Part IV

Bioreactor Engineering

Chapter 10 Ideal Bioreactors

There are two kinds of ideal bioreactors. One is perfectly
mixed bioreactor, including batch, fed-batch and continuous
operated bioreactors; and the other plug flow bioreactor. By
definition, there is no any gradient, including concentration,
temperature, etc. existing within perfectly mixed bioreactor.
Usually, small scale stirred vessels which are used for low
viscosity cell culture or fermentation systems can be listed
into this category. Conversely, the flow of biofluid through the
plug flow bioreactor is completely orderly without any element
of biofluid overtaking or mixing with any other element ahead
or behind, the residence time for all material is the same.
Usually, some tubular enzyme bioreactor when operated at
relatively high flow rate can be listed into this category.
2

10.1 Batch Operation of Perfectly Mixed Bioreactors

10.1.1 Enzyme Reaction

V, S, P

Figure 10.1 Flowsheet for

a batch bioreactor

General Mass Balance:

)
)
dM
= M i M o + RG RC
dt
Mass Balance for batch enzyme catalytic reaction

max S
d (VS )
=
V
dt
Km + S
Usually, V is constant, so we have:

S
dS
= max
dt
Km + S
4

Integrating:

dt =

Km + S
dS
max S

For initial condition S = S0 at t = 0:

Km
S0 S0 S f
tb =
ln
+
max S f
max
Enzymes are subject to deativation. Thus, the concentration of
active enzyme in the reactor, and therefore the value of max, may
change during reaction. When deactivation is significant, variation
of max with time can be expressed by:
5

 max = max 0 e

kdt

Therefore,

max 0 e k d t S
dS
=
dt
Km + S
Separating variables gives:

kd t

dt =

Km + S
dS
max 0 S

with initial condition S = S0 at t = 0,

6

Km
S0 S0 S f
1
tb = ln[1 k d (
ln
+
)]
kd
max 0
max 0 S f

10.1.2 Cell Cultures

Similarly to the analysis for enzyme catalytic reaction, we have:

d ( xV )
= xV k d xV
dt
For V constant,

dx
= ( k d ) x
dt

The initial condition x = x0 at t = 0, we have:

x = x0 e (max kd ) t
or

xf
1
tb =
ln
x0
max k d
If the rate of cell death is negligible, we have:

x = x0 e

maxt

and
tb =

1
max

ln

xf
x0

Mass balance for limiting substrate:

q

d ( SV )
= (
+ P + m ) xV
dt
Y X / S YP / S

If = max and V constant:

q
dS
= ( max + P + m ) x
dt
Y X / S YP / S
9


q
dS
= ( max + P + m ) x0 e max
dt
Y X / S YP / S
and

tb =

1
max

ln[1 +
(

1
YX / S

S0 S f
]
qP
m
) x0
+
+
maxYP / S max

If no product is formed or if production is directly linked with energy

metabolism, we have:

tb =

1
max

S0 S f
ln[1 +
]
1
m
+
(
) x0
Y X / S max
10

If maintenance requirements can also be neglected:

tb =

1
max

YX / S
ln[1 +
( S 0 S f )]
x0

Mass balance for product:

d ( PV )
= P xV
dt
If cell death is negligible and V constant,

dP
= P x0 emaxt
dt
11

If P is also constant, and with the initial condition P = P0 at t = 0

tb =

1
max

max
ln[1 +
( Pf P0 )]
P x0

10.1.3 Total Time for Batch Culture Cycle

In the above analysis, tb represents the time required for batch
culture or enzyme conversion. In practice, batch operations involve
very long unproductive periods. Following the fermentation or
enzyme reactions, time thv is taken to harvest the contents inside
the bioreactor and time tp is needed to clean, sterilize and otherwise
prepare the bioreactor for the next operation. For cell culture, a lag
time of duration t1 occurs after inoculation during which no growth
or product formation occurs. These time periods are illustrated
below for fermentation processes.
12

