Professional Documents
Culture Documents
Microbial Metabolism
J. A. ROELS, Laboratory for General and Technical Biology,
Department of Chemical Engineering, Delft University of
Technology, Julianalaan 67, 2628 BC Delft, The Netherlands
Summary
General expressions for mass, elemental, energy, and entropy balances are derived
and applied to microbial growth and product formation. The state of the art of the
application of elemental balances to aerobic and heterotrophic growth is reviewed
and extended somewhat to include the majority of the cases commonly encountered
in biotechnology. The degree of reduction concept is extended to include nitrogen
sources other than ammonia. The relationship between a number of accepted measures for the comparison of substrate yields is investigated. The theory is illustrated
using a generalized correlation for oxygen yield data. The stoichiometry of anaerobic
product formation is briefly treated, a limit to the maximum carbon conservation in
product is derived, using the concept of elemental balance. In the treatment of growth
energetics the correct statement of the second law of thermodynamics for growing
organisms is emphasized. For aerobic heterotrophic growth the concept of thermodynamic efficiency is used to formulate a limit the substrate yield can never surpass.
It is combined with a limit due to the fact that the maximum carbon conservation in
biomass can obviously never surpass unity. It is shown that growth on substrates of
a low degree of reduction is energy limited, for substrates of a high degree of reduction
carbon limitation takes over. Based on a literature review concerning yield data some
semiempirical notions useful for a preliminary evaluation of aerobic heterotrophic
growth are developed. The thermodynamic efficiency definition is completed by two
other efficiency measures, which allow derivation of simple equations for oxygen
consumption and heat production. The range of validity of the constancy of the rate
of heat production to the rate of oxygen consumption is analyzed using these efficiency measures. The energetics of anaerobic growth are treated-it is shown that
an approximate analysis in terms of an enthalpy balance is not valid for this case,
the evaluation of the efficiency of growth has to be based on Gibbs free energy
changes. A preliminary analysis shows the existence of regularities concerning the free
energy conservation on anaerobic growth. The treatment is extended to include the
effect of growth rate by the introduction of a linear relationship for substrate consumption. Aerobic and anaerobic growth are discussed using this relationship. A
correlation useful in judging the potentialities for improvement in anaerobic product
2458
ROELS
1. INTRODUCTION
2459
(1)
C.dV
rA.dV +
ja.dS
2460
ROELS
C=r,+@
rA = r a
(4)
If m independent reactions occur, r is an m-dimensional row vector,
in which the rates of chemical processes are specified. Each row
of the matrix OL expresses the stoichiometry of a reaction in terms
of the number of moles of each compound converted per unit reaction rate. It is an m x n matrix of the following structure:
rn
components
r*oL
+ Q,
(5)
-ra
(6)
2461
A practical implication of eq. (6) is that, for a steady state, the stoichiometry of a reaction pattern can, as far as the net rate of production of each component of the system is concerned, be studied
by observations of the exchange flows, @, with the environment.
This is a very useful observation, as these flows can in most cases
be readily studied experimentally.
ROELS
2462
rn
P
E =
aij
n
e
n
t
7 - J .
k chemical elements
vs
aij stands for the number of atoms of atomic speciesj present in one
mole of component i.
The change of the amounts (or for a system of constant volume
the concentrations) of each of the elements present in the culture
is obtained, if the right- and left-hand sides of eq. ( 5 ) are multiplied
by matrix E:
(CaE)
r.a.E + cP.E
(7)
The first term at the right-hand side of eq. (7) represents the net
production of each of the chemical elements in the transformation
processes in the system. By virtue of the definition of a conserved
quantity this term should always be zero:
=
ra-E = 0
(8)
Or, as eq. (8) must hold for any vector of reaction rates, r:
cw*E = 0
(9)
2463
ra.E
(10)
2464
ROELS
+ @.hT
(12)
Equation (12) states that the system's internal energy per unit volume, u , changes, due to the flow of heat to the system, j , , and the
flow of enthalpy, @-hT,associated with the various compounds exchanged with the environment. hTis the transpose of the row vector
in which the molar enthalpies of the compounds are specified. For
a system in steady state the classic equation for the calculation of
heat exchange from an enthalpy balance results:
j , = - @*h'
(13)
2465
The second law of thermodynamics allows the formulation of a restriction to the heat production in a process, and it can be derived
by the introduction of the Gibbs equation for a system of constant
volume:8
Ti =
- c a p '
(14)
c.p' = 0
(15)
(16)
The left-hand side of eq. (16) contains two contributions, the entropy
production, II,, being equal to - rwp'/Tand the entropy exchange
with the environment, cP.pT/T.The second law of thermodynamics
stipulates H, to be larger or equal to zero, hence it follows:
@*pT
20
(17)
Equation (17) is equivalent with the common notion, that for a chemical reaction to proceed at constant temperature and pressure a negative free-enthalpy change (Gibbs free-energy change) should be
associated with that reaction.
Minkevich and Eroshin's efficiency measure, q, is based on the
assumption that j , can never exceed zero. Hence, endothermic reactions, which are known to exist, are excluded. It is, formally
speaking, based on an incorrect statement of the second law, as is
clear from comparison of eqs. (17) and (13).
