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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.
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
CCC 0006-3592/92/080833-026$04.00
834
Table IIA.
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
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.
Yes
Yes
No
No
Yes
Yes
Yes
Yes
No
No
No
Yes
No
Yes
No
Yes
No
Yes
Yes
No
Yes
Yes
Yes
No
Yes
No
Yes
No
qBB
835
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
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
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
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'
.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
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;
5 815 C,O:-
- 0 2 NH,' + 1 CH,,O,,N,,
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
(-)
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
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
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)
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
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
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
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
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
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.
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)
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
YD
am
OD
I'
Error
1,
30%
'
400
800
1200
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.
847
Table VI.
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
(%)
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
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
heat
k J/C-rnol
biomass
Chemical
entropy
k J/C-mol
biomass
+687
+ 18(6 - c)'"
(1)
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).
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
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
- 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
- 5.815(-676)
- 5269
= -= 0.834
-6317
NOMENCLATURE
-5.815CzO42-
Thermodynamic Reference q F0
Combustion Reference
q p=
1 * 474.6 + 10.63 * 0
+ 0.2(0) + 0.8(0) + 1.857 * 0 + 5.42 * 0
474.6
= 0.311
1523.5
-0.896C2H60
= -702kJ.
qp
= 0.203
-880.8
1 I
qp=
,
.
I
-2.283C6HsO;-
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
obtains:
ll;
=
-
{l * (474.6) + 4.096
+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).
+ 1CHl.aOo.sNo.z
+ 1.876CzH302- + 0.0661C4H4042- + 0.504C~H60
+ 2.256HCO3- + 2.O91H2 + 3.083H'
=0
{l
Output Processes
+ CHix0o.sNo.z
1. -1C20:-
+ 0.802 = 0
AGF = +343 kJ
* (-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
---=+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.
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:
+ I.OCH1xOusNoz + 10.63HCO3- = 0
AGk = -1048 kJ
854
AG;' = +474.6 kJ
+ 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
':77
= -80.6/(972) = 0.083;
= 474.6/(1522) = 0.312
AG;' = -80.6 kJ
+ CH~sOosNoz+ 1.0502
+ 0.4H20 = 0
'
:
7
=0
+ CH~xOosNuz
4.6H20
0.2NH4'
+ CHix00sNoz
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-
+ CHl.8Oo.sNIj.z+ 0.666H20 = 0
AGF = -2.5 kJ
+ CH1,gOasNo.z+ 0.4HCO3-
AGE' = +6.7 kJ
+ CHl.8Oo.sNo.z
+ 2.5H20 = 0 AGE = -25.2
kJ
(+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
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
+ 1.98HCO3- 4-
1.98H' = 0
AG"' = -942 kJ
855
= 0.
-1.1904CsH1206
kJ
6.092402
+ 7.1424HCO3- + 7.14248'
=0
+ 4.0616HCO3- + 4.0616H'
+ 2.0308HzO = 0
* 0.086
Cz02-
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
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
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858