Cell concentration

xf

x0
tp

t1

tb

t hv

tp

t1

tb

t hv

Figure 10.2 The entire time period needed for batch cell culture

The total downtime tdn and operation time tT are:

t dn = t p + t1 + t hv

tT = tb + tdn
13

10.2 Fed-Batch Operation of Well-mixed Bioreactors

In fed-batch operation, intermittent or continuous feeding of
nutrients is used to supplement the bioreactor contents and provide
control over the substrate concentration. By starting with relatively
dilute solution of substrate and adding more nutrients as the
bioreaction proceeds, high growth rates are avoided. This is
important, for example, in cultures where the oxygen demand
during fast growth is too high for the mass-transfer capabilities of
the bioreactor. Alternatively, high substrate concentration may be
inhibitory or switch on undesirable metabolic pathways.

14

Fed-batch culture is used extensively in production of bakers yeast

to overcome catabolic repression and control oxygen demand; it is
also used routinely for penicillin production. Space must be allowed
in fed-batch bioreactors for addition of fresh medium; in some
cases a portion of the broth is removed before injection of
additional material. The flow rate and timing of the feed are often
determined by monitoring parameters such as dissolved-oxygen
level or exhaust gas composition. As enzyme reactions are rarely
carried out as fed-batch operations, we will consider fed-batch
bioreactors for cell cultures or fermentations only.

15

Feed stream
F
xi
Si
Pi
V, x, S, P

Figure 10.3 Flow-sheet for

a batch bioreactor

16

For fed-batch bioreactor, we have:

dV
=F
dt
A mass balance for cells:

d ( xV )
= Fxi + xV k d xV
dt
Expand the differential equation gives:

dV
dx
x
+V
= Fxi + ( k d ) xV
dt
dt
17

Therefore,

xF + V

dx
= Fx i + ( k d ) xV
dt

Dividing by V gives:

dx F
F
= xi + ( k d ) x
dt V
V
and

dx
= Dxi + ( k d D ) x
dt
18

Usually, the bioreactor is operated first in batch until a little high cell
density is achieved and the substrate virtually exhausted, then, fedbatch operation is started with medium flow rate F. As a result, cell
concentration x is maintained relatively high and approximately
constant so that dx/dt 0 and D, therefore, Monod expression
for cell growth can be written as:

D=

max S
KS + S

or

DK S
S=
max D

19

If no product is produced, or the product formation is directly

coupled with energy metabolism, we have:

x
dS
= D( Si S )
dt
YX / S
At high cell density, virtually all substrate entering the vessel is
consumed immediately; therefore, S << Si and dS/dt 0. Applying
these relationships with D, we obtain:

x Y X / S Si

20

For product synthesis directly coupled with energy metabolism, at

the same time assuming the feed does not contain product:

P YP / S Si
Even though cell concentration remains virtually unchanged with
time dx/dt 0, because the broth volume increases with time during
fed-batch culture, the total biomass within the bioreactor also
increases. Consider the rate of increase of total biomass in the
bioreactor dX/dt:

21

dV
dx
dX d ( xV )
=
=x
+V
= Y X / S Si F
dt
dt
dt
dt

Integrated with initial condition X = X0 at the start of feeding to give:

X = X 0 + (Y X / S Si F )tbf
This indicates that, if YX/S, Si and F constant, the total biomass in
fed-batch culture increases as a linear function of time.
Under conditions of high biomass density and almost depletion of
substrate, a quasi-steady-state condition previals in fed-batch
bioreactors in which dx/dt 0, dS/dt 0 and dP/dt 0, and x, S and
P are almost constant, but , V, D and X are changing with time.
22