3. CONCEIT OF ELEMENTAL BALANCE AND ITS
APPLICATION TO GROWTH AND PRODUCT FORMATION IN
MICROORGANISMS
3.1. General Expressions for Growth with Product Formation
ROELS
2466
1
a2
a3
E = a4
0
1
-0
bi
bz
b3
64
0
0
2
CI
di
c2
dz
c3
d3
c4
d4
2
2
I
0
0
0 .
In the present case there are seven flows and eq. ( 1 1) specifies four
equations between the flows, hence only three flows are independent
Fig. 1 .
2467
variables. Which flows are to be chosen independently strongly depends on the application one has in mind. First the case will be
the flow of substrate,
considered in which the flow of biomass,
aZ,and the flow of product, a3,are known. The standard routines
of linear algebra now allow eq. (1 1) to be solved for the remaining
flows, the results are given in Table I.
In the equations several conventions are adopted. The macroscopic yield factors Y h , the number of C mol biomass producedl
mol substrate taken up, and YLx, the number of mol biomass produced/mol product produced, are introduced. Yix and YLx are defined by:
YLx =
I@I/@3/
(20)
+ bl - 2 ~ 1- d1[(4U4+ 64 - 2~4)/d41
(21)
ys = (4Uz + bz - ~ C Z dz[(4U4 + b4 - 2~4)/d4]}/~2 (22)
yp = ( 4 ~ 3+ b3 - 2C3 - d3[(4~4+ b4 - 2~4)/d4]}/~3 (23)
yx =
nitrogen source
oxygen
carbon dioxide
water
@4
@5
Q6
@7
product
@,
-
independent
substrate
@*
dq
y
JX
1( d , - f
d +
independent
independent
Ypx
*)
biomass
Component
@I
Flow
TABLE I
Calculated Flows for the General Case
2469
to estimate the flows of biomass and product. By some rearrangements of the equations in Table I the following equations are obtained, if the nitrogen source is NH3 and product and substrate do
not contain nitrogen:
@I
@2[Ro(4 - yp.RQ) + a d y ,
Q3
Q2[-
R0(4
- ys)l/(yx - yP)
(24)
(25)
2470
ROELS
2471
N, are seen to vary very little. The relative standard error is less
than 5% in all cases. A good approximation to the average composition of biomass is the elemental composition formula CH, .*Oo.sNo.,.In calculations account has to be taken of the biomass fraction consisting of elements other than C, H, 0, and N. This fraction
will be termed ash and assumed to be 5% of the biomass DM. If
it is assumed that yx indeed is more or less constant, the equations
presented in Table I1 allow the formulation of a general equation
for the relationship between YLx and the yield on substrate Y%,
this relationship can be formulated as
YLx = (4/yx)*[q0/(l- qo)l
(26)
yxYYys
(27)
(28)
(3%)
s stands for the standard error of the estimate of yx (in parentheses the standard deviation expressed as percent of the average).
4.79
0.10
(2.1%)
s
5.78
s = 0.26
(4.5%)
4.19
Average
sa = 0.13
5.79
5.54
5.99
6.11
5.98
5.64
30
30
30
30
30
30
30
30
4.75
4.75
4.91
4.75
4.89
4.87
4.81
4.87
4.60
4.71
4.79
4.79
4.59
4.79
4.96
4.73
5.25
5.75
5.86
5.75
5.99
6.07
5.66
6.07
5.40
5.56
4.45
4.15
4.34
4.15
4.23
4.15
4.30
4.15
4.12
4.20
4.28
4.19
3.96
4.07
4.27
3.98
Candida utilis
C . utilis
C . utilis
C . utilis
Klebsiella aerogenes
KI. aerogenes
Kl. aerogenes
KI. aerogenes
Saccharomyces cerevisiae
S . cerevisiae
S. cerevisiae
Paracoccus denitrificans
P . denitrificans
Escherichia coli
Pseudomonas C12B
Aerobacter aerogenes
52
36
36
51
48
49
50
15
Ref.
NZ
Organism
HNO3
NH3
Elemental
formula
TABLE 111
Elemental Composition and Degree of Reduction for Biomass of Various Sources
h,
2473
efficiency
factor
qo
Fig. 2. Relation between biomass yield on oxygen and the efficiency factor, qo.
Theory and experimental results for growth with: glucose, NH, (0);acetate, NH3
(8);glycerol, NH3 (0);citrate, NH3 (H); ethanol, NH, ( x ) ; methanol, NH, (0);dodecane, NH, (e);
methane, NH, (@); gluconate, HN03 (A);pentane, HNO, (A);
methane, HNO, tr).
glucose
S . cerevisiae
E. coli
Penicillium chrysogenum
Pe. chrysogenum
Azotobacter vinelandic"
C . utilis
Pseudomonas jluorescens
Rhodopseudomonas spheroides S
Aspergillus awamori
Aspergillus nidulans
P . denitr$cans"'
C . utilis
Ps. jluorescens
A . aerogenes
A . aerogenes
Candida boidinii
C . utilis
Ps. jluorescens
C . boidinii
Klebsiella sp.
Methylomonas methanolica
Candida N-17
Hansenula polymorpha
"Bacteria" (I)
Candida lipolytica
Methane bacteria"'
Methylcoccus capsulatus
Methane bacteria
Nitrogen source is NH,, except for cases indicated by (I), HNO, and (2), N,.
pentane
dodecane
methane
methanol
glycerol
citrate
ethanol
gluconate
acetate
Substrate
Organism
0.59
0.62
0.52
0.56
0.30
0.59
0.44
0.52
0.62
0.70
0.45
0.42
0.32
0.66
0.34
0.61
0.61
0.43
0.52
0.47
0.60
0.46
0.45
0.47
0.41
0.38
0.63
0.56
yix
TABLE IV
Experimental Values of Yix, Yb, andY.,/.
