A Thermodynamically Based Correlation for Maintenance Gibbs Energy Requirements in Aerobic and Anaerobic Chemotrophic Growth

L. Tijhuis, M. C. M. van Loosdrecht, and J. J. Heijnen Department of Biochemical Engineering, Delft University of Technology, JulianaLaan 67, 2628 BC Delft, The Netherlands
Received November 11, 1992/Accepted March 8, 1993

A thermodynamic framework has been provided for the

description of maintenance requirements of microorganisms. The central parameter is the biomass specific Gibbs energy consumption for maintenance, mE (kJ/C-mol biomass . h). A large set of data has been used including (i) a large range of different organisms (bacteria, yeasts, plant cells), ( i i ) mixed cultures, ( i i i ) heterotrophic and autotrophic growth, (iv) growth under aerobic and anaerobic conditions, and (v) a large temperature range (5-75C).It appears that only the temperature has a major influence, with a n energy of activation of 69 kJ/mol. Different electron donors or electron acceptors only show a very minor influence on m E . On the basis of the data set, temperature correlations of mE have been derived for aerobic and anaerobic growth. The generalized concept for maintenance Gibbs energy is used to establish a correlation which allows the estimation of the biomass yield on electron donor a s a function of C-source, electron donor, electron acceptor, N source, growth rate, and temperature. The advantage of using the t n E parameter over other maintenance-related parameters (like p e rmoz, mD, Y D m o ) is discussed. 0 1993 John Wiley & Sons, Inc. Key words: Gibbs energy requirements chemotrophic growth maintenance anaerobic and aerobic

Nowadays many maintenance coefficients have been measured for many microbial growth systems, organic substrates, and under aerobicfanaerobic conditions from studies of the dependence of YDX on p in carbon-limited chemostat studies. Although there are some indications that the maintenance coefficient is dependent on p, this is still a matter of debate.3*0,46,64 Here maintenance is considered as a black box parameter which gives a very useful description of the dependence of YDX on p. Such a relation is needed to describe industrial fed-batch fermentations or to quantify sludge production in wastewater treatment systems, and hence knowledge of the maintenance coefficient is of great practical Furthermore, it is also clear that one should distinguish the maintenance requirement for microorganisms under optimal carbodenergy-limited growth from the increased maintenance requirement under suboptimal ~ o n d i t i o n s . ~ ~For ~example, the maintenance require,~ ,~* ment increases due to osmotic or solvent22 and due to the presence of undissociated acidsb9 or ~ n c o u p l e r ~ ~ which dissipates the transmembrane protonmotive force. Also it has been noted that calculated maintenance coefficients from YDX measurements can strongly be influenced by the occurrence of cellysis or a drop in cell viability,35 INTRODUCTION under presumably suboptimal conditions. A method to Recently a new thermodynamically based method has been distinguish cell lysis and maintenance has been provided35. provided to estimate the maximal biomass yield on electron In this article the above defined minimal maintenance donor, Ygy, for arbitrary chemotrophic growth systems requirements of microorganisms will be studied under under aerobic, denitrifying or anaerobic, carbodenergycarbon/energy-limited aerobic and anaerobic chemotrophic limited condition^.^^^^^ However, it is well known that growth under optimal conditions where such phenomena growth yields are influenced by the specific growth rate may be neglected. More specifically, a thermodynamic p . This is conventionally described using the concept of description will be presented based on the Gibbs energy Herbert32 of endogenous respiration or of Marr et al.37 requirements for maintenance. As such, this provides an and Pirt46 of substrate maintenance or of a combination extension of an earlier attempt to obtain a description for thereof as proposed by Rhamkrishna et al.48 and by Beeftink only the substrate maintenance requirement during aerobic et al.4 These descriptions define a so called maintenance heterotrophic g r o ~ t h . ~ , ~ The proposed thermodynamic coefficient, which is a measure of the required maintenance description is based on an extensive data set taken from energy. These coefficients may be provided as substrate, a literature which covers (1) a large range of different C 0 2 , 0 2 , or heat requirement or as an endogenous decay comicroorganisms (bacteria, yeast, plant cells), (2) mixed efficient, all of which are stoichiometrically ir~terrelated.~,~~ cultures, ( 3 ) growth on organic carbon sources and on C02, (4) growth under anaerobic and aerobic conditions, and ( 5 ) growth in a temperature range of 5-75C. * To whom all correspondence should be addressed.

Biotechnology and Bioengineering, Vol. 42, Pp. 509-519 (1993) 0 1993 John Wiley & Sons, Inc.

