate at optimal
Gibbs-energy efficiency is therefore invalid.
Battley's definition o f 'conservation efficiency'2 is
based on a definition of catabolism during growth that
is also biochemically incorrect: he assumes flail catab-
olism of all substrate whereas, in reality, part of the sub-
strate is converted into biomass and is not catabolized.
Therefore, although these proposed definitions o f
efficiency have all been used, with some success, to
correlate thermodynamic efficiency with biomass
yield, they cannot be regarded as representing the true
efficiency of the growth process.
Another approach to defining efficiency has been
proposed by McCarty 2,32. This is based on known
biochemical pathways o f anabolism, catabolism and
N-source incorporation, and focuses on the energy-
transformation efficiencies of each of these processes.
This method resembles the ATP-based approach o f
Stouthamer21; however if, as in the 'black-box
approach', detailed knowledge o f the stoichiometry
of the biochemical reaction is not available, McCarty's
method cannot be used reliably. If, however, the
stoichiometry of the biochemical reaction is available,
then the ATP-based approach is much more accu-
rate 21.
An alternative, measurable, thermodynamic par-
ameter that complies with the four requirements listed
above, that has none of the problems associated with
other methods, and which leads to a more accurate
prediction o f Ysx over a wide range of conditions, has
been proposed 5,1s. This parameter is Dsm/rx, the Gibbs
energy that must be dissipated to produce 1 C-mol of
biomass. From the equations in Box 2, it is clear that
Ysx and YDAcan be calculated if one knows D~l/r x. It
is known 1,22that, during growth, part o f the substrate
is used for biomass synthesis, and part is catabolized to
provide the energy required to maintain the integrity
o f the organism. In accordance with this idea, energy
dissipation can be attributed to growth-related and
maintenance-related components (Box 3) 1'22 .
The growth-related energy dissipation, (D~l/rx)g r,
depends only on the C source used 5,1s. The type of

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I an accuracy of 10-20% in the range Ys~ = 0.01-0.8
o.1[ im
F
0.01 ~
0.01 0.1
Ysx measured
Figure2
Comparison between measured and estimated values of Ysx-using
the DsOl/rx correlation. (Redrawnfrom Ref. 5.)
electron acceptor involved (02, N O 3 - , fermentation
where an internal electron acceptor functions) exerts
only a minor effect and may be ignored. It appears
that, for organic C sources, (Dsl/rx)gr increases for
C sources with fewer C-atoms, or whose degree of
reduction is different from that of biomass (Tx = 4.2
for NH4+ as N source). Furthermore, it has been
found that, for autotrophic growth on electron donors
(such as Fe2+ or NH4+), which requires reversed elec-
tron transport (RET), D~m/rx is very high. These find-
ings are easy to understand if one considers that a C
source with one C-atom needs much more metabolic
'tinkering' than a C source with six C-atoms for the
synthesis of biomass. In addition, a C source that is
either less, or more highly, reduced than biomass
requires additional oxidation (TD > Tx) or reduction
(TD < T~)- If required, R E T is an additional energy-
requiring process that must be taken into account. The
high values for D~m/rx simply reflect the larger num-
ber of chemical reactions and consequent energy dis-
sipation required to produce biomass from a given
C source. This result is also in broad agreement with
Stouthamer's approach, which shows that more ATP
is needed for 'poor' substrates 21.
The maintenance-related energy dissipation (mE)
depends primarily on the temperature, according to
an Arrhenius relationship with an energy of activation
of 6 9 k J m o P 1 (Box 3), and is independent of tile
C source or electron acceptor 22. Analysis of main-
tenance-related dissipation is optimal if based on Gibbs
energy of dissipation. The other approaches to quanti-
fying maintenance are based on concepts such as
biomass decay, substrate consumption or consumption
of electrons, but give much more variable results; for
IBTECHDECEMBER1994(VOL12)
example, a constant maintenance Gibbs energy pre-
dicts that the endogenous decay coefficient is three
times less for autotrophic than for heterotrophic
growth 22.
Using the correlations for calculating the dissipation
of Gibbs energy (Box 3) in combination with the
Ysx-equation (Box 2) enables Y~xto be predicted with
C-mol C-mol electron donor -1 for any C source,
electron donors-acceptors, N-source, temperature or
growth rate (Fig. 2) (Ref. 5). Box 4 shows an
example of such a calculation.
Application of this approach also indicates the con-
ditions that should be selected to optimize productivity.
For example, for anaerobic-product formation (YDa),
this could be achieved by selecting:
A 'poor' C source, which requires a large energy
dissipation to produce biomass.
An electron donor that is much more reduced than
the product (3'D > -TA) (for example, the anaerobic
production of acetate from methanol 29 and the pro-
duction of acetate from glycerol, Box 4).
A low growth rate, because this raises D?l/r x
(Eqn 1, Box 3; Box 4).
A poor electron donor-acceptor combination,
which leads to a low value of (A GeD-AGeA).
Anaerobic-product formation linked to catabolism
(Box 4) is related to biomass yields in a straightforward
manner. However, the thermodynamics of product
formation not directly linked to growth is a relatively
neglected area of research, and an obvious area for
further investigation.
Although this approach yields satisfactory results, it
should be stressed that the value of Ysxobtained is only
a preliminary estimate based on the average behaviour
of many growth systems. Even for the same C source
or electron donor, there is often more than one
catabolic route: for example, for gltlc0~se (glycolysis,
Entner-Doudoroff) or for methanol (serine, ribulose,
autotrophic route). Clearly, this influences Y~x- Con-
versely, if the predicted value of Y~x deviates strongly
from the measured value, then one might have found
an organism with an unusual biochemical route that
merits special attention.
Heat production during growth
The study of heat produced in biological systems
(calorimetry) in relation to metabolism has a long his-
tory (see P,.e~ 2 and 4 for overviews). The production
of heat is used as a characteristic energetic parameter of
growth, and can be used to calculate growth using
the enthalpy balance 4,23,33,34. In aerobic systems, heat
and O2-consumption are intimately linked (460kJ
mol O2-1). In anaerobic systems, heat production is still
a useful parameter to measure in relation to metabolism.
It is widely4, but incorrectly5, believed that growing
systems always produce heat: the second law of
thermodynamics only requires that the Gibbs energy is
dissipated; heat can therefore either be produced or
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Box 4. Example of the estimation of Ysx and anaerobic product yield YoA

