Download Chapter 4 Bioreactor Considerations for Suspension, Animal and

CHAPTER 4:
BIOREACTOR CONSIDERATIONS FOR
SUSPENSION, IMMOBILIZED, ANIMAL AND
PLANT CELL CULTURES
Choosing the Cultivation Method
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Batch or Continuous culture?
Do I need to modify the batch and continuous
reactors?
 Chemostat
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with recycle
 Multistage chemostat systems
 Fed-batch operation
Immobilization
Solid-state Fermentation
What are the advantages and
disadvantages of batch culture?
Advantages of continuous culture
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Growth rate can be controlled and maintained.
Effect of changes in physical or chemical
parameters can be examined.
Biomass concentration can be maintained by
varying the dilution rate.
Find out more on the advantages.
Dilution Rate, D
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D = F/V, the number of culture volumes passing through
the reactor per hour.
dX/dt=-DX + X = X(-D)
During steady state, dX/dt = 0 consequently  = D
So, by varying the medium supply, growth rate can be
varied.
This holds until D  m
Under these condition, the nutrient is no longer limiting
Therefore, the expression (-D) becomes -ve
Critical Dilution Rate
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The lowest dilution rate at which wash out occurs.
Dc is approximately equal to m.
At dilution rates approaching Dc, the chemostat
becomes less stable since slight fluctuations in the
flow rate.
A major drawback of chemostat is that they work
best at lower dilution rates where the changes in X
and S are small.
Modifications for batch and continuous
bioreactor
Choosing the cultivation method can affect:
1.
Product concentration and purity
2.
Degree of substrate conversion
3.
Yields of cells and products
4.
Capital cost in a process
Modifications that can be done:
 Chemostat with recycle
 Multistage chemostat systems
 Fed-batch operation
Chemostat with recycle
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To keep the cell concentration higher than the
normal steady-state level. Cells in the effluent can
be recycled back to the reactor.
Advantage of cell recycle:
 Increase
productivity for biomass production
 A chemostat can be operated at dilution rates higher
than the specific growth rate when cell recycle is used.
Multistage chemostat system
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Applicable to fermentations where the growth and
product formation need to be separated into
stages.
Fed-batch Operation
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Useful in antibiotic fermentation.
Reactor is fed continuously (or intermittentlly),
reactor is emptied periodically.
Purpose is to maintain low substrate concentration, S
Useful in overcoming substrate inhibition or
catabolic repression, so that product formation
increases.
Considerations for Microbial cell
Bioreactor
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Adequate mixing is essential to ensure adequate supply
of nutrients to the cells and removing any toxic
materials from their vicinity.
Mixing also affects the supply of oxygen by breaking
large bubbles into smaller ones and dispersing them in
the liquid so that their residence time in the reactor is
increased.
Accurate temperature control and efficient heat transfer
also require good mixing.
Mixing is also important to ensure quick dispersion of
any added solutions such as acids, base, nutrients, so
that there is no local high concentration buildup.
Considerations for Microbial cell
Bioreactor
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At high growth rates, there is a need for sophisticated pH
control and the addition of medium components, especially
glucose when the fermentation is in fed-batch mode.
High growth rates also result in a high output of metabolic
energy, which requires high energy input for cooling in large
fermenters.
It is therefore necessary to evaluate the maximal specific
heat production (cal/min-DCW) during a fermentation
process so that the cooling requirements may be precisely
determined.
Cultures containing cells that grow relatively slow are much
more susceptible to microbial contamination.
Considerations for Microbial cell
Bioreactor
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Most microorganism grow in the pH 5.5-8.8 range, with fungi optimum
at pH 5-7 and yeast pH 4-5.
pH 4-5 has been used for yeast fermentation to facilitate growth and
prevent contamination from other microorganisms.
Production of foam is very common in microbial fermentations. It arises
from the flow of air through the liquid medium and the subsequent
formation of small bubbles as a consequence of mixing.
If the bubble film is not strong enough, the bubble is easily destroyed
and no foam is formed. This strength is dependent mainly on the surface
tension of the liquid.
Compounds that contribute to lowering the surface tension include
proteins and protein hydrolysates, as well as oils and fats.
In large scale industrial fermentation, using yeast with high
concentration of corn steep liquor and molasses, the foaming is
problematic.
Considerations for Microbial cell
Bioreactor
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Even with less complex media, foam can form, usually in the last
stage of fermentation involving high air-flow, high-speed mixing,
and cell lysis.
Except for beer production, foam is usually considered to be
negative phenomenon because it can clog filters, which can cause
dangerous increase in pressure.
Wetting of the filters also stops them from functioning properly and
the fermentor is more susceptible to contamination.
