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Industrial Biotechnology lesson 6


Mass transfer Transfer of nutrients from the aqueous phase into the microbial cells during fermentation is relatively straightforward as the nutrients are normally ... – PowerPoint PPT presentation

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Title: Industrial Biotechnology lesson 6

Industrial Biotechnologylesson 6

  • The function of the fermenter or bioreactor is to
    provide a suitable environment in which an
    organism can efficiently produce a target
    productthe target product might be
  • Cell biomass
  • Metabolite
  • Bioconversion Product
  • The sizes of the bioreactor can vary over several
    orders of magnitudes.
  • The microbial cell culture (few mm3), shake flask
    ( 100 -1000 ml), laboratory fermenter ( 1 50
    L), pilot scale (0.3 10 m3) to plant scale ( 2
    500 m3) are all examples of bioreactors.

  • The performance of any fermenter depends on the
    following key factors
  • Agitation rate
  • Oxygen transfer
  • pH
  • Temperature
  • Foam production
  • The design and mode of operation of a fermenter
    mainly depends on the production organism, the
    optimal operating condition required for target
    product formation, product value and scale of
  • The design also takes into consideration the
    capital investment and running cost.

  • Large volume and low value products like
    alcoholic beverages need simple fermenters and do
    not need aseptic condition.
  • High value and low volume products require more
    elaborate system of operation and aseptic
  • The Designing of a Bioreactor also has to take
    into considerations the Unique Aspects of
    Biological Processes
  • A) The concentrations of starting materials
    (substrates) and products in the reaction mixture
    are frequently low both the substrates and the
    products may inhibit the process.

  • Cell growth, the structure of intracellular
    enzymes, and product formation depend on the
    nutritional needs of the cell (salts, oxygen) and
    on the maintenance of optimum biological
    conditions (temperature, concentration of
    reactants, and pH) within narrow limits.
  • B) Certain substances, inhibitors, effectors,
    precursors, metabolic products influence the rate
    and the mechanism of the reactions and
    intracellular regulation.
  • C) Microorganisms can metabolize unconventional
    or even contaminated raw materials (cellulose,
    molasses, mineral oil, starch, ores, wastewater,
    exhaust air, biogenic waste), a process which is
    frequently carried out in highly viscous media.

  • D) In contrast to isolated enzymes or chemical
    catalysts, mos adapt the structure and activity
    of their enzymes to the process conditions,
    whereby selectivity and productivity can change.
  • Mutations of the microorganisms can occur under
    sub optimal biological conditions.
  • E ) Microorganisms are frequently sensitive to
    strong shear stress and to thermal and chemical
  • F) Reactions generally occur in gas-liquid -solid
    systems, the liquid phase usually being aqueous.
  • G) Continuous bioreactors often exhibit
    complicated dynamic behavior.

  • H) The microbial mass can increase as biochemical
    conversion progresses.
  • Effects such as growth on the walls,
    flocculation, or autolysis of microorganisms can
    occur during the reaction.

Installation of a fermenter S-steam
C-condensate W-water A-air. The steam line
permits inplace sterilization of valves, pipes
and seals.
Requirements of Bioreactors
  • There is no universal bioreactor.
  • The general requirements of the bioreactor are as
  • A) The design and construction of bioreactors
    must keep sterility from the start point to end
    of the process.
  • B) Optimal mixing with low, uniform shear.
  • C) Adequate mass transfer, oxygen.
  • D) Clearly defined flow conditions.
  • E) Feeding substrate with prevention of under or
  • F) Suspension of solids.
  • G) Gentle heat transfer.
  • H) Compliance with design requirements such as
    ability to be sterilized simple construction
    simple measuring, control, regulating techniques
    scale-up flexibility long term stability
    compatibility with up- downstream processes
    antifoaming measures.

Fermenter Design
  • The basic points of consideration while designing
    a fermentor
  • Productivity and yield
  • Fermenter operability and reliability
  • Product purification
  • Water management
  • Energy requirements
  • Waste treatment
  • Other few significant things to be taken in
  • Design in features so that process control will
    be possible over reasonable ranges of process
  • Operation should be reliable
  • Operation should be contamination free

Fermenter design
  • To achieve these the fermenter should have
  • Heat and oxygen transfer configuration
  • Sterilization procedures
  • Foam control
  • Fast and thorough cleaning system
  • Proper monitoring and control system
  • Traditional design is open cylindrical or
    rectangular vessels made from wood or stone.
  • Most fermentations are now performed in close
    system to avoid contamination.
  • It should be constructed from non-toxic,
    corrosion-resistant materials.
  • Small fermentation vessels of a few liters
    capacity are constructed from glass and/or
    stainless steel.

