Biochemical Engineering II

Effect of impeller speed

► The shear forces that an impeller generates play a major role in determining bubble size. If
the impeller speed is to slow then the bubbles will not be broken down. In addition, if the
impeller speed is too slow, then the bubbles will tend to rise directly to the surface due to
their buoyancy.

Slow impeller speed Fast impeller speed

The bubbles will not be sheared Smaller bubbles will be generated and
into smaller bubbles and will tend these bubbles will move with throughout
to rise directly towards the surface the reactor increasing the gas hold up and
bubble residence time
impeller , contnd…

► Another consequence of too slow an impeller speed is a flooded impeller.

► Under these conditions, the bubbles will accumulate and coalesce under the impeller,
leading to the formation of large bubbles and poor oxygen transfer rates.

► A similar phenomenon will happen when aeration rate is too high.

► In this case, the oxygen transfer efficiency will be low

Agitation requirement in fermentation systems

The dimensions of what is called a “standard” stirred tank

bioreactor vessel with Baffles.
Geometric Ratios for a Standard Bioreactor Vessel
Impeller Di/Dt HL/Dt Li/Di Wi/Di Hb/Di Wb/Dt No. Baffles
Type

Flat-Blade 0.33 1.0 0.25 0.2 1.0 0.1 4

Turbine

Paddle 0. 3 3 1.0 - 0.25 1.0 0.1 4

impeller

Marine 0.33 1.0 pitch = Di 1.0 0.1 4

Propeller
Where:
Dt = tank diameter,
HL = liquid height
Di = impeller diameter
Hb = impeller distance from bottom of vessel
Wb = baffle width
Li = impeller blade length
Wi = impeller blade height
Sparger

► The air sparger is used to break the incoming air into small
bubbles.
► Although various designs can be used such as porous materials
made of glass or metal, the most common type of filter used in
modern bioreactors is the sparge ring:
Sparger, contnd…

► A sparge ring consists of a hollow tube in which small holes have been
drilled. A sparge ring is easier to clean than porous materials and is less
likely to block during a fermentation.

► The sparge ring must located below the agitator and will have approximately
the same diameter as the impeller.

► Thus, the bubbles rise directly into the impeller blades, facilitating bubble
break up.
Sparger, contnd…
Air flow rates

► Air flow rates are typically reported in terms of

► volume per volume per minute
or
vvm

which is defined as:

 Condenser
In small reactors, the exit air system will typically include a condenser.
 Condenser, contnd…

► The condenser is a simple heat exchanger through which cool water is

passed.

► Volatile materials and water vapour condense on the inner condenser

surface.

► This minimizes water evaporation and the loss of volatiles.

► Drying the air also prevents blocking of the exit air filter with water
Cleaning and sterilization facilities.

❖ Small scale reactors are taken apart and then cleaned before being
re-assembled, filled and then sterilized in an autoclave.
❖ However, reactors with volumes greater than 5 litres cannot be
placed in an autoclave and sterilized. These reactors must be
cleaned and sterilized "in place". This process is referred to "Clean
in Place”.
❖ CIP involves the complete cleaning of not only the fermenter but
also all lines linked to the internal components of the reactor.
Steam, cleaning and sterilizing chemicals, spray balls and high
pressure pumps are used in these processes. The process is usually
automated to minimize the possibility of human error.
Air sterilization system
❖ Sterilization of the inlet air is undertaken to prevent contaminating
organisms from entering the reactor.

❖ The exit air on the other hand is sterilized not only to keep contaminants
from entering but also to prevent organisms in the reactor from
contaminating the air.

❖ A common method of sterilising the inlet and exit air is filtration. For
small reactors (with volumes less than 5 litres), disk shaped hydrophobic
Teflon membranes housed in a polypropylene housing is used. are used.
Teflon is tough, reusable and does not readily block.
 Air sterilization, contnd…

For larger laboratory scale fermenters (up to 1000 litres), pleated membrane filters
housed in polypropylene cartridges are used.
 Air sterilization, contnd…

