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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
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Culture can be withdrawn into a sampling device or a reservoir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.
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.
The speed of the operation of the pump can be set manually or whole system
put under the computer control.
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.
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 .
Beckman fermenter
Fermenter design
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 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
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.
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:
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.
1)
Membrane filters
2)
Depth filters
Membrane filters
2)
Depth filters
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.
i)
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.
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.
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
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
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
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.
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
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