Journal of Biotechnology, 7 (1988) 113-130 113

Elsevier

JBT 00289

On material balance models

and maintenance coefficients in CSTRs
with various extents of biomass recycle

H.A. Haughney, D.M. Dziewulski and E.B. N a u m a n

Rensselaer Polytechnic Institute, Department of Chemical Engineering~ Troy, New York, U.S.A.
(Received 20 February 1987; accepted 18 September 1987)

Summary

This work considers the consumption of substrate as needed to maintain cell

populations in systems that are closed or partially closed with respect to biomass.
The maintenance coefficient is divided between mass and energy maintenance
terms. Analysis of data on anaerobic degradation of glucose to lactic acid and
ethanol shows the mass maintenance term to be negligibly small. The energy
maintenance term is large and variable. It is speculated that the energy is consumed
in a futile cycle and is not directly related to cell viability.

Maintenance coefficient; Material balance; Biomass recycle

Introduction

Our focus is on continuous flow systems which have achieved a steady state and
that are fully or partially closed with respect to biomass. Both aerobic and anaerobic
examples of such systems are possible. Total cell mass (as measured by dry weights
or turbidity) can be constant as can be the viable cell count (as measured by CFUs),
yet the carbon substrate is present in excess. Growth is suppressed by factors such
as product inhibition or nutrient deprivation; and in the limiting case of a

For abbreviations see p. 128.

Correspondence to: E.B. Nauman, Rensselaer Polytechnic Institute, Department of Chemical Engineering,
Troy, NY 12180, U.S.A.

0168-1656/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

114

long-lived, closed system, carbon consumption for growth can become negligibly
small. Under these circumstances of suppressed or negligible cell growth, it becomes
possible to examine the carbon balance quite closely. We propose a subdivision of
the traditional maintenance term into 2 components: energy maintenance and mass
maintenance. Energy maintenance will be represented by Me, while mass mainte-
nance will be represented by M m.
The energy maintenance coefficient measures the consumption of substrate as
used for the formation of energy, in effect ATP. The excreted products associated
with energy maintenance are the primary products of the energy producing metabo-
lism, e.g. carbon dioxide, ethanol, acetic acid, lactic acid and butanediol for the
glucose fermentations being considered here. The mass maintenance coefficient
measures the consumption of substrate associated with all excreted products other
than the primary metabolic products. It thus includes any substrate required to
maintain the structural and functional integrity of the cell population under
conditions of zero net growth. As defined here, it also includes substrate consump-
tion associated with secondary metabolic products, such as antibiotics. Further
refinements of the definition are obviously possible in systems where such products
are important.
The mass maintenance coefficient has particular significance in closed biomass
formation where a primary metabolic product is sought for commercial purposes. If
the mass maintenance coefficient is small, the yield of substrate to desired product
is high and no significant amount of substrate is consumed in maintaining the cell
mass. Mass maintenance could be further subdivided between secondary metabolic
products such as antibiotics and cell debris, but the present treatment combines
these contributions to M m. The mass maintenance term imposes an ultimate limit on
reaction efficiency when net growth is completely suppressed.
Both the energy maintenance and mass maintenance coefficients are related to
cell viability, although the relationship may be rather indirect. A primary purpose of
the present paper is to show that the mass maintenance coefficient is negligibly
small in a variety of systems which are closed or partially closed with respect to
biomass. The maintenance energy requirement (i.e. the production of desired
product) can remain high in such systems. The simplest explanation of these results
is that the energy is consumed in a futile cycle. Examples of futile cycles include
ATPase activity or inefficient synthetic pathways (Belaich et al., 1972).

Literature background

Material balances have been used for computer simulation of continuous cell
recycle reactors (Lee et al., 1983), as well as for estimation of yield and maintenance
coefficients (Ohleyer et al., 1985). This treatment considers substrate to be utilized
for production of biomass (both the mass and energy required for growth), energy
maintenance through product formation, and maintenance of biomass; therefore, a
substrate material balance should contain terms that represent each type of utiliza-
tion. Models in the literature may or may not include a term for 'substrate utilized
 115

