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Title: A1256655380OogvX


1
Industrial Microbiology INDM 4005 Lecture
14 23/03/04
2
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

3
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.

4
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

5
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

6
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.

7
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.

8
  • 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.
9
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.

10
Aspergillus niger mycelia
11
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.

12
4. Shake flasks
13
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.

14
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

15
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
16
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

17
Baffled flask
Unbaffled flask
18
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

19
5. Mechanically stirred bioreactors
20
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.

21
(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.

22
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23
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.

24
Bubble driven bioreactors
25
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

26
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.

27
Airlift bioreactors
Small bubbles lead to an increased surface area
for oxygen transfer.
28
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.

29
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

30
(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.

31
Airlift bioreactor
32
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.

33
(7.2) Airlift bioreactors - Disengagement zone
34
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.

35
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

36
8. Packed bed and trickle flow bioreactors
  • The topic of packed bed bioreactors was discussed
    in another lecture on immobilisation.

37
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.

38
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39
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.
40
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.

41
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.

42
9. Fluidised bed reactors
43
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.

44
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.

45
Fluidised bed reactors
46
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
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