Consider the anaerobic growth of a microorganism on glycerol, which only produces acetate at 4542. According to
Table III in Ref. 13, AGeD01-AGeA01 = 37.8 - 26.86 = 10.94 kJ e-molq. If one assumes that NH3 is the N source, then
the degree of reduction of biomass b/) = 4.2. The degree of reduction of glycerol is 4.66. Glycerol contains three car-
bon atoms; hence C = 3. The temperature is 4542, therefore T = 318 K. Box 3 (after substitution of C = 3, 7s = 4.66,
and T = 318 into Eqns 1-3) leads to the estimated total Gibbs-energy dissipation per C-mol of biomass as a function of
growth rate:
Dsi / rx = 428 + 2__6_6 kJ C- mol biomass -1
I I I I L tJ, J

Total Growth-related Maintenance-related

dissipation dissipation dissipation (Eqn 1)

At/~ = 1.0 h-1, one can calculate Ds01/f x = 454 kJ C-mo1-1, while at ~ = 0.05 h-1, one can calculate Dsm/rx = 948 kJ
C-mol q .
The biomass yield Ysx follows from Box 2, Eqn 3, using [AGeD01-AGeAol = 10.94, 7D = 4.66, 7x = 4.2]:
For/, = 1.0 h-1, one obtains Ysx = 0.10 C-mol C-mo1-1
For/, = 0.05 hq, one obtains Ys = 0.05 C-mol C-mo1-1

Using the value of (-~'k) = 4 (for the product, acetate), one obtains, from the YDA equation (Eqn 4) in Box 2, the follow-
ing yields of acetate on glycerol:

For t* = 1 h-1, one obtains YDA ---- 1.058 C-mol acetate C-mol glycerol q
For/, = 0.05 h-1, one obtains YDA= 1.111 C-mol acetate C-mol glycerol -1

Clearly, the product yield on carbon is >100%, which is due to the fixation of CO 2 because glycerol is more reduced
than acetate. In addition, one observes that the product yield increases at low growth rate as a result of maintenance
requirements.

