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Fermentation technology, Designs and features of industrial fermenters,…

- - - - Porous sparger
      - Orifies sparger
      - Nozzle sparger
    - - Disc turbines
      - Vaned discs
      - Open turbines of variable pitch
      - Propellers
    - - Operational steps:
        
        Sterilization of fermenter
        
        Sterilization of air supply
        
        Sterilization of exhaust gas
        
        Sealing of joints
- - - - Recombination
        
        Translocation
        
        Transformation
        
        Conjugation
        
        Transduction
        
        Cross over
        
        Protoplast fusion
      - Recombinant DNA technology
        
        Feedback regulation - biosynthesis of amino acids, nucleotides, vitamins:
        
        Feedback inhibition
        
        Feedback repression
      - Mutant selection
        
        Resistance selection methods
        
        Substrate utilizing method
        
        Replica planting technique
        
        Carcinogenicity test
- - - - Sealing of joints
        
        Vital step
        
        Seal of the joints, cap and the fermentation jar is very vital
        
        Maintain aseptic conditions
        
        Types of sealing: 'o' ring seal, gasket seal, lip seal - commonly used
        
        N-octyl or butyl rubber are commonly used for sealing
        
        Periodic monitoring
      - Sterilization of exhaust gas
        
        Vital step
        
        On the outlet pipe o,2 micrormeter filters are fixed
        
        Moisture and solid matter may plug the filter
        
        Filters checked regularly - no viable cells escape from fermenter
        
        Cyclone separator for solid & coalesce fro liquid droplets are included in upstream of 2 filters to prevent clogging
        
        Suitable test procedure should be established at the outlet
      - Sterilization of air supply
        
        Sterile air is essential in large amount for aerobic process
        
        Air sterilized by 2 methods: Heat (costly) & Filter (cartridge type)
        
        Steam jacketed air filters are used
        
        Concomitant use of steam and air supply through filters sterility can be maintained
      - Sterilization of fermenter
        
        Auntoclave
        
        Medium sterilized in vessel separately and added to the fermenter aseptically
        
        Medium preheated and added to the vessel and sterilize in situ
        
        Stem is introduced by coils steam jackets
        
        All pipes should be simple and straight
        
        Slope towards drainage points - steam effectively reached all parts of the fermenter
        
        Periodically maintenance of pipe and valves
- - - - Types:
        
        Transformation
        
        Bacteria take up DNA from their environment (recipient cells)
        
        The DNA remnants in the environment most commonly originate from death bacteria cells
        
        The DNA incorporating it into recipient own genome, where the cell is described as being competent
        
        Conjugation
        
        Bacterium connects itself to another through protein tube structure - pillus
        
        Genes are transferred from one bacterium to another through the pillus
        
        Transduction
        
        Bacterial DNA is exchanged thru a virus that infects bacteria - bacteriophage
        
        The transfer of DNA from one bacteria to another by bacteriophage
        
        Translocation
        
        Crossing over that occurs in non-homologous chromosomes
        
        When a chromosome segments detaches from one chromosome and move to a new position on another non-homologous chromosome's
        
        Protoplast fusion
        
        During fusion, two or more protoplasts come in contact and adhere with one another either spontaneously or in presence of fusion inducing chemicals
        
        The cytoplasm of the two protoplasts intermingle
        
        The fusion between non-antibiotic producing strains of two species has yielded a strain that produce indolizomycin, a new indolizine antibiotic
        
        Cross over
        
        Occurs during prophase 1 of meiosis
        
        The chiasma breaks and the broken chromosome segments get switched onto homologous chromosome
        
        The broken chromosome from the maternal chromosomes get joined to its homologous paternal chromosome
        
        Exchange of gene code
      - Formation of new gene combinations among those present in different strain
        
        Recombinant used for both genetic analysis & strain improvement
        
        To generate new products
    - - Feedback regulation - biosynthesis of amino acids, nucleotides, vitamins:
        
        Feedback inhibition
        
        The final product of the metabolite pathways, inhibits the action of the first enzyme of the pathway.
        
