F

So S
Xo=0
Po 0 P

Liquid Volume
-0
Working Volume
VR
S,X, P

Gas
Sporge
 6.4.3. The ldeal Chemostat

An ideal chemostat is the same as a

perfectly mixed continuous-flow, stirred-tank reactor
(CFSTR). Most chemostats require some control elements, such as pH and dissolved-
oxygen control units, to be useful. Fresh sterile medium is fed to the completely mixed
and aerated (if required) reactor, and cell suspension is removed at the same rate. Liquid
volume in the reactor is kept constant.
Figure 6.18 is a schematic of a simplified chemostat. A material balance on the cell
concentration around the chemostat yields
dX
FX- FX + VgH,X - VRk,X =V dt
(6.64)

Sec. 6.4 How Cells Grow in Continuous Culture 191

 Gas
Sparge
Figure 6.18. Simplified schematic of a chemostat.

where F is the flow rate of nutrient solution (1/h), Ve is the culture volume (1) (assumed
constant), X is the cell concentration (g/1), and u, and kj are growth and endogenous (or
death) rate constants, respectively (h). The reader should note that if cell mass is the pri-
mary parameter, it is difficult to differentiate cell death from endogenous metabolism.
When we use kj, we imply that endogenous metabolism is the primary mechanism for cel
mass decrease. With K, we imply that cell death and lysis are the primary mechanisms of
decrease in mass. The reader should also note that, if eq. 6.64 had been written in terms of
cell number, kj could only be a cell death rate. When balances are written in terms of cell
number, the influence of endogenous metabolism can appear only in the substrate balance
equation. Since most experiments are done by measuring total cell mass rather than num-
ber, we write our examples based on X. However, the reader should be aware of the ambi-
guity introduced when equations are written in terms of X.
Equation 6.64 can be rearranged as
dX
dt
=
DX, +(H -ka -D)X (6.65)

where D is dilution rate and D = FVR. D is the reciprocal of residence time.

Usually, the feed media are sterile, Xo = 0, and if the endogenous metabolism or
death rate is negligible compared to the growth rate (ka «< u) and if the system is at
steady state (dX/dt =
0), then
H=D (ifk = 0) (6.66)
In a chemostat, cells are removed at a rate equal to their growth rate, and the growth rate
of cells is equal to the dilution rate. This property allows the investigator to manipulate
growth rate as an independent parameter and makes the chemostat a powerful experimen-
tal tool.

192 How Cells Grow Chap. 6

 Since growth rate is limited by at least one substrate in a chemostat, a simple de-
scription of chemostat performance can be made by substituting the Monod equation (cq.
6.30) for u, in eq. 6.66:

HD=S
(6.67)
K,+S
where S is the steady-state limiting substrate concentration (g/). If D is set at a value greater
than 4 the culture cannot reproduce quickly enough to maintain itself and is washed out.
Equation 6.67 is identical to that for Michaelis-Menten kinetics and, as we discussed in
Chapter 3, a plot of 1/4, versus 1/S can be used to estimate values for , and K,
Using eq. 6.67, we can relate effluent substrate concentration to dilution rate for D<

S=
K,D
6.68)
-D

A material balance on the limiting substrate in the absence of endogenous metabolism

yields
ds
FS-FS-VH,X-VrAp
XIS YPIS d (6.69)

where So and S are feed and effluent substrate concentrations (g/), qp is the specific rate
of extraceilular product formation (g P/u, cells h), and Yys and Ypis are yield coefficients
Yyis denotes maximum value of
(g cell/g S and g Plg S). The use of the superscript M on a

the yield coefficient; such a superscript will be important in discussing the effects of
maintenance energy.
When extracellular product formation is negligible and the system is at steady state
(dSldt = 0),

DS-S)- (6.70)

Since , =D at steady state if k, =0,

X=Ys(S -S) 6.71)
the steady-state cell concentration can be expressed as
Using eq. 6.68,
K,D
X=YSo-D
m-D
(6.72)

coefficient (xis) 1s assumed to be constant, which is

In 6.71 and 6.72, the yield
eas.
metabolism has been neglected. Usually, Ye Varies
an approximation,
since endogenous
rate. Lnsider the eitect the inclusion of endogenous
with the limiting nutrient and growth
metabolism will have; eq.
6.66 becomes

