Bioreactors

Bubble Column
Alternatives to the stirred reactor include vessels with no mechanical
agitation. In bubble-column reactors, aeration and mixing are achieved
by gas sparging; this requires less
energy than mechanical stirring. Bubble columns are applied
industrially for production of bakers' yeast, beer and vinegar, and for
treatment of wastewater.
Bubble columns are structurally very simple. As shown in Figure 13.5,
they are generally cylindrical vessels with height greater than twice the
diameter. Other than a sparger for entry of compressed air, bubble
columns typically have no internal structures. A height-to-diameter
ratio of about 3:1 is common in bakers' yeast production; for other
applications, towers with height-to-diameter ratios of 6:1 have been
used.
Perforated horizontal plates are sometimes installed in tall bubble
columns to break up and redistribute coalesced bubbles.

Advantages of bubble columns include low capital cost, lack of

moving parts, and satisfactory heat- and mass-transfer performance.

As in stirred vessels, foaming can be a problem requiring mechanical

dispersal or addition of antifoam to the
medium.
Bubble-column hydrodynamics and mass-transfer characteristics depend entirely on the
behaviour of the bubbles released from the sparger. Different flow regimes occur

depending on the gas flow rate, sparger design, column diameter and medium properties such
as viscosity. Homogeneous flow occurs only at low gas flow rates and when bubbles leaving the
sparger are evenly distributed across the column cross-section.

In homogeneous flow, all bubbles rise with the same upward velocity and there is no backmixing
of the gas phase. Liquid mixing in this flow regime is also limited, arising solely from entrainment
in the wakes of the bubbles. Under normal operating conditions at higher gas velocities, large
chaotic circulatory flow cells develop and heterogeneous flow occurs as illustrated in Figure 13.6.
In this regime, bubbles and liquid tend to rise up the centre of the column while a corresponding
downflow of liquid occurs near the walls. Liquid circulation entrains bubbles so that some
backmixing of gas occurs.
Air lift reactor
An air-lift fermenter (Fig. 7.47) is a riser tube (liquid ascending)
connected to a downcomer tube (liquid descending). Figure 7.47a
shows an external riser and Fig. 7.47b an internal riser. Air or gas
mixtures are introduced into the base of the riser by a sparger during
normal operating conditions.

The driving force for circulation of medium in the vessel is produced by

the difference in density between the liquid column in the riser (excess
air bubbles in the medium) and the liquid column in the downcomer
(depleted in air bubbles after release at the top of the loop).
As in bubble columns, mixing in airlift reactors is accomplished without
mechanical agitation. Airlift reactors are often chosen for culture of
plant and animal cells and immobilised catalysts because shear levels
are significantly lower than in stirred vessels.
Stirred and Air-Driven Reactors:
Comparison of Operating Characteristics
For low-viscosity fluids, adequate mixing and mass transfer can be achieved in stirred tanks, bubble
columns and airlift vessels.

When a large fermenter (50-500 m3) is required for low-viscosity culture, a bubble column is an
attractive choice because it is simple and cheap to install and operate.

Mechanically-agitated reactors are impractical at volumes greater than about 500 m3 as the power
required to achieve adequate mixing becomes extremely high

If the culture has high viscosity, sufficient mixing and mass transfer cannot be provided by air-driven
reactors.

Stirred vessels are more suitable for viscous liquids because greater power can be input by mechanical
agitation.
Heat transfer can be an important consideration in the choice between
air-driven and stirred reactors.

Mechanical agitation generates much more heat than sparging of

compressed gas.

When the heat of reaction is high, such as in production of single-cell

protein from methanol, removal of frictional stirrer heat can be a
problem so that air-driven reactors
may be preferred.
Packed Bed
Packed-bed reactors are used with immobilised or particulate
biocatalysts. The reactor consists of a tube, usually vertical,
packed with catalyst particles. Medium can be fed either at the top or
bottom of the column and forms a continuous liquid phase between the
particles.