The stable foam can also cause entrapment of oxygen so that the
dissolved oxygen level increase substantially. However, the increased
dissolved oxygen level is immediately decreased when the foam is
broken. Such large and sudden changes in measured dissolved
oxygen can play havoc if the fermentation is automatically
controlled depending on the dissolved oxygen concentration.
Considerations for Microbial cell
Bioreactor
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In large bioreactors, it may be
advantageous to install
mechanical foam breaker.
In smaller bioreactors, chemical
antifoam agents is generally
used.
Considerations for Animal culture
bioreactor
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Products of animal cell culture: enzymes, hormones, vaccines,
monoclonal antibodies.
Animal cells do not have cell walls, but are surrounded by a
thin and fragile plasma membrane.
This structure results in significant shear sensitivity.
Mammalian cells grow at 37°C and pH 7.3.
Usually 5% CO2-enriched air is used to buffer the medium pH.
The culture medium needs to be gently aerated and agitated.
Some mammalian cells are anchorage dependent and must
grow on surfaces of glass or other support material. Some are
not anchorage dependent and can grow in suspension culture.
Considerations for Animal culture
bioreactor
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The reactor should be gently aerated and agitated. Some
mechanically agitated reactors operating over 20 rpm and
bubble column/airlift bioreactors operating at high aeration
rates may cause shear damage to cells. Shear sensitivity is
strain dependent.
Well-controlled homogeneous environmental conditions (T,
pH, DO) and supply of CO2-enriched air must be provided.
A large support material surface-volume ratio needs to be
provided for anchorage-dependent cells.
The removal of toxic products of metabolism, such as lactic
acid and ammonium, and high-value products such as Mab,
vaccines should be accomplished during cell cultivation.
Considerations for Animal culture
bioreactor
Lab-scale cultivation:
 T-flasks (25-100 ml) – for anchorage dependent cell
lines and shallow suspension culture.
 Spinner flask (100 ml – 1L) – with paddle type
magnetic stirrers.
 Roller bottles (50 ml – 5L) rotating at about 1-5 rpm
 Trays containing shallow liquid suspension culture.
These reactors are placed in a CO2 incubator at 37°C.
Considerations for Animal culture
bioreactor
Bioreactors for anchorage dependent cells:
 Hollow fiber reactors
 Ceramic matrix systems
 Weighted porous beads (immobilization)
Bioreactors for suspension culture:
 Modified stirred bioreactors
 Airlift bioreactors
 Bubble column reactors
Considerations for Plant cell Bioreactor
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In the Western world, over 25% of pharmaceuticals are
derived from extraction from whole plant.
Examples of plant products:
Pharmaceuticals: taxol, morphine, codeine
 Food colors: anthocyanins, saffron, shikonin
 Flavors: vanilla, strawberry, garlic
 Fragrances: jasmine, lemon, mint, rose
 Sweeteners: Miraculine, stevioside, thaumatin
Whether the product is a chemical or a new plant, the
biochemical engineer must become familiar with some basic
characteristics of plants and their implications for bioreactor
design.
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Considerations for Plant cell Bioreactor
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Plant cells are large, and when they are exposed to
turbulent shear fields where the eddy size approaches
the cell size, the cells can be exposed to a twisting
motion that can damage them.
Lower level of shear appear to affect cell surface
receptors and nutrient transport. Reactors of high shear
must be avoided.
Plant cells can withstand far more shear than animal
cells.
Stirred tanks designed for bacterial culture are not
good choices, but modified stirred tanks can be
suitable, eg change impellers.
Considerations for Plant cell Bioreactor
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Plant cell cultures can achieve high cell densities and
viscosities. Their reduced respiration rate compensates
in part of the need for vigorous agitation.
Airlift reactors are better for low or moderate cell
densities.
Reactor with paddle type or helical ribbon impellers for
high cell densities.
Mixing depends on combination of sparging and
mechanical agitation. Oversparging can be a problem
for plant cells, because plants make at least one
volatile hormone, ethylene, and its rapid removal can
affect productivity.
Challenges in plant cell fermentation
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Low growth rates of plant cells presents problems
for large-scale system:
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Maintenance of aseptic conditions for the 2-4 weeks of
fermentation is difficult.
Genetic instability of many cell lines.
Considerations for Solid State
Fermentation (SSF)
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Fermentation of solid substrates at low moisture levels
or water activities.
Most SSF are mold fermentation producing extracellular
enzymes on moist agricultural substrates.
Low contamination risk due to low moisture level.
Easy product separation.
Poor mixing characteristics
Difficult to control pH, DO, temperature within the
fermentation mash.
Usually a rotating-drug fermenter is used.
Considerations for Solid State
Fermentation (SSF)
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Many SSF are shear sensitive due to mycelia
disruption at high rotation speeds.
At low agitation rate, oxygen transfer and CO2
evolution rates become limiting.