BIOTECHNOLOGY AND BIOENGINEERING,

VOL. XIX, PAGES 425433 (1977)

An Analysis of Extended and

Exponentially-Fed-Batch Cultures

HENRY C. LIM, BILL J. CHEN, and CHRISTOPHER C.

CREAGAN, School of Chemical Engineering, Purdue University,
West Lafayette, Indiana 47907

Summary
Under certain conditions it is shown that an extended culture is equivalent
to an exponentially-fed-batch culture, that an exponentially-fed-batch culture
(and an extended culture) can be maintained a t a steady state and that an
exponentially-fed-batch culture may be mimicked by a continuous-flow culture
with a constant dilution rate. Operational conditions required to maintain
steady states are specified.

INTRODUCTION
Semicontinuous operation of batch cultures in which a medium
containing the growth-limiting substrate is fed continuously has
been developed empirically for such industrial applications as
penicillin‘-3 and bakers’ yeast productions3 and waste disposals.
This type of culture operation was termed by Yoshida et al.4 as
“Fed-Batch Culture,” who tried fed-batch fermentation of hydro-
carbon and reported that the cell/substrate yield during the linear
growth phase was about twice as high as that during the exponential
growth phase. Edwards e t aL5 gave a brief treatment of what
they termed “extended culture,” a fed-batch culture in which the
limiting substrate concentration is kept constant by manipulating
the feed rate of the substrate. They also gave various examples
of industrial practices of extended culture. Apparently, the fed-
batch culture may be operated at a constant feed rate, a programmed
feed rate, or a n exponentially increasing feed rate.4-6 I n fact, a
device had been proposed which delivers exponentially increasing
amounts of nutrients and allows precise control of bacterial growth
rate.6 Pirt’~8 presented arguments to show that when the specific
growth rate of organisms follows that of a Monod type, a fed-batch
culture may reach a “quasi-steady state,” which is defined as dx/dt
425
01977 by John Wiley & Sons, Inc.
426 LIM, CHEN, AND CREAGAN

0 and ds/dt G 0 and called this a “Quasi-Steady-State Fed-Batch

Culture.” Ryu and Humphreyg used substrate feeding to control
the oxygen demand of a culture while Calam and Russell10 discussed
a computer model of a penicillin fermentation. A means of over-
coming catabolite repression of product formation by substrate
feed rate has been considered by Demain.“ I n spite of the impor-
tance of fed-batch cultures and their unique features, theoretical
analysis of fed-batch cultures has not been considered fully, with the
exception of the work of Dunn and Mor,I2 who discussed and gave
analyses of constantly-fed-batch cultures including the analogy with
a continuous-flow culture of decreasing feed flow rate. Apparently
exponentially-fed-batch culture, in spite of its ability to precisely
control the bacterial growth rate, has not been analyzed fully.
Edwards et al.5 gave a brief treatment of extended culture. It is
the purpose here to consider theoretically extended cultures and
exponentially-fed-batch cultures and to show that: 1) under certain
conditions a n extended culture is a n exponentially-fed-batch culture,
2) operational conditions under which a n exponentially-fed-batch
culture and a n extended culture may be reduced to ((steady-state”
fed-batch cultures ( d z l d t = 0 = d s / d t ) , and 3) the performance of a n
exponentially-fed-batch culture may be mimicked by a continuous-
flow culture operating at a constant dilution rate.

ANALYSIS
We begin by writing pertinent material balance equations for
fed-batch cultures. Henceforth we shall assume that the cultures
are run under constant environmental conditions including pH,
temperature, and other nutrients except the limiting substrate.
We shall also assume that the culture is pure and there is only one
limiting substrate. For convenience we will not consider any product
formation and consider the biomass (cell) as the product as in the
case of single-cell protein production.
Fed-Batch Culture
Consider a batch culture into which a nutrient medium containing
a fixed concentration of a limiting substrate SF is fed continuously
at a flow rate of Ffb. Initially the culture volume is Vo and the
culture contains fixed concentrations of cells and substrate, zo and
so. The maximum culture volume is VmaX. The appropriate
balance equations during the filling time period, 0 5 t _< t,
(/fa= ~ I b ( r )dT _< - v,),are
vmaX
 EXTENDED AND EXPONENTIALLY-FED-BATCH CULTURES 427

and

where u is the specific substrate consumption rate, p is the specific

growth rate, s is the limiting substrate concentration a t any time t,
x is the cell concentration, and V f b is the fed-batch culture volume
a t any time t. Any change in density is assumed negligible. Sub-
stitution of eq. (3) into eqs. (1) and (2) yields

