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M.PhilMicrobiology-NEETHU
ASOKAN
o FERMENTOR
 Closed vessels
 Used for fermentation
 Production of large scale process
 Fermenter is a closed vessels now it is called as bioreactor
 Fermenter is a old process used for cultivate the
microorganisms
 Fermenter used for cultivate Prokaryotic cells (fungi
bacteria)
 Bioreactors used for cultivate eukaryotic cells
(mammalian ,,insects)
M.PhilMicrobiology-NEETHU
ASOKAN
Fermentation is a metabolic process
acid gas alcohol
sugar
convert
Occur by yeast and bacteria
M.PhilMicrobiology-NEETHU
ASOKAN
HISTORICAL
PERSPECTIVE
It is known and practiced by humankind prehistoric times since.
The production of beer, bread, wine etc., are established in ancient Egypt.
In 1857 Pasteur developed the fermentation process.
1897 Eduard Buchner discovered that sucrose could be fermented to alcohol by
yeast.
In 1984 first constructed bioreactor by Stanberry and Whitaker
M.PhilMicrobiology-NEETHU
ASOKAN
STRUCTURE OF FERMENTER
M.PhilMicrobiology-NEETHU
ASOKAN
Components' of Fermenter
Parts Working
Water jacket Circulating the cooling water and remove the heat and generate
the metabolic activity
Steam inlet Sterilizes the fermenter between fermentation
Heating plate Raise the temperature
Nutrient inlet Where nutrients enter the fermenter
Temperature and ph probe Maintain pH and temperature in neutral level
Stirring paddle Stirs the reaction microorganisms are mixed with the nutrients,
which stops them from sinking to the bottom and keeps the
temperature well
Harvesting outlet Where the products' are release
Steam outlet Where steam comes out let
Stainless steel Some microorganisms produce acid as waste which might
corrode other metals
Electric motor Connected centre shaft to rotate
M.PhilMicrobiology-NEETHU
ASOKAN
TYPES OF FERMENTOR
Continuous stirred tank bioreactor
Bubble Colum bioreactor
Air lift flurized bioreactor
Internal loop airlift bioreactor
External loop airlift bioreactor
Tower bioreactor
Photo bioreactor
Cyclone Colum bioreactor
Holl fibre bioreactor
Cylinder conical bioreactor etc.,
M.PhilMicrobiology-NEETHU
ASOKAN
Upstream
process
Fermentation
process
Downstream
process
M.PhilMicrobiology-NEETHU
ASOKAN
The pre-fermentation stage
Isolation
Improvement
Producing of microorganisms
Screening method; isolate microbes to produce dicered products
Two methods;
primary screening checking the quality of microbes done in agar
plate.
Secondary screening checking the quntative of microbes done in
liquid media
M.PhilMicrobiology-NEETHU
ASOKAN
Microbes isolated from natural sources thus is improved to get productive
strains by using
Recombination
Mutations
Cell fusion
Gene cloning
Media formulation : growth medium must have essential nutrients for
microbial growth for successful fermentation process
Two kind media :
Inoculum media : enrich the culture
Production media: contain carbon and nitrogen
Raw materials : corn molasses, cellulose, corn, streep liquor soybean,
sugar, beet molasses, malt extract etc.,
M.PhilMicrobiology-NEETHU
ASOKAN
Upstream processing includes formulation of the fermentation
medium, sterilization of air, fermentation medium and the Fermenter,
inoculum preparation and inoculation of the medium.
The fermentation medium should contain an energy source, a carbon
source, a nitrogen source and micronutrients required for the growth
of the microorganism along with water and oxygen, if necessary.
A medium which is used for a large scale fermentation, in order to
ensure the sustainability of the operation, should have the following
characteristics;
1. It should be cheap and easily available
2. It should maximize the growth of the microorganism, productivity
and the rate of formation of the desired product
3. It should minimize the formation of undesired products
M.PhilMicrobiology-NEETHU
ASOKAN
Usually, waste products from other industrial processes, such
as molasses, lignocelluloses wastes, cheese whey and corn
steep liquor, after modifying with the incorporation of
additional nutrients, are used as the substrate for many
industrial fermentations.
Sterilisation is essential for preventing the contamination with
any undesired microorganisms.
Air is sterilised by membrane filtration while the medium is
usually heat sterilised.