taken up during growth. Table I indicates the measured
biomass yield, the calculated Gibbs-energy dissipation,
the calculated heat production/consumption and the
calculated entropy-related dissipation, all per C-mol
produced biomass for three substrates (glucose, H 2 and
acetate) under aerobic and anaerobic conditions. It can
be seen that, for a given electron donor, the Gibbs-
energy dissipation is nearly the same under both
aerobic and anaerobic conditionss. The relative contri-
butions of heat and entropy are, however, markedly
different.
For aerobic growth on glucose, Gibbs-energy dissi-
pation and heat production are nearly equal, with very
little contribution from the entropy term. Under
anaerobic conditions, however, two-thirds of the
energy dissipated is due to an increase in entropy
(Table 1). As the overall dissipation does not change
significantly, the heat production per C-mol biomass
is only one-third of that associated with aerobic
growth.
For aerobic growth on H2, there is a significant
decrease in entropy, because small gaseous molecules
(H2, 02, CO2) are converted into large molecules
(biomass), and into liquid (water). This leads to 'extra'
heat production, because dissipation of Gibbs energy
is constant. This effect is even more pronounced
under anaerobic conditions, where 4 H 2 + C O 2 are
converted into C H 4 and H20, resulting in a huge
decrease in entropy and significant production of heat.
For H2, the heat production per C-mol biomass is
greater under anaerobic conditions than under aer-
obic conditions. For aerobic growth on acetate, nearly
all energy dissipated is due to heat production, with
very little contribution from changes in entropy.
However, for anaerobic growth (where acetate is con-
verted into C H 4 and CO2), there is such a large
increase in entropy that heat is taken up during
growth. Clearly, this shows that Gibbs-energy dissi-
pation (which must be positive) is the key thermo-
dynamic parameter that must be considered, whereas
heat can be produced or taken up during growth.
As shown above, Gibbs-energy dissipation and heat
production are nearly equal for aerobic systems. The
correlations in Box 3 therefore provide us with a direct
estimate of the expected heat production and the
O2-consumption (460 kJ mol O2-1), for aerobic
growth as a function of C source and growth rate. For
example, growth on glucose at/z = 0.5 h -1 produces
heat at a rate of 229 kJ C-tool biomass-1 at 25oc, and
1543 kJ C-mol biomass-1 at 90C. This is equivalent
to a biomass yield of 2 C-mol mol 02 -1 and of 0.30
C-molmol 02 -1 at 25C and 90C, respectively. The
difference results from maintenance-related energy
requirements.
Increased heat production resulting from main-
tenance requirements is obviously an important
factor, leading to greater heat production per C-mol
biomass at low/z, and lower heat production at high
/z. Because of the limited cooling capacity of a fer-
menter, processes designed to produce biomass [such
as single cell protein (SCP) processes] are usually
carried out at high growth rate 19. It should also be
realized that the efficiency of aerobic growth at high
temperatures suffers from extremely high maintenance

