Composición Microbiana Articulo Científico Roels PDF

Application of Macroscopic Principles to
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 meas-
ures 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 thermo-
dynamic 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 effi-
ciency 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 con-
sumption. Aerobic and anaerobic growth are discussed using this relationship. A
correlation useful in judging the potentialities for improvement in anaerobic product
Biotechnology and Bioengineering, Vol. XXII, Pp. 2457-2514 (1980)

0 1980 John Wiley & Sons, Inc. 0006-3592/80/OO22-2457$05.80
2458 ROELS
formation processes is derived. Finally the relevance of macroscopic principles to

the modeling of bioengineering systems is discussed.
1. INTRODUCTION
In the methods of analysis used in physics two broad groups of

approaches can be distinguished-the corpuscular and the contin-
uum description. The corpuscular description takes into account the
fact that particles or objects are the basic units of any physical sys-
tem. It is based on a more or less careful analysis of the properties
of and interactions between the objects composing the system. After
performing this analysis the methods of statistical mechanics are
invoked to calculate the properties of a system in which a large
number of individual objects are present.'-4
The corpuscular description is extremely versatile, but it requires
a rather detailed knowledge about the properties of the objects and
their interactions. Furthermore, the mathematics involved are quite
elaborate.
The continuum approach, which is more usual in engineering
studies, is the basis of the so-called macroscopic method for the
description of systems in which large numbers of objects are present.
It is based on the definition of variables, e.g., concentrations, pres-
sure, and temperature, which are continuous with respect to time
and length coordinates and it ignores the corpuscular structure un-
derlying the system.
In this report the possibilities of macroscopic methods with re-
spect to the transformation processes in living systems will be ana-
lyzed. Microorganisms are, as has been pointed out by several au-
thor~,'-~ so-called open systems, they are not in thermodynamic
equilibrium and exchange matter and energy with the environment.
Hence, a macroscopic description of their behavior should use the
formalism of thermodynamics of irreversible processes.
Excellent treatments of this formalism can be found in the liter-
ature.'.' The metabolic pattern of microorganisms is characterized
by a highly complex network of chemical reactions and, as has been
shown by, amongst others, Aris,' the treatment of such patterns is
facilitated by the use of matrix operations. Matrix notation will
hence be used in this paper. Readers unfamiliar with this formalism
are referred to one of the texts on this subject."
To conclude Sec. 1 a short survey of this report will be given. In
present day bioengineering a number of elements of macroscopic
theory, e.g., mass and elemental and energy balances, are becoming
BIOENGINEERING REPORT: MICROBIAL METABOLISM 2459
increasingly accepted. This report is an attempt to show the general

framework from which these principles derive. The results of the
applications of these principles to aerobic and anaerobic growth and
product formation will be treated and experimental evidence existing
in the literature will be analyzed using this formalism.
2. BALANCE EQUATIONS FOR EXTENSIVE QUANTITIES
2.1. Balance Equation for the Chemical State Vector

An important tool of the macroscopic method is the balance equa-
tion. Balance equations can be formulated for each of the extensive
properties of the system. Extensive properties are characterized by
the fact that they are additive with respect to parts of the system,
some examples are mass, energy, volume, and the number of moles
of the various components present in the system.
The structure of a balance equation is always the same; in words
it can be formulated as:
(accumulation -
- (in-out (conversion
in system) transport) + transformation) (1)
Equation (1) states that the total amount of an extensive quantity

changes by two kinds of processes-transformation processes inside
the system (e.g., chemical reactions) and exchange processes with
the system’s environment. A convenient starting point for the for-
mulation of balance equations for the amounts of the various chem-
ical species in the system is the formulation of the chemical state
vector, C , of the system. In the state vector, the concentrations of
each of the n chemical compounds present in the system are iden-
tified by one number, representing the number of moles of that com-
pound per unit of system volume. The verbal statement of the bal-
ance principle given by eq. ( I ) can, for the chemical state vector,
be represented by the following mathematical equation:
dt
C.dV = rA.dV + 1 ja.dS
r A is the vector of the net production rates of each of the compounds

in the system (mol/m3hr). j, is the vector of the net rates of trans-
port to the system (mol/m2 hr). S and V are the system’s surface
and volume, respectively.
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For a system of constant volume, in which no gradients occur,

eq. (2) transforms to'
C=r,+@ (3)
where CP is the vector of the net rates of transport to the system
expressed per unit of system volume (mol/m3hr).
The reaction pattern inside a system can be specified in terms of
a number of independent chemical reactions and their stoichiometric
equations.' The net rates of conversion of each of the chemical spe-
cies can be expressed in terms of the rates of these reactions and
their stoichiometry. It can be shown'~'*''that the following expres-
sion holds:
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 re-
action rate. It is an m x n matrix of the following structure:
rn
R components
Combination of eqs. (3) and (4) allows the formudion of a balance

equation for the chemical state vector of a system of constant vol-
ume:
C = r*oL + Q, (5)
Equation ( 5 ) is the starting point of the derivations which are to
follow. The concept of steady state is of fundamental importance
in the treatment of open systems.
In a steady state the time derivative of the state vector has become
zero, hence it follows from eq. (5):
Q, = -ra (6)
A practical implication of eq. (6) is that, for a steady state, the stoi-
chiometry of a reaction pattern can, as far as the net rate of pro-
duction 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.
2.2. Conserved Quantities: The Elemental Balance Equations

There is a group of extensive quantities, which have the property
that net production in the reaction pattern in the system does not
take place. These quantities are called conservative quantities. Most
biological systems, excluding man, have the property that elements
are conserved in all transformation processes possible to the system.
This observation results in the formulation of elemental balances,
which have become increasingly accepted in bioengineering in the
last decade. The earliest paper, known to this author, which contains
an explicit treatment of microbial metabolism in terms of stoichi-
ometry, is due to Hoover and Allison;” it was published as early
as 1940. Another early contribution is due to Battley.13To this au-
thor’s knowledge the pioneering articles in biotechnology are most
probably due to sol om on^'^ and Harrison.’S Important further de-
velopments and generalizations are due to Minkevich and Eroshin.I6
Mateles,I7Harrison et al. , I 8 H o l ~ e ,Sukatch
’~ and Faust,” Takahashi
et Kanazawa” and Hamer et al.23studied applications to the
single cell protein (SCP) production technology. Wang et al. ,24 Za-
briskie,” and Zabriskie and Humphrey26reported studies concerning
the bakers’ yeast production technology. The penicillin fermentation
was studied by Heijnen et al.27
Stouthame?8 applied the principles with special reference to the
study of microbial energetics. Erickson et al.” presented an appli-
cation to product formation. The state of the art was reviewed by
Herbert3’ and Cooney et aL3’
Roels and Kossen recently presented a rather general analysis of
the principle. I Important new generalizations were recently reported
by Erickson et al.32
In this report the various proposals for the formulation of the
concept of elemental balance will not be treated in detail, rather a
new, more general formulation will be derived directly using eq.
( 5 ) and the conservation principle for elements.
The derivation of an expression for the elemental balances is easily
achieved by the introduction of an elemental composition matrix E.
If elemental balances for k elements are constructed. this matrix has
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the following structure:
C
0
rn
P
E = aij 0
n
e
n
t
vs
7 - J .
k chemical elements
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)
Equation (9) is the general form of the principle of the conservation
of atomic species; it specifies rn x k relationships between the rn
x n stoichiometric constants of the system.
Direct application of eq. (9) to the stoichiometry of bioconversion
processes does, however, present some difficulties. In the derivation
of eq. (9) it was formally necessary to specify each component pres-
ent in the system in the system’s state vector C. Hence, application
of eq. (9) presupposes the elemental composition of each of these
components to be specified in the matrix E. In biosystems this pre-
sents some difficulty as several hundreds of components take part
in metabolism. Up to now, in the application of elemental balances
to microbial metabolism, this problem was avoided and only a fairly
limited number of compounds was considered in a so-called gross

stoichiometric equation of growth. Although this is intuitively valid,
it is interesting to analyze, if such an approach can be theoretically
justified.
To perform such an analysis eq. (7) is studied for the system’s
steady state, hence the left-hand side of eq. (7) becomes zero and
it follows:
@E = - ra.E (10)
And, by combination of eqs. (10) and (8):
cD*E = 0
Equation (1 1) presents a second statement of the principle of ele-
mental balance, which is restricted to a steady state. It has the ad-
vantage of formulating the elemental balance principle in terms of
the flows of matter leaving and entering the system. In the appli-
cation of eq. (1 1) components, which are present in the system, but
are not exchanged with the environment in significant amounts, need
not be considered.
The reasoning presented above formally validates the treatment
of microbial metabolism in terms of a gross stoichiometric equation
of growth. It has to be borne in mind that its application is only valid
for a system in steady state. Furthermore each component ex-
changed with the environment in non-negligible amounts needs to
be specified in the vector of flows.
2.3. Thermodynamic Treatment of Energy Transformations

The efficiency of the transformation of the energy in the substrate
to biomass on growth of microorganisms has received considerable
attention in the last decade. Related problems like the heat produc-
tion in microorganisms have also been studied.
An interesting measure for the efficiency of growth, YATp, has
been proposed by Bauchop and E l ~ d e nand ~ ~it was recently exten-
sively discussed by stout ha me^.^^*^^ Y A T p expresses the yield of bio-
mass/mol ATP consumed and it is based on knowledge of the amount
of ATP produced in the various catabolic pathways in the organism.
As such it is not a quantity of a purely macroscopic nature and its
merits will not be studied in this paper.
Various proposals for macroscopic efficiency measures have ap-
peared in literature and they are all more or less related. The quantity
Yavle, as proposed by Payne and his c o ~ o r k e r s expresses
~ ~ - ~ ~ the
yield, biomass dry matter (DM)/mol electrons in the substrate, which
2464 ROELS
are available for transfer to oxygen. A second measure proposed by

the same authors isYkcal,the yield of biomass DM/kcal heat of com-
bustion of the substrate. These two measures are related as the heat
of combustion of an organic compound is known to be proportional
to the electrons available for transfer to ~ x y g e n . ' ~The
, ~efficiency
~.~~
measure, q, as proposed by Minkevich and EroshinI6 is proportional
to Yavle as well as Ykcal. The authors, however, do put limits to q
based on thermodynamic considerations. Although the authors in
our opinion missed the correct formulation of the second law of
thermodynamics for open systems, this approach is very promising
and will be used in a somewhat modified form in this present paper.
The various efficiency measures have been recently reviewed by
NagaL4'
Heat produqtion is the second aspect of the energetics of growth,
which has been studied extensively. Cooney et al.41were among the
first to focus attention to the proportionality between heat produc-
tion and oxygen consumption. Minkevich and Eroshin greatly con-
tributed to the understanding of the phenomenon,'6 Imanaka and
Aiba,42Mou and C ~ o n e yand , ~ Wang
~ et al.44also made contributions
to the theoretical and experimental evaluation of the enthalpy bal-
ance. Two other important contributions to the thermodynamic
analysis certainly merit mentioning. The review due to Wilson and
gives a very detailed thermodynamic analysis of growth
and explicitly stipulates the correct formulation of the second law.
The treatment of M ~ C a r t ydirected
,~~ to the analysis of wastewater
treatment, gives a detailed analysis of the efficiency of growth in
thermodynamic terms. The argument in this report will be very sim-
ilar to that treatment.
It will not be attempted to integrate the various contributions in
literature into a general theory, rather a direct approach, based on
nonequilibrium thermodynamics will be used.
For open systems, such as microorganisms, the first law of ther-
modynamics' is formulated as:
U =jQ + @.hT (12)
Equation (12) states that the system's internal energy per unit vol-
ume, u , changes, due to the flow of heat to the system, j , , and the
flow of enthalpy, @-hT,associated with the various compounds ex-
changed 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)
The second law of thermodynamics allows the formulation of a re-

striction 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)
In this equation p r stands for the transpose of the row vector in
which the partial molar thermodynamic potentials of the system's
components are specified. s is the system's entropy per unit volume.
For a system in steady state, s and u become independent of time
and eq. (14) can be written
c.p' = 0 (15)
By combination of equations (15) and (3) it follows:
r w p T + @.pT= 0 (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 chem-
ical reaction to proceed at constant temperature and pressure a neg-
ative 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 re-
actions, 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