Fermenter design
  • Pilot scale and many production vessels are
    normally made of stainless steel with polished
    internal surfaces
  • Very large fermenters are often constructed from
    mild steel lined with glass or plastic, in order
    to reduce the cost.
  • If aseptic operation is required, all associated
    pipelines transporting air, inoculum and
    nutrients for the fermentation need to be
    sterilizable, usually by steam.
  • Most vessel cleaning operations are now automated
    using spray jets, and called cleaning in place
    CIP. And located within the vessel.
  • Associated pipe work must also be designed to
    reduce the risk of microbial contamination. There
    should be no horizontal pipes or unnecessary
    joints and dead stagnant spaces where material
    can accumulate otherwise this may lead to
    ineffective sterilization.

Fermenter design
  • Normally, fermenters up to 1000 L capacity have
    an external jacket, and larger vessels have
    internal coils.
  • Pressure gauges and safety pressure valves must
    be incorporated, (required during sterilization
    and operation).
  • For transfer of media pumps are used. Centrifugal
    pumps (generate high shear forces and path for
    easy contaminations), magnetically coupled, jet
    and peristaltic pumps.
  • Alternate methods of liquid transfer are gravity
    feeding or vessel pressurization.
  • In fermentations operating at high temperatures
    or containing volatile compounds, a sterilizable
    condenser may be required to prevent evaporation
  • Fermenters are often operated under positive
    pressure to prevent entry of contaminants.

Control of Physicochemical Parameters
  • A) Agitation
  • Agitation of suspended cell fermentations is
    performed in order to mix the three phases within
    a fermenter
  • liquid phase contains dissolved nutrients and
  • gaseous phase is predominantly oxygen and carbon
  • solid phase is made up of the cells and any solid
    substrates that may be present.
  • Mixing should produce homogeneous conditions and
  • a) Nutrient transfer
  • b) Gas transfer
  • c) Heat transfer
  • Heat transfer is necessary during both
    sterilization and for temperature maintenance
    during operation.

Control of Physicochemical Parameters
  • Transfer into liquid from the gaseous phase is
    enhanced by agitation It prolongs retention of
    air bubbles in suspension, reduces bubble size to
    increase the surface area for oxygen transfer,
    prevents bubble coalescence and decreases the
    film thickness at the gas-liquid interface.
  • Maintenance of suitable shear conditions during
    the fermentation is very important
  • Certain agitation systems develop high shear
    that may damage shear-sensitive cells.
  • Low shear systems can lead to cell flocculation
    or unwanted growth on surfaces, such as on the
    vessel walls, stirrer and electrodes.
  • The mixing of nutrients and gaseous exchange
    within any fermenter is influenced by

Control of Physicochemical Parameters
  • a. medium density and rheology,
  • b. size and geometry of the vessel
  • c. the amount of power used in system.
  • CSTRs have agitators with multiple impellers to
    give a well mixed homogeneous environment.
  • Nevertheless, in reality, non-uniform conditions
    normally prevail in vessel greater than 500
    liters capacity.
  • No direct contact exists between the cooling!
    Heating system and the fermentation medium.
  • The heat is conducted through the vessel wall,
    coils and baffles.
  • These systems are also used to sterilize the
    vessel and contents before inoculation, by the
    injection of pressurized steam contents before

Control of Physicochemical Parameters
  • Automatic temperature control during the
    fermentation is accomplished by injecting either
    cold or hot water into the outer jacket and/or
    internal coils.
  • In some circumstances alternative cooling media
    may be used, e.g. glycol.
  • Mass transfer
  • Transfer of nutrients from the aqueous phase into
    the microbial cells during fermentation is
    relatively straightforward as the nutrients are
    normally provided in excess.
  • A. Transport of Nutrients
  • The performance of the reactor is affected if the
    rate of the transport of the limiting nutrients
    is slower than the rate of utilization by the
  • Efficiency of the bioprocess could be increased
    by increasing the rate of transport of a limiting

Control of Physicochemical Parameters
  • B. Transport of Oxygen
  • Compressed air entering a fermenter is usually
    stripped of moisture and any oil vapors that may
    originate from the compressor.
  • To prevent the risk of contamination, gases
    introduced into the fermenter should be passed
    through a sterile filter.
  • A similar filter on the air exhaust system
    avoids environ-mental contamination.
  • Sterile filtered air or oxygen normally enters
    the fermenter through a sparger system, and
    airflow rates for large fermenters rarely exceed
    0.5-1.0 volumes of air per volume of medium per
    minute (v/v/m).
  • To promote aeration in stirred tanks, the sparger
    is usually located directly below the agitator.

