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Biochemical Engineering

CEN 551
Instructor: Dr. Christine Kelly
Chapter 9

Bioreactors

What two type of bioreactors have
we discussed in this course?

What are the characteristics of each
type of reactor?

Which type is more efficient?

Which type is more common?

Reactor Types

Batch and Chemostat (CSTR).

Batch: changing conditions - transient (S, X,
growth rate), high initial substrate, different
phases of growth.

Chemostat: steady-state, constant low
concentration of substrate, constant growth rate
that can be set by setting the dilution rate (i.e. the
feed flow rate) .

Chemostat more efficient.

Batch more common.

Choice of continuous vs.
batch production

Productivity

Flexibility

Control

Genetic stability

Operability

Economics

Regulatory
What do each
of these factors
mean?

Reactor Choices

Productivity: rate of product per time
per volume. Chemostat better for
growth associated products. Wasted
time in batch process.

Flexibility: ability to make more than
one product with the same reactor.
Batch better.

Control: maintaining the same
conditions for all of the product
produced. In theory, chemostat better,
steady state. In reality????


Genetic stability: maintaining the
organism with the desired
characteristics. Chemostat selects for
fast growing mutants that may not have
the desired characteristics.

Operability: maintaining a sterile
system. Batch better.

Regulatory: validating the process.
Initially, many process batch, too
expensive to re validate after clinical
trials.

Comparison of Productivity:
Batch vs. Chemostat
Consider production of a growth associated
product (like cell mass) in suspension
culture
F
S
0

X
0
F
S
0

X
0
F
S
X
F
S
X
air air
air air
? ?

Batch Reactor
t
cycle
t
growth
t
l
Batch cycle time is: Batch cycle time is:
where t
growth
is the time required for growth and
t
l
is the lag time + preparation and harvest time.
where t
growth
is the time required for growth and
t
l
is the lag time + preparation and harvest time.
t
cycle
1
max
ln
X
max
X
0
t
l
where X
0
is the initial concentration and X
max
is
the maximum concentration (carrying capacity).
where X
0
is the initial concentration and X
max
is
the maximum concentration (carrying capacity).

Batch Production Rate
So net biomass production rate is: So net biomass production rate is:
Pr
X
batch
·
Y
X/ S
S
0
1
µ
max
ln
X
max
X
0
+ t
l
Recall the definition of biomass yield: Recall the definition of biomass yield:
Y
X/ S
·
∆X
∆S
·
X
max
− X
0
S
0
−0
·
X
max
− X
0
S
0
Pr
X
batch
·
X
max
− X
0
t
cycle
(1) (1)

Chemostat
For negligible k
d
, negligible extracellular product
formation and steady state, Lec. Notes 16, Eq. (10)
gave:
For negligible k
d
, negligible extracellular product
formation and steady state, Lec. Notes 16, Eq. (10)
gave:
X · Y
X/ S
S
0

K
S
D
µ
max
− D
¸
¸

_
,

For optimum cell productivity (X•D), calculate
d(X•D)/dt, set equal to zero, and solve for D
opt
:
For optimum cell productivity (X•D), calculate
d(X•D)/dt, set equal to zero, and solve for D
opt
:
D
opt
· µ
max
1 −
K
S
K
S
+ S
0
¸
¸

_
,

(3) (3)
(2) (2)

Chemostat
Substituting Eq. (2) into Eq. (3) gives the value
of X at the maximum production rate. :
Substituting Eq. (2) into Eq. (3) gives the value
of X at the maximum production rate. :
( ) [ ]
S 0 S S 0 X/S opt
K S K K S Y ) D (at X + − + ·
Optimum productivity is D•X when D=D
opt
and X= X
(at D
opt
):
Optimum productivity is D•X when D=D
opt
and X= X
(at D
opt
):
( ) ( ) [ ]
S 0 S S 0
0 S
S
max X/S
chemo opt,
X
K S K K S
S K
K
1 μ Y Pr + − +
1
]
1

¸

+
− ·
(4) (4)
(5) (5)