Any nutrient component which is heat labile is filter-sterilised
and later added to the sterilised medium.
The fermenter may be sterilised together with the medium or
separately.
M.PhilMicrobiology-NEETHU
ASOKAN
Inoculum build up is the preparation of the seed culture in
amounts sufficient to be used in the large Fermenter vessel.
This involves growing the microorganisms obtained from the pure
stock culture in several consecutive Fermenter.
This process cuts down the time required for the growth of
microorganisms in the Fermenter, thereby increasing the rate of
productivity.
Then the seed culture obtained through this process is used to
inoculate the fermentation medium.
M.PhilMicrobiology-NEETHU
ASOKAN
Inoculum preparation procedure
Test tube
Pure culture
under good
condition
Test tube
Contain seed
culture
In Suitable
conditions
Conical flask
Containing
culture for
large scale
fermentation
Fermenter contain medium
and culture for mass culture
contain suitable conditions
Fermenter
M.PhilMicrobiology-NEETHU
ASOKAN
• The degradation of carbon compounds by cells or organisms
under anaerobic conditions.
Carbon
compounds
degraded Microorganisms
Complex into simple one
by
M.PhilMicrobiology-NEETHU
ASOKAN
Process
• Three cultural fermentation system used in
bioreactors
• Batch culture fermentation
• Continuous culture fermentation
• Feed batch culture fermentation
• Batch culture fermentation
• It is closed system filled fresh medium and
inoculated
• It is refilled for the next process of fermentation
M.PhilMicrobiology-NEETHU
ASOKAN
• Continuous reaction
• Fresh medium added
continuously
• Shut down less frequently
then batch system.
M.PhilMicrobiology-NEETHU
ASOKAN
• Feed batch
• Feed continuously added until
maximum liquid fermenter
volume reached.
• Then fermenter may be allowed to
continue or completed partially or
completely depending on the
process
M.PhilMicrobiology-NEETHU
ASOKAN
• Define different modes of fermentation and
known their limitation.
• Develop a suitable medium and perform a
material balance.
• Deter-mind fermentation productivity and yields.
• Fermentation take place in absence of oxygen.
• The science of fermentation is know as zymology
M.PhilMicrobiology-NEETHU
ASOKAN
 when fermentation is over, the desired product is recovered
from the growth medium.
Then the product is Extraction Purification and Packed of a
biotechnological product from fermentation is refered to as
DSP or product recovery or downstream processing.
The end products include Antibiotics, Amino acid, Vitamins,
Organic acid, Industrial enzyme, vaccines etc.
It is complex and important as fermentation process.
M.PhilMicrobiology-NEETHU
ASOKAN
MULTI
STAGE
OPERATION
5.
FORMULATION
1.
DISINTEGRATION
OR CELL-
DISRUPTION.
2.SOLID-LIQUID
OR
CLARIFICATION
3. CONCENTRATION
4. PURIFICATION
M.PhilMicrobiology-NEETHU
ASOKAN
DESIRED PRODUCT IN CULTURE
BROTH
INTRACELLULAR
PRODUC
EXTRACELLULAR
PRODUCT
OR
DISINTEGRATION
PHYSICAL, CHEMICAL
ENZYMATIC METHODS
BROTH WITH SOLIDS
AND LIQUID
SOLID-LIQUID
SEPARATION
FLOTATION,
FLOCCULATION,
FILTRATION,
CENTRIFUGATION
M.PhilMicrobiology-NEETHU
ASOKAN
CONCENTRATION
EVAPORATION,
DIALYSIS, EXTRACTION,
MEMBRANE
FILTRATION,
PRECIPITATION,
ADSORPTION,
DISTILLATION
PURIFICATION
ADSORPTION
CHROMATOGRAPHY,
ION- EXHANGE
CHROMATOGRAPHY,
GEL
CHROMATOGRAPHY, etc.