CCC 0006-3592/93/040509-11

A GIBBS ENERGY-BASED DESCRIPTION OF MAINTENANCE ENERGY REQUIREMENTS

In recent paper^^^,^' it has been shown that the formation of biomass form its carbon and nitrogen source is necessarily accompanied by a specific amount of Gibbs energy dissipation (in kJ/C-mol biomass), called (D:l/rAx)gr. One C-mole of biomass is the amount of biomass which contains 12 g C, which is about 26 g dry weight. The dissipation of Gibbs energy per C-mole produced biomass (under high growth rate conditions) was found to lie in the range of 200-3500 kJ/C-mol biomass and appeared to depend primarily on the nature of the carbon source and the occurrence or absence of the need for reversed electron transport (RET). The carbon source can be characterized by its carbon chain length, C , and its degree of reduction ys. Here ys is the number of electrons per C-mol of substrate upon complete oxidation. Its value is 8 for CH4 and 0 for C02. The following correlations have been obtained for organic substrate^^^, which do not require RET: -RET

There has also been derived3' a relation for Y T F :

(3)
where YFF is the maximal yield of biomass on electron acceptor in C-mole of biomass per mole of acceptor and Y A is the degree of reduction of electron acceptor. It is noted that, by definition, Y A is negative. For example, 0 2 has Y A = -430. Here ( - 7 ~AG:; is the Gibbs energy ) production of the redox reaction, but calculated per mole of electron acceptor. Now the following relations [eq. (4) and (S)] are well known5" for the calculation of the actual yield of biomass on electron donor or acceptor (YDX and Y A X )from the maximal yield values (YE?, YF","") and the maintenance coefficients ( m D , mA):

(4)

(5)
Equations (2) and (4) and (3) and (S), respectively, may now be combined by elimination of YE? and YAF, respectively. This leads to Eq. (6) and (7) for the calculation of the actual yields from the dissipation:

(D:l/rAx)gr 200 =

+ 18(6 - C)"*

exp ((3.8 - YS) }

*

2 0.16

(3.6 + 0.4C)

For substrates which do require reversed electron transport (Fe2', N02-, NH4+, etc.) +RET

(la)

( D : ' / ~ A= 3500 ~)~~

(1b) Now it is well known that there always holds the stoichiometric relation (8) between YDX and YAX.30 This relation follows directly from the balance of degree of reduction of the growth system:

This dissipation of Gibbs energy per C-mole of biomass, (D:l/rAx)gr, relates directly to the maximal biomass yield according to Eq. (2), which has been derived re~ently.~'

In this equation, ( D : ' / F - A is )the~Gibbs energy required ~ ~ to produce 1 C-mol biomass at high growth rates. Values are provided by Eqs. (la) and (lb). The terms yx and Y D are the degrees of reduction of biomass and electron donor (which is often identical to the C source); YE? is the maximal biomass yield on electron donor at high growth rate p ; and AG;: (in kJ/e-mol) is the available Gibbs energy of reaction for the redox reaction between electron donor and acceptor, but calculated per electron. Because there are Y D electrons per (C)-mol electron donor, it is clear that Y D AG:; is the Gibbs energy of the redox reaction between donor/acceptor per C-mole of electron donor. For any specific redox reaction, AG:; follows directly from thermodynamic tables6'. For aerobic growth on organic substrates AG;; is more or less constant for all organic substances, being 111 -C 5 kJ/e-m01.~" For anaerobic growth AG:: is much lower and is in the range of 2 to 30 kJ/emo13". A example of the calculation of AG;; is presented n in Appendix 1. More details are provided by Heijnen et al.30

This relation also holds for the maximal yield values YF? and YE?, as can be readily seen from Eqs. (2), (3), and (8). For aerobic growth ( y = -4) a well-known relation ~ is obtained between yield of biomass on substrate and oxygen.*' If one now combines Eqs. (6), (7), and (8), one obtains the equality5'
(9) Based on the definitions of mD, mA, y o , Y A , and AG;; it can readily be seen that both sides of this equality represent the rate of Gibbs energy which is dissipated per C-mole of biomass for maintenance purposes. If this maintenance rate of Gibbs energy is denoted as mE (kJ/C-mol biomass * h), one can write
~ D Y D AG:~ =

mA(-yA)

~ ~ , 9 f

(10) Clearly one can calculate mE from measured values for mD or mA and the known values of yo, Y A , and AG:;
mE = m D y D =

AG:;

mA(-yA)AG:;

510

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 4, AUGUST 5, 1993

(which follow from the known electron donor and acceptor, Appendix 1). It is now also possible to define the total Gibbs energy dissipation, D:'/rAx, needed for growth and maintenance, which is then related to the actual yields YDX and Y A X . Combination of the bracket terms in the denominator of Eqs. (6) and (7) together with Eq. (10) gives