The formalism developed in Sec. 2 is thus general, that in principle
very complex cases of growth and product formation can be treated.
This results, however, in complicated equations, which offer little
advantage over the more formal equations. Therefore only situations
of limited complexity will be studied in more detail. An organism
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is considered, which grows on one sole source of carbon and energy,

which may contain nitrogen. One sole source of nitrogen is supplied,
it may also contain carbon. One product is excreted, CO,, H20, and
0, are the only other components, which may be exchanged with
the environment.
In terms of the formalism developed in Secs. 2.1 and 2.2 the sys-
tem will be considered to be a given quantity of microorganisms
DM. Growth is assumed to be balanced.'' The organism exchanges,
macroscopically speaking, an exact replica of itself with the envi-
ronment; it is characterized by its gross elemental composition for-
mula CHb,O,,Nd,.The concept of the C mol of ~rganisrn,~'the
amount containing 1 mol carbon, is adopted.
Figure 1 gives a schematic representation of the system and the
possible flows to and from the system. Only the elements C, H, 0,
and N are considered and indeed these elements comprise in most
cases about 95% of the cellular mass and the various other exchange
flows. Equation (11) can be directly applied to this specific case
with:
and
-
1 bi CI di
a2 bz c2 dz
a3 b3 c3 d3
E = a4 64 c4 d4
0 0 2 0
1 0 2 0
-0 2 I 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 . System and flows for the macroscopic analysis of microbial growth.
variables. Which flows are to be chosen independently strongly de-

pends on the application one has in mind. First the case will be
considered in which the flow of biomass, the flow of substrate,
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 macro-
scopic yield factors Y h , the number of C mol biomass producedl
mol substrate taken up, and YLx, the number of mol biomass pro-
duced/mol product produced, are introduced. Yix and YLx are de-
fined by:
YLx = I@I/@3/ (20)

By analogy, macroscopic yield factors for C 0 2 , Y & and oxygen,
YAx can be defined.
In the equation for the oxygen flow the generalized degrees of
reduction of substrate, ys, of biomass, yx, and of product, yp, are
introduced. These are defined by analogy to the definition of degree
of reduction used by Eroshin and his coworker^,'^^^^ who defined
the value for growth with ammonia as a nitrogen source. In the
present case the degree of reduction is modified to apply to any
nitrogen source. The generalized degrees of reduction are given by:
yx = 4+ 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)
The rationale for the definition is the concept of available electrons

as introduced by Payne and his coworker^^^,^' and Eroshin and his
coworker^.'^,^^ As can be seen the degree of reduction of the biomass
is defined dependent on the nature of the nitrogen source used, this
is a convenient procedure, as it makes specification of the degree
of reduction of the nitrogen source and assumptions about the degree
of reduction of the nitrogen present in the biomass unnecessary.
The equations given in Table I can be used to calculate the flows
of oxygen, carbon dioxide, and nitrogen source, once the flows of
biomass, substrate, and product are known. However, the existence
of accurate gas analyzers often results in a situation where these
flows can be readily obtained. In that case a combination of these
flows with the often also measured flow of substrate can be used
TABLE I
Calculated Flows for the General Case
Flow Component Flow (in mourn3 hr)
@I biomass independent
@* substrate independent
@, product independent
- 1( d , - f
d + *)
@4 nitrogen source
dq y JX Ypx
@5 oxygen
Q6 carbon dioxide -
@7 water
to estimate the flows of biomass and product. By some rearrange-

ments of the equations in Table I the following equations are ob-
tained, if the nitrogen source is NH3 and product and substrate do
not contain nitrogen:
@I = @2[Ro(4 - yp.RQ) + a d y , - ys)l/(yx - yP) (24)
Q3 = Q2[- R0(4 - yx.RQ) - a2(yx - ys)l/[a3(yx - yp)l (25)
These equations show that knowledge of the respiratory quotient,
RQ, the ratio of the carbon dioxide production to the oxygen con-
sumption, and Ro, the ratio of the oxygen consumption to the sub-
strate consumption, allows direct estimation of the biomass pro-
duction rate and the production formation rate. The nature of the
equations shows, as yx - y p appears in the denominator at the right-
hand side of eqs. (24) and (25), that the method becomes less ac-
curate if yx approaches yp. Procedures of this kind have been pro-
posed for the detection of unwanted alcohol formation in aerobic
bakers’ yeast produ~tion.~’ There is a variety of other applications
of the equations presented in Table I, but their use in particular
applications will be left to the reader.
3.2. Aerobic Growth on a Sole Source of Carbon and Energy

without Product Formation
In this section the case of growth on a single source of carbon and
energy not containing nitrogen will be studied. Three simple nitrogen
sources are considered and product formation is excluded. In Table
I1 the equations for the various flows are given. The equations were
obtained from the general equations of Table I by substitution of
composition data for the nitrogen sources and putting YLx equal to
infinity. In the equations another version of the substrate yield factor
has been introduced, YZx the yield expressed as C mol biomass pro-
duced/C mol substrate, it can also be regarded as the fractional con-
servation of the substrate’s carbon in the biomass. The equations
for oxygen consumption appear to be the same, irrespective of the
nitrogen source. The reader should note, however, that the equations
are different, as yx depends on the kind of nitrogen source supplied.
The equations for yx in the three cases studied are also given in Table
11. Eroshin and his coworker^'^.^^ pointed out that the degree of
reduction with respect to NH, as the nitrogen source does vary little,
if biomass of various sources is compared. In Table 111 some lit-
erature data on biomass elemental composition are summarized and
the degrees of reduction calculated with respect to NH,, HNO,, and
2470 ROELS
N, are seen to vary very little. The relative standard error is less
than 5% in all cases. A good approximation to the average com-
position of biomass is the elemental composition formula CH, .*-
Oo.sNo.,.In calculations account has to be taken of the biomass frac-
tion 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)
In this equation qo is the efficiency factor of Minkevich and
Eroshin.I6 It is given by
qo = yxYYys (27)
The correlation is numerically different for the three nitrogen
sources as yx is dependent on the nitrogen source used.
In Figure 2 the relationship according to eq. (26) is shown for
growth with HNO, and NH, as the nitrogen sources. Some fairly
recent experimental data on substrate yield and oxygen yield, com-
piled in Table IV, are plotted in Figure 2. As can be seen there is
good agreement between theory and practice. It is interesting to
compare the relationship given in Figure 2 with the concept of yield
on available electrons, Yavl,as proposed by Payne and his cowork-
e r ~ . ~YaVl,
~ . ~is 'easily identified to be given by
Yav/e = ( Y Y y s ) * M x (28)
where M , is the molecular weight of the biomass g/C mole. As is
apparent from eq. (28) a plot of YLx vs. Yavieshould have the same
shape as the general correlation given in Figure 2. This was shown
to be the case by NagaL40 The property of Yav,, and of qo will be
discussed further in Sec. 4.
The general correlation presented in Figure 2 and also by eq. (26)
can be used in a variety of ways:
1) For the case of growth on a substrate of known degree of re-
duction with a known nitrogen source and known absence of product
formation the equation can be used to estimate oxygen consumption
if the substrate yield factor is known.
2) In the case of measured oxygen yield and measured substrate
yield the relationship can be used to check the consistency of the
data. In the case of discrepancy and, of course, positive indications
h,
TABLE 111 5
N
Elemental Composition and Degree of Reduction for Biomass of Various Sources

Degree of reduction (yx)
Elemental
Organism formula NH3 HNO3 NZ Ref.
Candida utilis 4.45 5.25 4.75 30
C . utilis 4.15 5.75 4.75 30
C . utilis 4.34 5.86 4.91 30
C . utilis 4.15 5.75 4.75 30
Klebsiella aerogenes 4.23 5.99 4.89 30
KI. aerogenes 4.15 6.07 4.87 30
Kl. aerogenes 4.30 5.66 4.81 30
KI. aerogenes 4.15 6.07 4.87 30
Saccharomyces cerevisiae 4.12 5.40 4.60 15 w
S . cerevisiae 4.20 5.56 4.71 48
S. cerevisiae 4.28 5.64 4.79 49
Paracoccus denitrificans 4.19 5.79 4.79 50
P . denitrificans 3.96 5.54 4.59 51
Escherichia coli 4.07 5.99 4.79 52
Pseudomonas C12B 4.27 6.11 4.96 36
Aerobacter aerogenes 3.98 5.98 4.73 36
Average 4.19 5.78 4.79

sa = 0.13 s = 0.26 s = 0.10
(3%) (4.5%) (2.1%)
a s stands for the standard error of the estimate of yx (in parentheses the standard deviation expressed as percent of the average).
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);do-
decane, NH, (e); methane, NH, (@); gluconate, HN03 (A);pentane, HNO, (A);
methane, HNO, tr).
that no measuring errors are present, the deviations may indicate

the presence of undetected other carbon sources or products. If the
amount of carbon converted to product is known, for example from
a total organic carbon measurement of the broth filtrate, the equa-
tions presented in Sec. 3.1 can be used to estimate the degree of
reduction of the product and hence can provide indications of its
chemical nature.
Again in complete analogy to the cases treated in Sec. 3.1, in those
cases where the oxygen flow and the carbon dioxide flow are known,
the theory presented here can be used to calculate biomass growth
curves. As in this case the number of independent variables is two,
the number of flows which has to be measured is only two. Any
TABLE IV
Experimental Values of Yix, Yb, andY.,/.
yix
(mol DM/C mol Yb* YW/,
Organism Substrate substrate) (mol DM/mol 02) (g DMlavle)
S . cerevisiae glucose 0.59 I .34 3.63 53
E. coli 0.62 1.61 3.81 54
Penicillium chrysogenum 0.52 1.93 3.20 55
Pe. chrysogenum 0.56 1.55 3.44 56
Azotobacter vinelandic" 0.30 0.50 1.85 57
C . utilis 0.59 I .64 3.63 58
Pseudomonas jluorescens 0.44 1.16 2.71 58
Rhodopseudomonas spheroides S 0.52 1.81 3.20
59 ;a
Aspergillus awamori 0.62 1.55 3.81 56
Aspergillus nidulans 0.70 2.64 4.31 56
P . denitr$cans"' gluconate 0.45 1.76 3.02 50 b~
C . utilis acetate 0.42 0.87 2.58 58
Ps. jluorescens 0.32 0.57 1.97 58
A . aerogenes glycerol 0.66 1.17 3.48 60
A . aerogenes citrate 0.34 1.25 2.78 34
Candida boidinii ethanol 0.61 0.82 2.50 61
C . utilis 0.61 0.82 2.50 58
Ps. jluorescens 0.43 0.51 1.76 58
C . boidinii methanol 0.52 0.47 2.13 61
Klebsiella sp. 0.47 0.70 1.93 62
Methylomonas methanolica 0.60 0.66 2.46 63
Candida N-17 0.46 0.49 1.89 64
Hansenula polymorpha 0.45 0.46 1.85 65
"Bacteria" (I) pentane 0.47 0.54 1.81 21
Candida lipolytica dodecane 0.41 0.54 1.64 66
Methane bacteria"' methane 0.38 0.27 1.17 67
Methylcoccus capsulatus 0.63 0.36 1.94 68
Methane bacteria 0.56 0.32 1.72 69
a Nitrogen source is NH,, except for cases indicated by (I), HNO, and (2), N,.
combination of two flows suffices to specify all the remaining flows.

