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Fermentation

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on 30 August 2013

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Transcript of Fermentation

Fermentation
Contents:
Introduction to Fermentation
Types of Fermentation processes
Components of Fermentation
Inocullum development
Preparation & Preservation of Culture media
Sterilization of Culture media & fermenter
Fermentation process
Types of fermenters
Product recovery & its purification methods
Fermentation of some specific agents:
Antibiotics:- Penicillin Streptomycin
Vitamins:- Vitamin B2, Vitamin B12
INTRODUCTION
Ferver = to boil
Traditionally it was used to describe catabolic action of yeast on sugars to produce alcohol accompanied with evolution of carbon dioxide bubbles, resembles to boiling appearance.
According modern terms, Fermentation is the growth of microbes in liquid media under either condtion (i.e. aerobic or anaerobic) to produce economically useful products.
Microbiologically, fermentation involves use of metabolic capacity (enzymes) of microorganisms on organic substrates under anaerobic conditions to produce useful metabolites.
Range of Fermentation process
Produce microbial cells (Yeast cells used in baking industry)
Produce Microbial enzymes
Produce microbial metabolites
Produce recombinant products
Also for transformation process
-
- A. Whiteshaker &
Nagori
Can also used in...
Generation of Bioenergy
Bioremediation
Biomining
In sort, Fermentation is the technique of cultivating microbes which serve as source of industrially & therapeutically useful bioproducts
Types of
Fermentation Processes
1. Depending upon the scheme:
2. Depending upon when product is made:
Depending upon the scheme of Fermentation:
Batch Fermentation
Fed Batch Fermentation
Continuous cultures
Cascade fermentation
3. Depending upon how the culture is kept pure:
Depending upon when the product is made:
Type I fermentation:
These are products of primary metabolism, prepared during
Tropohophase

Type II fermentation:
Prepared when the culture is still growing i.e. during undergoing primary metabolism

Type III fermentation:
These are products of secondary metabolism, prepared during
Idiophase
Depending upon how the culture is kept pure:
Aseptic/sterile fermentation:
In which the entry of
other
microbes is
prevented
...
Protected fermentation:
In which growth of unwanted microbes is prevented...
Consortium fermentation:
In which a group of microbes grown
together
, the growth of which is
dependent on one another
...

Components of Fermentation:
Components of fermentation can be divided into
Two groups:
Upstream processes:
Operations done before starting of fermentation
Downstream processes:
Operations done after fermentation has occurred
Upstream processes
Inoculum preparation
Preparation of culture medium
Sterilization of culture medium
Product recovery and its purification
Effluent treatment and waste disposal
Downstream processes
Fermentation Processes
Inoculum development
Characteristics of Microbes:
Can be fermented using cheapest culture medium
Should have an optimum temperature for growth & operation above 40, so that cooling cost are reduced
Genetically stable
Consistency in production
High yielding for the desired product
Permit easy recovery of the formed product
Should be resistant to infections, non-foaming, morphologically favorable, tolerant to low oxygen tension, etc.
Source of Microbes:
Solid, oceans, mountains, marine life, fruits, garbage, animal fecal matter, space or the entire universe
Preservation:
Lyophilization or freeze drying
Agar slope
Cryopreservation
Dried culture
The whole process comprises
Three
steps:
1. Culture collection
2. Isolation
3. Improvement

Development of Inoculum
Preparation of Culture medium
Ideal Characteristics of Culture medium
Should produce
maximum yield &