10.3 Continuous Operation of Well-mixed Bioreactors

From the view of engineering application, continuous operation of
bioreactors shows great advantage over batch and fed-batch
processes discussed above. Not only a long downtime required by
batch and fed-batch bioreactors is saved, but a steady condition can
also be realized easily within the bioreactors, enzyme or cells will be
maintained under optimum environments, therefore, the productivity
of aimed product or biomass can be improved obviously. One of the
biggest disadvantages of a continuous stirred-tank reactor
(CSTR) is the withdrawn of biocatalyst contained within the product
stream when used for freely suspended cell culture and enzyme
reaction systems. Only when the enzymes are inexpensive and can
be added continuously to maintain the required concentration, can a
C S T R b e app l i e d. On th e other hand, the high risk of
23

contamination also hinders CSTRs application in animal and plant

cell cultures and other fermentations, such as penicillin, in which
much lower growth rate of cells and complete nutrients increase the
opportunity for undesirable microorganisms to grow quickly within
the system. Different steady-state operating strategies are available
for a CSTR. In a chemostat, the liquid volume within the bioreactor
is maintained constant by setting the inlet and outlet flow rates
equal; the dilution rate is therefore constant and steady state is
achieved in the chemostat by adjusting itself to the feed rate. In a
turbidostat, the liquid volume is kept constant by setting the outlet
flow rate equal to the inlet flow rate; however, the inlet flow rate is
adjusted to keep the biomass concentration constant. Thus, in a
turbidostat the dilution rate adjusts to its steady-state value
corresponding to the set biomass concentration.
24

Feed stream
Product stream

F
xi
Si
Pi
V, x, S, P

F
x
S
P

Figure 10.4 Flowsheet for a CSTR

25

Characteristic operating parameters for CSTRs are the dilution

rate D and the average residence time . The following
relationship exists:
=

1 V
=
D F

For a given throughput, the bioreactor size V and associated capital

and operating costs are minimized when is made as small as
possible. Continuous bioreactor theory allows us to determine
relationships between or D and steady-state substrate, product
and cell concentrations.

26

10.3.1 Enzyme Reaction

Mass balance gives:

FS i FS

max S
V =0
Km + S

Dividing by V and applying the definition of dilution rate gives:

D( Si S ) =

max S
Km + S

27

If kinetic parameters including max and Km and substrate

concentration contained within the feed can be used directly to
calculate the dilution rate required to achieve a particular level of
substrate conversion, then, the steady-state product concentration
and productivity can be evaluated from stoichiometry.
For immobilized enzyme system, we have:
D( Si S ) =

T max S
Km + S

where T is the total effectiveness factor, S is the bulk substrate

concentration, and max and Km are intrinsic kinetic parameters.

28

10.3.2 Cell Culture

If cell death is also negligible compared with cell growth, the mass
balance of biomass gives D = .
When cell growth kinetics can be described by Monod equation:
S=

DK S
max D

The mass balance of substrate gives:

x=

D( Si S )

D
+ P +m
Y X / S YP / S

29

If no product synthesis other than biomass:

x=

D( Si S )
D
+m
YX / S

If, further, maintenance effects can further be ignored:

x = Y X / S ( Si S )
Therefore,

x = Y X / S ( Si

DK S
)
max D

30

The dilution rate for the highest biomass productivity is given by:

Dopt = max (1

KS
)
K S + Si

Now we consider the CSTR in which immobilized cells are used as

illustrated below.

31

Feed stream
F
xi
Si
Pi

Product stream

V, x im , S, P

F
xs
S
P

Figure 10.6 Flowsheet for a CSTR

with immobilized cells

32

Mass balance for biomass:

F xs + xsV + TximV = 0
Rearrange:

or

Dx s = ( x s + T xim )

T xim
D = (1 +
)
xs

33

Mass balance for substrate:

x s
T x im
V
V =0
F (Si S )
YX /S
YX /S
and

D( Si S ) =

YX / S

( x s + T xim )

also

D ( Si S )Y X / S
max S
=
K S + S ( Si S )Y X / S + T xim

34

100

Substrate conversion, %

80

xim = 0.1 g l1, T = 1.0

60

xim = 0.1 g l1, T = 0.3

40

xim = 0

20

Dcri

0
0

0.1

0.2

0.3

0.4

0.5

Dilution rate, D h1
Figure 10.7 Steady-state substrate conversion as a function
of dilution rate with and without immobilized cells
(calculated with max = 0.1 h1, KS = 103 g l1, YX/S = 0.5 and Si = 8 103 g l1)
35

We can find for any xim > 0, dilution rate D at steady state is greater
than . Accordingly, dilution rate is no longer limited by the
maximum specific growth rate max, as discussed before, that
means immobilized cell bioreactor can be operated at D
considerably greater than Dcrit for free cells without washout
happening. We also can find that, at a given dilution rate, presence
of immobilized cells improves substrate conversion and reduces
the amount of substrate lost in the product stream. However,
bioreaction rates with immobilized cells can be significantly affected
by the mass transfer in and around the particles.