0.50
I .64
1.16
1.81
1.55
2.64
1.76
0.87
0.57
1.17
1.25
0.82
0.82
0.51
0.47
0.70
0.66
0.49
0.46
0.54
0.54
0.27
0.36
0.32
1.81
1.64
1.17
1.94
1.72
1.85
3.63
3.81
3.20
3.44
1.85
3.63
2.71
3.20
3.81
4.31
3.02
2.58
1.97
3.48
2.78
2.50
2.50
1.76
2.13
1.93
2.46
1.89
I .34
1.61
1.93
1.55
YW/,
(g DMlavle)
Yb*
(mol DM/mol 02)
60
34
61
58
58
61
62
63
64
65
21
66
67
68
69
58
59
56
56
50
58
58
58
53
54
55
56
57
b~
;a
2475
(31)
2476
ROELS
From the foregoing it will have become clear that elemental balances are an invaluable tool in the description of the systems commonly encountered in bioengineering. It is as fundamental as stoichiometry in chemical reaction engineering systems. The theory
seems to be well developed and the field is open to the development
of specific applications. The strategy for the application of the tool
can be summarized as follows:
1) Verify that the system is defined such that a (pseudo) steadystate assumption is justified.
2) Construct a list of all n components and their elemental composition, which the system exchanges with the environment in nonnegligible amounts.
3) Determine the number of elements ( k ) for which elemental balances can be constructed.
4) Choose the most convenient set of n - k flows to be obtained
experimentally.
5 ) Apply the balance principle given by eq. ( 1 1) and obtain the
unknown flows by the solution of the matrix equation.
There is a significant further problem associated with the application of elemental balances. It applies to instances in which more
flows are measured than are minimally needed to calculate the remaining ones. In this case a statistical procedure can be applied to
a more optimal estimate of all measured and unknown flows. The
procedure can also be used to detect systematic errors in the measurements of one or more of the flows and may also be used for
identification of the existence of unremarked exchange flows with
the environment. It is beyond the scope of this report to treat this
2477
In this section some energetic aspects of the growth of microorganisms will be dealt with. Two simple cases will be treated, aerobic
heterotrophic growth on a single source of carbon and energy with
simple nitrogen sources and the anaerobic formation of products.
In both cases only two variables, for example, the yield factor for
biomass on substrate, Y k , and the biomass flow, a,, remain unknown after the application of the elemental balance principle.
The application of the first law of thermodynamics, the energy
balance introduces one new unknown flow, the heat flow j , , but
also introduces one new equation, the enthalpy balance according
to eq. (13). Hence, the introduction of the first law of thermodynamics does not diminish or increase the number of unknown flows.
For a system in steady state the enthalpy balance will not provide
any further information if two flows are already known. Equation
(13) allows one to calculate the heat flow if Yyx and a, are known.
On the other hand the heat flow in combination with any other of
the flows allows calculation of all remaining flows. The second law
of thermodynamics, e.g., eq. (17), specifies an inequality, which
must always be satisfied. This allows the calculation of a maximum
value Yix can never exceed. It thus also allows definition of the
thermodynamic efficiency of the growth process. It is in principle
possible to define other, less fundamental efficiency measures, for
example the one proposed by Minkevich and Eroshin,I6 where the
maximum yield is defined with respect to zero heat flow (or zero
oxygen flow) to the environment. These efficiency measures are
very useful for some types of calculations. In order to obtain values
for the maximum yield consistent with the second law and with a
positive heat flow to the environment thermodynamic data are
needed. In Table V some data are shown. The enthalpy values refer
to the pure components in the standard state and are expressed per
C mol of the various substances. The free enthalpy (also often
termed Gibbs free energy) values refer to 1M aqueous solutions of
the respective compounds. The procedure used for the calculation
of the data for biomass is summarized in Appendix I. Especially in
ROELS
2478
TABLE V
Thermodynamic Properties of Some Compounds in the Standard State
ho
(kJ/C mol)
Component
Acetic acid
Ammonia
C2H402
NH3
Ammonium ion
Biomass
Carbon dioxide
Bicarbonate ion
Citric acid
Dodecane
Ethane
Ethanol
Formic acid
Fumaric acid
Glucose
Glycerol
Lactic acid
Malic acid
Methane
Methanol
Nitric acid
Nitrogen
Oxalic acid
Oxygen
Pentane
Propane
Succinic acid
Water
NH,'
CHl.800.sNo.z
c02
HCOjC6H807
CIZH26
C2H6
C2H60
(33202
C4H.404
C6H1206
C3HsO3
C3H603
C&Os
CH4
CH4O
HNO3
N2
CZH204
- 244.8
- 46.3
- 80.9
- 133
- 91.4
- 394
- 692
(aq)"
(a4
(as)
(aq)
(g)
(ad
(as)
(1)
- 29.3
- 42.4
- 139
-410.6
(g)
(as)
(ad
(aq)
(as)
(1)
(as)
(ad
-211
- 222.3
- 75.0
- 239
- 173
0
-414
0
- 34.6
- 34.7
(g)
(1)
(ad
(9)
(aq)
0 2
(g)
C5H12
C3Hs
C4H604
HzO
H'
(as)
(g)
(a4
- 286
(1)
(PH
7)
IJ?
