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FERMENTER DESIGN AND

HIGH PERFORMANCE
BIOREACTER

Fermenter
Fermenter is usually cylindrical vessel with a domed
(or dished) tops bottom. It is made from steel so they do
not rust. The thickness of steel depends upon the rise of
the fermenter. It is a system which provide controlled
environmental condition for the growth of microbes (and or
product of specific metabolites) in liquid culture in liquid
cultures whilst preventing the entry and growth of
contaminating microbes from outside environment.

Fermenter

This main components of a fermenter and their


uses
Base component including drive motor, heaters, pumps,
gas control etc.
Vessel accessories
Peripheral equipment such as reagent bottles
Instrumentation and sensors

Fermentation operation mode


Batch culture fermentation
Fed culture fermentation
Continuous culture fermentation

Major component of a fermenter

Component parts of a typical vessel


i) Vessel
Single walled cylinder of borosilicate glass with a flat bottom
Glass jacketed system typically round bottom steel vessel
Steel vessel
ii) Top plate
Top plate is mode from 316 stainless steel and is compressed onto
the vessel flange by an easily released clamping system.
iii) Seal
A seal separate the vessel glass from the top plate.
iv) Port fittings
Port fittings of various sizes are provided for insertion of probes,
inlet pipes, exit gas cooler, cold fingers, samples pipes etc.

v) Inoculation port
A special inoculation port will have a membrane seal held in place
with a collar.
vi) Sampling device
Culture can be withdrawn into a sampling device or a reservoir
bottle by a sample pipe.
vii) Gas sparger
A gas sparger is fixed on the top plate and this terminates in a
special assembly which insures that incoming air is dispersed efficiently
within the culture by the flat bladed Rushton type impellors fixed to drive
shaft.
viii) Drive motor
It provides stirring power to the drive shaft and is usually fitted in
the drive hub on the vessel top plate.

ix) Exit gas cooler


It works like a simple Liebig condenser to remove as much moisture
as possible from the gas leaving the fermenter to prevent excessive liquid
losses during fermentation and wetting of the exit air filter.
x) Pt-100
A narrow platinum resistance temperature sensor, temperature
control is either by direct heating using a heater pad or by circulating warm
water around the vessel jacket.
xi) Cold finger
If a direct heating is used, a cold finger is used to cool the vessel
contents. It passes the a closed pipe or coil which passes through fermenter
top plate and allows cooling water to circulate, to act as heat exchanger with
the culture.
xii) pH electrode:
xiii) Rotameter
It indicates rate of gas flow into a fermenter.

Peripheral part and accessories:


a) Reagent pumps
Pumps are normally part of instrumentation system for measuring.
pH
Antifoam
Control
Peristaltic pumps are used and the flow rate is fixed with a time shot and
delay feed system of control.

Flow rate depends on the bore of the tubing used; peristaltic tubing links the
reservoir bottles with the vessel multiway inlet.
The tubing is clamped shut during autoclaving, already connected to vessel
and opened for active addition of reagent.

b) Medium feed pumps and reservoir bottles


Medium feed pumps are often variable speed to give the maximum possible
range of feed rates.

The speed of the operation of the pump can be set manually or whole system
put under the computer control.

The reservoir bottles are usually larger 5-20 litre.

Reservoir bottle may have changed several times during a long continuous
culture experiment.

Effluent pump is also used for to remove culture fluid from the fermenter
vessel into a storage reservoir bottle.
c) Rotameter/gas supply
Rotameter is used to control the air flow rate into a fermenter vessel. A
pressure regulator valve before the rotameter ensures safe operation.

A sterile filter (usually 0.22 m) is used as a bridge between tubing from the
rotameter that connected to the air sparger of the fermenter.

A second filter on the exit gas cooler stops microbes being released into
atmosphere.

d) Sampling device
This allow culture fluid is removed aseptically during fermentation at the
intervals decreased by users according to the need of the experiment.

Alternative Vessel Designs


Alternative vessel design tried when standard vessel configuration does
not allow adequate growth of the organism.
a) Air lift
This vessel design eliminate the need of a stirrer system.
A ring sparger is normally used to produce a column of air bubbles which
rises up. This is called air lift system.
This produce a down flow of a medium which is channeled by a draught
tube fixed in the centre of the fermenter.

b) Fluidized beds
The microbes/ cell are trapped in a physical medium and held in the vessel
by mesh.
The medium is reticulated via a pump and this can be easily adapted to give
a continuum/ semi continuous flow to allow the trapped cells to affect the
chemical changes on the contortions of the medium without being washed out
along with spent medium.
c) Hollow fibre
The cells are embedded in fibre contained in cartridge which is bathed in
circulating culture medium. e.g. used for toxicity or interactive studies and
mammalian cell culture.
d) In situ sterilizable fermenters
The use of autoclavable vessels of greater than 5-10 litre working volume are
not safe and vessels above this size an dually mode of stainless still and are
designed to be sterilized in situ using house steam or an electrical steam
generator
Heating is provided via a double Jacket .