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Table 1. Gibbs energy dissipation and heat production for aerobic and anaerobic growth a
YDx
(C-mol C-mol
Microbial system Conditions donor-Z)
Saccharomyces cerevisiae Glucose/02; 0.57
aerobic
S. cerevisiae Glucose; 0.14
anaerobic
H2 bacterium H2 + C02/02; 0.13
aerobic
Methanobacterium arborophilus H2 + C02/CH4; 0.015
anaerobic
Pseudomonas oxalaticus Acetate/02; 0.406
aerobic
Methanobacterium soehngenii Acetate/CH4; 0.024
anaerobic
aDatafrom Ref. 5.
requirements: this leads to very high Oi-consumption
per C-mol biomass. Only very low biomass concen-
trations can therefore be achieved in aerobic processes
at high temperatures, where a severe bottleneck in 0 2-
transfer in the reactor from the gas to liquid phase is
always a problem 19.
Thermodynamic approaches to understanding
growth kinetics
As described above, the Gibbs-energy dissipation
has been used to predict Y~x.The calculations of Gibbs
energy are generally based on standard conditions
(298 K, 1 mol 1-1, 1 bar, pH7). However, the con-
centration ofsubstrate in many culture systems is fre-
quently much lower (10-3-10 -6 mol 1-1). In addition,
the culture of organisms under extreme conditions
(temperatures up to 100C and pH values ranging
1-14) is attracting increasing interest. In such situ-
ations, conditions deviate considerably from the stan-
dard. The Y~x equation in Box 3 shows that these
deviations can be accounted for in the calculation of
(AGeD-AGeA). This value is directly related to the
Gibbs energy of reaction (AGp.) of the catabolic
process, and consideration of the effects of concen-
tration and temperature on the value of AGp. for the
catabolic process is sufficient to quantify their influ-
ence on Y~x, as has been demonstrated in a number
of cases.
% It has been f o u n d 23,25 that, for such anaerobic
Concentration effects in aerobic-growth kinetics
It has already been noted that, in the aerobic
catabolism of organic electron donors, (AGeD--AGeA)
is very large, and differences in concentration or tem-
perature therefore exert only a very small effect on
(AGer)-AG~A). However, during aerobic growth on
inorganic electron donors (for example, Fei+), the
value of AG~D-AGeA is large at pH = 1,.whereas, at
pH = 7, this difference is close to zero, leading to
Y,x = 0: this explains why microbial growth that is
dependent on Fe2+ oxidation only occurs at low pH.
IBTECHDECEMBER1994(VOL12)
Dissipation resulting from:
Total Chemical
dissipation Heat entropy
(kJ C-mol {kJ C-tool (kJ C-mol
biomass-Z) biomass-Z) biomass-Z)
332 +339 -7
270 +95 +175
1265 +1686 -421
1035 +3923 -2888
562 +593 -31
597 -90 +687
Concentration and temperature effects in
anaerobic-growth kinetics
For fermentative growth, or growth with 8042- as
the electron acceptor, the value ofA GeD-A GeA is quite
small (2-25 kJ e-mo1-1) and concentration effects then
become very important. In some natural microbial
communities, Hi-producing and Hi-consuming
species frequently grow in close association, and the
effect of Hi-partial pressure on the process of inter-
species Hi-transfer is very well known 24-26. The influ-
ence o f H i (stimulation/inhibition) and acetate (inhi-
bition) on the growth kinetics of Hi-producing and
Hi-consuming organisms is easily understood, con-
sidering its effect on AGp, of the catabolic process. It
may be calculated that the optimal thermodynamic H i
concentration, in this food chain, of a Hi-producer
and a Hi-consumer is such that the AGe. of the
catabolic reactions of both the Hi-producer and the
Hi-consumer are equal: this has been observed for
some systems27,28. The optimal Hi-concentration is
very low (10 -4 bar), and therefore interspecies H i
transport is promoted, as observed26, by direct physi-
cal contact.
Differences in H 2 partial pressure can also influence
product formation. For instance, the formation of
reduced products at increased Hi-pressures is thought
to be due to catabolic processes that operate close to
equilibrium (for example, the formate/H 2 system) 29.
growth processes, there appears to be a minimal
value of AGg of the catabolic process required to
sustain growth, and this lies in the range o f - 5 to
-15 kJmol Hi -1 (Table 2). The physiological basis of
such a minimum value probably lies in the quantiz-
ation of the cellular energy carriers: i.e. the Gibbs
energy for H+-pumping (proton-motive force) is
thought to be -10 to 15 kJ per mol H +. This minimal
Gibbs energy leads to threshold values of electron
donor concentration (for example, H2) to sustain
growth.
 491