(-+) (sp - s) - ux =
ds
-
dt
s(0) = so (4)

and

Provided that u and p depend on the limiting substrate concentration

under constant environment but not on the cell age distributions,
eqs. (3)-(5) describe the dynamics of fed-batch culture. Otherwise,
an additional cell age distribution equation as well as the precise
dependency of u and p on the age distribution must be known. We
assume that under constantly kept environmental conditions u and p
are dependent only on the limiting substrate concentration and
ignore the age distribution.
Exponentially-Fed-Batch Cultures
Apparently the fed-batch cultures are operated either at a constant
feed rate or a t an exponentially increasing feed rate.4-6 Constantly-
fed cultures have been thoroughly analyzed by Dunn and Morl*
and no further analysis is made here. Instead, exponentially-fed-
batch cultures are treated. For a fed-batch culture which is fed at
an exponential feed rate, F f b e = aebt, where a and b are arbitrary
positive numbers, eq. (3) reduces to
428 LIM, CHEN, AND CREAGAN

and the dynamics are given by eqs. (7) and (8),

b(SF - 8 ) ds
- -
( ( V o b - a)e-bf/a +1 ux =
dt
s(0) = so (7)

- bx dx
- px x(0) (8)
( ( V o b - a)e-bt/a +1 = -
dt
= 50

Thus, the dynamics of exponentially-fed-batch cultures is described

by eqs. (6)-(8). These equations are utilized below to analyze
limiting behaviors of exponentially-fed-batch cultures.
Extended Culture and Analogy to Exponentially-Fed-Batch Culture
An extended culture is a fed-batch culture in which the feed
flow rate is continuously manipulated to keep the limiting substrate
concentration in the culture constant at all times. Therefore, the
analysis of extended cultures can be made using the equations for
fed-batch cultures, eqs. (1)-(5) , with appropriate subscripts. Let
us denote by sex the desired limiting substrate concentration, F,, is
the flow rate required to keep the substrate concentration constant
a t sex,and V e xis the volume of extended culture. Then, the required
flow rate is obtained by setting ds/dt = 0 in eq. (4) :
Fex = Q X V ~ X / -
( ~ sFe x ) (9)
Even with this flow rate there would be a transient period in the
substrate concentration unless the initial concentration is also a t
the desired value, i.e., s(0) = sex. Therefore, we need to start
initially with the desired substrate concentration. If u depends on
only the limiting substrate concentration which is kept at sex, u
evaluated a t s = sex, u(sex)] is also constant. When the concentra-
tion of substrate in the feed is constant, eq. (9) suggests that the
flow rate F,, should vary in proportion to the total biomass in the
culture xVex,i.e.,
Fex = [u(sex)/(sF- sex)IzVex S(OX .= Sex = SO (10)
Hence, if the total biomass increases exponentially, the flow rate
must also be increased exponentially to keep the substrate concen-
tration constant. Under the assumption that the specific growth
and substrate consumption rates depend on the limiting substrate
concentration only, keeping the limiting substrate concentration
constant implies that a(seX) and p(s,,) are also constant. Therefore,
eq. ( 2 ) can be integrated to obtain
ZV,, = zoVoep(*e~)f sex = so (11)
 EXTENDED AND EXPONENTIALLY-FED-BATCH CULTURES 429

