Biochemical Engineering CEN 551

1
Biochemical EngineeringCEN 551

• Instructor Dr. Christine Kelly
• Chapter 9

2
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?

3
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.

4
Choice of continuous vs. batch production

• Productivity
• Flexibility
• Control
• Genetic stability
• Operability
• Economics
• Regulatory

What do each of these factors mean?
5
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????

6

• 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.

7
Comparison of Productivity Batch vs. Chemostat

• Consider production of a growth associated
  product (like cell mass) in suspension culture

F S0 X0
F S X
?
air
air
8
Batch Reactor
Batch cycle time is
where tgrowth is the time required for growth and
tl is the lag time preparation and harvest time.
where X0 is the initial concentration and Xmax is
the maximum concentration (carrying capacity).
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Batch Production Rate
So net biomass production rate is
Recall the definition of biomass yield
(1)
10
Chemostat
For negligible kd, negligible extracellular
product formation and steady state, Lec. Notes
16, Eq. (10) gave
(2)
For optimum cell productivity (XD), calculate
d(XD)/dt, set equal to zero, and solve for Dopt
(3)
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Chemostat
Substituting Eq. (2) into Eq. (3) gives the value
of X at the maximum production rate.
(4)
Optimum productivity is DX when DDopt and X X
(at Dopt)
(5)
12
Chemostat Productivity Rate
Noting that S0 is usually much larger than KS, we
have
(6)
Comparing the rates for batch production and
production in a chemostat
(7)
13
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 tlag5 hr
and Xmax/X020,
Even so, most industrial fermentation processes
occur in a batch reactor. Why?
14
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

15
Specialized Reactors

• Chemostat with recycle
• Multistage chemostat
• Fed-batch
• Perfusion

16
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?

17
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.

18
Operation of Chemostats at High Dilution Rates

• Chemostats cannot be operated if µmaxltD. Higher
  dilution rates can be achieved with recycle.

F S0 X0
(1a)F S,X
F X
aF S,bX
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Chemostat with Recycle

• Biomass balance on the chemostat

(8)
where avolumetric recycle ratio and bthe
concentration factor of the separator. At steady
state and with X00
(9)
(10)
Note that for bgt1, µltD.
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Substrate Mass Balance
(11)
At steady state
(12)
(13)
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Steady-state Values
Substituting µ given by Eq. (10) into Eq. (13)
(14)
We can get the expression for the substrate
concentration by equating the expression for µ
from Monod kinetics to Eq. (10)
22
Steady-state Values
(15)
or
(16)
So now we can get X entirely as a function of D
(17)
23
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.

24
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.

25
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.

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Fed-batch Operation
F, S0
F, S0
V0, X, S, P
Vw, X, S, P
V, X, S, P
Start fed-batch
Fed batch fill
Harvest
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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
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For a batch culture
(1)
Since liquid is being added, the volume is
changing
or
(2)
If the total amount of biomass (grams) in the
reactor is Xt then the concentration X is
(3)
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So the change in the biomass concentration with
time is
(4)
Using the definition of the growth rate
...the dilution rate
...and the expression for dV/dt
we have
(5)
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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
(6)
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At quasi-steady state, for this case we will have
(7)
So X is constant (but not Xt). Now we have
(8)
Assuming Monod growth kinetics, this gives (just
as in the case of a chemostat)
(9)
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If the total amount of substrate in the reactor
is St, then a substrate mass balance gives
(10)
which, for quasi-steady state gives
(11)
Returning to Equation (4), we have, at
quasi-steady state
(12)
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Integrating, we have
(13)
since X is constant (dX/dt0). Therefore, the
total biomass in a fed-batch reactor operated as
assumed here increases linearly with time.
Substituting the appropriate expression for X
(14)
Often, SltltS0 and X0ltltYX/SS0 and so
(15)
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Product Output
If the specific productivity (g product/g cells/
hr) is constant
or
(16)
where Pt is the total product concentration in
the reactor
Substituting
35
we have
(17)
Integrating this expression, we have
(18)
or in terms of concentration
(19)
36
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.

37
For the case of repeated fed-batch cultures
(20)
Where Vw is the volume just before harvesting, V0
is the volume after harvesting, DwF/Vw and
(21)
tw is the cycle time and is given by
(22)
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With this definition, we now have
(23)
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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

40
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

41
Major Limitation

• Mass transfer (diffusional) resistances
• Whole cells provide cofactors, reducing power,
  energy that many enzymatic reactions require.

Advantage over immobilized enzymes
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Types of Immobilization

• Active Immobilization similar to enzyme
  immobilization. Entrapment and binding.
• Passive Immobilization Biofilm multilayer
  growth on solid surfaces.

43
Diffusional Limitations

• Analysis similar to immobilized enzymes
• Damkohler number
• Effectiveness factor
• Thiele modulus

44
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.

45
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