Different type of instrumentation


Fermenter instrumentation can range from simple analogue control
modules arranged as stack to powerful, embedded micro processor which
operate as on board computers to directly operates the heaters, pumps,
valves and other control actuators of a fermenter.

Analogue control less- rack system


The individual modules for the measurement and control of each parameter
are made plug into a rack system for compact, flexible, instrumentation.
A bus system in the stabling transmits the signals from the module to
another allowed measured value to be passed to controllers and for one
controller to influence another e.g. oxygen level altering speed.

Analogue controllers- separate module in housing


Each measurement/ control module has separate housing and essentially
operates in isolation.
The modules may be stacked one on top of another and can be supplied
with Pass through power connection so that only one power socket is
needed.

Beckman fermenter
Fermenter design

Common Measurement and Control Systems


Speed control
Speed control relies on the feed back from a tachometer located within drive
motor
A digital display shows actual speed in rpm as determined by the tachometer
signal.
A power meter is used to maintain the set speed depends upon the Viscosity
or density of the culture fluid.
A DC low vow voltage motor is often used for safety reasons.

Temperature control
A thermo circulation system around a vessel jacket is used.
Heater pad is ferried setting the desired temperature and switching on
Cooling is normally via a cold finger and flow of cooling water is controlled
via the action of Solenoid value.
The Pt-100 sensor provides the feed back signal to the controller.
Heat radiation system appeared to the choice for laboratory bioreactors. The
source of sad ration is directed to the vessel, then the heating would be gentle
as that of water by Sun. the optimal location for the recitation is underarm
Neath the bottom of the vessel.

Control of gas supply


The supply of gas (normally air) to the ferment or vessel is provided from a
compressed air supply soil free which may provide oil for more than one
vessel depending upon its size & output.
A pressure regulation value ensures that air crashes the vessel at a
maximum of 1.5 bar.
The rotameter control the actual flow rate of air through the fermentor. This
should not exceed 1.5 vessel volumes per minute or.
The air passes through inlet filter which prevent any microbes from entering
the vessel.
The bubbles are immediately broken up and dispersed by impellor on the
drive shaft and the baffles which can be felted near the wall of the vessel.

Control of pH
pH control is achieved by the addition of either acid or alkali to correct
changes in the pH of the culture during growth.
The controller uses a pH electrode to sense the pH changes and provide a
feed back signal.
The electrode must be in direct contact with the medium and so must be
fixed in the fermenter wall.

Antifoam electrode
The control of a foam is based on its detection by a conductance probe in the
vessel head space, which leads to the controller delivering a dose of liquid
antifoam reagent via peristaltic pump.
The sensitivity of a probe to foam can be adjusted by the user to suit the
conditions prevailing in the fermenter.
Normally, metal top plate is used to provide the electrical circuit for the probe
to operate foam can be controlled in two ways

Chemical control foam


It involves the addition of Chemical substances which lower the
surface tension of the medium of case bubble to collapse.

Mechanical foam control


It uses a device called a foam bracers to break up foam by mechanical
action. The foam breaker must be positioned at the top of the fermenter in
order to deal with foam in lead space

Nutrient Concentration
It is often advantageous to limit the nutrient concentration in medium, so
they are not present in excess.
Electrode technology have made the monitoring the some nutrients
possible.
Ions selective electrode are now available for the measurement of NH3/ NH
glucose oxide) are available for the
4+. Enzyme electrodes (based on
measurement of glucose.

Stirred- Baffled system


In the stirred system the medium is stirred by paddles (impellers} driven by
an electric motor.
The motor is placed above the fermenter and it drives a shaft which passes
down into a fermenter and attached to the Impellers.
A common type of impeller is the disc -turbine impeller, which consists of
vertical vanes on a horizontal disc.