Chemostat Productivity Rate
Noting that S
0
is usually much larger than K
S
, we
have:
Noting that S
0
is usually much larger than K
S
, we
have:
Pr
X
opt, chemo
max
Y
X/ S
S
0
Comparing the rates for batch production and
production in a chemostat:
Comparing the rates for batch production and
production in a chemostat:
Pr
x
opt , chemo
Pr
x
batch
ln
X
max
X
0
max
t
l
(6) (6)
(7) (7)

Comparison
X
max
is always larger than X
0
and is typically 10-20
times larger, so the chemostat outperforms the
batch reactor. For E. coli growing on glucose, µ
max

is around 1/hr. Using t
lag
=5 hr and X
max
/X
0
=20,
X
max
is always larger than X
0
and is typically 10-20
times larger, so the chemostat outperforms the
batch reactor. For E. coli growing on glucose, µ
max

is around 1/hr. Using t
lag
=5 hr and X
max
/X
0
=20,
Pr
x
opt , chemo
Pr
x
batch
8
Even so, most industrial fermentation processes
occur in a batch reactor. Why?
Even so, most industrial fermentation processes
occur in a batch reactor. Why?

Reasons for Batch Popularity

Equations were for cell mass (or other
growth-associated product). Many
industrial applications are for non-
growth associated products.

Selective pressure of a chemostat is
detrimental to engineered organisms

Batch is more mechanically reliable

Batch system is more more flexible

Specialized Reactors

Chemostat with recycle

Multistage chemostat

Fed-batch

Perfusion

Chemostat with Recycle
Can we operate a chemostat with a dilution
rate greater than maximum growth rate?
Why or why not?
What conditions would we want to operate
a chemostat with a dilution rate higher
than the maximum growth rate?

High dilution rate

No

Because the cell growth cannot keep up
with how fast the cells are removed from
the reactor, and after some time the cells
would washout of the reactor.

We want a high dilution rate when we have
a high volume of feed with a low
concentration of substrate. Waste water
treatment has these characteristics.

Operation of Chemostats at
High Dilution Rates
Chemostats cannot be operated if
µ
max
<D. Higher dilution rates can be
achieved with recycle.
F
S
0

X
0
F
S
0

X
0
(1+α)F
S,X
(1+α)F
S,X
αF
S,βX
αF
S,βX
F
X’
F
X’

Chemostat with Recycle
Biomass balance on the chemostat:
( ) μVX FX α 1 αFβX FX
dt
dX
V
0
+ + − + ·
where α=volumetric recycle ratio and β=the
concentration factor of the separator. At steady
state and with X
0
=0:
where α=volumetric recycle ratio and β=the
concentration factor of the separator. At steady
state and with X
0
=0:
( ) 0 μX X
V
F
α 1 βX
V
F
α · + + −
( ) [ ]D β 1 α 1 μ − + ·
Note that for β>1, µ<D. Note that for β>1, µ<D.
(8) (8)
(9) (9)
(10) (10)

Substrate Mass Balance
V
dS
dt
· FS
0
+ αFS −V
µX
Y
X/ S
− 1 +α ( )FS
F
V
S
0
+ α
F
V
S−
µX
Y
X/ S
− 1 +α ( )
F
V
S · 0
X ·
D
µ
Y
X/ S
S
0
− S
At steady state: At steady state:
(11) (11)
(12) (12)
(13) (13)

Steady-state Values
Substituting µ given by Eq. (10) into Eq. (13): Substituting µ given by Eq. (10) into Eq. (13):
X
Y
X/ S
S
0
S
1 1
(14) (14)
We can get the expression for the substrate
concentration by equating the expression for
µ from Monod kinetics to Eq. (10):
We can get the expression for the substrate
concentration by equating the expression for
µ from Monod kinetics to Eq. (10):

Steady-state Values
µ ·
µ
max
S
K
S
+ S
· 1 +α 1 −β D
or: or:
S ·
K
S
D 1 +α 1 −β
µ
max
− D 1 +α 1 −β
(16) (16)
(15) (15)
So now we can get X entirely as a function of D: So now we can get X entirely as a function of D:
X ·
Y
X/ S
1 +α 1 −β
S
0

K
S
D 1 +α 1 −β
µ
max
− D 1 +α 1 −β

¸

1
]
1
(17) (17)

Special Cases - Chemostat

Recombinant product under the control of
an inducible promoter.

Recombinant strain and wild type grow at
the same rate if the recombinant product is
not expressed.