❖ By pleating the membrane, it is possible to create a compact filter with a very large
surface area for air filtration. Increasing the filtration area decreases the pressure
required to pass a given volume of air through the filter.
❖ Sterilization of the inlet and exit air in large bioreactors (> 10,000 litres) can present a
major design problem. Large scale membrane filtration is a very expensive process.
The filters are expensive as they are difficult to make and the energy required to pass
air through a filter can be quite considerable.
❖ Heat sterilization is alternative option. Steam can be used to sterilize the air. With older
style compressors, it was possible to use the heat generated by the air compression
process to sterilize the air. However, compressors are now multi-stage devices which
are cooled at each stage and disinfecting temperatures are never reached.
Air sterilisation, contnd…
Maintaining positive pressure at all stages of the fermentation setup and operation
is an important aspect of reducing the risk of contamination

Without aeration, a vacuum With aeration, positive pressure is

always maintained and contaminants
forms as the reactor cools.
are pushed away from the reactor
Air sterilisation, contnd…

► During sterilisation the concept of "maintaining positive pressure" will often be used.
► Maintaining positive pressure means that during sterilisation, cooling and filling and if
appropriate, the fermentation process, air must be pumped into the reactor.
► In this way the reactor is always pressurised and thus aerial contaminants will not be
"sucked" into the reactor.
► It is very important that positive pressure is maintained when the bioreactor is cooled
following sterilisation. Without air being continuously pumped into the reactor, a vacuum
will form and contaminants will tend to be drawn into the reactor.
pH control system

The pH control system consists of

∙a pH probe
∙alkali delivery system
∙acid delivery system
The pH probe is typically steam sterilizable
pH control , contnd…

• The neutralizing agents used to control pH should be non-corrosive. They

should also be non-toxic to cells when diluted in the medium.
• Potassium hydroxide is preferred to NaOH, as potassium ions tend to be
less toxic to cells than sodium ions. However KOH is more expensive
than NaOH. Sodium carbonate is also commonly used in small scale
bioreactor systems.
• Hydrochloric acid should never be used as it is corrosive even to stainless
steel.
• Likewise sulphuric acid concentrations should not be between 10% and
80% as between this range, sulphuric acid is most corrosive.
pH control , contnd…

• For fermentations that produce large amounts of acids, for example lactic acids
fermentation using media containing high sugar concentrations, high concentrations of
alkali (4 M and above) are preferred. This will prevent dilution of the medium due to
the addition of excessive addition of the alkali solution.
• For laboratory fermenters, a peristaltic pump is used to add the pH adjusting agents.
Silicone tubing is often used. However, note that silicone tubing will decay in the
presence of high alkali concentrations. Thick walled slicone tubing should be used.
• Alternatively Tygon or Neoprene tubing can be used. Tygon is not autoclavable but
can be sterilized by passing the NaOH through the tubing for about 1 hour. Neoprene is
autoclavable but is not transparent or translucent as is Tygon or silicone.
Setpoint and deadband
Setpoint and deadband, contnd…

► The pH control system (and indeed all other fermenter control systems) are designed to
have a deadband. A deadband is used to prevent excessive alkali and acid addition.
► The pH control deadband is shown in the following diagram:
Setpoint and deadband
► The setpoint is the pH at which the fermenter is being attempted to be controlled at. For
example, if the fermentation is to be run at a constant pH of 6.5, then the setpoint is set to
6.50.
► If for example, a 5% deadband is used, then the upper deadband limit will be
► 1.05 x 6.5 = 6.83
► and the lower deadband limit will be
► 0.95 x 6.5 = 6.18
► If the deadband is too small, then it is possible that pH will often overshoot and undershoot
the deadbands leading to excessive alkali and acid addition. The trade off is that a wide
deadband will lead to less precise pH control.
► As many fermentations tend to produce acids rather than substances that increase the pH, acid
addition is often not required. Indeed not all fermentations need continuous pH control.
Foam control system
► Foam control is an essential element of the operation of a sparged bioreactor. The following photograph shows the
accumulation of foam in a 2 litre laboratory reactor.
Foam, contnd…
• Excessive foam formation can lead to blocked air exit filters and to pressure build up in the
reactor.

• The latter can lead to a loss of medium, damage to the reactor and even injury to operating
personnel.

• Foam is typically controlled with aid of antifoaming agents based on silicone or on

vegetable oils.