reviews

A low Gibbs energy of catabolism has several kinetic Table 2. Minimal required Gibbs energy of catabolic
consequences, which originate in the effects of con- processes {AGR) to sustain growth
centration, temperature and the possible involvement
of precipitates (Box 5). The effect of concentration has Microbial process AGR{kJ per mol H2) Ref.
been dealt with above; examples of the role of pre-
cipitates can be found in sulphide-forming processes, H2-consuming acetogens -5 to -6 24
H2-consuming methanogens -9 to -12 24
where sulphide precipitates occur. The temperature H2-producing acetogens -5 to-10 26
effect is less well known 24,25,3 and is described in (propionate/ethanol)
Box 6, using the Van't Hoff relation. It is clear that
temperature has very significant effects on AGP` of
catabolism, particularly in anaerobic systems. Depend-
ing on ASp., the catabolic process can generate either Box 5. Kinetic consequences of catabolic processes with
much more, or much less, Gibbs energy at elevated low Gibbs energy of reaction (AGR)
temperatures. An interesting case is the production of
H e from the complete conversion of glucose. At 25C, Strong effects of concentrations (particularly pH) on AGR, and hence
this provides hardly any energy, but at 95C, the value on Ysxand product yields.
Strong temperature effect on AGR(see Box 6), and hence on Ysxand
of AGp. is high. This suggests the possibility of com- product yields.
pletely converting carbohydrate t o H 2 at high tem- Strong effect of precipitates on AGR (in sulphide-producing pro-
perature anaerobically (provided that there are no cesses, for example).
unforeseen biochemical bottlenecks). This effect of Threshold concentration for H2 in H2-consuming processes.
temperature on AG R also helps to explain the rise in Inhibition by, for example, H2 and acetate for H2-producing pro-
H2-threshold concentrations with temperature in the cesses using propionate or ethanol.
interspecies H2-transfer process24. Changes in product formation resulting from mass-action effects
(bicarbonate+H2/formate system).
Effects of highly irreversible reaction kinetics on growth
kinetics
The kinetic effects mentioned so far arise from the Box 6. Temperature effects on the Gibbs energy of
significant influence of concentration/temperature on reaction (AGR) of catabolic processes
A Gp`, if A G~ is close to zero, which means that the
reactions are reversible. Such an approach does not Van't Hoff relationship: AGR = AHR- T(ASR)
hold for aerobic systems, where catabolism is essen-
tially irreversible. In the past, attempts have therefore
been made to formulate thermokinetic models for Reaction Temp (C) AGR (kJ) Ref.
irreversible processes (see R.ef. 3 for an extensive
Glutamate- + 4 H20 --~ 25 -7 30
review) as an alternative to conventional kinetic mod- propionate + 2 HCO3- 55 -17
elling. This approach is based on the realization that + H+ + H2
many metabolic reactions are coupled so that energy
transfer is involved. Examples are chemiosmotic pro- Glucose + 6 H20 ---, 25 -26 25
ton extrusion, the H+/ATPase in mitochondria, vari- 6 CO 2 -I- 12 H2 95 -151
ous transmembrane transport processes driven by the
4 H2 + 2 CO 2 --* 25 -95 24
proton-motive force. Each of these coupled processes acetate- + H + 2 H20 90 -8
may be described as a linear Gibbs-energy transducer:
the process kinetics are simple, and thermodynamic
efficiencies agree with optimality criteria (i.e. maxi- Conclusions:
mal rate of energy production) 3. AGR becomes more negative with increasing temperature if ASR is
positive (gas-producing reactions).
This approach has also been applied to growth by AGR becomes less negative with increasing temperature if ASR is
incorporating growth considerations into a coupled negative (gas-consuming reactions).
process of anabolism and catabolism3. However, a
number of key difficulties with this approach were
recently outlineds,ls (see above). data set (heterotrophic/autotrophic growth under
Gnaiger 31 has also stressed the point that growth aerobic/anaerobic conditions) at 25C, a maximal
metabolism is composed of the coupled processes, electron transport rate of 1-2 e-mol C-mol biomass
linked either in series or in parallel. Accordingly, the h -1 was found with an Arrhenius temperature depen-
overall growth efficiency is a complex function of the dence according to Eact ~ 60 kJ mo1-1.
combined efficiencies of each coupled process. It is Another interesting result has been the thermo-
then not clear which optimality criterion should be dynamic description of maintenance, as pioneered
applied. by P-oels 1 and refined recently by Tijhuis et al. 22 (see
McCarty 32 has, however, proposed an approach for also the discussion above on maintenance).
predicting values o f / x m~x. He suggested that micro- In general, however, it must be concluded that the
organisms have a limited capacity for processing elec- thermodynamic basis of kinetic behaviour of growth
trons through the catabolic pathway. From his limited is, as yet, far less advanced than the thermodynamic

TIBTECHDECEMBER1994(VOL12)
 492
reviews
basis of the stoichiometry of growth like the biomass
yield. Furthermore, it is still unclear whether there is
any specific advantage in applying a thermodynami-
cally based kinetic description 3, compared with the
more usual mass-action approach to understanding the
kinetics of microbial systems1.
Conclusions
There now exists a well-established thermodynamic
approach, based on dissipated Gibbs energy, to under-
standing and predicting values of biomass yield for
microbial growth processes. It also appears that a
thermodynamic approach can be used to evaluate
maintenance-related requirements. Thermodynamic
considerations with respect to process design relate
primarily to being able to predict stoichiometric co-
effluents (particularly for heat, oxygen and anaerobic
products). In addition, it would be useful to be able
to estimate any possible effects of temperature or con-
centration ofsubstrate/media components on growth
processes with a low catabolic AGI,. value (i.e. anaer-
obic processes). The principal difficulties with ana-
lysing growth- and metabolic-network efficiencies
have been addressed extensively over the past few
years. By contrast, development of thermodynamic
methods to predict kinetic behaviour (for example,
values of/* m=') is far less advanced. Similarly, thermo-
dynamic analysis of product formation that is not
linked to catabolism has not been considered. Eluci-
dating the thermodynamics of product formation, and
the development of a thermodynamic framework for
the kinetic description of growth and product for-
marion is an essential area of research for attention in
the near future.
Acknowledgements
The author would like to thank E. H. Batdey, H. V.
Westerhoff, U. yon Stockar andJ. A. Roels for many
interesting discussions on the thermodynamics of
microbial growth in the past years.
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