Equations (6) and (7) can then be rewritten as

It can be seen that Eqs. (2) and (3), which give the maximal yields, are homologous to Eqs. (12) and (13), which give the actual measured yields. The only difference lies in the dissipation which must be used. The calculation of maximal yields must be based only on the growthassociated dissipation D!'/rAx, while the calculation of the actual yields [Eqs. (12) and (13)] requires the total dissipation ( D : ' / r A x ) g rwhich includes the growth-associated , and maintenance-associated dissipation [Eq. (11)]. In a previous article2' a simple correlation has been provided for ( D : ' / r A x ) g rIn this article a correlation for mE will be . pursued.

A CORRELATION FOR mE IN ANAEROBIC AND AEROBIC CHEMOTROPHIC GROWTH

The minimal maintenance Gibbs energy requirement m E , as defined in the introduction, is generally related to unavoidable leak and denaturation processes which occur in biomass. One can think here of protein denaturation/hydrolysis, other polymer hydrolysis, leak of protons, or other ions across membranes along their electrochemical gradient. All these processes must be compensated in order to maintain the desired steady state membrane gradients and intracellular concentrations. Hence repolymerization and transport processes must occur. Each of these processes requires a certain Gibbs energy dissipation, which add up to mE. Because microbial membranes and biomass of different organisms under optimal carbon and energy-limited conditions may be supposed to have comparable molecular composition, one might speculate that for many different microorganisms these rates are similar. The main parameter which would influence mE would then be the temperature, because this influences the rates of the said processes. Different electron donors or electron acceptors have in general a limited influence on biomass composition, and therefore, one could speculate that mE is independent of the electron donor or acceptor. Summarizing, the simple view of the minimal maintenance-related processes leads to speculation that mE mainly is dependent on temperature and that mE is not much influenced by the nature of the electron donor or acceptor.

Tables I and I1 contain a large number of maintenance coefficient values (mD and mA) taken from literature sources where growth was studied under carbodenergy limitations in a chemostat. The growth systems cover aerobic (Table I) and anaerobic (Table 11) systems, including organic and inorganic carbon sources. The mE may be calculated from the obtained mD and mA values using Eq. (lo). Both calculations should give theoretically the same mE value. For combined mA, mD data, only data sets were taken where both calculated mE values did not differ more than a factor 2. Although this might seem a large span, it is well known that in general large uncertainties are associated with the reported values of mD or mA. Confidence intervals of 5 5 0 % are quite common." The reason lies in the fact that the maintenance contribution is generally small in the normally studied range of growth rates of 0.05-0.50 h-'. Only recently the much lower growth rate regime of < 0.03 h-' has been studied to some extent.3,7,'0.28,56,64,67 The calculated mE values follow as the average of the values calculated from mD and mA using eq. (10) or using single mA or mD values. Based on this data set for M E , the following aspects will be discussed: (i) effect of temperature on m E ; (ii) effect of electron acceptor (aerobic versus anaerobic); (iii) effect of different C-sources on mE; (iv) effect of type of organism; and (v) mixed sludges versus pure cultures. The effect of temperature on mE is shown in Arrhenius plots for aerobic and anaerobic growth systems (Figs. 1 and 2). Although there is a large scatter, there is a clear Arrhenius type of temperature dependence. Both aerobic and anaerobic systems show an activation energy of 69 kJ/mol. This means a doubling of mE at 8C increase of temperature. This activation energy is in agreement with single culture behavior, e.g., Klebsiella pneumoniae, Bacillus stearothermophilus, Bacillus caldotenax, Escherichia coli (Table I), and other organisms reviewed by Esener et al. l8 Furthermore, it appears that anaerobic growth systems have a somewhat lower mE value than aerobic growth systems. Previously it was found that the growth associated dissipation (D;l/rAx)gr also was somewhat lower for anaerobic The relations in Figures 1 and 2 are given by Aerobic

mE

5.7 exp

lo4
Anaerobic kJ/C - mol mE = 3.3
3

(14)

kJ/C - mol

(15)

The confidence intervals are indicated and show that mE values from Eqs. (14) and (15) have confidence intervals of 41 and 32%, respectively. Although the difference in mE relations between aerobic and anaerobic growth is not very significant, the difference appears always to be present in the few systems where the same microorganisms have

TlJHUlS ET AL.: GlBBS ENERGY CORRELATION FOR MAINTENANCE REQUIREMENTS

51 1

2 N
A: G;
Substrate
YO

Table I. Maintenance Gibbs energy mE for aerobic growth.