FORMULATION
CHAMBER DRYER,
TUMBLER DRYER DRUM
DRYER, BELT DRER, THIN-
LAYER DRYER
M.PhilMicrobiology-NEETHU
ASOKAN
CELL -DISRUPTION OR
DISINTEGRATION
oMechanical methods:
Shear forces in solid matter and solution.
oNon mechanical methods:
Lysis
Physical – freezing and thawing : now high
osmotic pressure, shock
Chemical – surface active agents, solvents,
antibiotics etc.,
Enzymatic – lysozyme
Drying
Freeze-drying, Air drying, Pressure release,
Drying with solvents
M.PhilMicrobiology-NEETHU
ASOKAN
Primary operation
 Separation of whole cells
 Removal of cell debris
 Collection of protein precipitate
 Collection of inclusion bodies etc.,
SOLID-LIQUID SEPARATION
OR CLARIFICATION
M.PhilMicrobiology-NEETHU
ASOKAN
Solid-liquid separation (clarification)
Coagulation
 Colloids into small flocs using simple electrolytes
Flocculation
 Agglomeration of these small flocs into larger settle-able
particles using polyelectrolytes.
Flotation
 Enrichment of microorganisms
Filtration
 The separation of suspended particles from liquid
Centrifugation
 Gravitational force used for separate the particles
M.PhilMicrobiology-NEETHU
ASOKAN
CONCENTRATION METHODS
• The purity or concentration of metabolite
Evaporation –steam as heat source
Extraction – the cell mass and more or less clear solution
obtained
Adsorption – special polymer resins (chemical) used for
the isolation of hydrophilic metabolites that cannot be extract
with organic solvents.
Filtration – separation of biomolecules and particles [pore
size]
Precipitation – removal of product from the solvent
Dialysis – semi permeable membrane
M.PhilMicrobiology-NEETHU
ASOKAN
PURIFICATION
• Purify relatively low concentration of metabolic products
• The chromatography technique are used
• Purification is a main process in fermentation
• Desired product purification is important
• Many techniques are used for the purification
M.PhilMicrobiology-NEETHU
ASOKAN
FORMULATION
• Products is depended on the maintenance of its activity and stability
during distribution and storage.
• Concentrated solutions after removing most of the water.
Drying :
Withdrawal of water from the products
Crystallization :
Established method used for concentration of the bio-products.
Widely used for final purification
M.PhilMicrobiology-NEETHU
ASOKAN
MONITORING OF DSP
• To keep control over the presence and
concentration of target molecule
• Monitored by using sensor
• Mostly used in pharmaceuticals
• Monitor the UV-absorbance, conductivity, pH,
molecular size, protein, bio-specific binding
reactions etc.,
M.PhilMicrobiology-NEETHU
ASOKAN
MARKETING
• The formulated product is packed and sent to the
market for the consumers.
• The purified product is modified before
marketing.
• Product may be in aqueous form or in solid form.
• The nature of the product have 98-100% in pure
for seal in market.
• The purity of the final product depends upon the
successful operation of all these downstream
processes.
M.PhilMicrobiology-NEETHU
ASOKAN
Reference
 Text book of fermentation technology by
H.A. Modi
 Text book of biotechnology by R.C Duby
 Text book of biotechnology by Dr. Prakash S Lohar
 Net references
M.PhilMicrobiology-NEETHU
ASOKAN

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Upstream and down stream process

  • 2. o FERMENTOR  Closed vessels  Used for fermentation  Production of large scale process  Fermenter is a closed vessels now it is called as bioreactor  Fermenter is a old process used for cultivate the microorganisms  Fermenter used for cultivate Prokaryotic cells (fungi bacteria)  Bioreactors used for cultivate eukaryotic cells (mammalian ,,insects) M.PhilMicrobiology-NEETHU ASOKAN
  • 3. Fermentation is a metabolic process acid gas alcohol sugar convert Occur by yeast and bacteria M.PhilMicrobiology-NEETHU ASOKAN
  • 4. HISTORICAL PERSPECTIVE It is known and practiced by humankind prehistoric times since. The production of beer, bread, wine etc., are established in ancient Egypt. In 1857 Pasteur developed the fermentation process. 1897 Eduard Buchner discovered that sucrose could be fermented to alcohol by yeast. In 1984 first constructed bioreactor by Stanberry and Whitaker M.PhilMicrobiology-NEETHU ASOKAN