(W/C mol)
- 186.4
- 16.7
- 26.7
- 79.6
- 67.1
- 395
- 588
- 195
2Sb
- 16.4
- 90.9
- 335.2
- 151.3
- 153.1
- 163.0
- 173.0
- 141.0
- 50.9
- 176.5
- 110.7
0
- 338
0
- 3.9
7.8
- 172.8
- 238
- 40.5
In the case of aerobic heterotrophic growth without product formation eq. (17) allows the calculation of a maximum to Y:x, which
can never be surpassed due to the restrictions put by the second
law. The maximum value of YYx consistent with the second law will
be termed wr.
2479
Two other restrictive values will be used in the treatment to follow, we, the maximum value of Yyx consistent with heat transport
to the environment, and wo, the maximum value of Yyx consistent
with oxygen uptake from the environment. It has, however, to be
emphasized that there exist no a priori laws of nature forbidding
reactions with the uptake of heat or oxygen generation. In Table VI
the numerical values of of,we, and wo are given for a variety of
substrates and growth with the nitrogen sources NH,, HN03, and
nitrogen. In the calculations the elementary formula of the biomass
was assumed to be given by CH,.,Oo.sNo.,. All values refer to ashfree dry weight. An example of such a calculation is given in Appendix 11. In Table VII the general expressions for we and wo are
recorded as these will be used later on. The numerical values given
in Table VI allow the following conclusions:
0
The w, values do not deviate more than 10% from the we values
and hence the maximum yield according to the second law
does not deviate too much from that according to zero heat
exchange with the environment. This validates the approach
of Minkevich and Eroshin.'6 In terms of thermodynamics the
observation can be formulated as follows: For aerobic growth
TABLE VI
Calculated Values of of,we, and wo
Nitrogen source
HNO3
NH3
Substrate
Oxalic acid
Formic acid
Malic acid
Citric acid
Succinic acid
Gluconic acid
Lactic acid
Acetic acid
Glucose
G1ycero1
Methanol
Ethanol
Dodecane
Pentane
Propane
Ethane
Methane
Y.
Of
we
1
2
3
3
3.5
3.67
4
4
4
4.67
6
6
6.17
6.4
6.67
7
8
0.28
0.53
0.71
0.70
0.79
0.25
0.56
0.93
0.89
1.00
1.15
1.46
1.38
1.37
1.42
1.47
1.54
1.72
00
0,
0.24 0.24
0.48 0.47
0.72 0.62
0.72 0.61
0.84 0.69
0.88
0.96 0.81
0.90 0.96 0.78
0.97 0.96 0.87
1.14 1.12 1.00
1.50 1.44 1.27
1.42 1.44 1.21
1.40 1.48 1.20
1.45 1.53 1.24
1.53 1.60 1.29
1.62 1.67 1.34
1.84 1.91 1.50
0,
00
0.21
0.42
0.63
0.63
0.73
0.77
0.84
0.84
0.84
0.98
1.26
1.26
1.29
1.34
1.40
1.46
1.67
ROELS
2480
TABLE VII
Expressions for the Maximum Values of YYx Consistent with Various Restrictions
Restriction
Nitrogen
h,'
+ 394 +
302.5
143(bz/az)
1436, - 383dl
h,'
+ 394 + 143(b2/a2)
302.5
+ 143bl
2481
was assumed that the composition of the biomass could in all cases
be adequately represented by the average formula CH,.,Oo.,No.,. An
ash content of 5% was assumed.
In Table IX the data and the calculated efficiencies are summarized. The data are also shown graphically in Figure 3 where the
thermodynamic efficiency is plotted against the degree of reduction
of the substrate for growth with NH, as a nitrogen source. Although
Figure 3 shows the existence of appreciable variation the trend
seems to be adequately represented by the dotted line. The thermodynamic efficiency seems to be low for the highly reduced as
well as highly oxidized substrates, although the latter conclusion
rests on only very little data. The tendency for the thermodynamic
efficiency to be low for highly reduced substrates is, however, clear.
A reason for this drop in thermodynamic efficiency can be easily
obtained, if it is recognized that, due to carbon limitation, YYx can
never exceed 1, hence the thermodynamic efficiency is subject to
TABLE VIII
CoIrelations of of, o,,and oo with ys and 0,with wo
Nitrogen
source
NH3
Correlation
Of
0.045
+0.22~~
0.047
0.07
0, =
0.24~~
0.24~~
0,
00
~f
= 0.08
0,
+ 0.18~~
= 0.06 + O.l9y,
0,
00
W,
0,
0.039
1.18~~
0.041
+ 0.18y,
= 0.06 + O.l9y,
0.041
0, =
00
0.042
0.041
0, =
0.09
0.21y,
0.21y,
wo
Remarks
least-squares
estimate
least-squares
estimate
0.058
0.058
0.2oy,
0.17~~
0, =
N1
+ 0.21y,
we =
WO
HNO3
= 0.09
Residual standard
error of estimate
least-squares
estimate
least-squares
estimate
0.040
0.058
0.058
least-squares
estimate
least-squares
estimate
Azoiobacter vinelandii
C . uiilis
Ps. fluorescens
Pseudomonas sp.
