Introduction to Bioreactors and Bioreactor

designs

Dr. E . Y Fernando
Department of Biological Sciences
Rajarata University
Outline of the lecture and learning outcomes
• Introduction to bioreactors
• Bioreactor types for bioprocesses
• Controlling various parameters in bioreactors
• Basic understanding of mass transfer and cell growth kinetics

Learning outcomes – at the end of the lecture, the students should be

able to describe the types of bioreactors and their utility in various
bioprocesses and also have a basic understanding of cell growth
kinetics and mass transfer
Bioreactors and bioprocesses
• Bioreactor is a vessel for the growth of microorganisms
(fermentation).
• Bioreactors provide the aseptic condition for fermentation by not
permitting contamination.
• A bioreactor can be defined as an apparatus, such as a large
fermentation chamber, for growing organisms such as bacteria,
yeast, plant/animal cells or their enzymes, that are used in the
biotechnological manufacture of substances such as
pharmaceuticals, antibodies, or vaccines, or for the bioconversion
of organic waste.
The job of a bioreactor
• The function of a bioreactor is to provide a suitable environment in
which an organism or an enzyme system can efficiently produce a
target product.
• Cell biomass
• Metabolite
• Bioconversion Product
• Control conditions for the bioprocess
• Allow harvesting of the target product
• Allow downstream processing for the target product
Bioreactors- Introduction

◆Importance of considering process engineering factors

when culturing cells.

◆Biological factors include the characteristics of the cells,

their maximum specific growth rate, yield coefficient, pH
range and temperature range.

◆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.
 Bioreactors- Introduction
◆The oxygen demand of an industrial process is generally
satisfied by aeration and agitation
◆Productivity is limited by oxygen availability and
therefore it is an important factor that affects a
fermenters efficiency in supplying O2
◆O2 requirement, quantification of O2 transfer and factors
influencing the transfer of O2 into solution
 Bioreactors- Introduction
◆Mass transfer, in particular, oxygen transfer are
important factor which determined how a reactor
must be designed and operated.

◆Cost is also described as an important consideration.

The larger the reactor or the faster the stirrer speed
and aeration rate, the greater the costs involved.
The performance of any bioreactor depends on
the following key factors:
• Agitation rate
• Oxygen transfer
The design of a bioreactor should consider the
• pH
following factors:
• Temperature • the production organism
• Foam production • the optimal operating condition required for the
target organism
• product formation,
• product value
• scale of production.
• capital investment
Requirements of Bioreactors
• The design and construction of bioreactors must keep sterility from the start
point to end of the process.
• Optimal mixing with low, uniform shear.
• Adequate mass transfer, oxygen.
• Clearly defined flow conditions.
• Feeding substrate with prevention of under or overdosing.
• Retention of solids (especially biomass).
• Gentle and efficient heat transfer.
• ability to be sterilized for long term;
• simple construction; simple measuring, control,
• scale-up &flexibility;
Microbial growth in batch bioreactors
• When microbes are growing in a batch culture, several parameters
can characterize the accumulation of cell mass

Growth index – Is the relative estimation of formed biomass at any

given point of time, in comparison to the initial biomass of the
fermentation. Where in this equation, GI is growth index while Wf is
final cell mass and Wi is the initial cell mass. Both Wf and Wi are taken
either as fresh or dry weight
Microbial growth in batch bioreactors
• Specific growth rate - the specific growth rate (µ) refers to the steepness of a
curve, and it is defined as the rate of increase of biomass of a cell population per
unit of biomass concentration. It can be calculated in batch cultures, since
during a defined period of time, the rate of increase in biomass per unit of
biomass concentration is constant and measurable. This period of time occurs
between the lag phase and stationary phases. During this period, the increase in
the cell population fits a straight-line equation between ln x and t
Microbial growth in batch bioreactors
• Where, xo is the initial biomass (or cell density), x is the
biomass (or cell density) at time t, and µ is the specific
growth rate. Mu can be calculated from the above
relationship, which is the slope of the line between ln x and t.