As was recently pointed out, also the flow of the nitrogen source
can in some cases be advantageously
There is an obvious limit7' to the value of Y:x in the cases treated
in the present sections. The substrate is the only source of the ele-
ment C, hence Y&, the fractional conservation of the element C in
the biomass, cannot exceed unity, Y: 5 1. This restriction also
defines a maximum to the value of Y,, on substitution of Y'& 5 1
in eq. (26) it follows:
this equation only holds if ys > yx. For ys 5 yx Y:, can have any
value.
Equation (29) was derived earlier by the present using
a somewhat different approach.
3.3. Anaerobic Growth with Product Formation

As much as has been published concerning the application of el-
emental balances to aerobic growth as little has recently been pub-
lished concerning anaerobic growth. This is understandable as much
of the work done recently has been concerned with the study of SCP
production. Owing to the rising prices of crude oil a renewed interest
is growing with respect to the production of bulk chemical products
by the anaerobic metabolic routes in microorganisms. An example
is the production of ethanol. The anaerobic formation of one product
is easily analyzed using the concept of elemental balances. All flows
to the system are completely specified if two flows are known. An
equation for the rate of product formation can be obtained by putting
the oxygen flow, a5,of Table I equal to zero:
This equation can be used to estimate the rate of product formation

if the biomass yield factor on substrate and the rate of substrate
consumption are known. Equation (30) can be reformulated to an
equation for the fractional conversion of the substrates carbon to
product, ep, as a function of Y%:
ql = yslyp - Y X Y X / Y P ) (31)
Equation (3 1) leads to the conclusion that the maximum fractional
conversion of the substrates carbon to one single product can never
exceed the ratio of the substrate degree of reduction to that of the
2476 ROELS
product. For the anaerobic formation of ethanol from glucose this

results in a maximum conservation of 2/3, for the formation of meth-
ane 1/2. This maximum is reached if YYx approaches zero. The rea-
soning can also be made to apply to mixtures of products, if a C mol
averaged degree of reduction is used.
A common value for the substrate yield factor YYxin the anaerobic
formation of ethanol is 0.14 (C mol biomass/C mol produ~t).’~ The
fractional conversion of substrate to product is calculated to be 0.57
for that case, being 85% of the potential maximum. In view of the
already mentioned renewed interest in the anaerobic formation of
products a further study of the potentialities of macroscopic methods
in anaerobic product formation studies may prove rewarding. The
subject will be pursued somewhat further in the following sections.
3.4. Conclusion
From the foregoing it will have become clear that elemental bal-
ances are an invaluable tool in the description of the systems com-
monly encountered in bioengineering. It is as fundamental as stoi-
chiometry 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) steady-
state assumption is justified.
2) Construct a list of all n components and their elemental com-
position, which the system exchanges with the environment in non-
negligible amounts.
3) Determine the number of elements ( k ) for which elemental bal-
ances 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 appli-
cation of elemental balances. It applies to instances in which more
flows are measured than are minimally needed to calculate the re-
maining 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 meas-
urements 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
powerful method in further A field, where further devel-

opments may prove especially rewarding, is the realm of the ana-
erobic product formation.
4. ENERGETIC ANALYSIS OF MICROBIAL GROWTH AND

PRODUCT FORMATION
4.1. Introduction
In this section some energetic aspects of the growth of microor-
ganisms 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 un-
known 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 thermody-
namics 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
2478 ROELS
TABLE V
Thermodynamic Properties of Some Compounds in the Standard State
ho IJ?
Component (kJ/C mol) (W/C mol)
Acetic acid C2H402 (aq)" - 244.8 - 186.4
Ammonia NH3 - 46.3 - 16.7
(a4 - 80.9 - 26.7
Ammonium ion NH,' (as) - 133 - 79.6
Biomass CHl.800.sNo.z (aq) - 91.4 - 67.1
Carbon dioxide c02 (g) - 394 - 395
Bicarbonate ion HCOj- (ad - 692 - 588
Citric acid C6H807 (as) - 195
Dodecane CIZH26 (1) - 29.3 2Sb
Ethane C2H6 (g) - 42.4 - 16.4
Ethanol C2H60 (as) - 139 - 90.9
Formic acid (33202 (ad -410.6 - 335.2
Fumaric acid C4H.404 (aq) - 151.3
Glucose C6H1206 (as) -211 - 153.1
Glycerol C3HsO3 (1) - 222.3 - 163.0
Lactic acid C3H603 (as) - 173.0
Malic acid C&Os (ad - 141.0
Methane CH4 (g) - 75.0 - 50.9
Methanol CH4O (1) - 239 - 176.5
Nitric acid HNO3 (ad - 173 - 110.7
Nitrogen N2 (9) 0 0
Oxalic acid CZH204 (aq) -414 - 338
Oxygen 0 2 (g) 0 0
Pentane C5H12 (as) - 34.6 - 3.9
Propane C3Hs (g) - 34.7 - 7.8
Succinic acid C4H604 (a4 - 172.8
Water HzO (1) - 286 - 238
H' (PH = 7) - 40.5
a aq = aqueous; I = liquid; g = gaseous.
By extrapolation of data for lower hydrocarbons.
the case of the free enthalpy balance it should be emphasized that

the free enthalpy values at the concentrations actually encountered
in the process should be used so the values obtained can only be
considered an approximation.
4.2. Aerobic Heterotrophic Growth without Product Formation

In the case of aerobic heterotrophic growth without product for-
mation 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.
Two other restrictive values will be used in the treatment to fol-

low, 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 ash-
free dry weight. An example of such a calculation is given in Ap-
pendix 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
NH3 HNO3
Substrate Y. Of we 00 0, 0, 00
Oxalic acid 1 0.28 0.25 0.24 0.24 0.21 0.17 0.24 0.22 0.21
Formic acid 2 0.53 0.56 0.48 0.47 0.48 0.35 0.47 0.48 0.42
Malic acid 3 0.71 0.72 0.62 0.52 0.62 0.63
Citric acid 3 0.70 0.72 0.61 0.52 0.62 0.63
Succinic acid 3.5 0.79 0.84 0.69 0.61 0.70 0.73
Gluconic acid 3.67 0.88 0.63 0.77
Lactic acid 4 0.93 0.96 0.81 0.69 0.82 0.84
Acetic acid 4 0.89 0.90 0.96 0.78 0.77 0.69 0.78 0.78 0.84
Glucose 4 1.00 0.97 0.96 0.87 0.83 0.69 0.88 0.84 0.84
G1ycero1 4.67 1.15 1.14 1.12 1.00 0.97 0.81 1.01 0.98 0.98
Methanol 6 1.46 1.50 1.44 1.27 1.28 1.04 1.28 1.30 1.26
Ethanol 6 1.38 1.42 1.44 1.21 1.21 1.04 1.22 1.23 1.26
Dodecane 6.17 1.37 1.40 1.48 1.20 1.20 1.07 1.20 1.21 1.29
Pentane 6.4 1.42 1.45 1.53 1.24 1.24 1.11 1.25 1.25 1.34
Propane 6.67 1.47 1.53 1.60 1.29 1.31 1.15 1.30 1.32 1.40
Ethane 7 1.54 1.62 1.67 1.34 1.38 1.21 1.35 1.40 1.46
Methane 8 1.72 1.84 1.91 1.50 1.57 1.38 1.52 1.59 1.67
2480 ROELS
TABLE VII
Expressions for the Maximum Values of YYx Consistent with Various Restrictions
Restriction
Nitrogen
h,' + 394 + 143(bz/az)

302.5 + 1436, - 383dl
h,' + 394 + 143(b2/uz)
302.5 + 143bj + 30dl
h,' + 394 + 143(b2/a2)
302.5 + 143bl
the entropy flow associated with matter is small as compared

to that associated with heat.
o, is approximately proportional to the degree of reduction
of the substrate. This is shown in Table VIII where the values
of Table VI are analyzed statistically. The error of an estimate
assuming proportionality between o,and ysis not significantly
different from that of the least-squares estimates in each of
the three cases considered.
For the nitrogen sources N2 and NH3 the equation oo = we
is approximately valid, the residual error of that estimate not
differing significantly from that of the least-squares estimate
of we from the relationship with ys.
For HN03 the relationship o, = oo is not valid, the most
reliable proportionality relationship being we = 1.18 coo.
The consequences of this observation will be discussed later on.
The various expressions formulated in this section can now be used
to define a number of efficiency measures.
4.2.1. Thermodynamic efficiency of growth

The values of ofcalculated in Sec. 4.2 are the maximum values
of the yield allowed by the second law of thermodynamics. The
thermodynamic efficiency of the growth process can now be defined
as:
Equation (32) was used in the analysis of some recent yield data
which have been published in the literature. In the calculations it
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 summa-
rized. 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 ther-
modynamic 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 Residual standard
source Correlation error of estimate Remarks
NH3 Of = 0.09 + 0.21y, 0.045 least-squares
estimate
we = 0.07 +0.22~~ 0.047 least-squares
estimate
0, = 0.24~~ 0.058
WO = 0.24~~ -
0, = 00 0.058
HNO3 ~f = 0.08 + 0.18~~ 0.042 least-squares

estimate
0, = 0.06 + O.l9y, 0.039 least-squares
estimate
0, = 0.2oy, 0.041
00 = 0.17~~ -
0, = 1.18~~ 0.041
N1 W, = + 0.18y,
0.09 0.041 least-squares
estimate
0, = 0.06 + O.l9y, 0.040 least-squares
estimate
0, = 0.21y, 0.058
00 = 0.21y, -
0, = wo 0.058
TABLE IX
Experimental Values of Y:, and the Thermodynamic Efficiency, q t h , as well as Yav/rand ye
Organism Substrate 7s yyx qlh YW/. tl. Ref.

