240 Optimization for Cell Mass Production

S0 = S m

Substrate Conc., S
Flow Rate, F
Sm

Time Time
Amount of Cells, XV

Reactor Volume, V V0
X 0V0
0
Time Time
Figure 12.2. Time profiles for initial concentration S0 = Sm .

Case 1: The initial substrate concentration is on the singular arc, that is, it equals
the substrate concentration that maximizes the specific growth rate, S0 = Sm
and μ(Sm ) = μmax .

This is the optimal choice of the initial substrate concentration. Because the initial
substrate concentration is already on the singular arc, that is, the optimal substrate
concentration Sm that maximizes the specific grow rate μmax , the singular flow rate
Fsin is applied from time zero to maintain the substrate concentration at Sm , until
the bioreactor volume is full, tfull , when no feed is added and the reactor is operated
in a batch mode until the final time, t f . Therefore, the optimal feed policy can be
summarized as follows:

S0 = Sm at t = 0
⎛ ⎞
μmax (XV )0
⎜Fsin = Y (S − S ) exp(μmax t), S held constant, S = Sm , 0 < t ≤ tfull ⎟
F =⎝ X/S F m ⎠
Fb = 0 batch operation, S decreases from Sm to S f , tfull < t ≤ t f
(12.64)

Typical time profiles of the optimum feed rate and substrate concentration are shown
in Figure 12.2.
With this optimal feed policy, the total cell mass increases exponentially from
time zero because the singular flow rate is applied throughout the filling stage and is
obtained by integrating Eq. (12.1),

XV = (XV )0 exp(μmaxt ) (12.65)

 12.2 Maximization of Cell Mass at Fixed and Free Final Times 241

while the bioreactor volume is obtained by integrating Eq. (12.3) with the singular
feed rate (Eq. (12.62)):
t
V (t ) = V0 + μmax X0V0 exp(μmax τ )dτ /YX/S (SF − Sm )
0
= V0 [YX/S (SF − Sm ) − X0 + X0 exp(μmaxt )]/YX/S (SF − Sm ) = α + β exp(μmaxt )
(12.66)

According to Eq. (12.66), the bioreactor volume increases exponentially. The cell con-
centration is obtained by dividing Eq. (12.65) by Eq. (12.66):
XV
X = = X0YX/S (SF − Sm ) exp(μmaxt )/[YX/S (SF − Sm ) − X0 + X0 exp(μmaxt )]
V
(12.67)

According to Eq. (12.67), the cell concentration can increase, decrease, or remain con-
stant, depending on the sign of YX/S (SF − Sm ) − X0 ,

dX X0YX/S (SF − Sm )[YX/S (SF − Sm ) − X0 ]μmax exp(μmaxt )

= (12.68)
dt [YX/S (SF − Sm ) − X0 + X0 exp(μmaxt )]2

so that
⎧ ⎫
⎪< 0 if X0 > YX/S (SF − Sm ), cell concentration decreases with time⎪
dX ⎨ ⎬
=0 if X0 = YX/S (SF − Sm ), cell concentration remains constant (12.69)
dt ⎪
⎩> 0 ⎪
if X0 < YX/S (SF − Sm ), cell concentration increases with time ⎭

The cell concentration will decrease if the initial cell concentration is larger than the
maximum cell concentration that can be obtained, YX/S (SF − Sm ), while the cell concen-
tration increases if the initial cell concentration is smaller than the maximum obtainable
cell concentration. It is interesting to note that the cell concentration can remain con-
stant at X = X0 = YX/S (SF − Sm ) during the entire period of singular feed rate, if the
initial cell concentration and the feed substrate concentration are chosen properly to
match the condition, YX/S (SF − Sm ) = X0 . The substrate concentration is maintained
constant during the entire period of feeding. These phenomena were noticed in the
exponentially fed fed-batch operation in Chapter 4.
During the batch period, F = 0 and V = Vmax , tfull ≤ t ≤ t f , and the cell and
substrate concentrations are related by Eqs. (12.1) and (12.3),
d(SV ) dS μXVmax V dX
= Vmax = −σ XVmax = − = − max S(tfull ) = Sm (12.70)
dt dt YX/S YX/S dt
which may be integrated from tfull to t to yield

(Sm − S) = (1/YX/S )(X − Xfull ) tfull ≤ t ≤ t f (12.71)

Because the cell concentration is related to the substrate concentration during the
batch period by Eq. (12.71) and the volume remains constant, the substrate balance
equation (12.2) may be written as
dS μ(S)X μ(S)[Xm + YX/S (Sm − S)]
=− =− tfull ≤ t ≤ t f (12.72)
dt YX/S YX/S
242 Optimization for Cell Mass Production

which may be integrated numerically to obtain the time profile of substrate concen-
tration.

12.2.3.10 A Special Case of Constant Substrate Concentration

As noted earlier, it is possible to pick the feed substrate concentration, the initial
substrate concentration, and the inoculum concentration to satisfy X0 = YX/S (SF −
Sm ) and S(0) = Sm and apply the singular (exponential) feed rate, Eq. (12.62), to
maintain constant the substrate and cell concentrations throughout the course of
the singular feed rate period. Then, the cell and substrate concentrations remain
constant, and the reactor volume increases exponentially during the singular flow
rate period (entire filling period):

X (t ) = X0 = YX/S (SF − Sm ), S(t ) = Sm

μ X V exp(μmaxt ) F (t )
F (t ) = max 0 0 , V (t ) = for 0 < t ≤ tfull (12.73)
YX/S (SF − Sm ) μmax

Case 2: The initial substrate concentration is less than the substrate concentration
at which the specific growth rate is maximum, S0 < Sm .