T ("C)

Microorganism
YA

(kJ/e-mol)

mD [(C)-mol/C-mol

. h]

mA (mol Oz/C-mol . h)

mE (kJ/C-mol

. h)

Ref.

;I ; 0

E
-4 -4

Aerobacter aerogenes Bacillus acidocaldarius Saccharomyces cerevisiae

<

Thiobacillus acidophilus Penicillium chrysogenum

-4 -4 -4 -4 -4 -4

4 0

0.044 0.060 0.015 0.01 0.021 0.018 0.030

-

?lz

rn

rn

0.015 0.012 -

n z

Aspergillus niger Kluyveromyces fragilis Catharanthus roseus Nicotiana tabacum Trichoderma viride Rhodopseudomonas spheroides Bacillus coagulans Bacillus licheniformis Klebsiella aerogenes

! -

N P

0.058 0.021 0.065

-

z P

0.082 0.0 12 0.01 0.015 0.019 0.017 0.030 0.028 0.009 0.008 0.011 0.011 0.15 0.03 0.036 0.058 0.015 0.077 0.022
0.031 0.018
-

C 0 C

Candida lipolitica Bacillus sp. Nocardia sp. 239 Methylomonas methanolovorans Aeromonas punctata Bacillus megaterium D440 Bacillus stearothermophilus

v)

-4

VI

Hansenula polymorpha

A (D (D

Candida utilis

Pseudomonas oxalaticus

37 51 30 30 30 25 25 30 30 25 25 30 30 50 37 35 37 30 52 37 30 30 30 45 55 63 37 37 30 30 28 28 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4

glucose glucose glucose glucose glucose glucose glucose glucose glucose glucose glucose glucose glucose glucose glucose glucose glycerol n-alkanes methanol methanol methanol glycerol glycerol glucose glucose glucose methanol methanoVglucose glucose ethanol formate oxalate

118.6 118.6 118.6 118.6 118.6 118.6 118.6 118.6 118.6 118.6 118.6 118.6 118.6 118.6 118.6 118.6 116.6 111 115 115 115 116.6 116.6 118.6 118.6 118.6 115 118.6 118.6 109.3 126.6 131

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4.66 6 6 6 6 4.66 4.66 4 4 4 6 4 4 6 2 1

0.020 0.026 0.018 0.017 0.081 0.057

0.029 0.018 0.014 0.021 0.122 0.163 0.052 0.058 0.018 0.009 -

20.9 37.0 6.5 4.8 7.1 9.5 8.3 14.2 13.3 4.3 3.8 6.2 5.5 66.4 14 17.1 29.3 9.65 41.5 15 13.3 12.6 8.2 20.7 57.8 77.4 18.9 22.7 8.5 6.7 20.5 7.5

55 21 71 51 47 38 49 39 5, 6 62 62 9 44 14 10, 23 42 32 40 1 26 2 34 16 45 45 45 24 24 32 32 15 15

Table 1. (continued)
T ("C)

Microorganism
YD
-

Substrate
YA

AC;: (kJ/e-mol)
mD [(C)-mol/C-mol . h ] mA (mol 02/C-mol . h)

mE (kJ/C-mol . h)

Ref.

Bacillus caldotenax

-I L
0.059 0.076

c
0.044 0.018 0.014 0.019

Micrococcus denitrificans

v)

4 4 4 4 4 3.50 3.0

rn

--I

Paracoccus denitr$cans

!..

0.15 0.23 0.33 0.52 0.77 0.039 0.052 0.044 0.070 0.049

D
-

W W v)

Torula utilis
-4

rn z rn

-4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 0.014 0.014 0.012

-

G)

<

Escherichia coli

rn

0.039 0.029 0.047 0.026 0.016

2 z
~ 2 0 3 ~ ~ 2 0 3 ~ ~ 2 0 3 ~ ~ 2 0 3 ~ +

0.015 0.031

Klebsiella pneumoniae

e z

glucose glucose glucose glucose glucose succinate malate mannitol/methanol methanol formate mannitol succinate xylose ethanol glucose glucose glycerol malate lactose glucose glycerol glycerol glycerol glycerol glycerol glycerol 6 2 4.33 3.5 4 6 4 4 4.66 3 4 4 4.66 4.66 4.66 4.66 4.66 4.66 8 8 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 0.014 0.022 0.029 0.058 0.012 0.024

2 z
8 8 6
-

Thiobacillus denitrificans Thiobacillus ferrooxydans

50 55 60 65 70 30 30 35 35 35 35 35 30 30 30 40 40 40 40 35 30 40 25 30 35 43 25 30