• Excessive antifoam addition can however result in poor oxygen transfer rates.
► The antifoam requirement will depend on
∙ the nature of the medium.
Media rich in proteins will tend to foam more readily than simple media.
∙ the products produced by the fermentation.
Secreted proteins or nucleic acids released as a result of cell death and hydrolysis have
detergent like properties.
∙ the aeration rate and stirrer speed.
Increasing the aeration rate and stirrer speed increases foaming problems.
∙ the use of mechanical foam control devices
Foam control devices such as mechanical and ultrasonic foam breakers help to reduce the
antifoam requirement.
∙ The head space volume
The larger headspace volume, then the greater the tendency for the foam to collapse under its
own weight. For example, for fermentations in which high levels of foam is produced, a 50%
headspace volume may be required.
∙ Condenser temperature
In laboratory scale reactors, a cold condenser temperature can help to control the foam. The
density of the foam increases when it moves from the warm headspace volume to the cold
condenser region. This causes the foam to collapse.
Foam is typically detected using two conductivity
or "level" probes.

When the upper level probe is above the foam level, When the upper level probe is immersed in
no current will pass between the level probes and the foam layer, a current is carried in the
the antifoam pump remains turned off. foam. This causes the antifoam to turn on.
Foam, contnd…

• One probe is immersed in the fermentation liquid while the other

placed above the liquid level.

• When the foam reaches the upper upper probe, a current is

carried through the foam.

• The detection of a current by the foam controller results in the

activation of a pump and the antifoam is then added until the
foam subsides.
Modern method of dissolved oxygen (DO) measurement in the lab or field involves a DO
sensor connected to a meter that records calibration and measurement data.

There are two types of DO sensors—electrochemical and optical. Electrochemical DO

sensors, also known as amperometric or Clark-type sensors, measure dissolved oxygen
concentration in water based on electrical current produced.

Optical DO sensors, popularly known as luminescent DO sensors (LDO) but some are
called fluorescent sensors, measure dissolved oxygen concentration in water based on the
quenching of luminescence in the presence of oxygen. They can measure either the
intensity or the lifetime of the luminescence as oxygen affects both.
When placed in an electrolyte solution, the potential between dissimilar metals causes them
to self-polarize with the electrons travelling internally from the anode to the cathode.

The cathode (e.g., Ag or another noble metal) accepts electrons from the anode via an
internal circuit and passes them on to the oxygen molecules. It does not interfere in the
reaction. Thus, the anode (e.g., Zn, Pb, or another active metal) is oxidized and oxygen is
reduced at the surface of the cathode.
Both the cathode and anode are submerged in an electrolyte (e.g., NaOH, NaCl, or another
inert electrolyte) and enclosed in a cap fitted with thin hydrophobic, oxygen-permeable
membrane.
When galvanic DO sensor is immersed in water sample, oxygen that diffuses across the
oxygen-permeable membrane at a rate proportional to the pressure of oxygen in the water is
reduced and consumed at the cathode. This reaction produces an electrical current that is
directly related to the oxygen concentration. This current is carried by the ions in the
electrolyte and runs from the cathode to the anode.

Anode (Pb) – lead oxidation reaction: 2Pb ￫ 2Pb2+ + 4e-

Cathode (Ag) – oxygen reduction reaction: O2 + 4e- + 2H2O ￫ 4OH-
Overall reaction: O2 + 2H2O + 2Pb ￫ 2Pb(OH)2

The current produced is proportional to oxygen consumed and thus to the partial pressure of
oxygen in the sample.
Dual LED referencing system – blue LED emits light that excites the dye causing its
luminescence. The red LED emits light but simply reflected back by the dye and does not
cause luminescence. It serves as a reference to ensure accuracy.

Sensor film – a luminescent dye entrapped in a film. When exposed to blue light, the dye
becomes excited (electrons gaining energy) and emits light as the electrons return to their
normal energy state.

Photodetector – photodiode measures the intensity or lifetime of luminescence from the dye.

The sensor film is coated on the sensor cap while the LEDs and photodetector are housed in
the sensor body.
When an optical DO sensor is immersed in water sample, oxygen crosses the membrane and
interacts with the dye. This quenches or reduces the intensity and lifetime of the dye’s
luminescence, which is measured by the photodetector and used to calculate the DO
concentration.