Doubling time (td) is the time required for the

concentration of biomass of a population of
suspension cells to double. One of the greatest
contrasts between the growths of cultured plant
cells refers to their respective growth rates. The
doubling time (dt) can be calculated according to
the following equation
Microbial growth – Monod kinetics
• Consider the operation of the "Batch" system
shown in Figure 1. This container initially
contains a known growth substrate
concentration S. The container is well mixed
and therefore the dissolved oxygen
concentration O2 does not become a limiting
factor for microbial growth. Initially a known
concentration X of viable microbial cells (i.e.
inoculum) is added to the container and, with
time, growth substrate S is utilized for cell
growth. We therefore over time will observe a
decrease in S (negative dS/dt) and a
corresponding increase in X (positive dX/dt)
Microbial growth – Monod kinetics
• During the lag phase dX/dt and dS/dt
are essentially zero. However as
exponential growth phase begins it is
possible to measure dX/dt and dS/dt
values which are very useful for
defining important microbial kinetic
parameters. Using corresponding
observations of dS/dt and dX/dt
obtained just after the onset of
exponential growth phase, we can
compute the yield coefficient YXS and
the specific growth rate µ as
Microbial growth - Monod kinetics
• The batch experiment shown in Figure 1 can be repeated by varying initial
substrate concentration S over a wide range of values—resulting in observation
of individual µ values which correspond to each substrate concentration. An
arithmetic plot of µ vs S will exhibit the general behavior.
• The most widely used expression for describing specific growth rate as a function
of substrate concentration is attributed to Jaques Monod (1942, 1949). This
expression is:
 MASS TRANSFER and RESPIRATION
(a) Mass balance

Stoichiometry of respiration e.g glucose;

C6H12O6 + 6O2  6H2O + 6 CO2

Oxidation of 180 gms Glucose requires 192 gms O2

However, O2 is 6000 times less soluble in water than glucose.

◆A saturated oxygen solution (in water) contains only 8 mg dm-3 of oxygen
◆Impossible to add enough oxygen to a microbial culture to satisfy needs for
complete respiration
◆Oxygen must be added during growth at a sufficient rate to satisfy requirements
The Oxygen requirements of
industrial fermentations
 Oxygen demand dependant on conversion of Carbon (C) to
biomass
 Stoichiometry of conversion of oxygen, carbon and nitrogen into
biomass has been elucidated
 Use these relationships to predict the oxygen demand for a
fermentation
 Darlington (1964) expressed composition of 100g of dry yeast
C 3.92 H 6.5 O 1.94
O2 Requirements
6.67CH2O + 2.1O2 = C 3.92 H 6.5 O 1.94 + 2.75CO2 + 3.42H2O

7.14CH2 + 6.135O2 = C 3.92 H 6.5 O 1.94 + 3.22CO2 + 3.89H2O

where CH2 = hydrocarbon

CH2O = carbohydrate

From the above equations to produce 100g of yeast from

hydrocarbon requires three times the amount of oxygen than
from carbohydrate
FACTORS AFFECTING OXYGEN DEMAND