        This to prevent further synthesis of intermediates and products of the pathways
        
        It happens when the final product is sufficient quantities
        
        Example:
        
        High conc. of D will inhibit conversion of A to B
        
        D is specifically able to bind and inhibit enzyme convert A to B
        
        D thus acts as a negative allosteric effector or feedback inhibitor of the enzyme
        
        Feedback repression and attenuation
        
        Involves turn off of enzyme synthesis
        
        Happens in gene level
        
        When the sufficient amount of the product have been made and it starts to accumulate :arrow_right: the feedback regression take place
        
        The end-product of the pathway acts as co-repressor
        
        Example:
        
        In bacteria, the presence of the synthesized particular amino acid in the culture medium prevents new synthesis of the amino acid via repression
        
        A small molecule such as histidine or leucin, acting as co-repressor, can ultimately block synthesis of the enzymes involved in its own synthesis
        
        In the presence of co-repressor, an active repressor is formed which binds to the operator to prevent transcription by RNA polymerase and prevent enzyme synthesis
      - Genetic Engineering technology
        
        Used to isolate & clone the genes of interest
        
        Isolated gene will be constructs using appropriate genetic tech in lab condition
        
        The isolated gene will be cloned into a vector (plasmid of a bacteria)
        
        The vector will expresses in bacteria
        
        Bacteria which successfully received the vector will expressed new gene - desired product
        
        This bacteria is known as recombinant bacteria
        
        rDNA tech is used to:
        
        Produce recombinant product
        
        in metabolic engineering
        
        Recombinant protein - The cloned new rDNA MO produced the desired protein. - Eg. insulin, interferon
        
        Metabolite engineering - The metabolic activities of an organism are modified by introduction of a transgene.
        
        affects the enzymatic, transport, regulatory functions of its cells
        
        Eg. high production of amino acid isoluccine in C. glutamicum & ethanol by E. coli
    - - 4 methods to isolate mutants:
        
        Replica planting technique
        
        Used to detect auxotrophic mutants which differentiates between mutants and wild type strains on the basis of ability to grow in the absence of an amino acid
        
        Carcinogenicity test
        
        Used to identify the environmental carcinogens that cause mutation and induce cancer in organisms
        
        Based on detecting potential of carcinogens & testing for mutagenicity in bacteria
        
        Ames test - mutational reversion assay
        
        Salmonella typhimurium carry mutation on gene that encodes histidine - His-salmonella
        
        The bacteria loss the ability to synthesis histidine utilizing the ingredients of culture media - Histidine is required in growth media
        
        Culturing His-salmonella in a media containing certain chemical (mutagens), causes mutation in histidine encoding gene
        
        Bacteria regain the ability to synthesize histidine gene - Reverse mutation
        
        Substrate utilizing method
        
        Several bacteria utilize only a few primary carbon sources
        
        The cultures bacteria are seeded onto medium containing an alternate carbon sources
        
        Any colony that grows on medium can use the substrate and are possibly mutants
        
        Can be isolated
        
        Resistance selection methods
        
        The wild type cells are not resistant either to antibiotics or bacteriophages
        
        It is possible to grow the mutant bacterium in the presence of antibiotics or bacteriophage and look for survivors
      - 4 types:
        
        Spontaneous mutation
        
        Occurs without any specific treatment
        
        Naturally it happens due to external factors
        
        Happens during cell division
        
        Induced mutation
        
        Due to treatment with certain agents - chemical or physical agents
        
        Major mutation
        
        Extreme changes in biochemical characteristics
        
        Used in strain improvement
        
        eg. Streptomyces griseus & Streptomyces aurofaciens - produce 6-methyl tetracylcine in place of tetracycline
        
        Minor mutations
        
        Stepwise changes take place in the biochemical characteristic
        
        Eg. Penicillium chrysogenum - yield 55% more penicillin than the original strain
- - - - Production of lactic acid
        
        2 systems:
        