The intensity and lifetime of luminescence when dye is exposed to blue light is inversely
proportional to the amount of oxygen in the sample.
MATERIAL OF CONSTRUCTION Laboratory scale bioreactor:
• In fermentation with strict aseptic requirements it is important to select materials that
can withstand repeated sterilization cycles.
• On a small scale, it is possible to use glass and/or stainless steel.
• Glass is useful because it gives smooth surfaces, is non-toxic, corrosion proof and it is
usually easy to examine the interior of vessel. The glass should be 100% borosilicate,
e.g. Pyrex® and Kimax®.
• The following variants of the laboratory bioreactor can be made:
1. Glass bioreactor (without the jacket) with an upper stainless steel lid.
2. Glass bioreactor (with the jacket) with an upper stainless steel lid.
3. Glass bioreactor (without the jacket) with the upper and lower stainless steel lids.
4. Two-part bioreactor - glass/stainless steel. The stainless steel part has a jacket and
ports for electrodes installation.
5. Stainless steel bioreactor with peepholes. Vessels with two stainless steel plates cost
approximately 50% more than those with just a top plate
Pilot scale and large scale bioreactors:
When all bioreactors are sterilized in situ, any materials use will have to assess on their ability
to withstand pressure sterilization and corrosion and their potential toxicity and cost.
Pilot scale and large scale vessels are normally constructed of stainless steel or at least have a
stainless steel cladding to limit corrosion.
The American Iron and Steel Institute (AISI) states that steels containing less than 4%
chromium are classified as steel alloys and those containing more than 4% are classified as
stainless steel.
Mild steel coated with glass or phenolic epoxy materials has occasionally been used.
Wood, concrete and plastic have been used when contamination was not a problem in a
process.
Vessel shape: - /Typical tanks are vertical cylinders with specialized top plates and bottom
plates. In some cases, vessel design eliminates the need for a stirrer system especially in air lift
fermenter. A tall, thin vessel is the best shape with aspect ratio (height to diameter ratio)
around 10:1. Sometimes a conical section is used in the top part of the vessel to give the
widest possible area for gas exchange.
Stainless steel top plates.
The top plates are of an elliptical or spherical dish shape.
The top plates can be either removable or welded. A removable top plate provides best
accessibility, but adds to cost and complexity.
Various ports and standard nozzles are provided on the stainless plate for actuators and
probes. These include pH, thermocouple, and dissolved oxygen probes ports, defoming, acid
and base ports, inoculum port, pipe for sparging process air, agitator shaft and spare ports.
Bottom plates: Tank bottom plates are also customized for specific applications. Almost most
of the large vessels have a dish bottom, while the smaller vessels are often conical in shape or
may have a smaller, sump type chamber located at the base of the main tank. These alternate
bottom shapes aid in fluid management when the volume in the tank is low.
One report states that a dish bottom requires less power than a flat one. In all cases, it is
imperative that tank should be fully drainable to recover product and to aid in cleaning of the
vessel. Often this is accomplished by using a tank bottom valve positioned to eliminate any
“dead section” that could arise from drain lines and to assure that all content will be removed
from the tank upon draining.
 If the bioreactor has a lower cover, then the following ports and elements should be placed
and fastened there:
1. Discharge valve; 2. Sampling device; 3. Sparger; 4. Mixer's lower drive; 5. Heaters.
Height-to-diameter ratio (Aspect ratio).
2. The height-to-diameter ratio is also a critical factor in vessel design. Although a
symmetrical vessel maximizes the volume per material used and results in a
height-to-diameter ratio of one, most vessels are designed with higher ratio.
3. The range of 2-3:1 is more appropriate and in some situation, where stratification of
the tank content is not an issue or a mixer is used, will allow still higher ratio to be
used in design. The vessels for microbiological work should have an aspect ratio of
2.5- 3:1, while vessels for animal cell culture tend to have an aspect ratio closer to 1.
 Bioreactors mostly used in bioprocess technology:

(1) Continuous Stirred Tank Bioreactors

(2) Bubble Column Bioreactors

(3) Airlift Bioreactors

(4) Packed Bed Bioreactors

(5) Fluidized Bed Bioreactors

(6) Photo-Bioreactors.

(7) Membrane Bioreactor

(8) Immobilized Cell Bioreactor

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