Industrial Microbiology

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Industrial Microbiology INDM 4005 Lecture
14 23/03/04
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Tutorial on Bioreactors

• 1. Introduction 2. Methods of aeration 3.
  Surface aeration 4. Shake flasks 5.
  Mechanically stirred bioreactors (5.1) Sparged
  stirred tank bioreactors 6. Bubble driven
  bioreactors 7. Airlift bioreactors (7.1)
  Air-riser and downcomer (7.2) Disengagement
  zone 8. Packed bed and trickle flow bioreactors
  9. Fluidised bed bioreactors

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Bioreactors- Introduction

• Previous lectures have stress the importance of
  considering process engineering factors when
  culturing cells.
• Biological factors include the characteristics of
  the cells, their maximum specific growth rate,
  Monod constant, yield coefficient, pH range and
  temperature range.
• We have seen however that the productivity of a
  fermentation is determined by the mode of
  operation of the fermentation process eg. the
  advantages of fed-batch and continuous
  fermentations over batch fermentations.

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Bioreactors- Introduction

• Likewise mass transfer, in particular, oxygen
  transfer was highlighted as an important factor
  which determined how a reactor must be designed
  and operated.
• Cost was also described as an important
  consideration. The larger the reactor or the
  faster the stirrer speed, the greater the costs
  involved.
• In this lecture, we shall look into how
  bioreactors are designed to meet cost, biological
  and engineering needs

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2. Methods of Aeration

• A bioreactor is a reactor system used for the
  culture of microorganisms. They vary in size and
  complexity from a 10 ml volume in a test tube to
  computer controlled fermenters with liquid
  volumes greater than 100 m3. They similarly vary
  in cost from a few cents to a few million
  dollars.
• In the following sections we will compare the
  following reactors
• Standing cultures
• Shake flasks
• Stirred tank reactors
• Bubble column and airlift reactors
• Fluidized bed reactors

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3. Standing cultures

• In standing cultures, little or no power is used
  for aeration. Aeration is dependent on the
  transfer of oxygen through the still surface of
  the culture.

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Standing cultures

• The rate of oxygen transfer will be poor due to
  the small surface area for transfer. Standing
  cultures are commonly used in small scale
  laboratory systems in which oxygen supply is not
  critical. For example, biochemical tests used for
  the identification of bacteria are often
  performed in test-tubes containing between 5-10
  ml of media.
• T-flasks used in the small scale culture of
  animal cells are another example of a standing
  culture. T-flasks are normally incubated
  horizontally to increase the surface area for
  oxygen transfer.

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• The surface aeration rate in standing cultures
  can be increased by using large volume flasks.
• The following photograph shows a 250 ml
  Erlenmeyer flask containing 100 ml of medium and
  a 3 litre "Fernback" flask containing 1 litre of
  medium.

Note how the latter has a large surface area.
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Standing cultures

• Large Pyrex flasks are used for the small scale
  production of fermented products. One example is
  Kombucha tea which is a tea brewed by mixture of
  yeasts and acetic acid bacteria.
• Standing culture aeration is not restricted to
  the laboratory.
• In some countries, where the availability of
  electricity is unreliable, citric acid is
  produced using surface culture techniques.
• In these cultures, the Aspergillus niger mycelia
  are grown on the surface of liquid media in large
  shallow trays.
• The medium is neither gassed nor agitated.

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Aspergillus niger mycelia
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Standing cultures

• Aerobic solid substrate fermentations are another
  example of standing cultures. In these
  fermentations, the biomass is grown on solid
  biodegradable substrates such as water softened
  bran, rice or barley.
• The solids may be continuously or periodically
  turned over to improve aeration and to regulate
  the culture temperature. One example of a
  commercial scale, solid substrate fermentation is
  the production of koji by Aspergillus oryzae on
  soya beans which is part of the soya sauce
  process.
• Another is mushroom cultivation. Considerable
  research is currently being invested into the
  feasibility of producing biochemicals by solid
  substrate fermentations.

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4. Shake flasks
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Shake flasks

• Shake flasks are commonly used for small scale
  cell cultivation.
• Through continuous shaking of the culture fluid,
  higher oxygen transfer rates can be achieved as
  compared to standing cultures.
• Shaking continually breaks the liquid surface and
  thus provides a greater surface area for oxygen
  transfer.
• Increased rates of oxygen transfer are also
  achieved by entrainment of oxygen bubbles at the
  surface of the liquid.