maximum product or biomass formation
Should permit
rapid growth of desired biomass & product
The yield, concentration and rate of
undesired product
formation should be
minimal
Should be of
consistent quality
Should be
readily available
, should be
stable
Should
not create problem
during fermentation
Should be
cheap
& have considerable rheological property...
Constituents of Culture medium
Carbon source:
Starch,
Barley malt,
Sucrose,
Molasses,Lactose,
Cellulose,
Oils and fats, Hydrocarbons
Nitrogen source:
Ammonia gas,
Nitrates,
Urea,
Protein & amino acids
Minerals:
Megnasium Potassium
Sulphur Phosphorus
Calcium Choloride
Growth factors
Vitamins:
B complex, Calcium, Pentothenate, Biotin
Amino acids
Fatty acids or sterols
Chelators:
EDTA
Polyphosphates
Metabolic regulators:
Precursors: Phenyl acetic acid, Cyanide, Chloride
Buffers:
Calcium carbonate
Inhibitors: Sodium bisulfite, Alkali, Bromide
Inducers: Maltose, Pectin, Yeast mannan, Proteins
Antifoaming agents:
Alcohols: Stearyl alcohol
Esters:
Fatty acids: Glycerides, vegetable oils
Silicones:
Sulphonates:
Polypropyleneglycol
Water:
De-ionized water
Culture Media
Sterilization of Culture medium
Drawbacks of Unwanted microbes
Substrates in the medium will be consumed by the contaminated by microbes results into loss of productivity
Unwanted microbes may outgrow the desired microbe
Desired product can be contaminated
Extraction of desired product would be contaminated
Desired product may degraded by the action of the contaminating microbes, Ex.: Beta lactamase producing bacteria in Penicillin fermentation
Contamination by bacteriophage which can lyse the bacterial cultrue
Sterilization of culture medium
Mainly by two methods:
1. Batch Process:
2. Continuous process:
Sterilization of Fermentor
Required if medium has been sterilized separately