Control of Physicochemical Parameters
  • Sparger structures can affect the overall
    transfer of oxygen into the medium, as it
    influences the size of the gas bubbles produced.
  • Small bubbles are desirable because the smaller
    the bubble, the larger the surface area to volume
    ratio, which provides greater oxygen transfer.
  • However, spargers with small pores that are
    effective in producing small air bubbles are more
    prone to blockage and require a higher energy
  • The availability of the oxygen depends on
  • Solubility
  • Mass transfer rate of oxygen in the
    fermentation broth
  • Rate of utilization of DO by microbial biomass.

Control of Physicochemical Parameters
  • To enhance the rate of bioconversions, sometimes
    the inoculum concentration is increased.
  • This is adversely affects the oxygen availability
    to the cells.
  • High density of cells causes rapid depletion of
    dissolved oxygen in the fermentation media, as
    there is a misbalance between the oxygen
    consumption rate and the rate of oxygen transfer.
  • In such cases the rate of oxygen transfer from
    the gas phase into the liquid media need to be
    enhanced to improve the rate of bioconversion.

Control of Physicochemical Parameters
  • The major resistance in oxygen transfer to cells
  • Gas film resistance between the bulk gas and
    gas-liquid interface
  • Interfacial resistance at the gas-liquid
  • Liquid film resistance between the interface
    and bulk liquid phase
  • Liquid phase resistance for the transfer of
    oxygen to the liquid film surrounding a microbial
  • Liquid film resistance around cells
  • Intracellular resistance

Control of Physicochemical Parameters
  • The total oxygen transfer resistance is the sum
    of the individual resistance.
  • The gas film resistance is almost negligible.
  • Liquid film around a single cell has negligible
    resistance to the diffusion of oxygen but when
    the cells are in pellets form, then the liquid
    film resistance around the cell is significant.
  • Intracellular oxygen transfer resistance is
    usually negligible compared to other factors.
  • If there is pellet formation, intrapellet
    resistance may be important since oxygen has to
    diffuse through the intercellular space to
    available cells.
  • Size of pellet is important to avoid formation of
    anaerobic regions.
  • The critical size of the clumps (pellets) depends

Control of Physicochemical Parameters
  • a. The rate of consumption of oxygen,
  • b. Diffusivity of oxygen
  • c. The concentration of dissolved oxygen in the
  • The major resistance is due to the liquid film
    around the gas bubble.
  • In a well mixed fermenter, the concentration of
    dissolved oxygen in the bulk liquid phase is
    constant and the concentration gradient in the
    bulk liquid will thus be negligible.
  • When proper mixing in the fermentation media is
    difficult to achieve, there may be significant
    concentration gradient within the bulk liquid and
    hence the oxygen transfer resistance in the bulk
    liquid may not be negligible.
  • Bulk fluid mixing is thus taken into
    consideration in the design of aerobic fermenters
    to reduce the oxygen transfer resistance.

Oxygen mass transfer from an air bubble to a
microbial cell
Physical Factors Affecting Oxygen Transfer
  • Temperature Temperature affects the solubility
    and diffusivity of oxygen in the fermentation
  • The solubility of oxygen decreases but
    diffusivity increase with the rise in
  • Pressure The partial pressure of oxygen in the
    gas phase mainly affects the solubility of
    oxygen. In certain fermentation systems,
    increasing the total pressure of air supplied to
    the fermenter or else by operating the system
    under a constant high pressure head of air
    improve the rate of oxygen transfer.
  • In aerobic fermentors , oxygen is supplied to the
    fermentation medium by sparging air bubbles
    underneath the impeller of an agitated fermentor.
  • Oxygen from a rising air bubble is first
    dissolved in the fermentation medium and then
    taken up by the cells.