Pseudomonas oxalaiicus oxalate 1.00 0.07 0.26 1.72 0.28 74
formate 2.00 0.18 0.34 2.21 0.32 74
Pseudomonas sp. 0.18 0.34 2.21 0.32 74
Pseudomonas deniirificans citrate 3.00 0.38 0.54 3.12 0.52 74
A . aerogenes 0.34 0.49 2.78 0.47 34
Ps. deniirificans malate 3.00 0.37 0.52 3.03 0.51 74
Ps. jluorescens 0.33 0.46 2.71 0.46 58
Ps. denitrificans fumarate 3.00 0.37 0.53 3.03 0.51 74
Ps. denitrificans succinate 3.50 0.39 0.49 2.74 0.46 74
Pseudomonas sp. 0.41 0.52 2.88 0.50 74
Ps . dentrificans gluconate" ' 3.67 0.45 0.67 3.01 0.59 28
A . aerogenes lactate 4.00 0.32 0.34 1.97 0.33 34
Ps. jluorescens 0.37 0.40 2.27 0.38 58
Pseudomonas sp. acetate 4.00 0.44 0.49 2.71 0.49 74
C . uiilis 0.42 0.47 2.58 0.46 58
Ps. jluorescens 0.32 0.36 1.97 0.35 58
Candida tropicalis 0.36 0.40 2.21 0.40 75
S. cerevisiae glucose 4.00 0.59 0.59 3.63 0.61 53
S. cerevisiae 0.57 0.57 3.51 0.59 48
E. coli 0.62 0.62 3.81 0.64 54
Penicillium chrysogenum 0.52 0.52 3.20 0.54 55
0.56 0.56 3.44 0.58 56
Azoiobacter vinelandii glucose"' 0.30 0.33 1.85 0.36 57
C . uiilis 0.59 0.59 3.63 0.61 58
Ps. fluorescens 0.44 0.44 2.71 0.45 58
R . spheroides 0.52 0.52 3.20 0.54 59
A . awamori 0.62 0.62 3.81 0.64 56
A . nidulans 0.70 0.70 4.31 0.72 56
Pseudomonas sp. benzoate 4.29 0.48 0.48 2.75 0.47 74
A . aerogenes glycerol 4.67 0.66 0.57 3.48 0.57 60
C . tropicalis ethanol 6.00 0.61 0.44 2.50 0.43 75
C . boidinii 0.61 0.44 2.50 0.43 61
C . utilis 0.61 0.44 2.50 0.43 58
Ps. jluorescens 0.43 0.23 1.76 0.30 58
C . utilis 0.55 0.39 2.26 0.39 76
C . brajsicae 0.64 0.46 2.62 0.45 77
C . boidinii methanol 6.00 0.52 0.36 2.13 0.35 61
Klebsiella sp. 0.47 0.32 1.93 0.31 62
M. methanolica 0.60 0.41 2.46 0.40 63
Candida N-17 0.46 0.31 1.89 0.27 64
H . polymorpha 0.45 0.31 1.85 0.30 65
Pseudomonas C 0.67 0.46 2.75 0.45 74
Pseudomonas EN 0.67 0.46 2.75 0.45 74
Torulopsis 0.70 0.48 2.87 0.47 78
Methylomonas sp. 0.50 0.34 2.05 0.33 79
M. methanolica 0.64 0.44 2.62 0.43 80
C . tropicalis hexadecane 6.13 0.56 0.41 2.24 0.40 75
C . lipolytica dodecane 6.17 0.41 0.30 1.64 0.29 81
"Bacteria" pentane"' 6.40 0.47 0.38 1.81 0.38 21
Job 5 propane 6.67 0.71 0.48 2.62 0.46 74
ethane 7.00 0.71 0.46 2.50 0.44 74
Methane bacteria methane"' 8.00 0.38 0.25 1.17 0.24 67
M. capsulatus methane 0.63 0.37 1.94 0.34 68
Methane bacteria methane"' 0.62 0.41 1.91 0.39 82
0.56 0.33 1.72 0.30 69
0.50 0.29 1.54 0.27 83
M . methanooxidans 0.68 0.40 2.09 0.37 84
2484 ROELS
,+o. l SECOND LAW
r
0.9 -
c-‘
6 0.8 - \ C -LIMITATION
P2
U
0.7-
u
9
z 0.6-
0
t
B
K
w 0.5 -
E
0.4 -
0.3 - 0 0
a2-
0.1 -
1 I I L
0 1 2 3 4 5 6 7 8
DEGREE OF REDUCTION v,
Fig. 3. Thermodynamic efficiency of aerobic growth with one carbon source as
a function of the substrate’sdegree of reductionand theoreticallimits to the efficiency.
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.
The tendencies observed in Figures 3 and 4 can be summarized

as follows. For substrates up to a degree of reduction of about 4.2,
the degree of reduction of biomass, the energy content of the sub-
strate is insufficient to allow all substrate carbon to be converted
to biomass, even if the thermodynamic efficiency were unity. For
substrates with a degree of reduction higher than 4.2 all carbon could
be converted to biomass as far as energy requirements are concerned
and even CO, could be fixed; the extent to which the energy can
be stored in biomass becomes limiting. Hence, the thermodynamic
CARBON LIMITATION
0 0 0
0 0
I lAl IUN I
0' 0
1 8 I I
0 1 2 3 4 5 6 7 8
DEGREE OF REDUCTION (ys)

Fig. 4. Relationship between substrate yield factor Y:, and substrate degree of
reduction and the theoretical limits to YE.
2486 ROELS
efficiency has to decrease on growth on substrates of a high degree

of reduction. This has been discussed more extensively by the pres-
ent author.72For reasons which are not understood by the present
author, the carbon conservation efficiency, Yyx, never exceeds 0.7
and has a maximum of about 0.60 on the average. Furthermore, the
thermodynamic efficiency never exceeds 0.7 and has a maximum
value of 0.55 on the average. Taking these empirical notions into
account the following estimate of biomass yield for substrates of
various degrees of reduction on growth on NH, seems reasonable:
Yyx = 0.05 + 0.12 ys ys 5 4.58 (334
Yyx = 0.60 ys > 4.58 (33b)

The values predicted with the present semiempirical correlation can
now be compared with data from other sources, notably the review
of (see Table X). In general there is fair agreement between
the empirical equation and the theory.
Bells5explains the low values for lactic acid and acetic acid from
the growth-inhibiting nature of these compounds. The low values
for formic and oxalic acids may be due to the fairly large amount
of energy needed to transport these components across the cellular
membrane.86
Concluding, it can be stated that eq. (33) can be of value in a
preliminary estimation of growth yield for growth with NH3 as a
nitrogen source.
It is also possible to speculate about the effect of the nitrogen
source on the yield on substrate. If it is assumed that the thermo-
dynamic efficiency does not depend on the nitrogen source used,
then, by virtue of the fact that ofvalues for HN03 and N, are lower
by about 15% as compared to NH,, the yields are expected to be
lower by maximally 15%, if these nitrogen sources are used.
The efficiency of the conversion of a substrate to biomass has also
received considerable attention in the literature on wastewater treat-
ment.& A well-known correlation, which is frequently used, is that
due to Servizi and Bogan:”
g biomass produced = 0.39 g COD consumed
In this equation COD stands for the number of g oxygen consumed
on complete combustion of the substrate.
The COD of the amount of substrate used in the production of 1
C mol biomass can be expressed as:
g COD = 8ys/Yyx (34)
TABLE X
Compilation of Average Measured and Calculated Data for Yix and YaVIefor
Aerobic Growth on Various Carbon Sources
yi: YWk
av av av av Number
Substrate meas. calc. meas. calc. of data Source
______ ~
Glucose 0.54 0.53 3.33 3.26 26 85

Glucose 0.57 0.53 3.51 3.26 10 Table IX
Other sugars 0.52 0.53 3.20 3.26 15 85
Glycerol 0.61 0.60 3.21 3.16 7 85
Acetate 0.41 0.53 2.52 3.26 7 85
Acetate 0.38 0.53 2.34 3.26 4 Table IX
Lactate 0.43 0.53 2.64 3.26 3 85
Citrate 0.40 0.41 3.28 3.36 4 85
Malate 0.40 0.41 3.28 3.36 3 85
Fumarate 0.41 0.41 3.36 3.36 3 85
Succinate 0.42 0.47 2.95 3.30 3 85
Pyruvate 0.42 0.45 3.10 3.32 2 85
Pentadecane 0.56 0.60 2.27 2.41 6 85
Hexadecane 0.50 0.60 2.00 2.41 5 85
Heptadecane 0.47 0.60 1.90 2.41 3 85
Octadecane 0.51 0.60 2.06 2.41 5 85
Higher n-alkanes 0.53 0.60 2.16 2.41 5 85
Ethanol 0.57 0.60 2.34 2.46 5 Table IX
Methanol 0.56 0.60 2.30 2.46 10 Table IX
Propane 0.71 0.60 2.62 2.21 1 Table IX
Ethane 0.71 0.60 2.50 2.11 1 Table IX
Methane 0.59 0.60 1.81 1.85 4 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)
g biomass/g COD = 1.94/yS ys > 4.58 (35b)
For substrates with a degree of reduction exceeding 3, eq. (35) is
close to Servizi and Bogan’s correlation. For highly oxidized or
highly reduced substrates the correlation will be less reliable, for
ethanol (ys = 6) a coefficient of only 0.32 is expected, for methane
only 0.24. In wastewater treatment, however, degrees of reduction
extremely differing from 4 are hardly expected.
4.2.2. Heat production on aerobic growth
A quantity that is of great practical importance is the heat pro-
duction on microbial growth as this quantity may determine the pro-
2488 ROELS
ductivity of a fermentor in cases where the cooling capacity of the

fermentor system is limited.
A convenient method to calculate the heat flow can, in cases
without product formation and for the nitrogen sources NH,, HNO,,
and N,, be based on the efficiency q,. A stoichiometric equation is
formulated in which mol biomass are produced and @,/we mol
substrate are consumed. As we is the value of YYx at which the heat
generation equals zero, no heat will be generated in the process
according to that reaction. This stoichiometric equation is now sub-
tracted from the gross stoichiometric equation of growth and stoi-
chiometric equation representing total combustion of ( l h , -
l/YYx)@,.C mol substrate is obtained. The heat production in this
component equation, being the heat production on growth with a
substrate yield, Yyx is now easily shown to be given by
j~ = (l/Yy,r - l/we).@I[hso+ 395 + 119(bJa2)] (36)