Because the initial substrate concentration is less than the optimal substrate concen-
tration Sm at which the specific growth rate is maximum, μmax , the maximum feed
rate, Fmax , is applied from time zero to t1 to bring the substrate concentration up
to Sm , at which point the singular flow rate, Fsin , is applied until tfull to maintain the
substrate concentration at Sm until the bioreactor is full, followed by a batch period:

S0 < Sm at t = 0
⎧ ⎫
⎪
⎨Fmax 0 < t < t1 S increases from S0 to Sm ⎪⎬
F = Fsin t1 < t ≤ tfull S held constant, S = Sm (12.74)
⎪
⎩ ⎪
⎭
Fb = 0 tfull < t ≤ t f S decreases from Sm to S f

This sequence is generally known as the bang-singular-bang control as a feed rate

profile consists of a period of maximum rate followed by a period of singular
flow rate and a minimum feed rate of zero (a batch period). Typical time pro-
files of the optimal feed rate and the substrate and cell concentrations are shown in
Figure 12.3.

Case 3: The initial substrate concentration is greater than the substrate concentra-
tion at which the specific growth rate is maximum, S0 > Sm .

Because the initial substrate concentration is greater than the optimal substrate
concentration, Sm , that maximizes the specific growth rate, μmax , it must be brought
down to Sm to be on the singular arc. Thus, no feed is applied initially, and a batch
period is maintained until the concentration is reduced to Sm , at which point, the
singular feed rate, Fsin , is applied to maintain the substrate concentration at Sm , until
 12.2 Maximization of Cell Mass at Fixed and Free Final Times 243

S0 < S m

Substrate Conc., S
Fmax
Flow Rate, F

Sm

S0

Time Time

Reactor Volume, V
Amount of Cells, XV

X 0V0 V0

Time Time
Figure 12.3. Time profiles for initial concentration S0 < Sm .

the bioreactor volume is full. A batch period follows. This sequence is generally
known as the bang-singular-bang control:

S0 > Sm at t = 0
⎧ ⎫
⎨Fmin = 0 0 < t < t2
⎪ Batch operation, S decreases from S0 to Sm ⎪
⎬
F = Fsin t2 < t < tfull Singular operation, S remains constant, S = Sm
⎪
⎩ ⎪
⎭
Fb = 0 tfull < t ≤ t f Batch operation, S decreases from Sm to S f
(12.75)

Typical time profiles of the optimal feed rate and the substrate concentration are
shown in Figure 12.4. These feed rate profiles suggest that the initial substrate
concentration should be properly chosen so that it is on the singular arc, that is, to
coincide with Sm to obtain the optimal result. It should also be noted that all of the
preceding results are obtained assuming that there is no constraint in the residual
substrate concentration. In practice, however, the final substrate concentration may
have to be much less than Sm for better utilization of substrate or to minimize the
residual substrate concentration so that it would not lead to difficult separation steps.
Thus, there is a batch period during which the substrate concentration is reduced to
a desired value. This will also be the case if one is treating toxic waste and wishes to
reduce its concentration to a desired value.
Let us investigate the Hamiltonian now. At the final time, the volume is full so
that F = 0. Therefore, the last term in Eq. (12.35) vanishes, and in the absence of
cell lysis or death, the Hamiltonian reduces to

H ∗ (t f ) = λ1 (t f )μ(t f )X (t f )V (t f ) = μ(t f )X (t f ) > 0 (12.76)

Thus, the Hamiltonian on the optimal path is a positive constant.

244 Optimization for Cell Mass Production

S0 > S m

Substrate Conc., S
Fmax S0
Flow Rate, F

Sm

0
t full tf t1 t full t f
t1
Time Time
Amount of Cells, XV

Reactor Volume, V
Vm

X 0V0 V0

t1 t full t f t1 t full t f
Time Time
Figure 12.4. Time profiles for initial concentration S0 > Sm .

At this point, it is proper to consider the effects of various operational parame-

ters: the maximum feed rate, the final time, the initial substrate concentration, and
the inoculum size.

12.2.4 Effects of Operating Parameters

12.2.4.1 Effect of Constraint on Maximum Feed Rate
When the upper bound on the feed rate is sufficiently large, as assumed earlier,
the general sequence of feed rate is bang (upper or lower) → singular → batch
or singular → batch. However, if the upper bound is not sufficiently large, the
singular feed rate, being exponential or semiexponential, can reach the upper bound
before the reactor is full, and therefore, the singular feed rate is short-lived and the
upper bound is applied until the reactor is full. Therefore, the sequence may consist
of bang (upper or lower) → singular → upper bang → batch. When the initial
substrate concentration is on the singular arc Sm , the optimal starting condition, the
feed rate sequence is singular → upper bang → batch. These cases are illustrated in
Figure 12.5.

12.2.4.2 Effect of Final Time

When the final time is too short, it is possible to have a sequence of bang →
singular → upper bang → batch. Because one has to use the maximum volume
available, Vmax , the minimum final time is the time to fill the reactor with the max-
imum feed rate, t f,min = (Vmax − V0 )/Fmax . In fact, when the specified final time is
less than t f,min , then the feed rate sequence is an upper bang → batch (a rapid fill
followed by a batch). It is also possible that the specified final time is not enough
to continue applying the singular feed rate and must be followed by an upper bang.