36

10.4 Plug-Flow Bioreactor

No mixing occurs in an ideal plug-flow bioreactor (PFR); material
entering the bioreactor passes through and does not interact with
neighboring fluid elements. This is achieved at high flow rates
which minimize backmixing and variation in fluid velocity. Plug flow
is most readily achieved in column or tubular bioreactors which can
be operated in upflow or downflow mode or, in some cases,
horizontally.
PFR is mainly used for some enzyme catalytic reactions and
medium sterilization during which contaminant microorganisms are
killed by heating.

37

Product stream F S f

L
z
F S

Feed stream F S i
Figure 10.8 Flowsheet for a PFR

38

Mass balance for substrate:

max S
FS Z FS z + z
Az = 0
Km + S
Rearrange:

F ( S z + z S z )
A z

max S
=
Km + S

and

u( S z + z S z )
z

max S
=
Km + S

39

u( lim

z 0

S z + z S z
z

)=

max S
Km + S

That is:

max S
dS
u
=
dz
Km + S
Integrating with boundary condition S = Si at z = 0 gives:

Km
Si Si S f
ln
+
)
L = u(
max S f
max

40

For

V
V
L
= = A =
F
F
u
A
we have:

Km
Si Si S f
=
ln
+
max S f
max

41

Only a few enzyme reactions, for example, liquification of starch

slurry using -amylase before being saccharified to fermented
sugar, are applying this operation mode. If enzyme is immobilized,
mass-transfer effects should be considered, therefore:

S
dS
= T max
dz
Km + S

Generally, PFR operation is not suitable for cell culture unless the
biomass is recycled or there is a continuous inoculation operation.
However, for sterilization of medium, PFR is widely used and will
be discussed later. If cells are immobilized and packed within the
column, PFR modified properly can be applied.
42

10.5 Sterilization of Medium

10.5.1 Batch Heat Sterilization of Medium
Medium can be sterilized in batch in the vessel where it will be
used. The medium is heated to sterilization temperature by
introducing steam into the coils or jacket of the vessel; alternatively,
steam is bubbled directly into the medium. If direct steam injection
is used, allowance must be made for dilution of the medium by
condense which typically adds 10 ~ 20% water to the medium;
quality of the steam must also be sufficiently high to avoid
contamination of the medium by metal ions.

43

Holding
Heating

140
Temperature C

120
100

Cooling

80
60

t hd

40
20
0
0

2
Time (h)

Figure 10.9 Variation of temperature with

time for batch sterilization of medium

44

For operation of batch sterilization systems, we must be able to

estimate the holding time required to achieve the desired level of
cell destruction. As well as destroying contaminant organisms, heat
sterilization also destroys nutrients in the medium. To minimize this
loss, holding times at high sterilization temperature should be kept
as short as possible. Cell death occurs at all times during batch
sterilization, including the heating-up and cooling-down periods.
The holding time thd can be minimized by taking into account cell
destruction during these periods.

45

Number of viable cells

Heating
N0
N1
Holding
t hd
Cooling

N2
Nf
0

2
Time (h)

Figure 10.10 Reductioin in number of

viable cells during batch sterilization

46

In a batch vessel where cell death is the only process affecting the
number of viable cells, we have:
dN
= k d N
dt

During the holding period:

N1
ln
= k d t hd
N2
or
N1
N2
kd

ln
thd =

47

where

kd =

E
d
Ae RT

therefore
E

d
dN
= Ae RT N
dt

for heating
E

d
t1
N0
ln
= Ae RT dt
0
N1

when heat transfer from isothermal steam

T = Ts (1 +

T0 Ts
e
Ts

UAt
M mC p

)
48

when heating directly by sparging with stream

M s ht
M m C p T0
)
T = T0 (1 +
Ms
t
1+
Mm
electrical heating

T = T0 (1 +

Qt
)
M m C p T0

for cooling
E

d
tf
N2
ln
= Ae RT dt
t2
Nf

49

where
UA

M wC pwt
M wC pw
)]
T0 Tci [( M mC p )(1e
T = Tci 1 +
e

T
ci

50

Temperature

Heating

Holding

Cooling
Linear
Exponential

Hyberbolic
Linear
Exponential

Fermentation
temperature

Raw medium temperature

Time
Figure 10.11 Generalized temperature-time profiles for the batch sterilization

51

Normally, cell death below 100 C is minimal; however, when

heating and cooling are relatively slow, temperatures remain close
to the maximum for considerable periods of time, cell numbers can
be reduced significantly outside the holding period. Usually, holding
time is of the order of minutes whereas heating and cooling of large
medium volumes take hours.
The design procedures outlined in this section apply to batch
sterilization of medium when the temperature is uniform throughout
the vessel. However, if the medium contains contaminant particles
in the form of flocs or pellets, temperature gradient within the

52

particles may develop and the temperature at the center of the

particles will lower than that in the bulk medium. As a result, cell
death inside the particles is not as effective as in the bulk medium.
Longer holding time is required to treat solid-phase substrates and
media containing particles.
When batch sterilization is scaled up to larger volumes, much
longer times are needed to achieve the same sterilization results as
that of small-scale tanks, the destroy of nutrients is exacerbated
extremely. Therefore, continuous sterilization process is developed
for large-scale use.

53

10.5.2 Continuous Heat Sterilization of Medium

(a)

Vapor
Flash
cooler

Holding section

Heat
exchanger
Jet

Fermenter

Steam
Raw medium

54

(b)

Raw medium
Heat
exchanger

Holding section

Heat
exchanger
Fermenter

Condensed water

Steam

Figure 10.12 Continuous sterilization processes

55

Continuous sterilization, particularly a high-temperature, shortexposure-time process, can significantly reduce damage to medium
nutrients while achieving high levels of cell destruction. Other
advantages include improved steam economy and more liable
scale-up. The amount of steam needed for continuous sterilization
is only 20 ~ 25% that used in batch processes; the time required is
also obviously reduced because heating and cooling are virtually
instantaneous.

56

(b)

(a)

Holding
2 - 3 min

Steam
injection

Flash
cooling

Heat
exchange

Time

Temperature

Temperature

Holding
2 - 3 min

Heat
exchange

Time

Figure 10.13 Variation of temperature with time

in the continuous sterilisers of Figure 11.12

57

An

important variable affecting performance of continuous

sterilisers is the characteristics of medium flow in the system.

Ideally, all medium entering the system at a particular instant
should spend the same time in the steriliser and exit the system at
the same time also, that is plug-flow. Otherwise, if mixing occurs
within the holding pipe, a risk of contamination will be transferred to
the outlet of the sterilized medium from the inlet of raw material.
Deviation from plug-flow behavior is characterized by the degree of
axial dispersion along the pipe which will be further discussed later.

58

10.6 Comparison between Modes of Bioreactor Operation

Si
Large number of CSTRs
Four CSTRs of equal size
PFR or batch
Single CSTR
Sf
Figure 10.14 Substrate concentration changes
in PFR, batch, CSTR and CSTRs in cascade

59

Summary
After studying this chapter, you should:
understand the concept of well-mixed flow and plug-flow modes,
two extremely flowing conditions;
be able to predict batch bioreaction time required to achieve
designed substrate conversion for enzyme reaction and cell
culture;
be able to predict the performance of fed-batch bioreactors
operated at quasi-steady-state conditions;
for CSTR, know how to control its operation in order to avoid
washout of cells and to obtain the optimum cell productivity; and
finally
be able to compare the performance of batch, CSTR and PFR
and select proper operation method for a designed bioprocess.
60