118.6 118.6 118.6 118.6 118.6 107.3 112.3 117.7 115 126.6 117.7 107.3 118.6 109.3 118.6 118.6 116.6 112.3 118.6 118.6 116.6 116.6 116.6 116.6 116.6 116.6 99.5 99.5

71 109 156 247 365 19.4 24.5 20.5 31 15 7.1 7.4 6.7 6.1 5.7 18.5 15.8 15.8 12.3 7.6 7 15 7.6 11.9 15.8 31.5 9.6 19.2

35 35 35 35 35 65 65 66 63 63 64, 67 64, 67 59 59 59 72 72 72 72 64 20 20 18 18 18 18 8 8

Thermotrix thiopara

$?
N H ~ organics organics organics

30

rn

0.11 0.19 0.008

Nitrosomonas marina Activated sludge

4
-

P rn

0.008 0.064 0.036

5 z

Pseudomonas fluorescens

70 75 20 20 40 29

99.5 99.5 45.8 111 111 111

-4 -4 -4 -4 -4 -4

87.4 151 2.2 3.6 28.4 16.0

8 8 25 56 28 7

v)

Table 11. Maintenance Gibbs energy mE for anaerobic growth.

Microorganism
Aerobacter aerogenes Clostridium butyricum Acetogenium kivui Zymomonas mobilis Saccharomyces cerevisiae Bacillus polymyxa Bacteroides amylophilus Microbacterium thermosphactum

Temperature ("C) 31 35 66 35 30 30 30 39 5
9

Substrate glucose glucose glucose glucose glucose glucose glucose glucose glucose glucose glucose aspartate pyruvate formate
H2 H2

A G:: (kJ/e-mol)
9

YD

mD [(C)-mol/C-mol . h]

mE (kJ/C-mol

. h)

Reference 55 12 70 22, 61 50 51 3 33 52 52 52 36 41 11 19 19 54 54 54 53 51,58

Campylobacter sp. Desulfovibrio vulgaris Methanobacterium formicicum Methanobacterium thermoautotrophicum Methanococcus thermolithotrophicus Klebsiella aerogenes

25 30 35 38 65 65 36 36 36 36 35

mannitol fructose gluconate glycerol glucose

13.0 12.9 9.3 9.3 9.3 9.1 12.8 8.1 8.1 8.8 6 4.1 24.8 11.4 11.4 7.9 9.2 9.3 6.2 8.9

4 4 4 4 4 4 4 4 4 4 4 3 3.33 2 2 2 4.33 4 3.61 4.66 4

0.34 0.30 0.40 0.43 0.12 0.01 0.16 0.29 0.016 0.01 0.06 0.15 0.54 0.18 3.41 3.82 0.31 0.20 0.54 0.31 0.34

12.2 15.6 20.6 9.8 4.5 2.8 5.8 14.1 0.5 2.3 2.1 2.1 8.5 8.9 115 129 7.9 9.2 9.3 6.2 12.1

been studied aerobically or anaerobically (compare SacchaThe combined correlation for mE (for aerobic/anaerobic romyces cerevisiae, Aerobacter aerogenes, and Klebsiella growth) runs as aerogenes in Tables I and 11. The effect of different C-sources on mE appears generally to be absent. This can be seen directly from E . coli, Hansenula polymorpha, Candida utilis, Torula This correlation [Eq. (16)] can now be combined with utilis (Table I), and Kl. aerogenes (Table 11). On the Eqs. (1) and (11) to give the correlation [Eq. (17)] which other hand sometimes differences are found like Kl. aeroallows for heterotrophic growth the estimation of the total gens, Pseudomonas oxalaticus, Paracoccus denitrificans dissipationlc-mole biomass as a function of (1) growth rate (Table I). However, autotrophic substrates like Hz, S Z O ~ ~ - , and NH4' (Tables I and 11) all fall in the same region with the mE values as the heterotrophic substrates. Considering t ["CI the inaccuracies in the mE values, it appears that, to a 80 60 40 20 5 first approximation, mE is not influenced by the nature of the electron donor, which is in agreement with the earlier aerobic suggestion5' based on only aerobic growth on organic substrates. With regard to the effect of different classes of organisms (Tables 1 and I1 list yeasts, fungi, bacteria, plant cells, mixed cultures), there does not seem to be a very pronounced difference in mE values. In summary, it appears that mE values are very dependent on temperature and not very dependent on electron accuracy 41% ~m~\\ donor, electron acceptor, and type of organism. This is in agreement with the above-mentioned expectation.