Pseudomonas deniirificans
A . aerogenes
Ps. deniirificans
Ps. jluorescens
Ps. denitrificans
Ps. denitrificans
Pseudomonas sp.
Ps . dentrificans
A . aerogenes
Ps. jluorescens
Pseudomonas sp.
C . uiilis
Ps. jluorescens
Candida tropicalis
S. cerevisiae
S. cerevisiae
E. coli
Penicillium chrysogenum
Pseudomonas oxalaiicus
Organism
4.00
glucose
glucose"'
4.00
acetate
3.67
4.00
3.00
3.50
fumarate
succinate
'
3.00
malate
gluconate"
lactate
3.00
1.00
2.00
7s
citrate
oxalate
formate
Substrate
0.07
0.18
0.18
0.38
0.34
0.37
0.33
0.37
0.39
0.41
0.45
0.32
0.37
0.44
0.42
0.32
0.36
0.59
0.57
0.62
0.52
0.56
0.30
0.59
0.44
yyx
0.26
0.34
0.34
0.54
0.49
0.52
0.46
0.53
0.49
0.52
0.67
0.34
0.40
0.49
0.47
0.36
0.40
0.59
0.57
0.62
0.52
0.56
0.33
0.59
0.44
qlh
1.72
2.21
2.21
3.12
2.78
3.03
2.71
3.03
2.74
2.88
3.01
1.97
2.27
2.71
2.58
1.97
2.21
3.63
3.51
3.81
3.20
3.44
1.85
3.63
2.71
YW/.
TABLE IX
Experimental Values of Y:, and the Thermodynamic Efficiency, q t h , as well as Yav/rand ye
74
74
74
74
34
74
58
74
74
74
28
34
58
74
58
58
75
53
48
54
55
56
57
58
58
0.28
0.32
0.32
0.52
0.47
0.51
0.46
0.46
0.50
0.59
0.33
0.38
0.49
0.46
0.35
0.40
0.61
0.59
0.64
0.54
0.58
0.36
0.61
0.45
0.51
Ref.
tl.
M . methanooxidans
Methane bacteria
M. capsulatus
Methane bacteria
R . spheroides
A . awamori
A . nidulans
Pseudomonas sp.
A . aerogenes
C . tropicalis
C . boidinii
C . utilis
Ps. jluorescens
C . utilis
C . brajsicae
C . boidinii
Klebsiella sp.
M. methanolica
Candida N-17
H . polymorpha
Pseudomonas C
Pseudomonas EN
Torulopsis
Methylomonas sp.
M. methanolica
C . tropicalis
C . lipolytica
"Bacteria"
Job 5
4.29
4.67
6.00
6.00
6.13
6.17
6.40
6.67
7.00
8.00
benzoate
glycerol
ethanol
methanol
hexadecane
dodecane
pentane"'
propane
ethane
methane"'
methane
methane"'
0.52
0.62
0.70
0.48
0.66
0.61
0.61
0.61
0.43
0.55
0.64
0.52
0.47
0.60
0.46
0.45
0.67
0.67
0.70
0.50
0.64
0.56
0.41
0.47
0.71
0.71
0.38
0.63
0.62
0.56
0.50
0.68
0.52
0.62
0.70
0.48
0.57
0.44
0.44
0.44
0.23
0.39
0.46
0.36
0.32
0.41
0.31
0.31
0.46
0.46
0.48
0.34
0.44
0.41
0.30
0.38
0.48
0.46
0.25
0.37
0.41
0.33
0.29
0.40
3.20
3.81
4.31
2.75
3.48
2.50
2.50
2.50
1.76
2.26
2.62
2.13
1.93
2.46
1.89
1.85
2.75
2.75
2.87
2.05
2.62
2.24
1.64
1.81
2.62
2.50
1.17
1.94
1.91
1.72
1.54
2.09
0.54
0.64
0.72
0.47
0.57
0.43
0.43
0.43
0.30
0.39
0.45
0.35
0.31
0.40
0.27
0.30
0.45
0.45
0.47
0.33
0.43
0.40
0.29
0.38
0.46
0.44
0.24
0.34
0.39
0.30
0.27
0.37
59
56
56
74
60
75
61
58
58
76
77
61
62
63
64
65
74
74
78
79
80
75
81
21
74
74
67
68
82
69
83
84
2484
ROELS
,+o. l
SECOND LAW
0.9
0.8
c-
C -LIMITATION
P2
0.7-
0.6-
t
0
K
w
0.5
0.4
0.3
a2-
0.1
1
0
L
8
DEGREE OF REDUCTION
v,
the restrictions given by the right-hand part of the solid line in Figure
3. The left-hand part of the solid line is the restriction put by the
second law, the efficiency can never exceed unity. Figure 4 shows
the results presented in Figure 3 in a somewhat other way: Yyx, the
carbon conservation in biomass, is plotted against substrate degree
of reduction. The solid line again gives the restrictions due to the
second law and total carbon conservation. Both in Figures 3 and 4
the results are seen to be well within the limits posed by both the
second law and that due to carbon limitation.
2485
CARBON LIMITATION
I lAl IUN I
4
DEGREE
0'
1
0
0 0
OF
REDUCTION
(ys)
Fig. 4. Relationship between substrate yield factor Y:, and substrate degree of
reduction and the theoretical limits to YE.