D H-kj = Hoet (6.73a)

or
H D+k (6.73b)

in Continuous Culture
How Cells Grow 193
Sec. 6.4
By substituting eq. 6.73b into the steady-state substrate balance, assuming no extracellu-
lar product formation (eq. 6.70), we find

0
DS -S)-1/Yys(D+k, )X =

6.73c)
where Ys denotes the maximum yield coefficient (no endogenous metabolism or mainte
nance energy). Ys has a single constant value independent of growth rate. This approach
does not allow the direct conversion of S into maintenance enerey. Rather, S must be first
incorporated into the cel mass, where it is degraded by endogenous metabolism. This
viewpoint is the proper one when working with an unstructured model. With a structured
model, which explicitly recognizes intracellular S as a subcomponent of the biomass, the
direct consumption of S for maintenance functions could be modeled. However, with un-
structured models, substrate that is no longer extracellular becomes part of the biomass.
Equation 6.73c can be rearranged to

(6.74)
X

or
D k
=0 (6.75a)
XIS YIS XIS

1
ka yAP (6.75b)
YNs D YxIS

1
M ms
D (6.76)
Y YxIS

where

m, XIS
(6.77)

where m, is the maintenance coefjicient based on substrate S. Ys is the apparent yield.

When Yys is written, it should be interpreted as Ys While Ys is a constant, Y varies
with growth conditions if k,> 0.
Values of Ys and m, can be obtained from chemostat experiments by ploting
1/Ys against 1/D. The slope is m, and the intercept is 1/Ys (see Fig. 6.19).
In the presence of endogenous metabolism (Hne4S/(K, + S) - k). we can also
show that

S=A,0 + k)
-D-k, (6.78)

and

X=Ys1S) D Dk, (6.79)

In addition to the effects of maintenance, it is often appropriate to consider the

conversion of extracellular substrate into extracellular product. With an unstructured

194 How Cells Grow Chap.6

 3.0
Slope O.2 9glucose
g ceils-hr

2.5

2.0 Intercept
M2
or Yxus 0.5g cells/g glucose
L
O 2 3 5
1/D (hr)
Figure 6.19. Graphical approach to estimating Ys and m, for chemostat data for E. coli
growing on glucose as the limiting nutrient.

model, we must view this conversion as instantaneous. Otherwise, a perceptible period of

substrate uptake, prior to conversion to product, would cause a change in the amount of X.
This is unlike maintenance, since the period between substrate uptake and use for mainte-
we allow S to become incorporated into X and X is then
nance functions may be long, so
the folowing equations asSume instantaneous conversion of sub-
degraded when needed;
with the cell acting as a catalyst. The balance on product
strate to extracellular product,
formation is

DP=qpX (6.80)

6.16, 6.1 /, or 0.18. For

nongrowth -associated product for-
where qp is described by eqs. growtn-assoCiated products it is a function of
while for
is a constant (B),
mation, qp
6.69 becomes
For substrate balance, eq.

DS-S)= D+k,)X +-4pX (6.81)

YKs YPIs

from tne case bolism

With endogenous metabolism and yields
balance is
unchanged
The biomass

Continuous Culture 195

Grow in
How Cells
Sec. 6.4
 s-
S: K,(D+k) (6.78)
-D-k,

Equation 6.81 can be solved for X:

D
X=s-5) yM
YxIs (6.82)
D+ka*4P Yps
from DP and
he productivity of a chemostat for (Pr,) can be found
product and biomass
is found by differentiating
DX, respectively. The dilution rate that maximizes productivity of
to zero. The optimaB value
DP or DX with respect to D and setting the derivative equal
formation are
will on whether endogenous
metabolism and/cr product
D {Dopt) depend biomaSs pro-
considered. When k, 0 = and qp =
0, eqs. 6.71 and 6.68 apply. Then Dopt for
duction (DX) becomes

(6.83)
D K, +S0
usually much greater than K, Dopt will approach
is
D um 0r the washout point.=

Since So
the flow rate and liquid
Stable chemostat operation with D M,n is very difficult, unless
=

a value of D slightly less than

volume can be maintained exactly constant. Consequently,
also
between stability and biomass productivity. it should
Dope may be a good compromise will not necessarily be optimal for product
be apparent that Dopt for biomass formation
formation.
Examples 6.4 and 6.5 illustrate the use of these equations to characterize the perfor
mance of chemostats.