APPLICATION OF THE FOUND mE CORRELATION

In this article it has been found that the maintenance effect in microbial growth can, as a first approximation, be described by a maintenance requirement of Gibbs energy (m~), which is only temperature dependent.

rn,-5.7exp(69400/R'(i

/298-1/T))

0.1

2.0

3.0

3.2

3.4

3.6

1000/T [K-'1
Figure 1. Temperature dependence of aerobic mE values.

514

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 4, AUGUST 5, 1993

t ["CI
80
1000 '

60

40

20

0.00
m,-3.5exp(69400/R'( 1 /298-1/T))

0.10

0.20

0.30

0.40

0.1

2.0

3.0

3.2

3.4

3.6
Figure 3. Maintenance related Gibbs energy dissipation per C-mole produced biomass as function of growth rate and temperature.

1000/T [K-'1
Figure 2.
Temperature dependence of anaerobic mE values.

p , (2) temperature T , and (3) carbon source (ys, C ) :

Di'/rAx

200

+ 18(6 - C)'.'
- ys)
2 0.16

donor, Y o x , one obtains [using Eqs. ( 2 ) and (lo)] for the growth rate where Yox is halved, which is also called the endogenous decay constant p e ,

+ exp[{(3.8 +
- exp

(3.6

+ 0.4C)]
298 1

-6.94 x lo4

(;

)]

(18)
(17)

The first part of Eq. (17 represents the Gibbs energy 21 which ~ , dissipation for growth, (Ds/ r ~ ~ ) ~ ranges between 200 and 3500 kJ/C-mol produced biomass. It should be realized that one normally considers that YE?, and hence ( D i ' / r ~ ~ )nearly, independent of temperat~re.~' is ~ ~ The second part of Eq. (17) is the maintenancerelated Gibbs energy dissipation per C-mole of newly produced biomass. It is obvious that this part increases with higher temperature and lower growth rates. Figure 3 shows that at low temperatures (< 15OC) the maintenance contribution becomes of the same magnitude as the growth contribution (>200 kJ/C-mol biomass) only at p < 0.01 h-I. At intermediate temperatures (30C), maintenance becomes important already at p < 0.05 h-' , while at thermophilic conditions (>SOC) maintenance appears always significant over the whole range of growth rates. The total dissipation value from Eq. (17) leads then [using Eqs. (12) and (13)] to estimated values of Yox and YAx as a function of temperature ( T ) , growth rate ( p ) , carbon source/electron donor ( C , ys), electron acceptor (AG,9f), and biomass composition/N source (-yx). Appendix 2 shows a simple example. From Eqs. (4) or (5) one can calculate the growth rate p at which the actual yields YDX or YAX are SO% of the maximal yields. For the biomass yield on electron

For the biomass yield on electron acceptor one obtains [using Eqs. (3) and ( l l ) ] the growth rate where YAX is halved,

Clearly the growth rates where YDX and YAX are half their maximal values are not identical. Further it appears that the endogenous decay rate ( p , ) depends on temperature (through mE), but also depends on the C-source used [through eq. (1) for and the electron acceptor (through AG;:). From Eqs. (18) and (14) one obtains then for ( p . 0 . 5for )~ aerobic heterotrophic growth systems with NH3 as N source (average AG;; 1 1 1 kJ/e-mol and yx = 4.2).30

Figure 4 shows how pe varies for different C-sources [expressed by their ( D : l / r ~ values] and temperatures. ~)~~ Clearly one observes that pe strongly decreases for growth systems with large dissipation values. Autotrophic growth systems are such systems, which are indeed characterized by low endogenous decay rates compared to heterotrophic

TlJHUlS ET AL.: GIBBS ENERGY CORRELATION FOR MAINTENANCE REQUIREMENTS

515

CL
i

0'01 0.01

1
0 800

30C; mE=9.0 30C; mE=9.0

0.001 1600 2400

3200

Figure 4. Endogenous decay pe for aerobic growth as a function of temperature and growth related dissipation ( D g ' / r ~. ~ ) ~ ~