for organic product' (energy maintenance term) in substrate material balances. The
following discussion considers the various definitions of cell maintenance.
Some examples of cellular activities that require maintenance are resynthesis,
osmotic regulation, heat loss to the environment, turnover of cell wall substances
and other cell components, preservation of the ionic composition of the cell,
resynthesis of protein during turnover, and motility. The carbon utilized for both
mass and energy maintenance could follow either the path of viability maintenance
(i.e. primary metabolite formation for energy maintenance and secondary metabo-
lite maintenance for mass maintenance) or the path of futile cycles, or even both.
The model does not depend on the exact path, or paths, that are followed, since the
mass and energy maintenance terms included in the substrate material balance exist
no matter what maintenance path is used.
Belaich et al. (1972) found that, during the anaerobic batch growth of glucose by
Zymomonas mobilis, the manner in which glucose was dissimilated did not vary with
the growth conditions. During growth no storage products were formed, and thus it
was concluded that the maintenance energy was wasted by ATPase activity. This is
an example of a futile cycle. According to Dawes and Ribbons (1964), cells require a
supply of energy for the essential processes for survival. When cells are deprived of
their energy source (i.e. the substrate) endogenous metabolism will be used to
provide the cell with the necessary energy to survive. Mason et al. (1986) have
postulated that a major portion of the maintenance energy could be provided by
endogenous metabolism (death and cell lysis) and that the maintenance requirement
is small relative to the cell breakdown and death.
The concept of maintenance is considered by McGrew and Mallette (1962) to be
the substrate carbon used to maintain the status quo of the biomass. Their batch
experiments with Escherichia coli indicated that the organism, when starved, utilized
the substrate, glucose, for maintaining viability, rather than for growth. In their
experiments high concentrations of cells were starved and then glucose was periodi-
cally added. The viability of the culture declined and there was no growth. Nilson
(1960), in a similar experiment, found that adding a continuous amount of glucose,
at a rate that would match the maintenance requirement, caused the cells to remain
viable, but with no growth.
According to Marr et al. (1963) the maintenance requirement in a CSTR is
evidenced by a decrease in steady state biomass concentration with decreasing
dilution rates, since more of the carbon source should be needed for maintenance
when the dilution rate is decreased. The continuous experiments of Doelle and
Hollywood (1978) indicate that the maintenance requirement of E. coli increased
with the increase in glucose concentration. They also found that the glucose
concentration affects biomass formation for both aerobic and anaerobic cultures,
since above the same glucose concentration biomass concentration reaches a maxi-
mum, thus all of the glucose cannot be utilized. Chesbro et al. (1979) and Arbige
and Chesbro (1982) found in their work with both E. coli and Bacillus polymyxa
that using a 100% biomass recycle reactor did not provide a steady state biomass
concentration (the organic products did reach a steady state), thus the maintenance
requirement was not constant during these experiments.
116

The definition of maintenance energy given by Pirt is the portion of energy

source not used for biomass formation, which implies all organic products plus mass
maintenance. Pirt (1974) considered maintenance primarily in the sense of an energy
balance. Since our work considers maintenance in terms of a material balance, a
mass maintenance coefficient can be defined to exclude substrate consumption for
product formation as well as the usual exclusion for biomass formation. Our work
revises the equations of Ohleyer et al. (1985) and Lee et al. (1983) by including a
term for product formation in the substrate material balance, since substrate
utilization for product formation is not accounted for by any other term.
Goma et al. (1979) have applied the concept of the redefined maintenance term
to a batch experiment involving Saccharomyces cerecisiae. The maintenance require-
ment decreased to zero during the course of growth. Humphrey (1986) has stated
that the substrate during steady state conditions is used for growth, maintenance,
and secondary metabolite formation. The yield parameters and maintenance coeffi-
cient are constant only when temperature, pH, salts, product and substrate are
constant.

Material balance models

Fig. 1 illustrates the configuration of a CSTR which includes provisions for cell
recycle and biomass bleed. A CSTR with an open effluent stream can be repre-
sented by setting F = 0 and B = Fin. A CSTR with 100% cell recycle is represented
by setting B = 0 and F = Fin. An overall material balance gives:
Fin--F+ B (1)
The dilution rate is defined as
D = F~n/V (2)
where V is the reactor volume (1).
A combination of Eqs. (1) and (2) yields an expression for the quantity B / V in
terms of dilution rate ( D ) and F/Fin (= R) (Nipkow et al., 1986):
B / V = D(1 - F/Fin ) = D(1 - R ) (3)

F.
I n

S.I s x p
n

>F
P V s p

Fig. 1. Reactor configuration. Fin = flow rate into reactor; B = bleed flow rate; F = permeate flow rate
(flow rates in 1 h - l ) ; sin= inlet substrate concentration; s = s u b s t r a t e concentration; x = b i o m a s s
concentration; p = product concentration (concentrations in g l - l ) .
 117

Equation (3) applies to a variety of reactor types:

* for CSTRs with recycle and bleed, (3) holds
* for CSTRs with an open effluent stream, B ~ V = D
* for CSTRs with 100% biomass recycle, R = 1, B / V = 0
A cell balance, neglecting death, is made by considering cell accumulation to be
equal to the cell growth, minus the cells leaving the system, or
dx B
d-T =/~x - --~x (4)
where # is the growth rate (h-l).
When the system is at steady state, cell accumulation ( d x / d t ) is zero, and the
cell balance becomes:
~, = B / V (5)
For the substrate material balance, substrate accumulation is set equal to the
substrate entering the reactor minus the substrate leaving the reactor (through both
the bleed and permeate streams) minus the substrate that is consumed. We now
suppose that the substrate has a single or predominant carbon source. This will be
glucose in subsequent examples. We also postulate that energy requirements are
supplied by only one metabolic pathway. Thus there is a single organic product
(such as lactic acid) or stoichiometrically linked products (such as ethanol and
carbon dioxide in a one-to-one molar ratio) associated with ATP production. In this
situation, the total energy and mass requirements are combined in the substrate
balance. These requirements have four components: mass required for growth,
energy required for growth, mass required for maintenance, and energy required for
maintenance. Thus the substrate balance can be written as
ds Fin F B /~x /tx
dt v S i n - - ~ s - --~s (Yx/s)m (Yx/s)e M m x - M e x (6)
where the two 'yield coefficients' now have special definitions.
The mass coefficient, (Yx/s)m, is based solely on the carbon content of the cells.
Thus it is a measure of cell stoichiometry. For an average carbon content of 0.462 g
per g of dry biomass (Erickson, 1979), we have (Yx/s)m = 0.866 g g-1 (biomass per
glucose).
The energy coefficient, (Yx/s)e, measures the energy required to produce new
cells. This number cannot be obtained directly from the call stoichiometry but can
be inferred from the production of product during periods of cell growth.
The maintenance requirements are similarly divided between mass and energy.
They have units of grams of substrate per gram cells per unit time. Neither is
directly related to stoichiometry, but M e can be inferred from the production of
product during periods of cell maintenance.
Equations (1) and (2) can be used to simplify the substrate material balance, and
since product concentrations will be used to infer (Yx/s)e and Me, it is useful to
rewrite the substrate balance as
~ (Yp/x)e q ]
d s = O ( si" _ s ) - x l~ + + M m + - - (7)
dt (Yx/s)m Yp/s Yp/~
118

Here, Yp/s is the product yield for substrate consumed by the energy metabolism.
Thus it is known from stoichiometry: Yp/s = 1 g g-1 (lactic acid per glucose) and
Yp/s = 0.511 g g-1 (ethanol per glucose). The non-stoichiometric relationships are
now confined to the quantities (Yp/x)e, Mm, and q where (Yp/x)e = mass of product
formed per mass of cells grown; q = mass of product formed per mass of cells
maintained per unit time.
The numerators for the energy growth and maintenance terms can be combined
q'=q+g(Yp/x)e (8)
Equation (8) indicates that the specific product formation rate (q') is a linear
function of the specific growth rate (g). Following Pirt (1974) we can say that
product formation is growth associated if q = 0 and (Yp/x), > 0, and is non-growth
associated if q > 0 and (Yp/x), ~<0. However, the present paper is restricted to
products of the energy-producing metabolism. Thus we would always expect the
mixed case with both q > 0 and (Yp/x)e > 0.
After simplifying Eq. (7) with Eq. (8), the steady state substrate balance becomes:
_ q'
O(Sin--S) ' ~ "[-Mmq- -- (9)
x ( ~rxTs ) m Yp/s
The product mass balance is:
dp E B
dt ~'P - --vP + q'x (10)
Since Eqs. (1) and (2) can be applied, Eq. (10) simplifies to:
dp
dt = q'x - Dp (11)
At steady state the product mass balance reduces to:
q' = D p / x (12)
Using Eqs. (5) and (8) Eq. (12) becomes
q" = D p / x = q + / . t (Yp/x) e = q + (Yp/x) D(1 - R) (13)
Linear regression of D p / x versus D ( 1 - R) makes it possible to determine the
parameters q and (Yp/x)e, and consequently whether product formation is growth
associated, non-growth associated, or mixed growth and non-growth associated.
The steady state substrate balance [Eq. (9)] can be combined with the steady state
equations for biomass [Eq. (5)] and product [Eq. (12)]:
D ( s i . - s) D(1 - R) Dp1
-- - --I" m m (14)
X (Yx/s)m + Yp/s x
A multiple linear regression of D ( s i n - s ) / x against D ( 1 - R) and D p / x yields
1/(Yx/~)m, 1/Yp/,, and M m as regression coefficients.
Equation (14) is the steady state equation for a CSTR with partial biomass
recycle, and can be utilized to determine the biological constants for a given set of
 119

data. The equation is generalized so that it can be applied to other reactor

configurations: a CSTR with 100% biomass recycle or a CSTR with an open
effluent stream, simply by using the appropriate value of R. However, it is not
possible to use the same values of (Yx/s)m, Yp/s, and M m for different reactor
configurations with a given organism. The values for these parameters must be
calculated separately for each reactor type. Cells are under different conditions in
each of the 3 reactor systems, and it is reasonable to assume that for a given reactor
type and organism, there exists a unique set of parameters.
The exact matching of growth rate to bleed rate via Eq. (5) is not fortuitous. The
growth rate assumes its steady state value due to some limiting factor. Here, we
assume # to be a function of substrate concentration (s), cell concentration (x), and
product concentration (p). Lee et al. (1983) use the following equation to describe
the dependence of # on s, x, and p:

# =/%(1 --p//Pmax)n(1 -- X//Xmax)m[s//(gs + S)] (15)

where/~0 is a constant (h-1); Pm~x is maximum product concentration (g 1-1); Xmax

is maximum biomass concentration (g 1-1); K s is substrate saturation constant (g
1-1); rn is the biomass inhibition power; n is the product inhibition power.
Equation (15) contains a product inhibition term, (1 -P/Pmax)", which predicts
that cell growth will cease at the maximum allowable product concentration, Pin,x"
Since high cell concentrations can be attained in recycle reactors, it is necessary to
consider the effects of cell inhibition in the equation for cell growth rate; this term
has the same form as the product inhibition term, (1-X/Xmax)". The term
representing substrate has the typical form of Monod kinetics [s/(K s + s)] (Monod,
1949).
Steady state data (i.e. x, s i n - s, and p at various dilution rates and recycle
ratios) from CSTR's with partial biomass recycle, CSTR's with 100% biomass
recycle, and CSTR's with open effluent streams can be applied directly to the
combined steady state balance Eq. (14), the growth rate Eq. (15), and to Eq. (13) so
that the constants in these equations can be found by regression analysis. Once the
constants are determined, the equations can be used to predict sin - s, p, and x for
any given D and R. Simplification is possible when sin - s, p, or x is constant.
Examples from the literature are used to illustrate how steady state data from the
three reactor types can be applied to the modeling equations. Simplifying assump-
tions are implemented when possible. For the CSTR with partial biomass recycle, it
is assumed that the growth rate is dependent only upon product inhibition.
Equations (15) and (5) are combined, considering only product inhibition; n is
assumed to be one, and I% is redefined as tXo/Pmax yielding:

P =Pmax - 1-~-(B/V) =Pmax - 1 D ( 1 - R) (16)

/% /%

Linear regression makes it possible to calculate/~0 and Pmax- The substrate balance,
Eq. (14), can be used to determine (Yx/s)m, Mm, and Yp/s through multiple linear
regression; while the product balance, Eq. (13), can be used to find q and (Yp/x)e
120

through linear regression. For a simulation, the three expressions [(16), (14), and
(13)] are required to predict Sin - - S , X , and p for given D(1 - R).
The 100% biomass recycle CSTR steady state data indicates that for this
particular case, sin - s and p are constant. It is assumed that since p is a constant it
is at Pmax" Thus only one equation is needed to model this reaction. Since # = 0 for
this flow system, Eq. (15) does not apply. Equation (14) can be applied to a CSTR
with 100% biomass recycle by setting R = 1
D(s~.- s) 1 Dp
-- - - - - + M m (17)
x Yp/s x
A linear regression of D(Sin- S)/X versus Dp/x yields the parameters Yp/x and
n m.
The steady state product balance equation [Eq. (13)], reduces to Eq. (18) when
applied to this reactor type, since bt = 0.
q ' - De - q (18)
X

The quantity Dp/x is essentially constant (q) for these data, even though there is
some scatter in the data. The product must be non-growth associated, since the
growth rate is equal to zero for this reactor type.
The data set for the CSTR has a constant value for the substrate utilization term,
( s i n - s). There are two equations that can be used for these CSTR data, Eqs. (13)
and (14). Making the necessary substitutions for a CSTR, Eq. (14) becomes:

O(Sin--S) 1 9 . 4 _ ~ Vp +M m (19)
X (Yx/s) m Yp/s x
A multiple linear regression of D(Sin - s ) / x against D and Dp/x makes it possible
to find 1/(Yx/s) m, 1/Yp/s, and M m.
The steady state Eq. (14) can be modified to determine Xmax ( a n d / o r Pmax), and
Yx/s, Yp/s, and Mm, if the quantity (Sin- S) is essentially constant (Marr et al.,
1963). Equation (14) can be modified to:

D(sin-S) (Yx/s)m= D ( 1 - Rl (Yx/s)mq,

Yp/s + (Yx/s)mM m (20)

The term (sin- s)(Yx/s) m is equivalent to the maximum concentration of biomass,

Xm~x, when the quantity (sin - s) is constant. Making this substitution and rearrang-
ing, Eq. (20) becomes:
1 (1-R) + (Yx/s)m 1 q' Mm(Yx/s)m 1
. . . . . + (21)
X X max Yp/s X max D X ma x D
In a similar fashion, Eq. (17) can be written in terms of Pmax:
1 rp/s (l-R)+ 1 q' Umrp/s 1
- - + - - (22)
X (Yx/s) m Pmax Pmax D Pmax D
since Pmax = (Sin -- s)Yp/s"
 121