Physical Factors Affecting Oxygen Transfer
  • In CSTR, the rate of oxygen transfer varies with
    the power supplied for agitation of fermentation
    broth, hence estimation of the power requirement
    for effective agitation and oxygen transfer is
    essential for the design of aerobic bioreactors.
  • When high biomass concentrations are used to
    increase productivity it also creates an enormous
    demand for oxygen.
  • The operation of aerobic processes is generally
    more demanding, as it is difficult to prevent
    oxygen from becoming a rate-limiting factor.
  • Oxygen transfer is complex, as it involves a
    phase change from its gaseous phase to the liquid
    phase, and is influenced by the following
  • 1. the prevailing physical conditions
    temperature, pressure and surface area of
    air/oxygen bubbles

Physical Factors Affecting Oxygen Transfer
  • 2. the chemical composition of the medium
  • 3. the volume of gas introduced per unit reactor
    volume per unit time
  • 4. the type of sparger system used to introduce
    air into the fermenter
  • 5. the speed of agitation or
  • 6. a combination of these factors.
  • During aerobic fermentations molecular oxygen
    must be maintained at optimal concentrations to
    ensure maximum productivity.
  • The two steps associated with an oxygen mass
    balance are the rate at which oxygen can be
    delivered to the biological system (oxygen
    transfer rate, OTR) and the rate at which it is
    utilized by the microorganisms (critical oxygen

Physical Factors Affecting Oxygen Transfer
  • If the rate of oxygen utilization is greater than
    die OTR, anaerobic conditions will develop, which
    may limit growth and productivity.
  • OTR may be raised by elevating the pressure,
    enriching the inlet air oxygen, and increasing
    both agitation and airflow rates.
  • In order for oxygen to transfer from the gaseous
    phase to an individual cell or site of reaction,
    it must pass through several points of
  • 1. resistance within the gas film to the phase
  • 2. penetration of the phase boundary between the
    gas bubble and bulk liquid
  • 3. transfer from the phase boundary to the bulk
  • 4. movements within the liquid
  • 5. transfer to the surface of the cell

Physical Factors Affecting Oxygen Transfer
  • 6. entries into cell and
  • 7. transport to the site of reaction within the
  • The rate-limiting step (controlling factor) in
    oxygen transfer is the movement of oxygen from
    the gaseous phase to the gas-liquid boundary
    layer, particularly for viscous media,
  • Gaseous oxygen molecules move rapidly, due to
    their kinetic energy.
  • However, to enter the liquid they have to cross
    this boundary layer at the surface of the bubble.
  • This is composed of a thin layer of oxygen
    molecules that line the inside of the bubble and
    a thicker layer of water molecules coating the
    bubble surface.
  • Diffusion across this boundary is particularly
    influenced by temperature, solutes and

Physical Factors Affecting Oxygen Transfer
  • Once in the liquid, the rate of oxygen
    acquisition by cells depends on the oxygen
    gradient between the oxygen in the bulk liquid
    and at the site of utilization.
  • Movement in the bulk liquid is aided by good
  • The rate of use by the biological system will be
    determined by the affinity and saturation
    characteristics of the terminal oxidase.
  • As microorganisms exhibit different oxygen
    requirements, the level of aeration necessary
    will vary from fermentation to fermentation.

Transfer of Heat in Bioreactors
  • Microbial growth is usually accompanied by the
    release of metabolic heat into the fermentation
  • Metabolic activities can generate as much as
    100-200 BTU gal-1h-1 of thermal energy, while
    mechanical energy inputs of 0.5 and 2.5 HP per
    100 gal can generate an additional 10-60 BTU
  • To maintain a constant temperature in the
    fermenter, heat is either supplied or removed
    from the fermentation broth during the course of
  • Heat transfer takes place in well stirred
    fermenters by forced convection.
  • In fixed bed microbial reactors heat transfer
    takes place by natural convection or phase change

  • Heat Transfer Configurations
  • The primary heat transfer configurations in
    fermentation vessels are
  • i. External jackets
  • ii. Internal coils
  • iii. External surface heat exchanger
  • The internal coils though provide better heat
    transfer capabilities, but they cause problems of
    microbial film growth on coil surfaces,
    alteration of mixing patterns and fluid
  • The external surface heat exchangers, the media
    is pumped through an external heat exchanger
    where the heat transfer takes place through the
    surface of exchanger tubes.