• Rate of cell respiration

• Type of respiration (aerobic vs anaerobic)
• Type of substrate (eg. glucose vs hexadecane)
• Type of environment (e.g pH, temp etc.)
• Surface area/ volume ratio
large vs small cells (bacteria v mammalian cells)
hyphae, clumps, flocks, pellets etc.
• Nature of surface area (type of capsule etc)
 Delivering oxygen to cells
• Aeration in the bioreactor typically occurs
when:
1) Oxygen diffuses through overlay to the cell
culture medium interface.
2) Oxygen from the spargers dissolves in the
cell culture through convection with the
help of agitation.
• Agitation disperses the oxygen bubbles and
promotes mass transfer of the gas bubbles
through the gas-liquid (cell culture medium)
interface. The rate of oxygen transfer (OTR)
from gas to liquid interface is a function of
physicochemical properties of the cell culture
medium, the geometrical parameters of the
bioreactor, and presence of cells.
Delivering oxygen to cells in bioreactors
• Oxygen transfer rate (OTR) has to match the
Oxygen utilization rate (OUR) by the
microorganisms
• During batch cell culture, OUR (or OTR) is
initially low during the lag phase, where cells
are self-synthesizing and there is little gain of
cell density. As cell density increases during the
exponential phase, OUR increases until OTR
becomes a limiting rate, as determined by the
mass transfer of oxygen into the bulk liquid.
• The OTR and OUR rates are correlated by the
oxygen mass transfer coefficient, kLa.
Oxygen mass transfer coefficient
• Imagine a gas bubble in liquid. For this discussion the gas bubble
contains oxygen, and the liquid is the liquid in a bioreactor. kLa can be
represented by the following equation:
• kLa = kL × a
• Where kLa is the mass transfer coefficient from the gas to liquid
phase, given in sec-1
• kL = liquid side mass transfer coefficient (resistance in gas side film
can be neglected)
• a = bubble surface (available for diffusion)
Measuring oxygen transfer coefficient (kLa)
• The mass balance for the DO in a well-mixed liquid phase is described
in;

; And OTR = kLa (C* - CL)

• In the absence of biomass or with nonrespiring cells, when

biochemical reactions do not take place, OUR = 0. In this case, the
above Equation can be simplified as;

• Where, dC/dt is the accumulation rate of dissolved Oxygen in the

liquid medium, C* is the Oxygen saturation concentration, CL is the
dissolved oxygen concentration in the liquid medium
Measuring kLa
• If you are trying to measure kLa experimentally, the most convenient
method is called the dynamic gassing out method. Where, you will
make the bioreactor medium completely devoid of Oxygen by
sparging an inert gas (Nitrogen or Argon), and then supplying oxygen
(or air) at a fixed air flow rate and an agitation speed.

• While doing that, you will periodically measure dissolved Oxygen

concentration of the liquid medium at set-time points
• When taking the logarithm of the above equation, it becomes;
ln(C*- CL) = - KLa.t
Measuring kLa
• From the experiment, if we use the above equation, we get the following;

• Here, C1 is the concentration of oxygen at time t1 and C2 is the O2 concentration

at t2. kLA can be calculated by rearranging the formula
• Or, a plot of the In(C* - CL) against time of aeration, the slope of which equals -
KLa
 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 dollars 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
 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.
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.
◆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.
Standing cultures
• Large Pyrex flasks are used for the small scale production
of fermented products.
• 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.
Aspergillus niger mycelia
Standing cultures
◆Aerobic solid substrate fermentations are another example of standing
cultures. In these fermentations, the biomass is grown on solid
biodegradable substrates.

◆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.
Shake flasks
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.
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
Shake flasks O2 Transfer
kLa decreases with liquid volume kLa is higher when baffles are present

kLa kLa

k
L
a

kLa

kLa increases with liquid surface area

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
Unbaffled flask Baffled flask
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
Mechanically stirred bioreactors
 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.
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.
 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.
Bubble driven bioreactors
 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 8:1 and 20:1.
◆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
 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.
Airlift bioreactors
Small bubbles lead to an increased surface area for oxygen transfer.
 Airlift bioreactors
◆Equalize 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.
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
Airlift bioreactor - Air-riser and down-comer
◆An air-lift reactor is divided into three regions:
- the air-riser
- down-comer
- disengagement zone.
 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.
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.
 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 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
Packed bed and trickle flow bioreactors
◆The target cells are immobilized on an immobilization matrix and are
loaded into the reactor. Only the media is circulated. Sometimes, the
reactor is run in batch operation or, it can also be done in continuous
mode. Common cell immobilization matrices are alginate and agarose
beads. The cells are entrapped in them.
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 enzymatic 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.
 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.
 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.
 Trickle flow bioreactors

◆Because stirring is not used, considerable capital costs are saved.

◆However, oxygen transfer rates per unit volume are low
compared with spared 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.
Fluidized bed reactors
 Fluidized 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.
Fluidized bed reactors
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.