If the efficiency factor q, is defined by
qe = Yb/w, (37)
it follows:
j~ = @l'Ah*(l- qe)/qe (38)
where
Ah = A,dl + 143bl + 302.5 (39)
In eq. (391, A, = -383 for NH,; he = 30 for HNO,; A, = 0 for Nz.
In the derivation of these expressions the equations for we as given
in Table VII were used.
For an organism of composition formula CH,.,Oo.,No.,it follows:
NH3: Ah = +480
N*: Ah = +560
The values of the constants for HNO, and N, are much higher in-
dicating 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-
pected values of qc thus results for growth with ammonia as a ni-

trogen source:
qe = 0.55 if ys 5 4.55 (40a)
qe = 2.5ly, if yo > 4.55 (40b)
These equations allow a calculation of the heat production to be
expected on substrates of varying degree of reduction. Figure 5 sum-
marizes the results. The figure shows the expected heat production
with methane as a carbon source to be higher by a factor of 2.5 to
3 than that on growth with glucose.
As has been explained the high heat production with methane as
the carbon source is not due to peculiarities of the organisms growing
HEAT
I
(kJ/C-moleD
I
1.5
DEGREE OF REDUCTION y,
Fig. 5. Expected values of the heat production in relation to the substrate’s degree
of reduction and theoretical limits, NH, as the nitrogen source.
2490 ROELS
on methane but is essentially due to the high energy content of the

substrate as compared to the carbon content. This excess energy
has to be dissipated if no substrates of a low degree of reduction are
available. In Figure 5 the lower limits to the heat production due to
the restrictions of the second law and carbon conservation have also
been drawn. (It was assumed that zero heat production gives a suf-
ficiently close approximation to the maximum yield obtainable in
view of the second law.)
It is apparent from Figure 5 that even in the limit of complete
carbon conservation the minimum heat production on growth on
methane is higher than that which is practically achieved on the
average for substrates of a degree of reduction of 4. Indeed the
maximum qefor growth with glucose actually observed, 0.7, would
result in a heat production lower by a factor 2 than that which is
minimally achievable on growth with methane as the carbon source.
4.2.3 Relationship between heat production and oxygen

consumption on aerobic heterotrophic growth
Completely analogous to the reasoning in Sec. 4.2.2 the following
equation for the oxygen flow can be derived:
@o = @i*Ao*(I- qo)/qo (41)
where qo is an efficiency factor with respect to oxygen consumption,
it is equal to the thermodynamic efficiency proposed by Eroshin16
and Erickson et al.,32and it is given by
qo = y;,/oo (42)
The constant A . is given by
A 0 = -(4 + b1 - 2 ~ 1+ hod,)/4 (43)
where ho = - 3 for NH,; ho = 5 for HNO,; X o = 0 for N,.
Equation (41) can also be used to obtain an expression for Yavle.
By combination of eqs. (28) and (42), it follows:
Yavle = (qouoM,)/ys (44)
with the aid of the equation for oopresented in Table VII, it follows:
Yavl, = (qoM,)/y, (45)
This equation shows that the concept of YaVle is equivalent to the
efficiency factor qo, if yx and M , are considered constant.
For growth with N, or NH, as the nitrogen source qo is by a good
approximation equal to q, (Table VIII) and eq. (45) can be approx-

imated by
Yavle = ( q e M x ) / y x (46)
If the second law and carbon conservation limits to q, are introduced
the limits to Y,,,, shown in Figure 6 result. An expected average
dependence of Y,,,, on the substrate’s degree of reduction is ob-
tained by substitution of the empirical q, correlations, according to
eqs. (40a) and (40b). The resulting relationship is shown in Figure
6 and compared with data compiled in Table X. The observed drop
0 1 2 3 4 5 6 7 8
DEGREE OF REWCTION v,
Fig. 6. Expected values of and theoretical limits to Yavle.Experimental results
for growth with: glucose (0); other sugars (A); glycerol (0); acetate (v); lactate
(0); citrate (0);malate (A);fumarate (B); succinate (TI;pyruvate (e); higher n-alkanes
( x ) ; ethanol (0);methanol (a); propane (0);(a);
ethane methane (@).
2492 ROELS
in Yavle on growth on substrates with a high degree of reduction is

seen to follow directly from the reasoning presented above.
We will now derive a relationship between heat production and
oxygen consumption. If qe equals qo, comparison of eqs. (41) and
(38) results in the following relationship:
1
[ ~ Q / @=
o ( AdA o I (47)
As the approximation q, = qo is valid for the nitrogen sources NH,
and N,, eq. (47) will hold for these nitrogen sources. The propor-
tionality constant can be calculated from eqs. (43) and (39) and is
seen to be uniquely dependent on the composition of the biomass.
For the average composition of biomass the proportionality constant
is about 460 for N, as well as NH,. These values agree rather well
with the empirical value of 520 of Cooney et and with earlier
theoretical estimates of 451,16 and 455.,* This derivation can be gen-
eralized quite easily in cases of growth even in complex media and
when appreciable amounts of product are formed. It can be shown
that heat production and oxygen consumption are proportional as
long as all components present obey the relationships that their heat
of combustion is proportional to the oxygen consumption on com-
bustion. This is known to hold for most compounds commonly en-
countered in fermentation systems. One notable exception is HNO,,
whose heat of combustion does not follow the general rule; hence
the equality of qo and qedoes not hold for that nitrogen source. For
growth with HNO, the following relationship between oxygen con-
sumption and heat production can be shown to apply:
IjQ/@ol = l.l8IAh/AOl’(l - qJ(l - 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

Interesting applications of the concept of the energy and entropy
balance are possible in the treatment of anaerobic growth and prod-
uct formation processes, although, probably for the reasons dis-
cussed in Sec. 3.3, little seems to have been published on this sub-
ject. A noteworthy exception is the review of McCarty concerning
wastewater treatment.*
It is important to note that the convenient approximations, which
can, to a large degree of accuracy, be used in the case of aerobic

growth, being the use of an enthalpy approach instead of a free
enthalpy approach and the concept of energy content of a compound
being proportional to the available electrons, are no longer valid.
Anaerobic growth has to be treated using a free enthalpy balance,
using tabulated free enthalpy values.
It is beyond the scope of this treatment to present a general dis-
cussion of anaerobic processes, rather attention will be focused on
some special processes.
First the aerobic formation of various products from glucose will
be discussed. The nitrogen source for growth will be assumed to be
ammonia.
It is convenient to split the overall stoichiometric equation of an-
aerobic growth into two parts. In the first reaction the biomass is
formed with consumption of all of the nitrogen source. No product
is formed in the reaction.
In the second reaction the remaining substrate is converted with
production of product, carbon dioxide, and water.
The free enthalpy change of the reaction resulting in the formation
of biomass precursors can be calculated in a straighforward manner.
For glucose as substrate it comes out to be -25 kJ/mol biomass
produced. The free enthalpy change of the biomass synthesis re-
action is negative, although small, hence, as far as the second law
is concerned, this reaction could proceed without a second energy-
generating reaction. But apparently this free enthalpy change is too
small for efficient growth, hence the energy-generating reaction in
which the product is formed is needed to provide the additional
dissipation. In Table XI the free enthalpy change of various anaer-
obic product formation reactions starting with glucose are summa-
rized. As can be seen the free enthalpy changes on formation of
methane, ethanol, and acetic acid are largest, hence these products
are efficient as far as the anaerobic generation of free energy for the
growth reaction is concerned. The formation of for example glycerol
as the sole product is less beneficial.
The formation of products with a degree of reduction lower than
that of the biomass would require CO, fixation, hence the anaerobic
formation of these compounds as sole products is not excepted. As
is also clear from Table XI the enthalpy change of the product for-
mation process can be largely different from the free enthalpy change
and hence becomes an incorrect quantity for judging dissipation in
terms of the second law.
In the treatment of aerobic growth the thermodynamic efficiency
2494 ROELS
TABLE XI
Free Enthalpy and Enthalpy Change for Various Anaerobic Product Formation
Reactions Starting with Glucose
Free enthalpy change Enthalpy change
Product (kJ/C mol glucose) (W/C mol glucose)
Methane - 67 - 24
Methanol - 16 + 16
Ethanol - 38 - 13
Glycerol - 9 + 5
Acetic acid - 53.6 - 34
of the growth process was shown to be an interesting variable that

followed some regularities, the thermodynamic efficiency being in
most instances about 0.55 except for substrates of a high degree of
reduction for which carbon conservation is the governing factor. It
is interesting to see if such general tendencies also exist in anaerobic
processes. The thermodynamic efficiency of anaerobic growth will
be defined analogous to that of aerobic growth, the free energy of
combustion conserved in the biomass relative to the sum of the free
energy of combustion conserved in the biomass and the dissipation
in the process. Using the stoichiometry considerations treated in
Sec. 4.2, this quantity can be calculated from a free enthalpy balance
if the substrate yield factor Yjxis known.
Table XI1 summarizes the results of such calculations for a fairly
limited number of literature data on anaerobic growth. The proce-
dure of calculation is essentially the same as for aerobic growth
(Appendix 11). As can be seen, the thermodynamic efficiency does
not vary much if one exception, growth on formate, is left out. The
efficiency is in all cases about 0.70 although the substrate yield factor
varies from 0.057 to 0.26. Again the concept of the thermodynamic
efficiency can most probably be advantageously used for a prelim-
inary estimation of growth yields in anaerobic product formation
processes. The thermodynamic efficiency on anaerobic growth
seems to be 10 or 15% higher than the aerobic growth efficiencies.
An enthalpy balance can serve to calculate heat production on
anaerobic growth. As an example the alcohol fermentation will be
treated. On aerobic growth of the yeast Succharornyces cerevisiue
on glucose with ammonia as a nitrogen source a typical growth yield
Yyx = 0.57 is obtained, this corresponds to a qc value of 0.59 and
a heat production per unit biomass produced of 334 kJ/C mol biomass
produced. On anaerobic growth a typical yield factor of 0.14 is ob-
tained, this corresponds to a heat production of 90 kJ/C mol pro-
TABLE XI1
Growth Yield, Thermodynamic Efficiency, and Carbon Conservation in Products on Anaerobic Growth
Y, EP
(C mol DM/C (C conservation
mol in products
Energy-generating reaction Ref. substrate) qth other than CO,) 3
Glucose -+ ethanol + COz (2 : 1)” 34 0.12 1-0.159 0.62-0.68 0.57 (0.67)’
Glucose + lactate 34
0.129-0.242
Glucose -+ acetic acid + ethanol 34 0.69-0.83 0.57-0.86 (1)
+ formic acid ( I : I : I )
Glucose -+ propionic acid + acetic acid 46 0.238 0.72 0.67 (0.89)
+ CO2 (6:2: I )
Glucose -+ methane + acetic acid 46 0.260 0.71 0.55 (0.75)
+ CO, (1:2:1)
Acetic acid -+ methane + CO, (1 : I ) 46 0.057 0.70 0.47 (0.50)
Methanol -+ methane + CO, (3: 1) 46 0.219 0.60 0.64 (0.75)
Formate -+methane + CO, ( 1 : 3) 46 0.043 0.31 0.23 (0.25)
Propionic acid -+methane + CO, (1.4: 1) 46 0.077 0.74 0.54 (0.58)
a Numbers in parentheses are the ratios of the C mol of the various C containing compounds formed in the product formation reaction.
’Numbers in parentheses are the theoretical maxima of the carbon conservation in products other than CO,.
2496 ROELS
duced. These values make clear that anaerobic growth results in a