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Shake flasks

• Although higher oxygen transfer rates can be
  achieved with shake flasks than with standing
  cultures, oxygen transfer limitations will still
  be unavoidable particularly when trying to
  achieve high cell densities.
• The rate of oxygen transfer in shake flasks is
  dependent on the
• shaking speed
• the liquid volume
• shake flask design

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Shake flasks O2 Transfer
kLa decreases with liquid volume
kLa is higher when baffles are present
kLa
kLa
kLa
kLa
kLa increases with liquid surface area
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Shake flasks O2 Transfer

• The kLa will increase with the shaking speed.
• At high shaking speeds, bubbles become entrained
  into the medium to further increases the oxygen
  transfer rate.
• The presence of baffles in the flasks will
  further increase the oxygen transfer efficiency,
  particularly for orbital shakers.
• The following photographs show how baffles
  increase the level of gas entrainment in a shake
  flask being shaken in an orbital shaker at 150
  rpm

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Baffled flask
Unbaffled flask
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Shake flasks O2 Transfer

• Note the high level of foam formation in the
  baffled flask due to the higher level of gas
  entrainment.
• The same improvement in oxygen transfer is not as
  evident with horizontal reciprocating shakers.
• The appropriate liquid volume is determined by
  the flask volume. For example, for a standard
  250ml flask, the liquid volume should not exceed
  70 ml while for a 1 litre flask, the liquid
  volume should be less than 200 ml.
• Larger liquid volumes can be used with wide based
  flasks

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5. Mechanically stirred bioreactors
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Mechanically stirred bioreactors

• For aeration of liquid volumes greater than 200
  ml, various options are available.
• Non-sparged mechanically agitated bioreactors can
  supply sufficient aeration for microbial
  fermentations with liquid volumes up to 3 litres.
  
• However, stirring speeds of up to 600 rpm may be
  required before the culture is not oxygen
  limited.
• In non-sparged reactors, oxygen is transferred
  from the head-space above the fermenter liquid.
  Agitation continually breaks the liquid surface
  and increases the surface area for oxygen
  transfer.

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(5.1) Mechanically stirred reactors - Sparged
stirred tank bioreactors

• For liquid volumes greater than 3 litres, air
  sparging is required for effective oxygen
  transfer.
• The introduction of bubbles into the culture
  fluid by sparging, leads to a dramatic increase
  in the oxygen transfer area.
• Agitation is used to break up bubbles and thus
  further increase kLa.
• Sparged fermenters required significantly lower
  agitation speeds for aeration efficiencies
  comparable to those achieved in non-sparged
  fermenters.
• Air-sparged fermenters can have liquid volumes
  greater than 500,000 litres.

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6. Bubble driven bioreactors

• Sparging without mechanical agitation can also be
  used for aeration and agitation. Two classes of
  bubble driven bioreactors are bubble column
  fermenters and airlift fermenters.
• Bubble driven bioreactors are commonly used in
  the culture of shear sensitive organisms such as
  moulds and plant cells. An airlift fermenter
  differs from bubble column bioreactors by the
  presence of a draft tube which provides better
  mass and heat transfer efficiencies.
• Airlift fermenters are however considerably more
  expensive to construct than bubble column
  reactors. There are several designs for air-lift
  fermenters although the most commonly used design
  is one with a central draft tube.

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Bubble driven bioreactors
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Bubble driven bioreactors

• An airlift fermenter differs from bubble column
  bioreactors by the presence of a draft tube which
  provides
• better mass and heat transfer efficiencies
• more uniform shear conditions.
• Bubble driven fermenters are generally tall with
  liquid height to base ratios of between 81 and
  201.
• The tall design of these fermenters leads to high
  gas hold-ups, long bubble residence times and a
  region of high hydrostatic pressure near the
  sparger at the base of the fermenter.
• These factors lead to high values of kLa and Co
  thus enhanced oxygen transfer rates

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7. Airlift bioreactors

• An airlift fermenter differs from bubble column
  bioreactors by the presence of a draft tube.
• The main functions of the draft tube are to
• Increase mixing through the reactor The presence
  of the draft tube enhances axial mixing
  throughout the whole reactor
• Reduce bubble coalescence. This presumably
  occurs due to circulatory effect that the draft
  tube induces in the reactor. The circulation
  occurs in one direction and hence the bubbles
  also travel in one direction.

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Airlift bioreactors
Small bubbles lead to an increased surface area
for oxygen transfer.
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Airlift bioreactors

• Equalise shear forces throughout the reactor.
  Major reason why the productivity of cells grown
  in airlift bioreactors have higher productivities
  than those grown in stirred tank reactors.

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Airlift bioreactors

• The major disadvantages of air-lift fermenters
  are
• - high energy requirements
• - excessive foaming
• - cell damage due to bubble bursting
  particularly with animal cell culture

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(7.1) Airlift bioreactor Air-riser and
down-comer

• An air-lift reactor is divided into three
  regions
• - the air-riser
• - down-comer
• - disengagement zone.