Sterilization
Most industrial operates using pure and thus using aseptic conditions.
The season for this:
a) Competition for substrate
b) Inhibition of growth or product formation
c) Destruction of the product
For these season pure culture are usually used. Fermenter and the
medium must be sterilized before inoculation. It is can be done:

A) Sterilization by heat: Heat is used to sterilize the fermenter.


i)

Sterilization in situ:

In this Method, the fermneter is filled to the required level with medium and
whole thing is heated.
Heating is carried out by passing pressurized steam around a jacket or coil
built into the fermenter wall.
A jacket normally covers the vessel walls up to the head space.
For sterilization, steam under pressure in passed around the jacked or coil
and the medium is heated.
At boiling point all the valves on the fermenter re closed and system become
superheated.

B) Continuous heat sterilization


To overcome the problems of sterilization in situ, continuous heat sterilization
can be used.
In continuous heat sterilization, medium is passed in a stream through
devices in which it is heated up very quickly to 140oC, held at the temperature
for one minute, then cool down again very quickly.

ii) Sterilization process


Another method is to use heat exchanges. For steam sterilization a spiral
heat exchanger is often used.
In this system, steam under pressure passes in one direction round the
system, while the medium passes around the spiral in other channel in
opposite direction.
Medium then travel through a holding tube. This is a coiled pipe packed in
insulation material so that temperature does not drop.
This system is designed so that it takes the medium one minute to flow
through holding tube.
Medium is then cooled down by passing it through another heat exchange.

iii) Sterilization by filtration


Some media, especially those which are rich in proteins, can not be
heat sterilized at all, in such cases medium must be sterilized by filtration.
There are two basic type of filters used in sterile filtration.
1)

1)

Membrane filters

2)

Depth filters

Membrane filters

Membrane filters are composed of a thin short of material fixed in a


casing (or housing). Liquid is pumped through housing and is forced through
the filter material. The filter material such as cellulose, nitrate, cellulose acetate
or poly carbonate.

2)

Depth filters

Depth filter composed of a pod of fibrous material fixed in a casing.


This type of filter does not work by sieving, but by adsorption, the
microorganisms stick on to the fibre when medium is passed between fibre.
Filter Design
For large scale filtration, filter material is normally packaged in disposable
cartridges.
In these, a rectangular piece of filter material is fluted, joined and packaged in
perforated plastic surround.

The cartridge fits into a stainless steel housing, when medium is pumped
through the filter housing, the medium has pass through the filter material
in order to reach the outlet.
Depth filters are sometime used in plate frame filters. In this type of
device disposable filter sheets are placed between plates, which are
locked together in a frame.
medium is pumped through filter, the medium passes through the
compartments in parallel. For large scale filtration, the medium is passed
through a depth filter to remove most microorganisms and derbis.

Filter Design
For large scale filtration, filter material is normally packaged in disposable
cartridges.
In these, a rectangular piece of filter material is fluted, joined and
packaged in perforated plastic surround.
The cartridge fits into a stainless steel housing. When medium is pumped
through the filter housing, the medium has pass through the filter material
in order to reach the outlet.
Depth filters are sometime used in plate frame filters. In this type of
device disposable filter sheets are placed between plates, which are
locked together in a frame.
Medium is pumped through filter, the medium passes through the
compartments in parallel. For large scale filtration, the medium is passed
through a depth filter to remove most microorganisms and derbis.

Fundamentals of Fermenter Design


The adaptation of submerged culture techniques for the production of
primary and secondary metabolite, have attracted the combined attention of
microbiologist, biochemists and engineers to resolve a variety of problems in
order to improve.
A variety of problems in order to improve both yield and process
efficiency. The two aspects are:

i)

Fermenter Design and Requirement of the microbial system

The material of construction must be such that they will not adversely
affected by the desired microbial activity, either interaction with fermentation
medium or by harbouring unwanted organisms.
The fermenter must be resistant to corrosion by the nutrient medium and
products and to effect of sterilization.
There must be provision for the regulation of temperature and air supply for
charging and discharging the vessel contents.
In continuous culture system additional facilities must be provided to
control culture volume and medium flow rate.

The effect of organism on the macroenvironment which may be reflected in


concomitant changes in the microenvironment, arises mainly a)
Rheological character b) change in interfacial tension.
It would desirable to relate fermenter design and control systems to micro
environment in practice only the macroenvironment can be influenced
directly. The factor with in the control of the designer and operator are:
a)
b)
c)
d)
e)
f)
g)
h)
i)

System geometry
Aeration rate
Intensity of agitation
Temperature
Pressure
Nutrient supply
pH and other parameters involving specific ions
Dilution rate in continuous flow system and
Foaming.