  • 6. Components' of Fermenter Parts Working Water jacket Circulating the cooling water and remove the heat and generate the metabolic activity Steam inlet Sterilizes the fermenter between fermentation Heating plate Raise the temperature Nutrient inlet Where nutrients enter the fermenter Temperature and ph probe Maintain pH and temperature in neutral level Stirring paddle Stirs the reaction microorganisms are mixed with the nutrients, which stops them from sinking to the bottom and keeps the temperature well Harvesting outlet Where the products' are release Steam outlet Where steam comes out let Stainless steel Some microorganisms produce acid as waste which might corrode other metals Electric motor Connected centre shaft to rotate M.PhilMicrobiology-NEETHU ASOKAN
  • 7. TYPES OF FERMENTOR Continuous stirred tank bioreactor Bubble Colum bioreactor Air lift flurized bioreactor Internal loop airlift bioreactor External loop airlift bioreactor Tower bioreactor Photo bioreactor Cyclone Colum bioreactor Holl fibre bioreactor Cylinder conical bioreactor etc., M.PhilMicrobiology-NEETHU ASOKAN
  • 9. The pre-fermentation stage Isolation Improvement Producing of microorganisms Screening method; isolate microbes to produce dicered products Two methods; primary screening checking the quality of microbes done in agar plate. Secondary screening checking the quntative of microbes done in liquid media M.PhilMicrobiology-NEETHU ASOKAN
  • 10. Microbes isolated from natural sources thus is improved to get productive strains by using Recombination Mutations Cell fusion Gene cloning Media formulation : growth medium must have essential nutrients for microbial growth for successful fermentation process Two kind media : Inoculum media : enrich the culture Production media: contain carbon and nitrogen Raw materials : corn molasses, cellulose, corn, streep liquor soybean, sugar, beet molasses, malt extract etc., M.PhilMicrobiology-NEETHU ASOKAN
  • 11. Upstream processing includes formulation of the fermentation medium, sterilization of air, fermentation medium and the Fermenter, inoculum preparation and inoculation of the medium. The fermentation medium should contain an energy source, a carbon source, a nitrogen source and micronutrients required for the growth of the microorganism along with water and oxygen, if necessary. A medium which is used for a large scale fermentation, in order to ensure the sustainability of the operation, should have the following characteristics; 1. It should be cheap and easily available 2. It should maximize the growth of the microorganism, productivity and the rate of formation of the desired product 3. It should minimize the formation of undesired products M.PhilMicrobiology-NEETHU ASOKAN
  • 12. Usually, waste products from other industrial processes, such as molasses, lignocelluloses wastes, cheese whey and corn steep liquor, after modifying with the incorporation of additional nutrients, are used as the substrate for many industrial fermentations. Sterilisation is essential for preventing the contamination with any undesired microorganisms. Air is sterilised by membrane filtration while the medium is usually heat sterilised. Any nutrient component which is heat labile is filter-sterilised and later added to the sterilised medium. The fermenter may be sterilised together with the medium or separately. M.PhilMicrobiology-NEETHU ASOKAN
  • 13. Inoculum build up is the preparation of the seed culture in amounts sufficient to be used in the large Fermenter vessel. This involves growing the microorganisms obtained from the pure stock culture in several consecutive Fermenter. This process cuts down the time required for the growth of microorganisms in the Fermenter, thereby increasing the rate of productivity. Then the seed culture obtained through this process is used to inoculate the fermentation medium. M.PhilMicrobiology-NEETHU ASOKAN
  • 14. Inoculum preparation procedure Test tube Pure culture under good condition Test tube Contain seed culture In Suitable conditions Conical flask Containing culture for large scale fermentation Fermenter contain medium and culture for mass culture contain suitable conditions Fermenter M.PhilMicrobiology-NEETHU ASOKAN
  • 15. • The degradation of carbon compounds by cells or organisms under anaerobic conditions. Carbon compounds degraded Microorganisms Complex into simple one by M.PhilMicrobiology-NEETHU ASOKAN
  • 16. Process • Three cultural fermentation system used in bioreactors • Batch culture fermentation • Continuous culture fermentation • Feed batch culture fermentation • Batch culture fermentation • It is closed system filled fresh medium and inoculated • It is refilled for the next process of fermentation M.PhilMicrobiology-NEETHU ASOKAN