If the recombinant product is expressed, the
recombinant strain grows much slower.

Design a continuous reactor system to
produce this product efficiently.

Mulistage chemostat

First chemostat is fed with a non-inducing
growth substrate, allowing the recombinant
strain to be produced.

The effluent from the first chemostat feeds a
second chemostat that is fed inducer, and
the product is produced.

Note: new recombinant cells are continually
added to the second chemostat not allowing
take-over by a fast growing mutant.

Fed-batch Operation

Fed-batch reactors gain some advantages
of a CSTR, retain some disadvantages of
batch.

Reduces substrate inhibition or
catabolic repression, allows for high
conversion, and the extension of
stationary phase.

Semi-batch nature usually leads to
higher operations cost and batch
variability.

Fed-batch Operation
F, S
0
F, S
0
V
0
, X, S, P V
0
, X, S, P
Start fed-batch Start fed-batch Fed batch fill Fed batch fill Harvest Harvest
V
w
, X, S, P V
w
, X, S, P V, X, S, P V, X, S, P
F, S
0
F, S
0

Fed-batch Operation

Fed-batch cultures are started as batch
cultures and grown to an initial cell
concentration X, after which fed-batch
operation begins.

Notation:
S
0
= initial substrate concentration of batch
V
0
= initial volume of batch
F= constant flow rate of addition stream during fed-batch
X
0
= initial concentration of batch
S
0
= initial substrate concentration of batch
V
0
= initial volume of batch
F= constant flow rate of addition stream during fed-batch
X
0
= initial concentration of batch

Since liquid is being added, the volume
is changing:
Since liquid is being added, the volume
is changing:
dV
dt
· F
V · V
0
+ Ft
X · X
0
+Y
X/ S
S
0
− S ( )
For a batch culture: For a batch culture:
or: or:
If the total amount of biomass (grams)
in the reactor is X
t
then the
concentration X is:
If the total amount of biomass (grams)
in the reactor is X
t
then the
concentration X is:
X X
t
/ V
(1) (1)
(2) (2)
(3) (3)

So the change in the biomass
concentration with time is:
So the change in the biomass
concentration with time is:
dX
dt
·
V
dX
t
dt
¸
¸

_
,

− X
t
dV
dt
¸
¸
_
,
V
2
Using the definition of the growth rate: Using the definition of the growth rate:
...the dilution rate: ...the dilution rate:
...and the expression for dV/dt: ...and the expression for dV/dt:
µ ·
1
X
t
dX
t
dt
D ·
F
V
dV
dt
· F
we have: we have:
dX
dt
· µ − D ( )X
(4) (4)
(5) (5)

Now, consider the case when the fed-
batch is started from a culture in the
initial substrate concentration was S
0
and
nutrient feed is begun at flow rate F and
concentration S
0
. Just as nutrient feed
begins:
Now, consider the case when the fed-
batch is started from a culture in the
initial substrate concentration was S
0
and
nutrient feed is begun at flow rate F and
concentration S
0
. Just as nutrient feed
begins:
Quasi-steady State

Substrate is consumed at the same rate it is
added.
X · X
0
+Y
X/ S
S
0
− S
(6) (6)

At quasi-steady state, for this case
we will have:
At quasi-steady state, for this case
we will have:
dX
dt
· 0
So X is constant (but not X
t
).
Now we have:
So X is constant (but not X
t
).
Now we have: µ · D
Assuming Monod growth kinetics,
this gives (just as in the case of a
chemostat):
Assuming Monod growth kinetics,
this gives (just as in the case of a
chemostat):
S ·
K
s
D
µ
max
− D
(7) (7)
(8) (8)
(9) (9)

If the total amount of substrate in the reactor
is S
t
, then a substrate mass balance gives:
If the total amount of substrate in the reactor
is S
t
, then a substrate mass balance gives:
dS
t
dt
· FS
0

µX
t
Y
X/ S
which, for quasi-steady state gives: which, for quasi-steady state gives:
FS
0
·
µX
t
Y
X/ S
Returning to Equation (4), we have, at
quasi-steady state:
Returning to Equation (4), we have, at
quasi-steady state:
dX
t
dt
·
X
t
V
dV
dt
¸
¸
_
,
· XF
(10) (10)
(11) (11)
(12) (12)