Chemical engineering aspects of fermenter design


The objective of fermenter design and operation ensure that the
desired activity of the microorganisms concerned shall not be restricted by
the characteristics of the equipment. The physical process might limit the
activity are:

A)

Mass Transfer

The transfer of mass within the system is the fundamental to the whole
operation.
Mass Transfer
More or less uniform distribution of
substrate and product molecule in
the bulk of fluid

Transfer of the bulk of the fluid


and microbial cell

First aspect is largely governed by the forced convection and turbulence


produced by agitation and the flow of gas.
Second aspect is determined by diffusional forces.

Heat Transfer
Heat is generated in submerged microbial systems, partly by the metabolic
activity of the organisms and partially by the mechanical work performed by the
agitator and gas bubble.
Some heat is lost as a result of increased humidification of gas stream in its
passages through culture and some by radiation from the outer surface of the
fermenter.

Mixing effects
A mixing serves to minimize local variation in concentration and temperature.
It causes random redistribution of elements of the culture suspension in the
impeller zone and from the random interaction, with the bulk of suspension, of
stream leaving the impeller.
In a given system rate of mixing in direct. This may be expresses as mean
circulation rate. Alternately one may consider the mean circular time. This will also
represent a average mean time.

Scaling up
The three parameters are
Geometry
Power input per unit volume
Superficial air velocity
The overall effect is to reduce the average shear rate and relative volumetric flow
of the suspension through impeller, without increasing turbulence and maximum
shear rate.

Application
Fermenter preparation and use
Disassembly of the vessel
Fermenter is shutdown from the control units cable connection and transfer
lines to be removed.
Vessel should be reautoclaved after fermentation
Inlets and outlets to prepared for that
Culture should be disposed of as per safety reason
Chips, bolts/clamps of vessel top plate to undone to turn whole assembly
spring free
The top plate can be lifted upwards away from the vessel taking care that air
sparger/ drive shaft/ impellors and Pt-100 temperature probes completely clear
the vessel.

Cleaning
pH and dissolved O2 electrodes should be removed and stored in a suitable
reagents according to the manufactures instructions

Periodic cleaning and regeneration of the electrodes is also important for


their maintaince and cost effective.
Vessel should be rinsed several times in distilled water to remove any loose
culture residue.

Cleaning of growth of culture on walls of culture on walls of vessels with


light brushing is done.
Chipping/ crack in glass vessel should be avoided/ replacement is to be
done.
Vessel should be stored clean and dry.
Contact between top plate and liquid with high chloride contents should be
avoided to prevent corrosion
Pump head and must thoroughly clean.

Preparation for autoclaving


Vessel seal should be removed and lightly greased with suitable silicon
grease.
Vessel is filled with medium to maximum of 80% full. (space vital for gas
exchange)
Maximum amount of medium volume is to be cover electrodes adequately.
Replacing of the top plate on vessel.
Clamps/bolts to tighten firmly.
Electrodes to be pushed in port fitting directly and pipes are fitted in same
way.
pH electrodes should be calibrated in appropriate buffers i.e. in pH 7 then
either pH 4 or pH 9 for high reference- low reference

pH and DO electrode to be fitted with utmost care not to damage them.


Both must be tightly capped to prevent moisture getting into electrical
contacts.
Pt- 100 must be fitted and capped.
Outlet pipes of reagent bottles should be clamped and shut.
Inoculation port is closed by cap
Exit gas cooler must be left open.
Drive hub is covered with foil.
Tubing is clamped so that no liquid can escape during autoclaving.

Autoclaving
A final check up is made that at least one route is available for air to enter
and leave the vessel.
All the lines dipping into liquid are clamped closed.
Autoclaving at 121oC for 30 minute.
Vessel and accessory must be allowed to cool, completely before handling.
Reduce the volume of the medium upto 10 % of the total

Set up Following Autoclaving


Air sparger line unclipped and connects to rotameter.
Exit gas cooler is connects to H2O supply
Tubing from the reagent bottle connected to multiway inlet and relevant
peristaltic pump.
Drive motor on top plate is connected to drive shaft.
Pt. 100 is connected to control module and pH electrode by removing cap and
DO electrode to appropriate cable.
Main electricity to mentation module. Is switched on allowing some hours (2
minute and 24 hours max) for OD electrodes to be polarized properly after that.
DO is calibrated and air supply is turned on maximum stirred speed to be
used, maximum air flow is needed on rotameter are set.
Fermenter is ready to inoculate.