ROELS
2486
Yyx = 0.60
+ 0.12 ys
ys 5 4.58
(334
ys > 4.58
(33b)
(34)
2487
TABLE X
Compilation of Average Measured and Calculated Data for Yix and YaVIefor
Aerobic Growth on Various Carbon Sources
YWk
yi:
av
meas.
Substrate
av
calc.
______
Glucose
Glucose
Other sugars
Glycerol
Acetate
Acetate
Lactate
Citrate
Malate
Fumarate
Succinate
Pyruvate
Pentadecane
Hexadecane
Heptadecane
Octadecane
Higher n-alkanes
Ethanol
Methanol
Propane
Ethane
Methane
0.54
0.57
0.52
0.61
0.41
0.38
0.43
0.40
0.40
0.41
0.42
0.42
0.56
0.50
0.47
0.51
0.53
0.57
0.56
0.71
0.71
0.59
0.53
0.53
0.53
0.60
0.53
0.53
0.53
0.41
0.41
0.41
0.47
0.45
0.60
0.60
0.60
0.60
0.60
0.60
0.60
0.60
0.60
0.60
av
meas.
av
calc.
Number
of data
3.26
3.26
3.26
3.16
3.26
3.26
3.26
3.36
3.36
3.36
3.30
3.32
2.41
2.41
2.41
2.41
2.41
2.46
2.46
2.21
2.11
1.85
26
10
15
7
7
4
3
4
3
3
3
2
6
5
3
5
5
5
10
Source
3.33
3.51
3.20
3.21
2.52
2.34
2.64
3.28
3.28
3.36
2.95
3.10
2.27
2.00
1.90
2.06
2.16
2.34
2.30
2.62
2.50
1.81
1
1
85
Table IX
85
85
85
Table IX
85
85
85
85
85
85
85
85
85
85
85
Table IX
Table IX
Table IX
Table IX
Table IX
If the semiempirical equation [eq. (33)] for YYx is adopted and C mol
biomass are converted to g biomass it follows:
g biomass/g COD = O.16/ys + 0.37
ys 5 4.58
(35a)
ys > 4.58
(35b)
A quantity that is of great practical importance is the heat production on microbial growth as this quantity may determine the pro-
ROELS
2488
+ 119(bJa2)]
(36)
Yb/w,
(37)
it follows:
j~ = @l'Ah*(l- qe)/qe
(38)
where
Ah = A,dl
+ 143bl + 302.5
(39)
Ah
+480
N*:
Ah
+560
The values of the constants for HNO, and N, are much higher indicating that at a given value of q, heat production will be higher
on growth with HN03 and N, as compared to NH,.
As we is for most applications very close to w, the trends observed
on an analysis of experimental material with respect to qthwill also
be valid with respect to qe.The following approximation to the ex-
2489
pected values of qc thus results for growth with ammonia as a nitrogen source:
qe =
qe =
0.55
2.5ly,
if
if
ys 5 4.55
(40a)
(40b)
yo > 4.55
HEAT
(kJ/C-moleD
1.5
DEGREE OF REDUCTION
y,
Fig. 5. Expected values of the heat production in relation to the substrates degree
of reduction and theoretical limits, NH, as the nitrogen source.
ROELS
2490
(41)
y;,/oo
(42)
+ b1
2 ~ 1+ hod,)/4
(43)
Yavle
(qouoM,)/ys
(44)
with the aid of the equation for oopresented in Table VII, it follows:
Yavl,
= (qoM,)/y,
(45)
2491
Yavle = ( q e M x ) / y x
(46)
DEGREE OF REWCTION
v,
(a);
(0);(a);
2492
ROELS
[ ~ Q / @=
o
( AdA o I
(47)
1.18 qe)
(48)
As is clear from eq. (48) for growth with HNO, the proportionality
constant between heat production and oxygen consumption depends
on qe. For qe = 0.55 (a common value) it is equal to 590 or 28%
higher than the value for the nitrogen sources N, and NH,.
4.3. Anaerobic Product Formation
2493
ROELS
2494
TABLE XI
Free Enthalpy and Enthalpy Change for Various Anaerobic Product Formation
Reactions Starting with Glucose
Product
Methane
Methanol
Ethanol
Glycerol
Acetic acid
Enthalpy change
(W/C mol glucose)
- 24
16
- 13
+ 5
- 34
0.55 (0.75)
0.47 (0.50)
0.64 (0.75)
0.23 (0.25)
0.54 (0.58)
0.71
0.70
0.60
0.31
0.74
0.260
0.057
0.219
0.043
0.077
46
46
46
46
46
Numbers in parentheses are the ratios of the C mol of the various C containing compounds formed in the product formation reaction.
-+
0.67 (0.89)
0.57-0.86 (1)
0.57 (0.67)
0.72
0.69-0.83
0.62-0.68
0.238
0.129-0.242
0.12 1-0.159
qth
EP
(C conservation
in products
other than CO,)
46
34
34
34
Ref.
Numbers in parentheses are the theoretical maxima of the carbon conservation in products other than CO,.