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Airlift bioreactor
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Airlift bioreactor

• The region into which bubbles are sparged is
  called the air-riser. The air-riser may be on the
  inside or the outside of the draft-tube. The
  latter design is preferred for large scale
  fermenters as it provides better heat transfer
  efficiencies.
• The rising bubbles in the air-riser cause the
  liquid to flow in a vertical direction. To
  counteract these upward forces, liquid will flow
  in a downward direction in the down-comer. This
  leads to liquid circulation and thus improved
  mixing efficiencies as compared to bubble
  columns.
• The enhanced liquid circulation also causes
  bubbles to move in a uniform direction at a
  relatively uniform velocity. This bubble flow
  pattern reduces bubble coalescence and thus
  results in higher kLa values as compared to
  bubble column reactors.

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(7.2) Airlift bioreactors - Disengagement zone
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Airlift bioreactors - Disengagement zone

• The roles of the disengagement zone are to
• add volume to the reactor,
• reduce foaming and
• minimise recirculation of bubbles through the
  down comer.

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Airlift bioreactors - Disengagement zone

• The sudden widening at the top of the reactor
  slows the bubble velocity and thus disengages the
  bubbles from the liquid flow.
• Carbon-dioxide rich bubbles are thus prevented
  from entering the downcomer.
• The reduced bubble velocity in the disengagement
  zone also leads to a reduction in the loss of
  medium due aerosol formation.
• The increase in area will also helps to stretch
  bubbles in foams, causing the bubbles to burst.
  The axial flow circulation caused by the draft
  tube also helps to reduce foaming

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8. Packed bed and trickle flow bioreactors

• The topic of packed bed bioreactors was discussed
  in another lecture on immobilisation.

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Packed bed bioreactors

• The rate of mass transfer between the cells and
  the medium depends on the flow rate and on the
  thickness of the biomass film on or near the
  surface of the solid particles.
• Packed bed reactors often suffer from problems
  caused by poor mass transfer rates and clogging.
  Despite this they are used commercially with
  enzymatically catalysts and with slowly or
  non-growing cells.
• They are also used in the anaerobic treatment of
  high strength wastewaters (eg. food processing
  wastes). Large plastic blocks are used as solid
  supports for the cells. These blocks have a large
  surface area for cell immobilization and when
  packed in the reactor are difficult to clog.

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Trickle flow bioreactors

• Trickle bed reactors are a class of packed bed
  reactors in which the medium flows (or trickles)
  over the solid particles. In these reactors, the
  particles are not immersed in the liquid.

The liquid medium trickles over the surface of
the solids on which the cells are
immobilized They are used widely in aerobic
treatment of sewage.
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Trickle flow bioreactors

• Oxygen transfer is enhanced by ensuring that the
  cells are covered by only a very thin layer of
  liquid, thus reducing the distance over which the
  dissolved oxygen must diffuse to reach the cells.

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Trickle flow bioreactors

• Because stirring is not used, considerable
  capital costs are saved.
• However, oxygen transfer rates per unit volume
  are low compared with sparged stirred tank
  systems.
• Trickle flow systems are used widely for the
  aerobic treatment of sewage.
• They are used to polish effluent from the
  activated sludge or anaerobic digestion process
  and for the nitrification of ammonia.

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9. Fluidised bed reactors
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Fluidised bed reactors

• Fluidised bed bioreactors are one method of
  maintaining high biomass concentrations and at
  the same time good mass transfer rates in
  continuous cultures.
• Fluidised bed bioreactors are an example of
  reactors in which mixing is assisted by the
  action of a pump. In a fluidised bed reactor,
  cells or enzymes are immobilised in and/or on the
  surface of light particles.
• A pump located at the base of the tank causes the
  immobilised catalysts to move with the fluid. The
  pump pushes the fluid and the particles in a
  vertical direction. The upward force of the pump
  is balanced by the downward movement of the
  particles due to gravity. This results in good
  circulation.

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Fluidised bed reactors

• For aerobic microbial systems, sparging is used
  to improve oxygen transfer rates.
• A draft tube may be used to improve circulation
  and oxygen transfer. Both aerobic and anaerobic
  fluidised bed bioreactors have been developed for
  use in waste treatment.
• Fluidised beds can also be used with microcarrier
  beads used in attached animal cell culture.
• Fluidised-bed microcarrier cultures can be
  operated both in batch and continuous mode. In
  the former the fermentation fluid is recycled in
  a pump-around loop.

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Fluidised bed reactors
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Summary

• Looked at methods of aeration in different
  bioreactors
• Aeration in standing cultures
• Oxygen transfer in shake flasks
• Advantages and applications of mechanically
  stirred bioreactors
• Bubble driven bioreactors
• Airlift bioreactors
• Packed bed and trickle flow bioreactors
• Fluidised bed bioreactors