Example 6.4.

strain of yeast is being considered for biomass production. The following

data were
A new
concentration of 800 mg/l and an excess of
obtained using a chemostat. An influent substrate
at a pH of 5.5 and T 35°C. Using the following data, calculate m K,
=

oxygen were used

Ys ka and m,, assuming Hnet mS(K, +S) ka
-

Carbon substrate Cell

Dilution rate (h") concentration (mg/l) concentration (mg/l)

0.1 16.7 366

0.2 33.5 407
0.3 59.4 408
0.4 101 404
0.5 169 371
0.6 298 299
0.7 702 59

Solution The first step is to plot 1/Ys versus 1/D. Ys is calculated from X/(S, -S).The in
tercept is 1/YN = 1.58 or Ys= 0.633 g X/g S. The slope is 0.06 g S/g X-h, which is the value
for m, Recall m, = k,/Ys then k= m,YXs =0.06g S/g X-h 0.633 g X/g S=0.038 h.

196 How Cells Grow Chap.6

 For the second step, recall that

D=" - k, = - ka
K, +S (a)
Then

-=,K,1
D+k S (b)
0.8 h, The slope is 1 0
We now plot 1/(D + k)1/S. The intercept is 1.25 h
versus or =

/(mg/l). Thus, K,/4,m = 100 or K, = 80 mg/

Example 6.5.
The specific growth rate for inhibited growth in a chemostat is given by the following equation:

HK, +S+ IK,IK, (a)

where
KS=0.18cells
S= 10 g K, = 1 g I= 0.05 g 8 subs

Xo=0 K= 0.01 g/l 0.5 h k=0

a. Determine X andSas a function of D when I =0.
determine the effluent substrate concentration
b. With inhibitor added to a chemostat,
and X as a function of D.
the cell productivity, DX, as a function of dilution rate.
c. Determine

Solution
S=D-_D

a) -D 0.5-D
D
X=Ysis(So-S) =0.1 10 -
0.5-D
inhibitor
b) In the presence of

K1
S=- 0.5-D
m-D

6D
S0.5-D
6D
X =Yis(S,-5)=0.110-o5-D

Continuous Culture 197

in
Sec. 6.4 How Cells Grow
 6
)Pr, = DX =
DYys(S, -S) 0.1D
= 10
0.5-D)

6.4.4. The Chemostat as a Tool

The chemostat can be used as a tool to study the mutation and sclection of cultures and
also to study the effect of changes in the environment on cell physioloy. The molecular

aspects of mutation and selection will be discussed in Chapter 8.

Natural or induced mutations can take place in a chemostat culture. Errors in DNA
replication take place with an average frequency o f about 10 to 10 * gene per generation.

With a cell concentration of 10° cells/ml in culture, the probability is high in a chemostat
that a wide variety of mutant cells will be formed. The vast majority of naturaB mutations
in a chemostat are of little significance, unless the mutation alters the function of a protein
involved in growth in the chemostat environment. If the specific growth rate of the mutant
is larger than that of the wild type, then the mutant outgrows the wild type in a chemostat.
This selection for a variant cell type can be accomplished by creating a more favorable en-
vironment for growth of the mutant organism.
A chemostat culture can be used for the selection of special organisms. Selection or
enrichment nutrient media need to be used for this purpose. For example, if it is desired to
select an organism growing on ethanol, a nutrient medium containing ethanol and mineral
salts is used as a feed to a chemostat culture. An organism capable of oxidizing some
toxic refractory compounds can be selected from a mixed culture by slowly feeding this
compound to a chemostat. A thermophilic organism can be selected from a natural popu-
lation by operating a chemostat at an elevated temperature (e.g., 50° to 60o°C). Selection in
chemostats also presents significant problems in the culture of cells containing recombi-
nant DNA. The most productive cells often grow more slowly and are displaced by less
productive cells. We will discuss this problem in more detail in Chapter 14.