E q u a t i o n (21) [or (22)] can b e used in a m u l t i p l e regression scheme, where 1/x is

regressed against 1/D a n d q'/D, for a c o n s t a n t R. T h e p a r a m e t e r s which c a n b e
d e t e r m i n e d are: Xma~, Pmax, Mm, Yx/s, a n d Yp/s.
E q u a t i o n (21) becomes, for a C S T R :

1 1 + __(Yx/s)m 1 q' Mm(Yx/s)m 1 (23)

X = Xma-----~ Yp/s Xmax D + Xmax D

T h e s t e a d y state p r o d u c t m a t e r i a l balance, Eq. (13) can b e m o d i f i e d for a C S T R

as follows:
Dp
x
=q+ ( Yp/x)mD (24)

T h e constants, q a n d (Yp/x)m, can b e f o u n d t h r o u g h a linear regression of Dp/x

a n d D. E q u a t i o n (19) or (23) a n d Eq. (24) are used to simulate the cell a n d p r o d u c t
c o n c e n t r a t i o n for a C S T R when ( s i n - s ) is constant.

Results and Discussion

O h l e y e r et al. (1985) have p r e s e n t e d b i o p r o c e s s d a t a (see T a b l e 1) for the

p r o d u c t i o n of lactic acid f r o m glucose in a C S T R with b i o m a s s recycle. T h e process
was p e r f o r m e d b o t h with a n d w i t h o u t a cell b l e e d stream. T h e o r g a n i s m used was
Lactobacillus delbrueckii, which is a h o m o f e r m e n t a t i v e lactic a c i d p r o d u c e r . A

TABLE 1
STEADY STATE DATA FOR CSTR WITH RECYCLE (OHLEYER ET AL., 1985)
D B sin x p s
0.59 0.012 58 64 57 0
0.43 0.023 59 46 58 0
0.43 0.032 61 44 59 0
0.43 0.035 85 36 57 27
0.59 0.037 59 48 56 2
0.59 0.065 60 36 50 7
0.43 0.065 83 22 49 32
0.59 0.080 63 26 48 14
0.25 0 85 17 58 26
0.36 0 60 38 58 0
0.40 0 83 40 57 23
0.59 0 60 41 57 0
0.90 0 85 51 59 27
1.06 0 60 72 59 0
1.67 0 60 115 57 0
2.45 0 60 106 59 0
2.55 0 62 118 59 0
2.80 0 62 140 57 4
122

TABLE 2
RESULTS OF M A T E R I A L B A L A N C E M O D E L F O R CSTR W I T H P A R T I A L BIOMASS R E C Y C L E
( O H L E Y E R ET AL., 1985)

Equation (16) parameters:

Pm~ = 61.88 g 1-1
/~o = 0.0057 g 1-1 h -1 (product per volume per time)

Correlation coefficient for regression:

R 2 = 0.8596

Equation (14) parameters:

M m = 0.035 g g - 1 h - 1 (substrate per biomass per time)
(Yx/s)m = 0.646 g g - 1 (biomass per substrate)
Yp/s = 1.138 g g - 1 (product per substrate)
Correlation coefficient for regression:
R 2 = 0.9954

Equation (15) parameters:

q = 0.370 g g - 1 h - i (product per biomass per time)
(Yp/,,) e = 8.293 g g - 1 (product per biomass)
Correlation coefficient for regression:
R 2 = 0.9232

cross-flow filtration unit was used to facilitate the recycle of biomass. According to
Ohleyer et al. (1985) the net biomass growth during steady state was negligible. The
cell viability was between 88-96% during steady state. The nitrogen requirement
during the steady state period for the 100% biomass recycle condition was low,
which indicates that growth duringthis period was negligible.
The data in Table 1 for a CSTR with bleed and for a CSTR with 100% biomass
recycle (B = 0) were treated separately with the material balance model equations
appropriate for each reactor configuration. The model predictions for the CSTR
with partial biomass recycle are presented in Table 2, while the predictions for the
CSTR with 100% biomass recycle are presented in Table 3.
The model can be tested by comparing the predicted parameters with literature
values. The expected value of (Yx/s)m is 0.866 g g - t (biomass per glucose), based on
an average carbon content of 0.462 g g-1 (carbon per dry biomass). (Goma et al.
(1979) used a value of 0.83 for Yx/s in their calculations.) The biomass yield (Yx/~)m
predicted by the material balance model for a CSTR with biomass recycle is 0.646,
which is in reasonable agreement with the literature value.
According to the stoichiometry of lactic acid production:

C 6 H 1 2 0 6 ---) 2 C 3 H 6 0 3 (25)
the maximum value of Yp/s is 1.0 g g-1 (lactic acid per glucose). The model
predictions for Yp/s in the CSTR with partial cell recycle, 1.138, and in the CSTR
with 100% cell recycle, 0.984, are relatively good predictions for this parameter.
 123

TABLE 3
RESULTS OF MATERIAL BALANCE MODEL F O R CSTR W I T H 100% BIOMASS RECYCLE
(OHLEYER ET AL., 1985)

sin - s = constant = 59.7 g 1-1

P = Pmax = 58 g 1-1

Equation (17) parameters:

M m = 0.0108 g g-1 h - 1 (substrate per biomass per time)
Yp/s = 0.984 g g - 1 (product per substrate)
Correlation coefficient for regression:
R 2 = 0.9941

Ohleyer et al. (1985) used their data to predict the maintenance coefficient ( M )
and the biomass yield (Yx/s). The results of their model are presented in Table 4.
The equations used by Ohleyer et al. (1985) are rewritten using the nomenclature
defined in this work:
CSTR with biomass recycle:
D[Sin - S - ( nsID)]/x = llYx/s B + M (26)
CSTR with 100% biomass recycle:
D[sin - s ] / x was plotted vs. D, with M as intercept (27)
where B = B~ V
Equation (26) contains an error - the ( B s / D ) term should not be in the equation
[compare with Eq. (14)]. In Eq. (27) the relationship between D ( s i n - s) and D is
not valid for a CSTR with 100% biomass recycle, since Eq. (26) predicts that
D ( s i n - s ) / x is e q u a l t o M , when there is 100% recycle (B = 0). The relationship in
Eq. (27) applies to a CSTR which is open with respect to biomass (B = D), not to a
CSTR with 100% biomass recycle.
The maintenance coefficient ( M ) and the biomass yield (Yx/s) for a continuous
recycle reactor with biomass bleed, using Eq. (26) without the ( B s / D ) term, are,
respectively, 0.36 g g-1 h-1 (glucose per cells per time) and 0.113 g g-1 (cells per
glucose), correlation coefficient R 2 = 0.9421. The Ohleyer et al. (1985) model
predicts a total maintenance coefficient, M = M e + Mm, which is an order of
magnitude greater than the mass maintenance coefficient found in the present
analysis. The material balance model of the present work defines a mass mainte-

TABLE 4
PARAMETER PREDICTIONS O F OHLEYER ET AL., 1985

Parameter CSTR with biomass recycle CSTR with 100% biomass recycle
M 0.33 0.6
Yx/s 0.114 -
124

nance coefficient that represents the amount of substrate required to maintain the
cell mass at a steady state and does not include the substrate utilized for energy
maintenance through product formation. Ohleyer et al. (1985) do not consider the
separate contributions of energy required for growth [l~x/(Yp/s)e] and mass re-
quired for growth [/tx/(Yp/s)m] in their substrate material balance, and this
accounts for the difference in biomass yield coefficients.
Ohleyer et al. (1985) reported a maintenance coefficient of 0.6 g g-1 h-1 (glucose
per cells per time) for a CSTR with 100% biomass recycle. The steady state substrate
utilization [(Sin- S)= 59.7 + 1.2] and the steady state product concentration [p =
58 + 0.9] are approximately constant over the given range of dilution rates. Equa-
tion (17) is applied to this data set. It is also possible to apply Eq. (20) to the data,
with R = 1, since the reactor is a CSTR with 100% biomass recycle, and with
(Sin- S)Yp/s =Pmax, since the quantity ( s i n - s) is constant. The appropriate sub-
stitutions lead to:
__
1 = _ _1 + M-m Y -p / s x
(28)
P Pmax Pmax Dp
Since the steady state product concentration is a constant for these data, it is
reasonable to assume p = Pmax, so that M m = 0. The experimental value, determined
by regression analysis of Eq. (17), M m = 0.0108, is not significantly different from
zero.
The product material balance (13), when regressed, yields q and (Yp/x)e as
parameters. These quantities can be compared to determine how much energy is
required for growth and maintenance. For the CSTR with partial biomass recycle,
q = 0.370 g g-a h-1 (product per biomass per time), the energy required for
maintenance is greater than the energy required for growth, (Yp/x)e/t0 = 0.047 g g-1
h-a (product per biomass per time).
Jobses et al. (1985) have transformed glucose to ethanol by Zymomonas mobilis
in a CSTR. Ethanol production, with this organism, is highly efficient, while there is

TABL E 5
S T E A D Y STATE DATA F O R CSTR (JOBSES ET AL., 1985)