Integrating, we have: Integrating, we have:
X
t
· X
0
t
+ FXt
since X is constant (dX/dt=0). Therefore, the
total biomass in a fed-batch reactor operated as
assumed here increases linearly with time.
Substituting the appropriate expression for X:
since X is constant (dX/dt=0). Therefore, the
total biomass in a fed-batch reactor operated as
assumed here increases linearly with time.
Substituting the appropriate expression for X:
( ) [ ] t S S Y X F X X
0 X/S 0
t
0
t
− + + ·
Often, S<<S
0
and X
0
<<Y
X/S
S
0
and so: Often, S<<S
0
and X
0
<<Y
X/S
S
0
and so:
X
t
· X
0
t
+ FY
X/ S
S
o
t
(13) (13)
(14) (14)
(15) (15)

If the specific productivity (g product/g cells/
hr) is constant:
If the specific productivity (g product/g cells/
hr) is constant:
Product Output
1
X
t
dP
t
dt
· q
p
dP
t
dt
· q
p
X
t
where P
t
is the total product concentration in the
reactor:
where P
t
is the total product concentration in the
reactor:
or: or:
Substituting: Substituting:
X
t
· VX · V
0
+ Ft X
(16) (16)

we have: we have:
dP
t
dt
· q
p
X V
0
− Ft
Integrating this expression, we
have:
Integrating this expression, we
have:
P
t
· P
0
t
+ q
p
X V
0
+
Ft
2
¸
¸
_
,
t
or in terms of concentration: or in terms of concentration:
P · P
0
V
0
V
+ q
p
X
V
0
V
+
Dt
2
¸
¸
_
,
t
(17) (17)
(18) (18)
(19) (19)

Repeated Fed-batch
Usually, fed-batch cultures are taken
through many feeding cycles, with
each feeding cycle followed by a
harvest cycle during which the
volume is drawn back down to V
0

and the cycle begun again.

For the case of repeated fed-batch
cultures:
For the case of repeated fed-batch
cultures:
P
w
· γP
0
+ q
p
X γ +
D
w
t
w
2
¸
¸
_
,
t
w
Where V
w
is the volume just before harvesting,
V
0
is the volume after harvesting, D
w
=F/V
w

and:
Where V
w
is the volume just before harvesting,
V
0
is the volume after harvesting, D
w
=F/V
w

and:
γ ·
V
0
V
w
t
w
is the cycle time and is given by: t
w
is the cycle time and is given by:
t
w
·
V
w
−V
0
F
·
V
w
− γV
w
F
·
1 − γ
D
w
(20) (20)
(21) (21)
(22) (22)

With this definition, we now
have:
With this definition, we now
have:
( )
2
w
p
0 w
γ 1
2D
X q
γP P − + ·
(23) (23)

Perfusion Culture

Animal Cell Culture

Constant medium flow

Cell retention

Selective removal of dead cells

Removal of cell debris, inhibitory by
products

High medium use, costs raw materials and
sterilization

Immobilized Cell Systems

High cell concentrations

Cell reuse

Eliminates cell washout at high
dilution rates

High volumetric productivities

May provide favorable
microenvironment

Genetic stability

Protection from shear damage

Major Limitation
Mass transfer (diffusional) resistances
Whole cells provide cofactors, reducing
power, energy that many enzymatic
reactions require.
Advantage over immobilized
enzymes

Types of
Immobilization

Active Immobilization: similar to
enzyme immobilization. Entrapment
and binding.

Passive Immobilization: Biofilm –
multilayer growth on solid surfaces.

Diffusional Limitations

Analysis similar to immobilized
enzymes

Damkohler number

Effectiveness factor

Thiele modulus

Immobilized Bioreactors

Packed-column: feed flows through a
column packed with immobilized cells.
Similar to a plug flow reactor. Can be
recycle chamber.

Fluidized-bed: feed flows up through a
bed of immobilized cells, fluidizing the
immobilized cell particles.

Airlift: air bubbles suspend the
immobilized cell particles in a reactor.

Solid-state Fermentations

Fermentations of solid materials

Low moisture levels

Agricultural products or foods

Smaller reactor volume

Low contamination due to low moisture

Easy product separation

Energy efficiency

Differentiated microbiological structures