which states that the total biomass increases exponentially. Sub-

stitution of eq. (11) into eq. (10) yields
- sex)]er(sex)t= FexOer(sex)f
Fex = [(T(s,,)zOVO/(S~ (12)
Therefore, when (T and p depend only on the limiting substrate
concentration, the extended culture, in which the substrate concen-
tration is kept constant at its initial value, so = sex, is equivalent
to an exponentially-fed-batch culture with the flow rate given by
eq. (12). Note that Edwards et al.5 in their interpretation of the
observation made by Martin and Felsenfeld6 assumed that there
was a n arbitrary exponential function of the form Fex = F o p t ,
while the above analysis shows definite restrictions on the parameters
Fo and a to keep the limiting substrate concentration constant
at sex for all time. Indeed, the initial flow rate FexOdepends on
the initial amount of inoculum zOVO, the initial substrate concen-
tration sex = so, the initial specific substrate consumption rate
c(seX),and the feed substrate concentration S F .
The cell concentration history is obtained by solving eq. (11) for z,
z = zo(Vo/V)e”a~”t
= zo/[(l - c)e-’(sex)t + c] (13)
where
Q(S e x ) zo -
- (FexolVo)
c = Sex = SO
p(Sex)(Sp - sex) p(sex)

where Y ( s e x ) is the biomass/substrate yield coefficient evaluated

a t s = sex. Equation (13) suggests that the cell concentration can
increase, decrease, or remain constant a t ZO depending upon the nu-
merical value of c. When c is negligibly small, the cell concentration
can increase exponentially since the increase in the culture volume
over the initial volume is negligible over a short time interval.
Physically, this can happen when the initial dilution rate required
t o keep the substrate concentration a t sex as predicted by eq. (12)
is negligibly small as compared to the initial specific growth rate
(FeXo/Vo << p ( s e x ) ) . Alternatively c is negligibly small when the
potential cell production, Y(s,,)(sF - sex) /: Fex d7, is much
greater than that which is needed t o keep the cell concentration
at ZO, zo Fex dr. When the initial dilution rate is greater than the
initial specific growth rate of cells, i.e., c > 1, the cell concentration
430 LIM, CHEN, AND CREAGAN

decreases as it should and reaches asymptotically xo/c. A very

interesting situation can arise if c can be chosen to be unity. This
case is unique and will be considered in the next section.
Extended Culture and Analogy to Steady-State Fed-Batch Culture
According to eqs. (13) and (14), if one can match the initial
dilution rate of the exponential feed Fexo/Vowith the initial specific
growth rate p(so), which can happen when Y ( s e x ) ( s-~ sex) = ZO,
then c = 1 and not only the substrate concentration but also the
cell concentration may be kept constant a t the initial value x0.
I n other words, one can feed the limiting substrate at such a rate
that the cell generated and substrate consumed will keep up with
the increasing culture volume in such a way as to keep the cell and
substrate concentrations constant. Under this condition the ex-
tended culture becomes a steady-state fed-batch culture. Since x =
xo and s = so = sex,the flow rate is
Fex(t)= p(sex)Voe'(de')t= p ( ~ o ) V ~ e " ( ~ " ) ~ (15)
and the cell and substrate balance equations (eqs. (4) and (5))
require that
20 = c((sex)(sF- sex)/a(sex) = y(sex)(sF - Sex)

= Y(SO)(SF - so) (16)

which is a constraint among xo, sF, and so. Not all arbitrary initial
conditions are allowed. With the above feed rate and starting
initial conditions the culture volume increases exponentially,

V = Vo + 1:
F e x ( r )dr = Voec(80)1

the cell and substrate concentration remain invariant, x = zo and

(17)

s = so, and the dilution rate is constant,

FexIVex = ~(80) (18)
Thus, the extended culture of initial volume V o and the initial cell
and substrate concentrations x0 and so, respectively, which meet
the constraint of eq. (16), when fed with the exponential feed rate
of eq. (15), reduce to a steady-state fed-batch culture in which the
substrate and cell concentrations remain constant a t the initial
values. This type of operation not only keeps the environmental
factors constant but also keeps the cell concentration constant,
eliminating any potential interaction factor of changing cell densities.
 EXTENDED AND EXPONENTIALLY-FED-BATCH CULTURES 431