smaller heat production than aerobic growth, even if the heat pro-
duction is expressed per unit biomass produced. This conclusion is
expected to be of a fairly general validity for most cases of anaerobic
growth. This phenomenon is of particular importance in the bakers’
yeast production process, where, due to the Crabtree or the Pasteur
effects, metabolism may become partially anaerobic. In that case
heat production per unit biomass produced has to be expected to
become lower with increasing fermentative action. This contrasts
the conclusions of a recent publication of Wang et a1.* These authors
report increasing heat production with increasing fermentation ac-
tivity.
4.4. Conclusion
In this section the relation between various efficiency measures
proposed in literature, the concept of Yavlc and Minkevich and
Eroshin’s’6 q were analyzed in terms of thermodynamics of open
systems. It was shown that the quantities q and YaVl,are proportional
and contain to a good degree of approximation the same information
as the thermodynamic efficiency, q t h , which is derived from the for-
mally more correct free enthalpy balance. It is shown that the for-
mulation of efficiency measures different from qth,notably qe,the
efficiency with respect to enthalpy transformation, and lo,an effi-
ciency with respect to oxygen exchange, allows the formulation of
simple expressions from which oxygen consumption and heat pro-
duction can be easily calculated. These concepts also allow an anal-
ysis of the validity of the principle of constancy of the ratio of the
rate of heat production to the rate of oxygen consumption, which
is shown to be not valid for growth with the nitrogen source HNO,.
An analysis of literature data shows some regularities to exist.
The thermodynamic efficiency of aerobic growth is shown to be
about 0.55 except for highly reduced substrates, where carbon lim-
itation takes over. The maximal carbon conservation seems to be
0.6 on the average. As far as carbon conservation, heat production,
and oxygen consumption are concerned substrates with a degree of
reduction between 4 and 6 are, for growth with ammonia as a ni-
trogen source, to be considered optimal for biomass production
(SCP).
Straightforward application of these observations to the case of
wastewater treatment results in a relationship between sludge pro-
duction and chemical oxygen demand as generally accepted in lit-
erature on the subject. The concept of the thermodynamic efficiency
of growth is also of value in the treatment of anaerobic processes.

A limited literature survey indicates that the efficiency of anaer-
obic growth is in most cases around 0.7. It is also shown that an
enthalpy balance gives an incorrect expression of dissipation in an-
aerobic processes. Heat production, even when expressed per unit
of biomass dry matter produced, is, compared with aerobic growth,
lower in anaerobic processes.
In future, further development of the application of energy and
entropy balances mainly has to be directed to the treatment of an-
aerobic growth, the treatment of aerobic growth being well ad-
vanced. Important questions to be solved in the treatment of aerobic
growth are the why of a maximum carbon conservation of about 0.6
and a thermodynamic efficiency of 0.55. It is the belief of this author
that macroscopic methods can provide no clues to those matters;
it is a subject where biochemical considerations come into the pic-
ture.
The thermodynamic treatment of anaerobic growth has to be ex-
tended to cover a larger amount of data. Furthermore, uncertainties
exist as far as the free enthalpy change at the concentrations oc-
curring in actual practice are concerned. This may affect the cal-
culations, the present treatment refers to free enthalpy changes at
unit molality of the reactants.
5. LINEAR RELATIONSHIP FOR SUBSTRATE CONSUMPTION
Since the advent of continuous culture various workers have em-

phasized 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 max-
imum 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
2498 ROELS
theoretical aspects of microbial energetics and the constancy of Y ,

and m , has been doubted. However, for technological applications
eq. (49) only needs to be considered a fairly good approximation to
the relationship between r s and r x , the physiological significance is
much less important. In a number of cases significant amounts of
a product are formed from the substrate and eq. (49) is no longer
adequate in all cases. For these cases eq. (49) can be generalized
fo1,91.
- r s = r x / Y S x+ r p / Y s p+ msCx (50)
Equation (50) defines a yield constant for product, it is the maximum
amount of product that is formed per unit substrate converted. For
a system in steady state, eq. (50) can be translated into a relationship,
which applies to the various flows of compounds to the system:
as= -@xlYsx - + m,C, (51)
Equation (51) was .obtained from eq. (50) by application of the
steady-state condition given by eq. (6).
If eq. (51) is combined with the equations for the relationships
between the various flows in a system with product formation (Sec.
3.1, Table I), expressions for the flows of oxygen, carbon dioxide,
nitrogen source, and water can be derived. The two most important
equations, those for oxygen consumption and carbon dioxide pro-
duction, take the form:
Qo = -@xlYox - + moCx (52)
QC = @JYCx+ - 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 con-
venient to make use of eqs. (38) and (41) for an estimation of heat
production and oxygen consumption. As the quantities o, and wo
TABLE XI11
52
0
Relationships between the True Yield Factors and Maintenance Factors P
m
Compound True yield factor for biomass True yield factor for product Maintenance factor
Oxygen y, = 4 Y S A Y , - yxYsx) Y , = 4Y&, - %Y,) mo = y,m,/4
$r!
Carbon y,, = YJ(1 - a4dJazd4) Y, = Y&1 - a,d2/a2d4) $!a
dioxide - YJl - a4d,/d4)1 - Ysp(l - a4d,/a,d,)l m , = m,(l - a,dz/a2d4)
??
F
Fl
2500 ROELS
are not affected by the postulation of the linear law, a combination

of eq. (51) (with Y , = 03) and the definitions for qc and qo given
by eqs. (37) and (42) results in the following expressions:
q e = pY,/[wc(p + msYsJ1 (54)
qo = F Y s x ~ b o ( p+ msYsx)l (55)
SPECIFIC GROWTH RATE 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 pro-
duced as a function of specific growth rate. Methane as the substrate.
BIOENGINEERING REPORT: MICROBIAL METABOLISM
SPECIFIC GROWTH RATE p (h-' 1

(b)
Fig. 7 (continued from previous page)
On substitution of qe and qo given by eqs. (54) and (55) in eqs. (38)
and (411, the oxygen flow and the heat flow result. Using repre-
sentative values of rn, and Ysx.' for growth on the substrate glucose
and methane, the plots presented in Figure 7 were obtained. As can
be seen heat production and oxygen consumption per unit biomass
produced do dramatically increase if the growth rate becomes less
than 0.1 hr-'. Several interesting conclusions can be drawn from
this treatment:
1) Productive fermentations, aimed at the production of a maxi-
mum amount of biomass, should be operated at high growth rates,
as in that case not only the substrates costs per unit product do
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 bio-
mass produced (sludge) is a nuisance, the growth rate should be as
low as possible, hence recirculation of biomass would be an advan-
tage.
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 sub-
strate can hence be expressed as
CPs -CPx/Ysx + msCx
= (56)
The expression for the rate of product formation becomes:
QP = [(ys - ~ x Y s x ) / ( y , Y s , ) -l ~ (ysmsIyp).C,
~x (57)
Equation (56) shows, that in the case of the anaerobic formation of
one product (or a constant ratio mixture), there does not appear a
separate contribution for the rate of product formation in the flow
of substrate CP,. This problem was discussed earlier by Roels and
Kossen' using a different approach. The result can be interpreted
as follows. On anaerobic product formation, energy generation is
directly coupled to the formation of the product and as energy is
assumed to be used for two purposes-the construction of new bio-
mass and cellular maintenance-the rate of product formation is
proportional to the rates of these processes. Equation (57) shows
that the rate of product formation thus contains two contributions,
the first proportional to the rate of biomass production, the second
proportional to the amount of biomass present. An equation of this
type has been proposed for the lactic acid f e r m e n t a t i ~ nand
~ ~ it was
generalized later on to cover all product formation p r o c e ~ s e s ; ~ ~ * ~ ~
it specifies a growth-associated and a non-growth-associated term
in product formation processes. A rationale behind these equations
is provided by the present stoichiometric treatment in terms of the
linear equation for the consumption of substrate. By combination
of eqs. (57) and (56) an interesting equation for the fractional con-
version of substrate carbon to product carbon, eP,can be obtained:
Equation (58) can be more compactly written
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 ref-
erence 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 infin-
ity..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 cere-
visiae, 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 ATP’s in yeast’s.” If it is
assumed that this causes the yield to decrease by a factor of ap-
proximately 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)
where qo is the efficiency factor as defined by eq. (42).
The situations represented by the processes, compiled in Table
XII, are shown in a plot according to eq. (61) in Figure 9. It has to
be pointed out that the values of eP do not represent measured values
2504 ROELS
EFFICIENCY FACTOR 7&
Fig. 8. Carbon conservation in products on anaerobic product formation.
but are calculated from stoichiometry considerations. Figure 9

shows that for most processes there is room for considerable im-
provement as far as the conservation of carbon in products is con-
cerned. This approach may become quite relevant to judge the po-
tentialities of various anaerobic product formation processes as a
substitute for the oil-based production of chemical feedstocks.
6. INTEGRATION OF ELEMENTAL BALANCES AND

KINETIC EQUATIONS IN THE CONSTRUCTION OF A
KINETIC MODEL
In order to construct a simple, as far as the biomass is concerned,

unstructured model for a fermentation process at least one of the
reactions taking place in the culture has to be specified in kinetic
terms. A constitutive equation has to be formulated that expresses

the dependence of the rate of that reaction on the concentrations
of the various chemical components in the system, for example,
specified by the chemical state vector C, and the relevant intensive
parameters, like temperature, T, or pressure, p.
In general a complete set of constitutive equations for each of the
m chemical reactions taking place in the culture can be written as
follows:
= r(C, p , T ) (62)
EFFICIENCY FACTOR qo
Fig. 9. Carbon conservation in products in various anaerobic processes on for-
mation 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 ) .
2506 ROELS
The net conversion rates of each of the components present in the

system follow with the aid of eq. (4):
r A = r ( C , p , T).a (63)
For a system in steady state the net production rates are equal to
minus the flow to the system, as is clear from eq. (6). Furthermore
the elemental balance principle according to eq. (1 1) specifies k re-
lationships between the flows ( k is the number of chemical elements
considered) and hence only n - k net conversion rates can be chosen
independently. From this reasoning it becomes clear that the number
of independent kinetic equations to be postulated cannot be chosen
at will, it is completely specified by the number of elemental balances
and the number of components in the system.
The procedure for the construction of a simple unstructured model
is outlined in Figure 10. Note that only in stages 1 and 4 creative
thinking is called for, the rest remains routine.
Now some specific examples of the principles will be outlined
briefly.
a) The aerobic growth without formation of product is considered,
only one carbon and energy source is used as well as one nitrogen
source and the only other exchange flows are oxygen, carbon diox-
ide, and water, i.e., the case treated in Sec. 3.2. The number of
compounds relevant to the system’s description can be considered
to be six. Hence the number of kinetic equations to be specified
becomes two, if elemental balances for four elements are consid-
ered. One equation, which can often be used, is the linear equation
for the consumption of substrate treated in Sec. 5:
-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 con-
centrations of the components of the state vector:
r s = AC,, ~ ,O ,C , , C c ,Cws)
C N HC (65)
Often a much more simple approach is used:
r , = AC,, C , ) (66)
As an example the Monod equation could be adopted
+ C,)
- r s = rs.maxCsCx/(Ks (67)
Equation (67) states the rate of substrate consumption to be a func-
tion of the substrate concentration and the concentration dry matter.
These kinds of models form the base of the classical continuous
D E C I D E WHICH N COMPONENTS
ARE RELEVANT TO THE
SYSTEM'S DESCRIPTION FOR
THE PURPOSE OF THE
MODELING EXERCISE
I
I I
DRAW UP A TABLE OF THE
ELEMENTARY COMPOSITIONS '< LITERATURE
EXPERIMENTS
OF THE COMPONENTS
CONSTRUCT THE If ELEMENTARY