Packed-bed reactors have been used commercially with immobilised cells

and enzymes for production of aspartate and fumarate, conversion of
penicillin to 6-aminopenicillanic
acid, and resolution of amino acid isomers.
Mass transfer between the liquid medium and solid catalyst is facilitated at high
liquid flow rates through the bed; to achieve this, packed beds are often operated
with liquid recycle as shown in Figure 13.8. The catalyst is prevented from leaving the
column by screens at the liquid exit. The particles should be relatively incompressible
and able to withstand their own weight in the column without deforming and
blocking liquid flow. Recirculating medium must also be clean and free of debris to
avoid clogging the bed.

Aeration is generally accomplished in a separate vessel; if air is sparged directly into

the bed, bubble coalescence produces gas pockets and flow channelling or mal-
distribution. Packed beds are unsuitable for processes which produce large quantities
of carbon dioxide or other gases which can become trapped in the packing.
Trickle bed reactor
The trickle-bed reactor is another variation of the packed bed.
As illustrated in Figure 13.10, liquid is sprayed onto the top of
the packing and trickles down through the bed.

Air may be introduced at the base; because the liquid

phase is not continuous throughout the column, air and other
gases move with relative ease around the packing. Trickle-bed
reactors are used widely for aerobic wastewater treatment.
Fluidised bed reactor
When packed beds are operated in upflow mode with catalyst
beads of appropriate size and density, the bed expands at high
liquid flow rates due to upward motion of the particles. This is
the basis for operation of fluidised-bed reactors as illustrated in
Figure 13.9. Because particles in fluidised beds are in constant
motion, channelling and clogging of the bed are avoided and
air can be introduced directly into the column. Fluidised-bed
reactors are used in waste treatment with sand or similar
material supporting mixed microbial populations. They are
also used with flocculating organisms in brewing and for
production of vinegar.
Perfusion system
An alternative to fed-batch culture is a perfusion system. Such systems
are used most often with animal cell cultures. The basic characteristic is
constant medium flow, cell retention, and in some cases selective
removal of dead cells. High cell density can be achieved. Cell retention
is usually achieved by membranes or screens or by a centrifuge capable
of selective cell removal.
The potential advantages of a perfusion system is the potential removal
of cell debris and inhibitory by-products, removal of enzymes released
by dead cells that may destroy product, shorter exposure time of
product to potentially harsh production conditions (compared to batch
or fed-batch operation), high per-unit volumetric productivity (due to
high cell density and metabolism), and a rather constant environment.
The primary disadvantage is that a large amount of medium is typically
used and the nutrients in the medium are less completely utilized than
in batch or fed-batch systems. High medium usage is expensive, owing
not only to the high cost of raw materials but also to the costs to
prepare and sterilize the medium. Additionally, costs for waste
treatment increase.
Mammalian cell retention devices
for stirred perfusion bioreactors
During a typical perfusion run inoculated at 105
cells mL−1, the cell concentration will increase by
two orders of magnitude or more over a span of
approximately 10 days. As in any culture, some
nonviable cells and debris will accumulate
throughout the run due to apoptosis and necrosis
and cell lysis will release intracellular
macromolecules into the culture medium. These
dynamic characteristics of long-term perfusion
culture present a challenge to the robust
operation of cell retention devices. Ideally, a cell
retention device should operate satisfactorily for
the required duration without replacement or
maintenance in order to minimize additional risk
of contamination.
The device should not adversely affect cell viability or productivity and
it should separate close to 100% of the viable cells from the effluent
stream, regardless of the cell concentration, in order to maintain high
viable cell concentration in the reactor. Finally, although the build-up of
nonviable cells and debris can be mitigated by the use of a bleed
stream (whereby the suspension is drawn directly from the reactor
without cell separation), an ideal retention device should selectively
retain viable cells while allowing the nonviable cells to pass through.
Bioprocess Considerations in Using
Animal Cell Cultures
Animal cells vary in size (10 to 30 mm) and shape (spherical,
ellipsoidal). Animal cells do not have a cell wall, but are surrounded by
a thin and fragile plasma membrane that is composed of protein, lipid,
and carbohydrate. This structure results in significant shear sensitivity.
The surface of an animal cell is negatively charged, and cells tend to
grow on positively charged surfaces, such as Sephadex or collagen
(anchorage-dependent cells).
Many cells possess specific cell surface receptors that adhere to ligands
on the surface. For example, the binding to collagen may be nonspecific
or may be mediated by specific cell surface receptors. The degree of
cell adhesiveness is usually greater if attachment is
receptor mediated. Some animal cells such as hybridomas are non-
anchorage dependent and grow in suspension culture.
Predominant damage mechanism in stirred tank and air lift reactors was due to
sparging and the break up of bubbles on the medium surface.