g r o ~ t h . ~ ' wastewater treatment systems a factor of 2-3 For is suggested at 10-20C. For autotrophic and heterotrophic growth typical dissipation values for growth are 3000 and 500 kJ/C-mol biomass. Figure 4 shows then that p e values for heterotrophic and autotrophic growth differ by a factor 3. Another interesting aspect is to study the behavior of ( p 0 . 5 )for the same carbon source [meaning a constant ~ value for ( @ ' / ~ - A X ) ~ ~but with different electron acceptors ] (meaning a variable AG::). Equation (18) clearly shows that ( ~ 0 . 5 increases for lower AG:: values. A typical ) ~ example is the aerobic and anaerobic (ethanol production) growth on glucose. Using mE = 8 kJ/C-mol biomass * h and ( D g ' / t - ~ = )300 kJ/C-mol biomass one obtains for ~ ~~ aerobic growth (AG:; = 118.6) and for anaerobic growth (AG:; = 9.3) values for ( p 0 . 5 )of 0.012 and 0.033 h-'. ~ Obviously, despite the same maintenance Gibbs energy requirement, cell yields drop at higher growth rates for growth systems with low AG:: values. Hence a faster drop in growth yield with decreasing p does not necessarily mean a higher mE value. The average substrate requirement for maintenance m D follows from Eq. (10) and the correlation (16), leading to Eq. (21):

donor must be used to generate the same maintenance energy requirement. Tables I and I1 show that for aerobic heterotrophic growth (AG:; = 111 H/e-mol) at about 30C (for substrates with = 4 ) a typical value of mD = 0.03 C-mol/C-mol * h is found, while anaerobically (AG:: = 10 kJ/e-mol), mD = 0.3 C-mol/C-mol * h. Figure 5 shows how yDmD varies with temperature and AG:; values [Eq. (21)]. Clearly at higher temperatures very high maintenance rates are observed which, at all growth rates, will constitute a major part of the substrate metabolism. For aerobic growth systems AG:; is nearly constant around 111 kJ/e-mol. Equation (21) shows that yDmD is only a function of temperature and not of the nature of the substrate. Here yDmD is equivalent to the electron consumption rate for maintenance, which has been hypothesized before" to be constant for aerobic growth at comparable temperatures for different substrates, which is confirmed here. This is, however, not so for anaerobic growth where AG:; can vary widely, and hence yDmD varies. Because from Eq. (9) one can calculate that for aerobic growth ( Y A = -4, mA = mo2)mo2 = 1/4yDmD, it is obvious that also mo2 appears to be only temperature dependent. From the above-mentioned discussion it is obvious that the effect of growth rate on biomass yield can be described using a variety of coefficients like mD, p e , mA, yDmD, and m o 2 . All of these coefficients are known to be related mathernati~ally,~~ therefore there seems to be and no preference for a specific coefficient. However, it has been shown here [Eqs. (18), (19) and (21)] that all these parameters depend, besides on temperature, on the substrate [through Y D and ( D ~ ' / ~ A and ~ , the electron acceptor X ) on used (through AG:;). The parameter mE however, mainly depends on temperature. Therefore, it seems advantageous to use the mE concept to describe the effect of growth rate on biomass yield.

ti

.. -.

This equation shows that, at a constant temperature, is much less for a more reduced substrate (where Y D is higher; notably varies from 1 to 8 for organic or inorganic substrates). This is logical because more Gibbs energy per C-mole of substrate is available upon combustion for the more reduced substrates. Also it follows that mD increases for growth systems with decreased AG:; values. This is also logical because the Gibbs energy of the donor/acceptor redox reaction per (C)-mole of donor decreases, and hence more electron
mD

20

40

60

80

100

120

AGavo' [kJ/e-moll
Figure 5. Maintenance requirement of available electrons ( y ~ m as ) ~ a function of temperature and energy content per electron (expressed as

A@,!).

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Table 111. Uncoupler stimulated additional maintenance energy dissipation rate.

Microorganism
Klebsiellu uerogenes,a 3 5 T , p = 0.15 h-' Succharuromyces cerevisiue,

Acid

Concentration of Acid (mM)

0 0.25
1

402

(mmol/g
4.1 6.9 10.8 2.5 5.9 15.3 19.5

. h)

A m
(mmol/g

. h)

Additional Maintenance Energy (kJ/C-mol . h)

Minimal Maintenance Energy (kJ/C-mol . h)

17 17 17 6
6

DNP

2.8 6.7
-

34.6 82.8

benzoate

42 158 210

30C. p

0.1 h-I

2
7.5 10

3.4 12.8 17

6 6

Reference 43. Reference 68, 69.

COMPARISON OF OPTIMAL AND SUBOPTIMAL MAINTENANCE REQUIREMENTS

In the introduction it was mentioned that additional maintenance energy requirements may occur under suboptimal conditions due to increased osmotic stress or the effect of uncouplers which dissipate the transmembrane proton-motive force. These contributions can be very large, as compared to the minimal maintenance rates. This follows from the additional 0 2 consumption which was found during aerobic growth of K. uerogenes [using dinitrophenol (DNP) as uncoupler] or S. cerevisiue (using benzoate) (Table 111). This oxygen consumption is considered as maintenance energy, because its value is independent of growth rate, but only depends on uncoupler concentration. In reading Table 111, it should be remembered that the minimal maintenance rates are low for comparable temperatures (range 5- 15 kJ/C-mol h). Hence maintenance requirements can be increased under suboptimal conditions very significantly due to the presence of relatively low uncoupler concentrations.