BALANCES
t
SELECT (N-K) KINETIC
EQUATIONS
CONSTRUCT BALANCE
EQUATIONS FOR THE STATE-
VECTOR
Fig. 10 Flow sheet of the construction of a model.
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 com-
ponents 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.
2508 ROELS
b) The same reasoning applies to the anaerobic production of one

sole carbon-containing product from one carbon source.
Here again two independent kinetic equations are needed. These
can again be the linear law for substrate consumption (Sec. 5) and
a kinetic equation for the rate of consumption of the substrate. As
an example these notions can be applied to anaerobic production
of ethanol in which case 1 often has to apply a kinetic equation
allowing for product inhibiti~n,'.~'for example.
-rs = ~ s . I n a x C s K p C A ( K s+ C M p + Cp)l (68)
Many other examples can be added to the ones presented above,
but the main purpose of this section is to emphasize one important
principle: the number of independent kinetic equations used in any
model of a process can, as soon as the problem is specified in terms
of its state vector and the number of elemental balances, be no longer
chosen at will, it is determined by the restrictions put by macro-
scopic theory. To the author's opinion this has not always been fully
recognized in literature.
7. CONCLUSION
In this report the possibilities of macroscopic balancing methods

like elemental, energy, and entropy balances to growth and product
formation for aerobic as well as anaerobic growth processes have
been investigated. The theory is shown to be very useful in the in-
terpretation of data from a variety of fields of biotechnology. Aero-
bic processes have received considerable attention up to now; less
attention has been paid to anaerobic growth. The principles can also
be shown to be useful for providing a strategy for the kinetic mod-
eling of fermentation processes.
APPENDIX 1: CALCULATION OF THE ENTHALPY AND FREE ENTHALPY OF

FORMATION FOR BIOMASS
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 2 -+ C02 + 0.9 H20 + 0.1 N2
The enthalpy balance leads to
hbiomasr= + 560 - 257.4 - 394 = - 91.4 kJ/C mol
The free enthalpy of formation of biomass from glucose with gaseous ammonia as
a nitrogen source is calculated at about -30 kJ/C mol by Morowitz.’ The stoichi-
ometry of the reaction is given by
1.05 CH,O + 0.2 NH, -+ C H , 8 0 0 5No.z + 0.45 HZO + 0.05 COz
The free enthalpy balance results in:
&oma,, = - 30 - 160.7 - 3.3 + 107.1 + 19.8 = - 67.1
APPENDIX 11: CALCULATION OF W, AND 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 + 0.2 NH,’ + [1/(4Y:,)
- 1.05]02-tCH1,8005NO~+ (l/YL - I)HCOC
Equation (17) results in the following inequality using the free enthalpy values of
Table V:
-338/Y:’, + 0.8(-40.5) + [1/(2YZ) - 0.4]*(-238) + 0.2(-79.6) - 1.(-67.1)
- (l/Yix - I)(-%) 20
This can be written

131/YyT2 474
or
Nomenclature
constant in expression for heat production (kJ/C mol DM)
constant in expression for the oxygen consumption (mol/C mol
DM)
a; number of carbon atoms in 1 mol component i (dimensionless)
a ij number of atoms of atomic species j present in a molecule of
component i (dimensionless)
number of hydrogen atoms in one molecule of component i (di-
mensionless)
C state vector of the system (C mol/m’)
C, concentration of component i in the system (e.g., C ,, C,) (C moll
m’)
number of oxygen atoms on one molecule of component i (di-
mensionless)
2510 ROELS
COD chemical oxygen demand (g oxygen)

d, number of nitrogen atoms in one molecule of component i (di-
mensionless)
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)
system’s surface area (m2)
system’s entropy per unit volume (Him’ K)
time (hr)
system’s internal energy per unit volume (kJ/m’)
system’s 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 substrate’s 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)
Yi, true yield factor for product on component i (e.g., Y,, Yo,) (C
mol/C mot)
Greek
a stoichiometry matrix (dimensionless)
a;, number of moles of component j produced per unit rate of re-
action of reaction i (dimensionless)
generalized degree of reduction of component i (e.g., v,, 7),
(dimensionless)
fractional conservation of the substrate’s C in products other than
COz (dimensionless)
vector of rates of flow to the system (C mol/m3 hr)
rate of flow of component i to the system (e.g., a,, as)(C mot/
m3 hr)
efficiency factor with respect to heat flow (dimensionless)
efficiency factor with respect to oxygen flow (dimensionless)
efficiency factor in anaerobic product formation (dimensionless)
BIOENGINEERING REPORT: MICROBIAL METABOLISM 251 1
thermodynamic efficiency (dimensionless)

vector of specific molar free enthalpies (kJ/C mol)
specific growth rate (hr-’)
specific molar free enthalpy in the standard state at unit molality
(kJ/C mol)
entropy production rate (kJ/m’ “K hr)
maximum substrate yield factor with respect to heat flow (C mol
DM/C mol)
maximum substrate yield factor with respect to the second law
(C mol DM/C mol)
WO maximum substrate yield factor with respect to oxygen flow (C
mol DM/C mol)
Subscripts
1, x biomass
2, s substrate
3,P product
4 nitrogen source
5, 0 oxygen
6, c carbon dioxide
7. w water
The author is indebted to Professor L. E. Erickson for his constructive evaluation

of a draft of this report.
References
1 . J. A. Roels and N. W. F. Kossen, Prog. Ind. Microbiol., 14, 95 (1978).
2. T. L. Hill, An Introduction to Statistical Thermodynamics, 2nd ed. (Addison
Wesley, Reading, 1962).
3. D. M. Himmelblau and K. B. Bischoff, Process Analysis and Simulation: De-
terministic Systems (Wiley, New York, 1%8), p. 59.
4. A. G. Fredrickson, D. Ramkrishna, and H. M.Tsuchiya, Math. Biosci., 1,327
(1967).
5. P. Glansdorff and I. Prigogine, Thermodynamic Theory of Structure, Stability
and Fluctuations (Wiley-Interscience, New York, 1971).
6. E. Schrodinger, What Is Life? (Cambridge U. P., New York, 1944).
7. H. J. Morowitz, Energy Flow in Biology (Academic, New York, 1968).
8. I. Prigogine, Introduction to the Thermodynamics of Irreversible Processes,
3rd ed. -(Wiley, New York, 1967).
9. R. Ark, Introduction to the Analysis of Chemical Reactors (Prentice Hall,
Englewood Cliffs, NJ, 1965).
10. F. Ayres Jr., Schaum’s Outline Series. Theory and Problems of Matrices
(McGraw-Hill, New York, 1974).
11. A. G . Fredrickson, R. D. Megee 111, and H. M. Tsuchiya, Adv. Appl. Mi-
crobiol., 13, 419 (1970).
12. S. R. Hoover and F. E. Allison, J . Biol. Chern., 134, 181 (1940).
13. E. H. Battley, Physiol. Plant., 13, 192 (1960).
14. G. L. Solomons, Process Biochem., 1, 307 (1966).
2512 ROELS
IS. J. S. Hamson, Process Biochem., 2(3), 41 (1967).