Fermentation Process
procurement of desired strain form culture collection
Isolation of natural variant from soil
Improvement of Wild type
Preservation
Cryopreservation using liquid nitrogen
Storing on agar slopes at 5.
Freeze drying sublimation
Drying a soil culture
Development
Stock/Master culture
Vegetative/Germination culture
Maintenance culture
Seed culture
Inoculum
Production of Culture
(Spores lyophilized with milk/lactose)
(Agar slope incubated every half to weekly intervals at 28 C)
(One to few or several stages: to increase inoculum volume)
(Final seed culture: 3-10% by volume of production culture)
(Final seed culture: 3-10% by volume of production culture)
(100's to 100 thousands of liters)
Source
Fermenter: Design
Should be such as to prevent the entry of extraneous microbes into the medium i.e. it should be operable aseptically
Should prevent escape of microbe employed for fermentation
Should have adequate provision for -
Aeration
Agitation
Temperature control
pH Control
Sampling facilities
Should have low requirement for power
Should have minimal evaporation losses
Its operation, harvesting, cleaning and maintenance should be easy and labor non-intensive
Should be suitable for a range of processes
Should have smooth internal surfaces
Its material of construction should be cheap
There should adequate service provisions: compressed/sterile air, hot/cold water, steam/condensate, electricity/generator, drainage/storage, monitoring/maintenance equipment, extraction/recovery equipment, etc...
Fermenter: Design
Types of Fermenter
Submerged fermenters or suspended growth system:
Surface fermenters or supported growth system:
Types of Fermenter (Acc. Design)
1. Stirred tank fermenter
2. Airlift or Loop fermenter
3. Micro-carrier fementers
4. Membrane fermenters
1. Stirred Tank Fermenter
3. Micro-carrier fermenter:
4. Membrane fermenter
2. Air lift fermenter:
Process Parameters
Physical
Temperature
Pressure
Current flow
Agitation speed
Chemical
pH Nutrient concentration
Redox potential
Gas analysis for CO2, O2
Product concentration
Metabolite concentration
State of culture
Cell concentration/density
Power input
Rheology
AMount of mixing
Foaming
Biological
Product Recovery & its purification
Step 1: Separation
Involves separation of the crude product from other constituents of broth such as biomass, substrates, metabolic waste products etc.
Can be achieved by filtration or centrifugation to remove microbial cells, debris, fragments, particles (virus).
Step 3: Purification
Step 2: Concentration
Removal of most of the broth water - Dewatering.
Due to this handling volume will decreased
During this, most contaminants are removed
Ion exchange method
Adsorption on solids
Solvent extraction
Distillation/fractionation
Sedimentation
Centrifugation
Flotation
Reverse osmosis
Ultrafiltration
Methods for Concentration
Involves isolating the product in its purest form, free from all trace contaminants and variant forms of molecules, if any.
Enzymes can also used for further purification.
"Harvesting" word is used for biomass
Precipitation
Crystallization
Polymer addition
Heat denaturation
Methods for Concentration
Dialysis
Supercritical fluids
Chromatography
Step 4: Cell disruption
If the desired product remains inside the cell, then it becomes necessary to first disrupt the cells to recover the product.
Mainly proteins, enzymes and other large molecules are generally found in side the cell...
Methods for Cell disruption
Mechanical method
High pressure homogenizers
Blenders
Solid shear - Hughes press
Enzymatic method
Lysozyme for bacterial cell wall
Chitinase for fungal cell wall
Gluconase for fungal cell wall
Cellulose for plant cell wall
Chemical method
Detergents
Quaternary ammonium compounds
Sodium lauryl sulphate
Sodium dodecyl sulphate
Triton X - 100
Alkali
High salt concentration
Organic solvents
Freeze Thaw Method
Autolysis
Ultrasonication
Method 1: Physical
Dewatering by filtration
Sedimentation
Centrifugation to remove solids waste
Incineration (to burn something till formation of Ash) of solid waste
Solid waste being composted
Solid waste being used fertilizer
Method 2: Chemical
Coagulation process
Digesting
Oxidation by Microbes
Effluent treatment & Waste disposal
Used to reduce amount of organic matter i.e. to reduce the BOD (Biological Oxygen Demand) of waste.
Also removes phosphates & nitrates...
Methods for Effluent Treatment
Method 3: Chemical
Method 4: Remedial
Pumping of oxygen equal to BOD in the water reservoir to help microbes oxidize the organic matter without causing imbalance
Fermentation of some specific agents
Antibiotics
Penicillin
Streptomycin
Vitamin B
Vitamin B
Vitamins
2
12
Fermentation of Penicillin
Fermentation of Streptomycin
Fermentation of Riboflavin
Fermentation of Cyanocobalamin
Upstream processes
Inoculum development
The Microbe:
Culture medium
Corn steep liquor
Lactose/Glucose
Phenyl acetic acid (For Penicillin G) OR
Phenoxy acetic acid (For Penicillin V)
Lard oil/ Vegetable oil
Calcium carbonate
Potassium Dihydrogen Phosphate
Ammonium sulphate
Micro & macro elements
Distilled water
Sterilization
Medium is sterilized using steam under pressure
Penicillium chrysogenum or Penicillium notatum
Vegetative medium:
Corn steep liquor
Sucrose/glucose
Calcium carbonate
Inorganic salts
Distilled water
Shake flask culture
Aeration and agitation of culture is must
pH should be adjusted to 5.5 - 6.0
Fermentation
Production by
Submerged culture method
Production volume: 40000-200000 Litres
Aerobic processes: Airation - 0.5 - 1 Lt air/min
Agitation - Turbine type impellers
Temperature: 23 - 25 C
Optimum pH: 7 - 7.5
During process: pH rises from 5.5-6 to 7-7.5 & at the end it will be around 8
Glucose starvation is required for penicillin production
At the end of fermetation process, due to lack of Lactose,
autolysis
of myecelium
Time period for completion:- 5 - 6 days
Downstream processes
Recovery & Purification
Two methods: One is carbon method (Obsolete)
Another (industrial) method: Solvent Extraction process:
Filtration using rotary vacuum filtres
Acidification using phophoric or sulphuric acid to a pH 2 - 2.5 for to convert it to ionic form
Extraction by organic solvents - amyl acetate, methyl isobutyl ketone or butyl acetate
Treatment with activated charcoal
Basification using KOH or NaOH for salt formation
Extract salt in water
Acidification of aqueous solvent resulting in precipitation of penicillin
Crystallization of pure penicillin as K or Na salt
Drying using horizontal belt filter
Upstream processes
Fermentation
Production by
Submerged culture method
Production volume: 100 - 200 Litre
Aerobic processes: Airation - 0.25 Lt/Hour
Agitation - two impellers, at 600 RPM
Temperature: 25 - 30 C
During process: pH rises from 6.5 - 7.5 - 8
Time period for completion:- 5 - 7 days
Growth of Streptomycin shows few phases:
First Phase/Growth Phase/Tropohophase:
Second Phase/ Production phase/Idiophase:
Third Phase/ Death phase
Streptomycin production: 1.2 Gm/Liter
Recovery & Purification
Many patented methods are there...
One involves fixation of streptomycin on Cationic exchange resins followed by elution using mineral acids:
Filtration for removal of mycelium from broth
Pretreatment of the filtered broth involving acidification, filtration and neutrilization
Fixation to cation exchange resin by Amberlite IRC-50
Elution in solution and passage through different column
Decolorization with carbon
Elution with diluted hydrochloric acid
Concentration under vacuum almost to dryness
Dissolve in Methanol & filter
Precipitation & washing using acetone
Dissolution in Methanol
Inoculum development
The Microbe:
Culture medium
Glucose
Soybean meal
Distiller's dried solubles
Corn-steep liquor
Potassium Dihydrogen Phophate
Sterilization
Medium is sterilized by autoclaving at 120 C for 90 minutes
Streptomyces griseus
Vegetative medium:
Glucose
Dextrin
Soy bean flour
Potassium dihydrogen phosphate
Incubation temperature: 26 C
Culture to be agitated at 250 RPM
Incubation period: 48 Hours
pH should be adjusted to 7.3 - 7.5
Upstream processes
Fermentation
Production by
Submerged culture method
Production volume: 200 - 300 Gallons
Aerobic processes: Airation - 0.25 volume air/volume of medium/ minute*
Temperature: 28 - 30 C
Optimum pH: 6 - 7.5
Production of Riboflavin shows few phase:
First phase/ Growth Phase:
Second phase/ Production phase:
Third phase/ Death Phase:
Time period for completion:- 4 - 5 days
Yield: 2-4 Gm/Liter
Recovery & Purification
If the product is required for feed supplement, then broth is concentrated to syrupy consistency by evaporation using Drug dryer at pH 4.5.
If a purified product is desired then many patented methods are available, from which one is as follow:
Solvent Extraction:
Butanol as a solvent followed by petroleum ether/ Acetone
Precipitation:

by Reducing agent titanium chloride, followed by Diatomaceous earth
Adsorption:
on Silica gel, followed by elution with aldehyde/ketone/alcoholic solution of an organic base
Microbial:
using reducing bacteria -
Streptomyces faecalis
Upstream processes
Inoculum development
The Microbe:
Propionibacterium
Medium contains:
KCN
Sodium nitrite
Inoculum development
The Microbes:
Culture medium
Glucose
Corn steep liquor
Animal Stick liquor
Antifoaming agent
Distilled water
Sterilization
By employing steam under pressure at a temperature of 135 C for 5 minutes
Clostridium acetobutylicum
Stock culture for A. gossypii medium:
Peptone
Yeast extract
Malt extract
Glucose
Agar
Distilled water
Shake flask culture
pH is adjusted to 6.5
Incubation temperature: 26 - 30 C
pH should be adjusted to 4.5
Note: Glucose-Ammonium sulfate-Soybean in optimum concentration increases the yield of product
Ammonium sulfate
Calcium carbonate
Sodium chloride
Four more ingredients which can stimulate the Growth:
Ammonium sulfate
Calcium carbonate
Sodium chloride
Soy bean oil
Distilled water
Myoinositol
Scelerine
Barbital
Factor A
Note: Glucose is sterilized separately
Candida flareri, Bacillus subtilis, Ashbya gossypi & Eremothecium ashbyii
Glucose
Peptone
Corn steep liquor
Animal stick liquor
Distilled water
Seed culture for A. gossypii medium:
Downstream processes
Downstream processes
Presented by:
Ganatra Tejas H.
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