-+
Energy-generating reaction
Y,
(C mol DM/C
mol
substrate)
TABLE XI1
Growth Yield, Thermodynamic Efficiency, and Carbon Conservation in Products on Anaerobic Growth
2496
ROELS
2497
Since the advent of continuous culture various workers have emphasized that the yield constant, the amount of biomass produced
per unit of substrate consumed, is dependent on the specific growth
rate. This led to the introduction of the concept of maintenance
energy by Herbert@and later on by Pirt." The introduction of the
maintenance concept resulted in the following linear relationship for
the dependence of the substrate consumption rate on the culture's
growth rate:
-rs
rxlY,
+ m,Cx
(49)
The equation defines the true yield coefficient Y,, being the maximum amount of biomass produced per unit substrate consumed and
the maintenance coefficient, m *, the amount of substrate consumed
for maintenance purposes per unit time and per unit biomass present
in the culture.
The physiological significance of the constants Y , and m , has
been the subject of much d i s c ~ s s i o between
n ~ ~ ~ ~workers
~ ~ ~ on the
ROELS
2498
- r s = r x / Y S x+ r p / Y s p+ msCx
(50)
as=
+ m,C,
-@xlYsx -
(51)
Qo = -@xlYox QC
@JYCx+
+ moCx
(52)
mcCx
(53)
The constants Y,, Y,, mo, Y,, Y,, and m , can be expressed in
terms of Y,, Y,, and m,.The equations are given in Table XIII.
The derivation presented above shows that once the linear law
for substrate consumption is adopted, linear relations result for the
flows of oxygen and carbon dioxide, water and as a matter of fact
also heat, as can be shown from an enthalpy balance. Hence in the
fairly general system of Sec. 3.1 the linear laws for the flows present
no new information and are already implicit in the postulation of the
linear law for the consumption of the substrate. These matters have
been studied earlier by amongst others Roels and Kossen, Heijnen
et al., and Heijnen and Roels. The equations for aerobic growth
without the formation of products are obtained from the general
equations if Y , is put equal to infinity. It is, however, more convenient to make use of eqs. (38) and (41) for an estimation of heat
production and oxygen consumption. As the quantities o, and wo
y,
y,,
Carbon
dioxide
YJ(1
a4dJazd4)
- a4d,/d4)1
YJl
4 Y S A Y , - yxYsx)
Oxygen
Compound
Y,
=
Y&1
Y , = 4Y&,
- a,d2/a2d4)
Ysp(l - a4d,/a,d,)l
- %Y,)
TABLE XI11
Relationships between the True Yield Factors and Maintenance Factors
y,m,/4
m , = m,(l
mo
a,dz/a2d4)
Maintenance factor
52
Fl
??
$!a
$r!
2500
ROELS
+ msYsJ1
qo = F Y s x ~ b o ( p+
(54)
(55)
msYsx)l
fA
(h-')
(a)
Fig. 7. (a) Substrate yield, oxygen consumption, and heat production per unit
biomass produced as a function of specific growth rate p. Glucose as the substrate.
(b) Substrate yield, oxygen consumption, and heat production per unit biomass produced as a function of specific growth rate. Methane as the substrate.
SPECIFIC
GROWTH RATE
p (h-'
(b)
2502
ROELS
decrease, but also oxygen consumption and heat production per unit
biomass are as low as possible.92
2) For processes like wastewater treatment, where often the biomass produced (sludge) is a nuisance, the growth rate should be as
low as possible, hence recirculation of biomass would be an advantage.
A special case of the general equations for product formation is
the case of anaerobic growth with production of a single product
(or a number of products in a constant ratio). For this case the flow
of oxygen must equal zero. In this way, using eq. (52), the flow of
product, CPp, can be expressed in terms of the flow CP, and the amount
of biomass C,. Combination of this result with eq. (51) results in an
equation in which no separate contribution for CPp is present any
longer. For the case of anaerobic product formation the flow of substrate can hence be expressed as
-CPx/Ysx + msCx
The expression for the rate of product formation becomes:
(56)
- ~ x Y s x ) / ( y , Y s , ) -l ~ (ysmsIyp).C,
~x
(57)
CPs
QP = [(ys
2503
where
qp
rxYsx~ys
(60)
From eq. (59) it can be concluded that the fractional conversion, ep,
reaches an upper limit, yslyp,if the growth rate approaches zero; if
the growth rate goes to infinity, a lower limit, y,/yp - (y,/yp), Y,,
is reached.
In Figure 8, eq. (59) is illustrated graphically, (yJys) * eP is plotted
against qp, m s Y s x / pbeing the parameter.
As an example the alcoholic fermentation will be treated and the
parameter values Y , = 0.14 and rn, = 0.127 will be assumed73to
apply to the reference situation. The specific growth rate in the reference situation is assumed to be 0.1. In that case m,Y,,/p comes
out to be 0.18, qp is about 0.15, hence it follows from eq. (59) that
ypep/ysis equal to 0.875 and the carbon conservation in alcohol is
0.583. It can be increased by about 14%, if the specific growth rate
is decreased to zero or the maintenance factor is increased to infinity..The first goal can be reached by going to very low dilution rates
in continuous culture or by recirculation of biomass, the second goal
can be approached by increasing the fermentation temperature.
Another possibility is to use an organism with a lower substrate yield
andlor a higher maintenance coefficient than Saccharomyces cerevisiae, e.g., Zymomonas mobilis or Zyrnomonas anaerobia, which
use a pathway for anaerobic alcohol formation which produces only
one ATP as compared to the normal two ATPs in yeasts. If it is
assumed that this causes the yield to decrease by a factor of approximately 2 and the maintenance to go up by that same factor,
growth at a specific growth rate of 0.1 would result in a carbon
conservation in product of close to 95% of the attainable maximum.