        Homolactic fermenters
        
        Utilize the glycolytic pathway
        
        Directly reduce their pyruvate to lactate with the enzyme lactate dehydrogenase
        
        Used by commercial production
        
        Hetrolactic fermenters
        
        Produce substantial amounts of other products - CO2 & ethanol
        
        Utilize phosphoketolase pathway
        
        Anaerobe process - no aeration required
        
        Starter culture is maintained in skimmed milk
        
        Medium:
        
        10-15% dextrose
        
        10% calcium carbonate
        
        small amounts of nitrogenous substances - malt sprouts, 0.25%
        
        Vitamin B & some amino acids
        
        Incubation temp: 43-45°C 48 hour
        
        Free lactic acid production must be neutralized as it is toxic to the bacteria
        
        pH: 5.5-6.5 by addition of calcium carbonate or calcium hydroxide for maintaining
        
        Agitation is essential fro smooth mixing - for calcium hydroxide to react with lactic acid
      - Recovery of lactic acid
        
        After 48-72 hours of fermentation
        
        Broth is boiled to coagulate protein
        
        Filtrate containing calcium lactate is concentrated under vacuum
        
        Purify the substance
        
        Lower quantity raw materials require even more extensive purification steps, including pre-purification & crystallization
        
        Can use solvent extraction or esterification with methanol and subsequent distillation - costly
  - - - Production of alcohol
        
        Carbon source: pure sugar, crude sugar, 10-18% molasses
        
        Nitrogen sources: ammonium sulphate
        
        Growth factor: molasseses
        
        pH: 4-4.5
        
        Temperature: 21-26°C 30-72 hours (depends on yeast strain)
        
        Must be continuous fermentation system
        
        No aeration
        
        Agitation is required
        
        Yield: 90% of carbohydrates can be converted into alcohol
        
        Inoculum size: 4% from the total mass of broth
      - Recovery of alcohol
        
        Mass separated via centrifugation
        
        Use distillation process to obtain alcohol
        
        Distillation:
        
        Separation process for a mixture of the interested compounds
        
        Relies on boiling point of the interested components
        
        Broth heated up to 78.5°C to vaporized the ethanol
        
        Ethanol vapors are collected and condensed in another flask
        
        At end: 95.6% ethyl alcohol & 4.4% water can be separated
        Harvest :arrow_right: Milling :arrow_right: Filtration :arrow_right: Fermentation :arrow_right: Distillation (10-90%) :arrow_right: Test dehydrate <99.6% :arrow_right: Mix with 3% gas :arrow_right: Bio-Gas
  - - - Production of vit B12
        
        Continuous process
        
        Salmonella typhimurium used for synthesis
        
        20 genes of S.typhimurium cob locus allowed production of vit B12 in E. coli
        
        Propionic acid is by-product of vit B12 cultivation
        
        Production is controlled via expanded bed adsorption bioreactors
        
        CObalt ions, glycine, amino-levulinic acid, amino-propanol added as precursor
        
        Medium: glucose / molasses
        
        Propionibacterium strains
        
        Temp:30°C
        
        pH: 6-7
        
        Required aeration & agitation
        
        Cobalt chloride - 2-10ppm is added as precursor
        
        Fermentation takes 3-4 days
        
        Most vit B12 remains with microbial cells until autolysis sets in
      - Recovery of vit B12
        
        Fermented broth, heated at 80-120°C for 10-30mins with pH 6.5-8.5
        
        Cyanide or thiocyanide added during this heating process to obtain cynocobalamin
        
        Zinc hydroxide added to separate the vit B12 alone from the mixture
        
        Tannic acid or cresol added into the solution to obtain 80% vit B12
        
        Dried for further usage
  - - - Production of Streptokinase
        
        Culture: Grown on blood agar media
        
        Continuous fermentation culture
        
        Fermentation media - Liquid state fermentation
        
        Ingredients of media:
        