NOMENCLATURE
biomass yield on electron donor, C-mol/(C)-mol biomass yield on electron acceptor, C-mol/mol degree of reduction of electron donor, dimensionless degree of reduction of C-source, dimensionless degree of reduction of electron acceptor, dimensionless degree of reduction of biomass, dimensionless available Gibbs energy in the electron donor/acceptor couple, kJ/e-mol dissipation of Gibbs energy per C-mole produced biomass, kJ/C-mol biomass specific maintenance rate of electron donor, (C)mol/C-mol . h biomass specific maintenance rate of electron acceptor, mol/C-mol . h biomass specific maintenance rate of Gibbs energy dissipation, kJ/C-mol . h temperature, K growth rate, h-' endogenous decay rate, h-' gas constant (8.314), J/mol K growth rate where biomass yield on the donor is half the maximal yield, h-' growth rate where biomass yield on acceptor is half the maximal yield, h-l

CONCLUSION
Maintenance requirements have been found to be correlated well on the basis of Gibbs energy maintenance requirement mE (kJ/C-mol biomass * h) for a large number of different microbial growth systems under aerobic and anaerobic conditions on organic and inorganic C-sources. The parameter mE appears (as a first approximation) to be mainly dependent on temperature with an activation energy of 69 kJ/mol. The influence of a different electron donor (heterotrophic or autotrophic) is absent. The value of mE appears to be somewhat lower for anaerobic growth, compared to aerobic growth. It was also found that mE appears to be a much more characteristic parameter of maintenance requirement than, e.g., the endogenous decay rate p e or the substrate maintenance rate mD or the available electron maintenance rate yDmD. Using the present correlation for mE and the previous correlation for (D:l/rAr)gr,one can estimate expected biomass yields for arbitrary growth systems as a function of temperature, growth rate, electron donor/acceptor, and N source (Appendix 2).

Superscript
max maximal yield values

Subscripts
gr growth associated

APPENDIX 1: CALCULATION OF AG,qf AND 70

Consider the microbial system which uses glucose as electron donor and 0 2 as electron acceptor. The redox reaction can then be written as -C6H1206 602

+ 6HC03- + 6H+ = 0

For AGR one can calculate a value of -2846 kJ, which is -474.4 kJ/C-mol glucose. This means that per C-mole of electron donor the redox reaction liberates 474.4 kJ. Glucose liberates four electrons per C-mole. Hence by definition Y D = 4. Then as AGti follows +474.4/4 = 118.6 kJ/e-mol.

TlJHUlS ET AL.: GlBBS ENERGY CORRELATION FOR MAINTENANCE REQUIREMENTS

517

APPENDIX 2: CALCULATION OF GROWTH YIELD O N SUBSTRATE FOR ARBITRARY GROWTH SYSTEM AT ARBITRARY TEMPERATURE AND GROWTH RATE
Consider the anaerobic growth of a microorganism on glycerol, which produces acetate only at 45C. According to Table I11 in ref. 30 AG,9f = 37.8 - 26.86 = 10.94 kJ/emol. If we assume NH3 as the N-source, then the degree of reduction of biomass yx = 4.2. The degree of reduction of glycerol ys = 4.66. Glycerol contains three carbon atoms; hence C = 3. The temperature is 45C; hence T = 318 K. Equation (17) (after substitution of C = 3, y s = 4.66, and T = 318) leads to the estimated Gibbs energy dissipation per C-mole of biomass as a function of growth rate: 26 @'/rAX = 428 + kJ/C-mol biomass
El.

At p = 1.0 h-' one can calculate Dg'/rAx = 454 kJ/Cmol, while at p = 0.05 h-' one can calculate D;'/rA, = 948 kJ/C-mol. The biomass yield YDX follows from Eq. (12) (AG;: = 1 0 . 9 4 , ~ ~4.66, y x = 4.2): = For p For , u
=

1.0 h-'

one obtains YDX = 0.10 C-moll C-mol one obtains YDX = 0.05 C-mol/ C-mol

0.05 h-'

Part of this research was supported by the Technology Foundation STW, project DCH 88-1548, the NOVEM, project number 51120/021and RIZA and STOWA, within RW212000, project number 324413.

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