16. I. G. Minkevich and V. K. Eroshin, Folia Microbiol., 18, 376 (1973).
17. R. I. Mateles, Biotechnol. Bioeng., 13, 581 (1971).
18. D. E. F. Hamson, H. H. Topiwala, and G. Hamer, Fermentation Technology
Today-Proceedings of the 4th International Fermentation Symposium, Tokyo ( I n ,
Tokyo, 1972), p. 491.
19. W. A. Holve, Process Biochem., 11(10), 2 (1976).
20. D. A. Sukatsch and U. Faust, DECHEMA Monogr., 81, 197 (1977).
21. J. Takahashi, N. Uemura, and K. Ueda, Agric. Biol. Chem., 34, 32 (1970).
22. M. Kanazawa, in Single Cell Protein II, S. R. Tannenbaum and D. I. C. Wang,
Eds. (MIT, Cambridge, MA, 1979, p. 438.
23. G. Hamer, D. E. F. Hamson, J. H. Harwood, and H. H. Topiwala, in Single
Cell Protein 11, S . R. Tannenbaum and D. 1. C. Wang, Eds. (MIT, Cambridge, MA,
1975), p. 357.
24. H. Y. Wang, C. L. Cooney, and D. I. C. Wang, Biotechnol. Bioeng., 19, 69
(1977).
25. D. W. Zabriskie, Ph.D. thesis, University of Pennsylvania, Philadelphia, PA,
1976.
26. D. W. Zabriskie and A. E. Humphrey, AIChEJ., 24, 138 (1978).
27. J. J. Heijnen, J. A. Roels, and A. H. Stouthamer, Biotechnol. Bioeng., 21,
2175 (1979).
28. A. H. Stouthamer, Antonie van Leeuwenhoek, 43, 351 (1977).
29. L. E. Erickson, S. E. Selga, and U. E. Viesturs, Biotechnol. Bioeng., 20, 16
( I 978).
30. D. Herbert, in Continuous Culture 6 , Applications and New Fields, A. C . R.
Dean, D. C. Ellwood, C. G. T. Evans, and J. Melling, Eds. (Society of Chemical
Industry, London, 1976), p. 1.
31. C. L. Cooney, H. Y. Wang, and D. I. C. Wang, Biotechnol. Bioeng., 19, 55
(1 977).
32. L. E. Erickson, I. G. Minkevich, and V. K. Eroshin, Biotechnol. Bioeng., 20,
1595 (1978).
33. T. Bauchop and S. R. Elsden, J. Gen. Microbiol., 23, 457 (1960).
34. A. H. Stouthamer, Symp. SOC.Gen. Microbiol., 27, 285 (1977).
35. A. H. Stouthamer, in Microbial Biochemistry, J. R. Quayle, Ed. (University
Park Press, Baltimore, 19791, Vol. 21, p. 1 .
36. W. R. Mayberry, G. J. Prochazka, and W. J. Payne, J . Eacteriol., 96, 1424
(1968).
37. W. J . Payne, Ann. Rev. Microbiol., 24, 17 (1970).
38. W. J. Payne and W. J. Wiebe, Ann. Rev. Microbiol., 32, 155 (1978).
39. N. S. Kharasch, Bur. Stand. J. Res., 2, 359 (1929).
40. S. Nagai, Adv. Biochem. Eng., 11, 49 (1979).
41. C. L. Cooney, D. I. C. Wang, and R. I. Mateles, Biotechnol. Bioeng., 11, 269
(1968).
42. T. Imanaka and S. Aiba, J . Appl. Chem. Biotechnol., 26, 559 (1976).
43. D. G. Mou and C. L. Cooney, Biotechnol. Bioeng., 18, 137 (1976).
44. H. Wang, D. I. C. Wang, and C. L. Cooney, Eur. J . Appl. Microbiol. Bio-
technol., 5 , 207 (1978).
45. P. W. Wilson and H. H. Peterson, Chem. Rev., 8, 427 (1931).
46. P. L. McCarty, in Advances in Water Pollution Research, Proceedings of the
Second International Conference, Tokyo, J. K. Baars, Ed. (Pergamon, New York,
1963, Vol. 2, p. 169.
47. S. Aiba, S. Nagai, and Y. Nishizawa, Biotechnol. Bioeng., 18, 1001 (1976).
48. H. E. de Kok and J. A. Roels, Biotechnol. Bioeng., 22, 1097 (1980).
49. H. Y. Wang, D. G. Mou, and J. R. Swartz, Biotechnol. Bioeng., 18, 1811
( 1976).
50. A. H. Stouthamer, Antonie van Leeuwenhoek, 43, 351 (1977).
51. T. Shimizu, T. Furuki, T. Waki, and K. Ichikawa, J. Ferment. Technol., 56,
207 (1978).
52. S. Bauer and E. Ziv, Biotechnol. Bioeng., 18, 81 (1976).
53. H. K. von Meyenburg, Arch. Mikrobiol., 66, 289 (1969).
54. K. L. Schulze and R. S. Lipe, Arch. Mikrobiol., 48, 1 (1964).
55. R. C. Righelato, A. P. J. Trinci, S. J. Pirt, and A. Peat, J. Gen. Microbiol.,
50, 399 (1%8).
56. H. R. S. Mason and R. C. Righelato, J . Appl. Chem. Biotechnol., 26, 145
(1976).
57. S. Nagai and S. Aiba, J. Gen. Microbiol., 73, 531 (1972).
58. E. Hernandez and M. J. Johnson, J. Bacteriol., 94, 996 (1967).
59. Y. Nishizawa, S. Nagai, and S. Aiba, J. Ferment. Technol., 52, 526 (1974).
60. D. Herbert, in Recent Progress in Microbiology, Symposium of the 7th Inter-
national Congress on Microbiology, G . Tunevall, Ed. (Almqvist and Wiksell, Stock-
holm, 1959), Vol. 7, p. 381.
61. J. L. Luecke, U. Oels, and K. Schuergerl, Chem. Ing. Technol., 48,573 (1976).
62. N. Nishio, Y. Tsuchiya, and M. Hayashi, J. Ferment. Technol., 55, 151 (1977).
63. M. Dostalek and N. Molin, in Single Cell Protein II, S . R. Tannenbaum and
D. I. C. Wang, Eds. (MIT, Cambridge, MA, 1979, p. 385.
64. M. Kuraishi, N. Matsuda, I. Terao, A. Kamibayashi, and T. Fujii, in Microbial
Growth on CI Compounds (Proceedings of the International Symposium on Microbial
Growth on C , Compounds) (Society of Fermentation Technology, Osaka, 1979, p.
231.
65. C. L. Cooney, in Ref. 64, p. 183.
66. M. Moo-Young, T. Shimizu, and D. A. Withworth, Biotechnol. Bioeng., 13,
741 (1971).
67. B. T. Sheenan and M. J. Johnson, Appl. Microbiol., 21, 511 (1971).
68. J. H. Harwood and S. J. Pirt, J. Appl. Bacteriol., 35, 597 (1972).
69. M. Bewersdorff and M. Dostalek, Biotechnol. Bioeng., 13, 49 (1971).
70. L. E. Erickson, V. D. Kuvshinnikov, 1. G. Minkevich, and V. K. Eroshin, J .
Ferment. Technol., 56, 524 (1978).
71. J. J. Heijnen and J. A. Roels, Biotechnol. Bioeng., accepted for publication.
72. J. A. Roels, Biotechnol. Bioeng., 22, 33 (1980).
73. S. Aiba, M. Shoda, and M. Nagatani, Biotechnol. Bioeng., 10, 845 (1%8).
74. J. D. Linton and R. J. Stephenson, FEMS Microbiol. Lett., 3, 95 (1978).
75. M. Gallo and E. Azoulay, Biotechnol. Bioeng.,17, 1705 (1975).
76. S. Goto, A. Kitai, and A. Ozaki, J. Ferment. Technol., 51, 582 (1973).
77. Y. Amano, 0. Yoshida, and M. Kagami, J. Ferment. Technol., 53,264 (1975).
78. H. Asthana, A. E. Humphrey, and V. Moritz, Biotechnol. Bioeng., 13, 923
(1971).
79. J. H. Kim and D. Y. Ryu, J. Ferment. Technol., 54, 427 (1976).
80. Y. Amano, H. Sawada, N. Takada, and G. Terui, J. Ferment. Technol., 53,
315 (1975).
81. M. Moo-Young, T. Shimizu, and D. A. Whitworth, Biotechnol. Bioeng., 13,
741 (1971).
82. S. Nagai, T. Mori, and S. Aiba, J. Appl. Chem. Biotechnol., 23, 549 (1973).
2514 ROELS
83. S. Fukuoka, H. Hachiya, S. Motomatsu, K. Takeda, K. Suzuki, and Y. Tak-

ahara, in Microbial Growth on C , Compounds (Proceedings of the International Sym-
posium on Microbial Growth on C I Compounds) (Society of Fermentation Technol-
ogy, Osaka, 1975), p. 241.
84. J. F. Wilkinson, in Ref. 83, p. 45.
85. G. H. Bell, Process Biochem., 7(4), 21 (1972).
86. L. Dijkhuizen, M. Knight, and W. Harder, Arch. Microbiol., 116,77 (1978).
87. J. A. Servizi and R. H. Bogan, J. Sanit. Eng. Div. Proc. ASCE, 89(SA3), 17
(1963).
88. S. J. Pirt, Proc. R. SOC.Lond., Ser B , 163, 224 (1965).
89. 0. M. Neyssel and D. W. Tempest, Arch. Microbiol., 107, 215 (1976).
90. A. H. Stouthamer and C. W. Bettenhausen, Arch. Microbiol., 111, 21 (1976).
91. A. E. Humphrey and P. R. Jefferis 111, ZVth International Conference, GIAM
Meeting, Sao Paulo, B r a d (Brasilian SOC.Microbiol., Sao Paulo, 1973), Vol. 4, p.
767.
92. B. J. Abbott and A. Clamen, Biotechnol. Bioeng., 15, 117 (1973).
93. R. Luedeking and E. L. Piret, J. Biochem. Microbiol. Technol. Eng., 1, 393
( 1959).
94. T. Kono and T. Asai, Biotechnol. Bioeng., 11, 19 (1969).
95. T. Kono and T. Asai, Biotechnol. Bioeng., 11, 293 (1%9).
96. A. H. Stouthamer, Yield Studies in Microorganisms (Meadofield Press, Dur-
ham, 1976).
97. T. YamanC and S. Hirano, J. Ferment. Technol., 55, 156 (1977).
98. S. Aiba and M. Shoda, J. Ferment. Technol., 47, 790 (1969).
Accepted for Publication March 21, 1980

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Application of Macroscopic Principles to

J. A. ROELS, Laboratory for General and Technical Biology,

Biotechnology and Bioengineering, Vol. XXII, Pp. 2457-2514 (1980)

formation processes is derived. Finally the relevance of macroscopic principles to

In the methods of analysis used in physics two broad groups of

increasingly accepted. This report is an attempt to show the general

2. BALANCE EQUATIONS FOR EXTENSIVE QUANTITIES

2.1. Balance Equation for the Chemical State Vector

Equation (1) states that the total amount of an extensive quantity

r A is the vector of the net production rates of each of the compounds

For a system of constant volume, in which no gradients occur,

Combination of eqs. (3) and (4) allows the formudion of a balance

2.2. Conserved Quantities: The Elemental Balance Equations

the following structure:

limited number of compounds was considered in a so-called gross

2.3. Thermodynamic Treatment of Energy Transformations

are available for transfer to oxygen. A second measure proposed by

The second law of thermodynamics allows the formulation of a re-

3. CONCEIT OF ELEMENTAL BALANCE AND ITS

3.1. General Expressions for Growth with Product Formation

is considered, which grows on one sole source of carbon and energy,

variables. Which flows are to be chosen independently strongly de-

YLx = I@I/@3/ (20)

yx = 4+ bl - 2 ~ 1- d1[(4U4+ 64 - 2~4)/d41 (21)

yp = ( 4 ~ 3+ b3 - 2C3 - d3[(4~4+ b4 - 2~4)/d4]}/~3 (23)

The rationale for the definition is the concept of available electrons

to estimate the flows of biomass and product. By some rearrange-

3.2. Aerobic Growth on a Sole Source of Carbon and Energy

Elemental Composition and Degree of Reduction for Biomass of Various Sources

Average 4.19 5.78 4.79

that no measuring errors are present, the deviations may indicate

combination of two flows suffices to specify all the remaining flows.

3.3. Anaerobic Growth with Product Formation

This equation can be used to estimate the rate of product formation

product. For the anaerobic formation of ethanol from glucose this

powerful method in further A field, where further devel-

4. ENERGETIC ANALYSIS OF MICROBIAL GROWTH AND

the case of the free enthalpy balance it should be emphasized that

4.2. Aerobic Heterotrophic Growth without Product Formation

Two other restrictive values will be used in the treatment to fol-

h,' + 394 + 143(bz/az)

the entropy flow associated with matter is small as compared

4.2.1. Thermodynamic efficiency of growth

HNO3 ~f = 0.08 + 0.18~~ 0.042 least-squares

Organism Substrate 7s yyx qlh YW/. tl. Ref.

,+o. l SECOND LAW

The tendencies observed in Figures 3 and 4 can be summarized

DEGREE OF REDUCTION (ys)

efficiency has to decrease on growth on substrates of a high degree

Yyx = 0.05 + 0.12 ys ys 5 4.58 (334

Yyx = 0.60 ys > 4.58 (33b)

Glucose 0.54 0.53 3.33 3.26 26 85

ductivity of a fermentor in cases where the cooling capacity of the

j~ = (l/Yy,r - l/we).@I[hso+ 395 + 119(bJa2)] (36)

pected values of qc thus results for growth with ammonia as a ni-

on methane but is essentially due to the high energy content of the

4.2.3 Relationship between heat production and oxygen

approximation equal to q, (Table VIII) and eq. (45) can be approx-

in Yavle on growth on substrates with a high degree of reduction is

4.3. Anaerobic Product Formation

can, to a large degree of accuracy, be used in the case of aerobic

of the growth process was shown to be an interesting variable that

duced. These values make clear that anaerobic growth results in a