CHAPTER 4:

BIOREACTOR CONSIDERATIONS FOR

SUSPENSION, IMMOBILIZED, ANIMAL AND PLANT
CELL CULTURES

Choosing the Cultivation

Method

Batch or Continuous culture?

Do I need to modify the batch and
continuous reactors?
Chemostat

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

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

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

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

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

Applicable to fermentations where the

growth and product formation need to be
separated into stages.

Fed-batch Operation

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

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

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
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

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

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

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 37C 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

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
37C.

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

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.

Considerations for Plant cell Bioreactor

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

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

Low growth rates of plant cells presents

problems for large-scale system:

Maintenance of aseptic conditions for the 24 weeks of fermentation is difficult.

Genetic instability of many cell lines.

Considerations for Solid State

Fermentation (SSF)

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)

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.