D Sin x p $
0.0078 50.0 0.217 24.3 < 0.01
0.0096 49.4 0.22 23.9 < 0.01
0.020 50.5 0.26 24.3 < 0.01
0.032 49.9 0.36 24.2 < 0.01
0.035 49.0 0.47 23.3 < 0.01
0.039 50.0 0.47 22.2 < 0.01
0.049 48.6 0.49 20.8 < 0.01
0.049 52.5 0.58 25.5 < 0.01
0.075 51.0 0.65 22.1 < 0.01
0.122 50.1 0.86 23.2 < 0.01
0.133 49.8 0.96 21.4 < 0.01
0.237 51.4 1.44 22.1 < 0.01
 125

TABLE 6
RESULTS OF MATERIAL BALANCE MODEL FOR CSTR (JOBSES ET AL., 1985)
si, - s = constant = 50.2 g 1-1

Equation (19) parameters:

M m = 0.017 g g-I h-1 (substrate per biomass per time)
(Yx/s) m= 0.229 g g-1 (biomass per substrate)
Yp/s = 0.485 g g- 1 (product per substrate)
Correlation coefficient for regression:
R 2 = 0.9900
Equation (23) parameters:
Xm~x = 5.95 g 1-1
(Yx/s) m= 0.119 g g-1 (biomass per substrate)
Yp/s = 0.504 g g - 1 (product per substrate)
M m = 0.015 g g - i h-1 (substrate per biomass per time)
Pmax = 25.29 g 1-1
Correlation coefficient for regression:
R 2 = 0.9968

Equation (24) parameters:

q = 1.143 g g-1 h-1 (product per biomass per time)
(Yp/x) e = 11.172 g g- 1 (product per biomass)
Correlation coefficient for regression:
R 2 = 0.8128

a low conversion o f glucose to b i o m a s s a n d o t h e r p r o d u c t s . T h e s t e a d y state d a t a

for a c o n t i n u o u s c u l t u r e of Z. mobilis at 30 o C are p r e s e n t e d in T a b l e 5. T h e s e d a t a
were a p p l i e d to the m a t e r i a l b a l a n c e m o d e l a p p r o p r i a t e to a C S T R with o p e n
effluent, R = 0. T h e results are p r e s e n t e d in T a b l e 6.
B o t h Eqs. (19) a n d (23) have b e e n u s e d to regress the d a t a of Jobses et al. (1985).
E q u a t i o n (23) uses the a s s u m p t i o n t h a t the q u a n t i t y ( s i , - s ) is c o n s t a n t , a n d it
gives the b e t t e r c o r r e l a t i o n coefficient.
T h e m o d e l p r e d i c t i o n o f (Yx/s)m [0.229 using Eq. (19)] is c o n s i d e r a b l y lower t h a n
the e x p e c t e d value of 0.866. However, Z. mobilis is k n o w n to have a low efficiency
for the c o n v e r s i o n o f glucose to b i o m a s s (Jobses et al., 1985) a n d thus, the m o d e l
result m a y b e reasonable. T h e c o n v e r s i o n of glucose to e t h a n o l is r e p r e s e n t e d b y the
following s t o i c h i o m e t r i c e q u a t i o n :
C6H1206 ---) 2 C 2 H , O + 2 CO 2 (29)
T h e maximum value o f Yp/s for the f o r m a t i o n f r o m glucose to e t h a n o l is 0.511 on a
m a s s basis. T h e p r o d u c t yield, p r e d i c t e d b y the m o d e l [Eq. (19)], is 0.485, which is
c o m p a r a b l e to the m a x i m u m value for this p a r a m e t e r .
Jobses et al. (1985) use a relationship similar to t h a t used b y O h l e y e r et al.
(1985):
D(sin - s ) / x = (1/Yx/s)D+ M (30)
126

to determine the parameters (Yx/s) and M. The values determined by Eq. (30) for
(Yx/s) and M are, respectively, 0.0316 g g - t (cells per glucose) and 3.0 g g-1 h-1
(glucose per cells per time). Eq. (30) does not differentiate between energy and mass
maintenance or the energy and mass required for growth. This simplification causes
the difference between the parameters predicted by Jobses et al. (1985) and those
predicted by the present work.
The parameters q and (Yp/x)e/~ can be compared to determine how much energy
is required for growth and maintenance. For the CSTR the energy required for
maintenance, q = 1.143 g g-1 h-1 (product per biomass per time), is greater than
the energy required for growth, (Yp/x)e /~, at dilution rates lower than 0.10 and is
less than the energy required for growth for dilution rates above 0.10.
The cell death rate has been assumed zero. Under this assumption, the mass
maintenance term measures substrate utilization by viable cells to maintain their
total mass. An alternative assumption is a finite death rate followed by rapid cryptic
growth that reused most of the dead cell mass. The mass maintenance coefficient
would then correspond to the washout of cellular material before it could be
reutilized. The model is unable to distinguish between these possibilities, although
the assumption of zero death rate has the merit of simplicity.
The material balance model equations can be rearranged to predict the product,
substrate, and biomass concentration for a given dilution rate in a reactor. It is
necessary to know the biological parameters that apply to the reactor model
equations when performing this type of simulation. Lee et al. (1983) use a similar