This type of damage causing cell death might be reduced by increasing the
height to diameter ratio in the vessel, increasing the bubble size, decreasing the
gas flow rate or by adding protective agents.

Other damage mechanisms in stirred fermenters are caused by cell-microcarrier

and microcarrier-microcarrier interactions. Damage of this type may be reduced
by reducing the impeller speed, impeller diameter, microcarrier size and
concentration or by increasing the viscosity of the medium
Mammalian cells are large (10 to 20 mm diameter), slow growing
(doubling time 10 to 50 h), and very shear sensitive. Moreover, some
animal cells are anchorage dependent and must grow on surfaces of
glass, specially treated plastics, natural polymers such as collagen, or
other support materials; some are not anchorage dependent and can
grow in suspension culture. Product concentration (titer) is usually very
low (mg/ml), and toxic metabolites such as ammonium and lactate are
produced during growth. These properties of animal cells set certain
constraints on the design of animal cell bioreactors. Certain common
features of these reactors are the following:
• 1. The reactor should be gently aerated and agitated. Some mechanically
agitated reactors operating at agitation speeds over 20 rpm and bubble-
column and airlift reactors operating at high aeration rates may cause shear
damage to cells. Shear sensitivity is strain dependent.
• 2. Well-controlled homogeneous environmental conditions (T, pH, DO,
redox potential) and a supply of CO2-enriched air need to be provided.
• 3. A large support material surface–volume ratio needs to be provided for
anchorage dependent cells.
• 4. The removal of toxic products of metabolism, such as lactic acid and
ammonium, and the concentration of high-value products, such as MAb’s,
vaccines, should be accomplished during cell cultivation.
Reactors
1. Stirred tank reactor
2. Perfusion reactor
3. Hollow fibre reactor
4. Air lift reactor
5. Fluidized bed reactor
6. Packed bed reactor
The use of microcarriers for the cultivation of anchorage-dependent mammalian
cells is an attractive approach. For example, microcarriers such as DEAE-Sephadex
beads, which provide large surface per unit volume of reactor, allow high cell
concentrations in the medium. The microcarrier beads with cells are suspended in a
stirred bioreactor.

Many shear protecting agents (such as serum or Pluronic F-68) work by preventing
cells from accumulating at the gas–liquid interface.

The immobilization of mammalian cells in gel beads (agar, alginate, collagen,

polyacrylamide) and the use of such systems in a packed- or fluidized-bed
configuration are possible.
Microencapsulation is another method used for the immobilization of
animal cells. Hybridoma cells have been encapsulated within spherical
membranes of polylysine–alginate for production of MAb’s.

Hollow-fiber reactors have also been used to provide a high growth

surface–volume ratio and, therefore, high cell concentrations. Cells are
immobilized on the external surfaces of hollow fibers, and nutrients
pass through the tubes.
Bioprocess Considerations in Using
Plant Cell Cultures
The factory production of chemicals from plant cell tissue culture offers
a number of important advantages:
1. Control of supply of product independent of availability of the plant
itself.
2. Cultivation under controlled and optimized conditions.
3. Strain improvement with programs analogous to those used for
microbial systems.
4. With the feeding of compounds analogous to natural substrates,
novel compounds not present in nature can be synthesized.
Potential Advantages and Problems of Large-scale Immobilized Plant Cell Cultures

Potential advantages

Continuous operation facilitated

High cell concentrations
Cell reuse may lead to increased efficiency
Cells can be protected from shear
Once immobilized, the slow growth and strain instability of plant cells are no longer problems
Media can be easily changed, which would be important for processes that require a series of media for optimal
production
Continuous removal of inhibitory metabolites may enhance the overall cellular metabolism or unmask biochemical
pathways
May be able to better exploit the biological relationships between aggregation, morphological differentiation, and
secondary metabolite production
• Potential problems
• Large-scale aseptic immobilization procedures must be developed
• Mass transfer limitations may significantly affect cell metabolism
(positively and adversely)
• Products must be released from the cell into the medium
• Experience in the scale-up of immobilized-cell systems is limited