Indeed, this situation is analogous t o a steady-state chemostat

with the exception that there is a potential difference in age dis-
tribution as will be discussed later.
It should be possible to utilize this situation which is unique to
exponentially-fed-batch culture as one would in taking advantage
of the quasi-steady state for constantly-fed-batch cultures. How-
ever, it should be noted that with constantly-fed-batch cultures the
steady state may be reached only after a suitably long time, while
with exponentially-fed-batch cultures the steady state can be estab-
lished for all times.
Analogy Between Exponentially-Fed-Batch Culture and
Continuous-Flow Culture
The analogy between constantly-fed-batch cultures and dynamic
continuous flow cultures was discussed by Dunn and Mor.12 Interest-
ing observations unique to exponentially-fed-batch cultures that
have not been pointed out previously will be considered here.
As pointed out by Dunn and Mor,12eqs. (4) and (5) also represent
unsteady-state substrate and biomass equations for a continuous-
flow culture provided that the dilution rates are equal, i.e.,
Fc/Vc = Ffb/Vfb (19)
For a constantly-fed-batch culture, Ffb/Vfb decreases continuously
with time so that in order for the analogy t o hold the flow rate
to the continuous-flow culture must also be made t o decrease
with time. Therefore, it can be stated that the transients are
caused by the initial concentrations xo and so and also b y the decreas-
ing flow rate F,.
For the exponentially-fed-batch culture with the flow rate given
by Fibs = aebt, the corresponding flow rate for the continuous
culture is given by

F, =
bVc
(Vob - a)edbt/a +1 (vmaX
- + 1) (20)

The flow rate starts a t a V , / V o and approaches bV, .according t o

eq. (20). Unlike constantly-fed-batch cultures which can be
mimicked by continuous-flow cultures with a decreasing flow rate,
the performance of exponentially-fed-batch cultures can be mimicked
by continuous-flow cultures with decreasing as well as increasing or
constant flow rates, depending upon the quantity Vob - a. When
Vob - a > 0, the flow rate must increase with time, while the
flow rate must decrease when Vob - a < 0. When Vob = a, the
432 LIM, CHEN, AND CREAGAN

corresponding flow rate t o the continuous-flow culture must be con-

stant. Thus the performance of the exponentially-fed-batch culture
with the feed rate given by

can be mimicked by the use of a continuous-flow culture of constant

volume V c and a constant flow rate

Unlike the mimicking of constantly-fed-batch cultures by continuous-

flow cultures, in this situation the transients in the continuous-flow
culture are caused strictly by the initial substrate and biomass
concentrations and not by the flow rate (therefore, the dilution rate)
which must be kept constant.
It is apparent that at least in theory the dynamics of any fed-batch
culture, a constant-feed fed-batch culture, a n extended culture,
a n exponential-feed fed-batch culture, or a steady-state fed-batch
culture may be mimicked by the performance of equivalent
continuous-flow culture as long as the feed rate t o the fed-batch
culture as well as the initial conditions are known. Of course, the
earlier assumptions that u and p are functions of the limiting sub-
strate must hold. Otherwise the analogy breaks down.

DISCUSSION
Various types of fed-batch cultures were analyzed and certain
equivalences were established under the assumption that u and p
are functions of the limiting substrate concentration only. Under
specified conditions it was shown that an extended culture may be
equivalent to an exponentially-fed-batch culture. Operational
conditions were also specified which should force a n exponentially-
fed-batch culture (and therefore also an extended culture) to behave
as a steady-state fed-batch culture in which the biomass and sub-
strate concentrations remain constant for all times. I n practice,
however, establishment of these conditions requires exact knowledge
of u and p a priori.
Under the same assumption on u and p the substrate and cell
concentration dynamics of fed-batch cultures may be mimicked
by constant-volume continuous-flow cultures in which the transients
are caused by the initial subst,rate and cell concentrations and/or
the feed flow rate which may decrease, increase, or remain constant.
 EXTENDED AND EXPONENTIALLY-FED-BATCH CULTURES 433

It is particularly interesting to observe that a continuous-flow

culture with a constant dilution rate can mimic the performance
of an exponentially-fed-batch culture. However, if the mixing
in the continuous-flow culture is not “perfect,” i.e., instantaneous
and homogeneous, then the age distribution in the continuous-flow
culture from which the cells are continually removed may be different
from that in the fed batch from which no cells escape. Under this
situation the difference in cell age distributions could alter the specific
substrate consumption and specific growth rates and make each
culture uniquely different. Conversely, the differences, if any,
between the fed-batch culture and the corresponding continuous
culture could be attributed to the differences in the properties of
cell populations with different cell age distributions.
This work waa supported in part by a grant from the National Science Founda-
tion, Grant No. ENG 75-17796.

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Accepted for Publication October 25, 1976