The data presented in Table XI1 on the observed Yyx values in a
number of anaerobic fermentation processes can also be interpreted
in terms of eq. (59), albeit that the Y , and nz, factors are not known,
but only the overall yield factor Y:,. Equation (59) can also be written
(Yp/Ys)Ep
= 1 - qo
(61)
2504
ROELS
EFFICIENCY
FACTOR
7&
2505
= r(C, p , T )
EFFICIENCY FACTOR
qo
Fig. 9. Carbon conservation in products in various anaerobic processes on formation of ethanol (A), lactate (o),propionic/acetic acid (o),
methane/acetic acid
(o),from glucose; methane from acetic acid (u);methane from methanol (r);
methane
from formic acid @I; methane from propionic acid ( x ) .
ROELS
2506
-rs =
rx/Ysx+ m s C x
(64)
Now only one further equation is needed. For example, the rate of
substrate conversion could be expressed as a function of the concentrations of the components of the state vector:
r s = AC,,
C N HC
~ ,O ,C , , C c ,Cws)
(65)
(66)
(67)
Equation (67) states the rate of substrate consumption to be a function of the substrate concentration and the concentration dry matter.
These kinds of models form the base of the classical continuous
2507
D E C I D E WHICH N COMPONENTS
ARE RELEVANT TO THE
SYSTEM'S DESCRIPTION
FOR
'<
LITERATURE
EXPERIMENTS
OF THE COMPONENTS
t
SELECT (N-K)
KINETIC
EQUATIONS
CONSTRUCT BALANCE
EQUATIONS FOR THE STATEVECTOR
culture theorym and of some of the models for growth in fed batch
culture.'.97It is important to emphasize that the postulation of a
separate kinetic equation for the conversion of one of the other components in the system is not only unnecessary; it may also lead to
erroneous results, if the net rate of conversion, obtained from the
kinetic equation, is not equal to that obtained from the elemental
balances.
ROELS
2508
~ s . I n a x C s K p C A ( K s+ C M p
+ Cp)l
(68)
The enthalpy of formation of biomass is conveniently calculated, using the fact that
the heat of combustion of biomass is about 560 kJ/C mol biomass.' The combustion
reaction can be written
CH,,,Oo,,No,2 + 1.2 0
-+
C02
hbiomasr=
The free enthalpy of formation of biomass from glucose with gaseous ammonia as
2509
a nitrogen source is calculated at about -30 kJ/C mol by Morowitz. The stoichiometry of the reaction is given by
1.05 CH,O
+ 0.2 NH, -+
C H , 8 0 0 5No.z
- 30 - 160.7 - 3.3
W.
The calculation of w, can be straightforwardly based upon eq. (13) withj, equals
zero. The enthalpy values of Table V and the equations for the flows recorded in
Table 11, are used to calculate the value of Yyr, consistent with zero heat flow, the
general equations of Table VII were thus obtained. The calculation of wf is slightly
more involved, account has to be taken of the fact that charged species may participate
in the reactions, this influences the calculations to some extent as the free enthalpy
of hydrogen is taken at a pH of 7 instead of unit molality (pH = 0). As an example
the calculation for oxalic acid will be given. The stoichiometric equation of growth
is given by:
(l/Yyx)COz-
+ 0.8 H + [1/(2Y:,)
- 0.41 HzO
Equation (17) results in the following inequality using the free enthalpy values of
Table V:
-338/Y:,
(l/Yix - I)(-%)
- 1.(-67.1)
20
Nomenclature
a;
a ij
C
C,
2510
COD
d,
Yi,
ROELS
chemical oxygen demand (g oxygen)
number of nitrogen atoms in one molecule of component i (dimensionless)
elemental composition matrix (dimensionless)
vector of molar specific enthalpies (kJ/C mol)
standard molar specific enthalpy of substrate (kJ/C mol)
vector of flows (C moVm* hr)
heat flow to the system (kJ/m hr)
Michaelis constant or inhibition constant (e.g., K,, K,) (C moV
m)
molecular weight of the biomass (g/C mol)
maintenance coefficient with respect to component i (e.g., m,,
mo)(C moVC mol DM hr)
vector of chemical reaction rates (C mol/m hr)
vector of the rates of production of components (C moVm hr)
rate of production of component i (e.g., rr, r , ) (C moVm hr)
maximum rate of substrate conversion (C moI/m hr)
oxygen consumption per unit substrate consumption (moYmol)
respiratory .quotient (dimensionless)
systems surface area (m2)
systems entropy per unit volume (Him K)
time (hr)
systems internal energy per unit volume (kJ/m)
systems volume (m)
yield factor for biomass on ATP (g DM/mol)
yield factor for biomass on available electrons (g DM/mol)
yield factor for biomass on the substrates heat of combustion
(g DM/kcal)
yield factor for biomass on substrate (C mol DM/C mol)
yield factor for biomass on component i (e.g., Y:*, YLJ (C mol
DM/mol)
true yield factor for biomass on component i (e.g., Y,, Y,) (C
mol DM/C mot)
true yield factor for product on component i (e.g., Y,, Yo,) (C
mol/C mot)
Greek
a
a;,
WO
251 1
Subscripts
1, x
2, s
3,P
4
5, 0
6, c
7. w
biomass
substrate
product
nitrogen source
oxygen
carbon dioxide
water
References
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2512
ROELS
2513
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