JENIS-JENIS

BIOREAKTOR
Ir. Maya Sarah, ST, MT, PhD
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
What do each
▪ Control
of these factors
▪ Genetic
stability mean?
▪ Operability
▪ Economics
▪ Regulatory
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 F
?
S0 S
X0 X

air air
Batch Reactor
Batch cycle time is: tcycle = t growth + tl

where tgrowth is the time required for growth and

tl is the lag time + preparation and harvest time.

1 Xmax
tcycle = ln + tl
max X0

where X0 is the initial concentration and Xmax is

the maximum concentration (carrying capacity).
Batch Production Rate
So net biomass production rate is:
Xmax − X0
(PrX )batch =
t cycle
Recall the definition of biomass yield:
X Xmax − X0 Xmax − X 0
YX / S = = =
S S0 − 0 S0

YX / S S0
(PrX )batch = 1 X (1)
ln max + t l
max X0
 Chemostat
For negligible kd, negligible extracellular product
formation and steady state, Lec. Notes 16, Eq. (10)
gave:
 KS D 
X = YX / S  S0 −  (2)
 max − D 

For optimum cell productivity (X•D), calculate

d(X•D)/dt, set equal to zero, and solve for Dopt:

 KS 
Dopt = max  1 −  (3)
 KS + S0 
 Chemostat
Substituting Eq. (2) into Eq. (3) gives the value
of X at the maximum production rate. :


X(at D opt ) = YX/S S0 + K S − K S (S0 + K S )  (4)

Optimum productivity is D•X when D=Dopt and X=

X (at Dopt):

(PrX )opt, chemo


= YX/S μ max 1 −
KS 

 S0 + K S − K S (S0 + K S )
K S + S0 


(5)
 Chemostat Productivity
Rate
Noting that S is usually much larger than K , we
0 S
have:

(PrX )opt, chemo  maxYX / S S0 (6)

Comparing the rates for batch production and

production in a chemostat:

(Prx )opt, chemo Xmax

= ln + max tl
(Prx )batch
(7)
X0
Comparison
Xmax is always larger than X0 and is typically 10-20
times larger, so the chemostat outperforms the
batch reactor. For E. coli growing on glucose, µmax
is around 1/hr. Using tlag=5 hr and Xmax/X0=20,

(Prx )opt, chemo

=8
(Prx )batch
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

µmax<D. Higher dilution rates can be
achieved with recycle.

F (1+a)F
S0 S,X
X0
F
X’
aF
S,bX
 Chemostat with Recycle
Biomass balance on the chemostat:
dX
V = FX 0 + αFβX − (1 + α )FX + μVX (8)
dt
where a=volumetric recycle ratio and b=the
concentration factor of the separator. At steady
state and with X0=0:
F F
α βX − (1 + α ) X + μX = 0 (9)
V V
μ = 1 + α(1 − β )D (10)

Note that for b>1, µ<D.

 Substrate Mass Balance
dS X
V = FS0 + aFS − V − (1 + a)FS (11)
dt YX / S

At steady state:
F F X F
S0 + a S − − (1 + a) S = 0 (12)
V V YX / S V

D
X= YX / S (S0 − S) (13)

 Steady-state Values
Substituting µ given by Eq. (10) into Eq. (13):
YX / S (S0 − S)
X= (14)
1 + a(1 − b)

We can get the expression for the substrate

concentration by equating the expression for
µ from Monod kinetics to Eq. (10):
 Steady-state Values
max S
= = 1 + a(1 − b)D (15)
KS + S
or:
K S D1 + a(1 − b)
S=
max − D1 + a(1 − b) (16)

So now we can get X entirely as a function of D:

YX / S  K S D1 + a(1 − b) 

X= S0 −
1 + a(1 − b) max − D1 + a(1 − b) (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, S0 F, S0

V0, X, S, P V, X, S, P Vw, X, S, P
Start fed-batch Fed batch fill Harvest
 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:

S0= initial substrate concentration of batch

V0= initial volume of batch
F= constant flow rate of addition stream during fed-batch
X0= initial concentration of batch
For a batch culture:
X = X0 + YX / S (S0 − S) (1)

Since liquid is being added, the volume

is changing:
dV V = V0 + Ft
=F or: (2)
dt

If the total amount of biomass (grams) in

the reactor is Xt then the concentration
X is:
X = X /V t (3)
 So the change in the biomass
concentration with time is:
 dX t  t  dV 
V −X  
dX  dt  dt (4)
=
dt V2
1 dX t
Using the definition of the growth rate: = t
X dt

...the dilution rate: F

D=
V
dV
...and the expression for dV/dt: =F
dt

we have: dX
= ( − D)X (5)
dt
 Quasi-steady State
▪ Substrate is consumed at the same rate it is
added.
Now, consider the case when the fed-
batch is started from a culture in the
initial substrate concentration was S0 and
nutrient feed is begun at flow rate F and
concentration S0. Just as nutrient feed
begins:
X = X0 + YX / S (S0 − S) (6)
At quasi-steady state, for this case
we will have: dX
=0 (7)
dt

So X is constant (but not Xt).

Now we have: =D (8)

Assuming Monod growth kinetics,

this gives (just as in the case of a
chemostat): S=
Ks D
max − D (9)
If the total amount of substrate in the reactor
is St, then a substrate mass balance gives:
dS t X t
= FS0 − (10)
dt YX / S

which, for quasi-steady state gives:

X t
FS0 = (11)
YX / S

Returning to Equation (4), we have, at

quasi-steady state:
dX t X t  dV  (12)
=   = XF
dt V dt
 Integrating, we have:

X = X + FXt
t t
0 (13)
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:
X = X + FX 0 + YX/S (S0 − S) t
t t
0 (14)

Often, S<<S0 and X0<<YX/SS0 and so:

X t = X0t + FYX / S So t (15)

 Product Output
If the specific productivity (g product/g cells/
hr) is constant:
t
1 dP
t = qp
X dt
or: t (16)
dP
= qp X t

dt
where Pt is the total product concentration in the
reactor:
Substituting:
X t = VX = (V0 + Ft )X
 t
we have: dP
= q p X (V0 − Ft ) (17)
dt

Integrating this expression, we

have: Ft
P = P + q p X  V0 +
t t t
(18)
0
2
or in terms of concentration:
V0  V0 Dt  (19)
P = P0 + q p X  +  t
V V 2
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 V0 and the cycle begun
again.
For the case of repeated fed-batch
cultures:
 Dwtw 
Pw = P0 + q p X   +  tw (20)
2

Where Vw is the volume just before harvesting,

V0 is the volume after harvesting, Dw=F/Vw
and: V0
= (21)
Vw
tw is the cycle time and is given by:
Vw − V0 Vw − Vw 1 − 
tw = = = (22)
F F Dw
With this definition, we now
have:
qpX
Pw = γP0 +
2D w
(1 − γ )
2
(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

Advantage over immobilized

enzymes
Whole cells provide cofactors, reducing
power, energy that many enzymatic
reactions require.
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