80
• ° p

60

×- 4 0

20

0 I
0.05 0.10
B/V (h - l )

Fig. 2. CSTR with partial biomass recycle: product and biomass concentrations. D = 0.43. Symbols
represent data of Ohleyeret al., 1985; solid lines represent simulation.
 127

80

• - Sin - s

60

v
u~
i 40
c
u~

20

o I
0.05 O.lO
BIV (h "l )

Fig. 3. CSTR with partial biomass recycle: substrate utilization. D = 0.43. Symbols represent data of
Ohleyer et al., 1985; solid lines represent simulation.

30.

• - p
m - x

2O"

¢m

0 I I
0.1 0,2 0
0 (h "I )

Fig. 4. CSTR: product and biomass concentrations. Symbols represent data of Jobses et a|., 1985; solid
lines represent simulation.
128

approach in their mathematical model of steady state bioreactors, although the

equations used in their paper do not consider the contributions of the mass and
energy maintenance terms in their substrate material balance.
The parameters that were calculated for the data of Ohleyer et al. (1985) for a
CSTR with biomass recycle (Table 2) have been applied to the appropriate material
balance equations. The dilution rate is fixed in this simulation, and the concentra-
tion of product, biomass, and substrate utilized are plotted against the bleed rate
( B / V ) . Figs. 2 and 3 illustrate the simulation and include experimental data points
for comparison. In a similar fashion, the appropriate equations for a CSTR have
been used to simulate the experimental data of Jobses et al. (1985); these results are
presented in Fig. 4. The rearranged model equations appear to provide a reasonable
fit of the experimental data.

Conclusions

The computer simulation of Lee et al. (1983) and the parameter estimation of
Ohleyer et al. (1985) and Jobses et al. (1985) do not separate the energy mainte-
nance and the mass maintenance terms in their substrate material balances. The
present work has separated them and shows the mass maintenance term to be an
order of magnitude smaller than the energy maintenance term. Although our
regression analysis gave positive values for M m, they are not significantly different
from zero. Thus the efficiency of substrate conversion to primary metabolic prod-
ucts approaches 100% in a closed biomass system. The energy maintenance term,
Me = q/Yp/s, was found to vary with operating conditions even though an ap-
proximately steady state of viable cells was achieved in all cases. Thus the relation-
ship between M e and viability seems indirect. A simple explanation is that most of
the energy is consumed in a futile cycle.
The growth term is separated into mass required for growth and energy required
for growth in the substrate balance. This separation makes it possible to find the
energy t e r m (Yp/x)e (or the mass of product formed per mass of cells grown) as well
as (Yx/s)m, the mass coefficient for cell formation.
The material balance equations can be simplified when the quantity ( s i n - s) is
approximately constant. The revised substrate balance predicts Xm~ and Pmax, as
well as (Yx/s)m, Yp/~, and M m. Simplifying assumptions should be made, so that the
best equation can be used to predict the biological parameters.

Abbreviations

B = bleed flow rate (1 h-1)

D = dilution rate = F~./V ( h - a)
Fin = flow rate into reactor (1 h -a)
F = permeate flow rate (1 h-1)
Ks = substrate saturation constant (g 1-1)
 129

M = total maintenance (g g-1 h - l ) (substrate per cells per time)

Me = q~ Yp/s = energy maintenance coefficient (g g-1 h - l ) (substrate per cells
per time)
Mm = mass maintenance coefficient (g g-1 h - l ) (substrate per cells per time)
m = biomass inhibition power
n = product inhibition power
P = product concentration in reactor (g l - l )
Pmax = maximum product concentration in reactor (g 1-1)
q = mass of product formed per mass of cells maintained per unit time (g
g-I h-l)
q' = specific production rate (g g-1 h - l ) (product per biomass per time)
R = fraction of biomass recycled, F/Fin
V = reactor volume (1)
Sin = inlet substrate concentration (g 1-1)
S = substrate concentration in reactor (g 1-1)
X = cell concentration in reactor (g 1-1)
Xmax = maximum biomass concentration (g 1- l )
Yp/s = product yield for substrate consumed by energy metabolism (g g-1)
(Yp/x)~ = mass of product formed per mass of cells grown (g g - l )
(Yx/s)e = energy required to produce new cells (g g - l )
(Yx/s) m = carbon required to produce new cells (g g - l )
= specific growth rate (h -1)
~o = constant in growth rate equation (h-t).

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