Inoculation of vessel
Port fitted with a membrane with in capped off before autoclaving.
Inoculum 5-10 % of total culture volume aseptically transferred to a sterile
disposable syringe of suitable size.
Port fitting is removed
Needle is quickly pushed through the membrane and inoculum is transferred
in the vessel.
Needle is quickly withdrawn and silicon membrane is reseals.

For added security 70 % ethanol can be placed on membrane surface


before piercing.

Sampling from vessel


Sampling starts with a sample pipe which should be dip into the bulk of
culture liquid.
Sample pipe plugged and covered at open end.
After autoclaving, plug is removed and opened end dipped in a container of
a 70 % ethanol
When sample is to be taken syringe is quickly coupled to the tubing and
sample is withdrawn into it.
Aeration is stopped during this process to prevent surging of culture into
syringe.
Second approach is to use sampling device.
This is a bottle with two metal needles/ permanently fixed of its cap
One pipe has 0.22 m air filter and 2nd is linked to sample pipe
Syringe is lifted to air filter after autoclaving.
Sample device is attached to the plate.

Additional sensors
Redox
This refers to reduction/oxidation potential of the system and can be applied to
biological cultures as well as chemical reactions.
Redox values are given in millivolt and it refers to a standard hydrogen half cell.

Air flow
A simple system would use a magnetic sensor and variable area flowmeter to
measure the current air flow and adjustant amotorized valve between 0 and 100 %
open or closed to control the air flow

Weight
It is possible to mount whole fermenter on a balance and tare out its weight.
A more precise approach is to use a system with a small load cell mounted in
such a that that the only the vessel and its component are measured.

Pressure
The use of the pressure as a control is more common in situ sterlizable fermenter.
The pressure measurement is provided by piezo electric sensor which can be
mounted into a port closure and fitted to the vessel top plate.
The control element is proportional valve which restricts the flow of the gas out of
the fermenter and thereby crate a back, pressure.

On line measurement
A direct measurement of number of the organisms in a culture is to be
desirable for
Feed rate
Oxygenation
Process optimization
Measurement of total number of cells, wet weight, and viable cells counts
would be made by taking sample of culture at set time and analysis is
performed in laboratory.
Fluorescence Microscopy has been used for the measurement of the viable
cell density either by a probe or externally through the vessel glass.
Metteler- Toldeo FCS turbidity system
Capacitance/conductance-based biomass monitor

The main components of the fermenter and their Use


Provide aeration free from contamination maintain a specific temperature
Provide adequate mixing and aeration
Control the pH of the culture
Allow monitoring and or/ Control of dissolved oxygen
Allow feeding of nutrients and reagents
Privides acess points for inoculation and sampling
Use fitting and geometry relevant to scale up
Minimize liquid loss from the vessel
Facilitate the growth of a wide range of the organism

Fermenter construction
A complete description of methodologies used for its development,
installation and operation
Validiation of the system with respect to the hardware, operation and its
application
Documentation of specific validable activities
Evidence confirming that each element of hardware an software perform
its function reliably and in accordance with documentation specifications
The following companies offer software programmes with a wide range of
applications
APPLIKON
B. BRAUN BIOTECH
INCELTECH AND INCELTECH SOFT
NEW BRUNSWICK SCIENTIFIC

Configuration and software


All are fully object oriented softwares running under WINDOWS 95
The data files can be processed under different operating systems (MS-Dos,
WINDOWS 3.1, WINDOWS 95, UNIX, etc..)

Data acquisition on the microcomputer


screen for lab scale reactor

Example of window available to the user


on the prefermenter

Example of the configuration on the


display unit during a fermentation

Example of the screen view during a


fermentation in the producer

Summary and conclusion


Fermentors are composed of a number of different components which can be
grouped by their functions, i.e. temperature control, speed control, continuous
culture accessories, etc. A wide range of peripheral devices can enhance the
basic facilities of the fermentor, e.g. feed pumps, exit gas analysers, etc.
Fermentor instrumentation can be of several different types, depending on
the age, cost and sophistication of the fermentor. Microprocessor-based
system offer advantages in terms of the flexibility or control and the ability to
store operational protocols.
Many different types of vessels exist such as air lift, fluidized bed, hollow
fibre and specially modified stirred tank reactors for containment of in situ
sterilization.
Practical steps for autoclaving use, inoculation and sampling ensure safe
and reliable operation for any make or type of fermentor.
Routine maintenance of electrodes, vessel, glass, etc. is repaid in longevity
of components and safe operation.
The range of organisms which can be adapted for growth in fermentors is
wide and includes microbes, plant and animal cells.