        Glucose 1g
        
        CaCO3 25mg
        
        yeast extract 10g
        
        KH2PO4 0.25g
        
        Corn Steep Liquor 0.1-0.8%
        
        Distilled water 500mL
        
        Growth condition: 37°C 120rpm 24hr at pH7
        
        Streptokinase extracted: centrifugation at 10000rpm for 20mins at 0°C
        
        Maximum streptokinase enzymatic activity at 0.3% of corn steep liquor in liquid state fermentation
      - Recovery of Streptokinase
        
        The activity is measured by 2 hours intervals
        
        Activity of secreted streptokinse was determined by solid & liquid colometric methods
        
        Production can stop by rapid cooling at 4°C and by addition of hexyl resorcinol
        
        Centrigued for 25mins at 10000g
        
        Filtered through a o.2 micromilli cellulose acetate filter
        
        Filtrate was concentrated by ultrafiltration
        
        pH of the concentrate was adjusted, cooled, and the fraction precipitating by 30% cold methanol was harvested
- - - - Production of penicillin
        
        The culture medium consists of carbon source like lactose
        
        Ingredients of media:
        
        Corn steep liquor solids 3.5%
        
        Lactose 3.5%
        
        Glucose 1%
        
        Calcium carbonate 1%
        
        Potassium dihydrogen phosphate 0.4%
        
        Edible oil 0.25%
        
        Corn steep liquor supplies fungus with nitrogen and growth factors
        
        High level of glucose repress penicillin
        
        Fermentation conditions: temp. 25-27°C 4-7days at pH 6-7.5
        
        Continuous supply of air and agitation
        
        Inoculated - Penicillum Chrusogenum
        
        The mold grows as pellets
        
        Vegetative growth phase (~40 hours) - Trophophase
        
        Followed by penicillin production phase (150-180 hr) - idiophase
      - Penicillin recovery
        
        Fermentation broth is first chilled
        
        Passed thru rotary filter to remove biomass
        
        Phosphoric or sulfuric acid added to lower the pH 2-2.5 - to convert the penicillin to the anionic form
        
        Penicillin in the acid (anion) form is solvent extractable, and the antibiotic as dissolved in an organic solvent, can be back-extracted as a salt into aqueous solution aqueous solution
        
        The broth is immediately extracted in Podbieniak counter current solvent extractor with an organic solvent such as amyl acetate, methyl isobutyl ketone, or butyl acetate
        
        The penicillin is then back-extracted into water from the organic solvent by adding potassium or sodium hydroxide to form a salt of the penicillin
        
        the above step is repeated and the antibiotic is purified by crystalllization
        
        Extract is crystallized by addition of sodium or potassium hydroxide or acetate
        
        Penicillin GK + salts crystals are washed with acetone by centrifugation
        
        dried under vacuum
        
        Penicillin crystals obtained are more than 90% pure
        
        Kept in dried environment
  - - - Production of Streptomycin
        
        Originally derived from Streptomycin griseus
        
        Currently procedure is mutant of Streptomyces with yield as 25000 units/mL
        
        Streptomyces mutants are genetically unstable
        
        The spores are maintained as soil stock or lyophilized
        
        Inoculate in the sporulation medium and then transferred into the fermenter for production
        
        With suitable conditions - able to yield 1-3g/L of the fermentation broth
        
        Fermentation conditions:
        
        Glucose, starch, dextrin, soy meal, corn steep liquor, sodium sulphate
        
        High aeration and agitation
        
        Temp: 28-30°C, pH 7.6-8 5-7days
        
        Involved 3 phases of production:
        
        Production of protease - Digest soybean meal & release ammonia, carbohydrate. Increase biomass in 24hr
        
        Idiophase - Streptomycin produced in 24hr - 6days
        
        Death phase - Cell lysis occured. Fermentation broth harvested before this phase
      - Recovery of Streptomycin
        
        Mycellium is separated from broth - Filtration process
        
        Remaining liquid is then seep into through cation exchange resin column
        
        Streptomycin gets absorbed
        
        Finally eluted by washing with buffer as Streptomycin sulphate
        
        Impurities are removed by treating it with sodium hyposhlorite, EDTA and activated carbon
        
        Purified Streptomycin sulphate solution is concentrated under vacuum
        
        Dried aseptically