  • 17. • Continuous reaction • Fresh medium added continuously • Shut down less frequently then batch system. M.PhilMicrobiology-NEETHU ASOKAN
  • 18. • Feed batch • Feed continuously added until maximum liquid fermenter volume reached. • Then fermenter may be allowed to continue or completed partially or completely depending on the process M.PhilMicrobiology-NEETHU ASOKAN
  • 19. • Define different modes of fermentation and known their limitation. • Develop a suitable medium and perform a material balance. • Deter-mind fermentation productivity and yields. • Fermentation take place in absence of oxygen. • The science of fermentation is know as zymology M.PhilMicrobiology-NEETHU ASOKAN
  • 20.  when fermentation is over, the desired product is recovered from the growth medium. Then the product is Extraction Purification and Packed of a biotechnological product from fermentation is refered to as DSP or product recovery or downstream processing. The end products include Antibiotics, Amino acid, Vitamins, Organic acid, Industrial enzyme, vaccines etc. It is complex and important as fermentation process. M.PhilMicrobiology-NEETHU ASOKAN
  • 22. DESIRED PRODUCT IN CULTURE BROTH INTRACELLULAR PRODUC EXTRACELLULAR PRODUCT OR DISINTEGRATION PHYSICAL, CHEMICAL ENZYMATIC METHODS BROTH WITH SOLIDS AND LIQUID SOLID-LIQUID SEPARATION FLOTATION, FLOCCULATION, FILTRATION, CENTRIFUGATION M.PhilMicrobiology-NEETHU ASOKAN
  • 24. CELL -DISRUPTION OR DISINTEGRATION oMechanical methods: Shear forces in solid matter and solution. oNon mechanical methods: Lysis Physical – freezing and thawing : now high osmotic pressure, shock Chemical – surface active agents, solvents, antibiotics etc., Enzymatic – lysozyme Drying Freeze-drying, Air drying, Pressure release, Drying with solvents M.PhilMicrobiology-NEETHU ASOKAN
  • 25. Primary operation  Separation of whole cells  Removal of cell debris  Collection of protein precipitate  Collection of inclusion bodies etc., SOLID-LIQUID SEPARATION OR CLARIFICATION M.PhilMicrobiology-NEETHU ASOKAN
  • 26. Solid-liquid separation (clarification) Coagulation  Colloids into small flocs using simple electrolytes Flocculation  Agglomeration of these small flocs into larger settle-able particles using polyelectrolytes. Flotation  Enrichment of microorganisms Filtration  The separation of suspended particles from liquid Centrifugation  Gravitational force used for separate the particles M.PhilMicrobiology-NEETHU ASOKAN
  • 27. CONCENTRATION METHODS • The purity or concentration of metabolite Evaporation –steam as heat source Extraction – the cell mass and more or less clear solution obtained Adsorption – special polymer resins (chemical) used for the isolation of hydrophilic metabolites that cannot be extract with organic solvents. Filtration – separation of biomolecules and particles [pore size] Precipitation – removal of product from the solvent Dialysis – semi permeable membrane M.PhilMicrobiology-NEETHU ASOKAN
  • 28. PURIFICATION • Purify relatively low concentration of metabolic products • The chromatography technique are used • Purification is a main process in fermentation • Desired product purification is important • Many techniques are used for the purification M.PhilMicrobiology-NEETHU ASOKAN
  • 29. FORMULATION • Products is depended on the maintenance of its activity and stability during distribution and storage. • Concentrated solutions after removing most of the water. Drying : Withdrawal of water from the products Crystallization : Established method used for concentration of the bio-products. Widely used for final purification M.PhilMicrobiology-NEETHU ASOKAN
  • 30. MONITORING OF DSP • To keep control over the presence and concentration of target molecule • Monitored by using sensor • Mostly used in pharmaceuticals • Monitor the UV-absorbance, conductivity, pH, molecular size, protein, bio-specific binding reactions etc., M.PhilMicrobiology-NEETHU ASOKAN
  • 31. MARKETING • The formulated product is packed and sent to the market for the consumers. • The purified product is modified before marketing. • Product may be in aqueous form or in solid form. • The nature of the product have 98-100% in pure for seal in market. • The purity of the final product depends upon the successful operation of all these downstream processes. M.PhilMicrobiology-NEETHU ASOKAN
  • 32. Reference  Text book of fermentation technology by H.A. Modi  Text book of biotechnology by R.C Duby  Text book of biotechnology by Dr. Prakash S Lohar  Net references M.PhilMicrobiology-NEETHU ASOKAN