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(Production of primary & secondary metabolites), Lactic acid Alcohol…

- - - - 1780 - souring of milk
        
        Lactic acid is the first organic acid produced microbiologically in 1881
        
        Highly hygroscopic, syrup liquid and is technically available in various grades
        
        Lactococcus, Streptococcus, Pediococcus, Eunococcus, Leuconostoc, Lactobacillus isolate from natural habitats
        
        Materials: glucose syrup, sucrose, lactose, maltose
        
        Lactobacillus delbreukii - used to produce lactic acid from whey
        
        Lactobacillus delbreukii & Lactobacillus pentosus - produce lactic acid when glucose and sulphite (from waste substance in liquor production) are used as substrate
        
        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
    - - Ethanol fermentation
        
        Sugar (glucose. fructose, sucrose) convert energy + ethanol + carbon dioxide
        
        Anaerobic fermentation by yeast
        
        Organisms used:
        Bacteria - Zymomonas mobilis, Closteridium aetobutylicum, Klebsiella pneumoniae
        Yeast - Saccharomyces cervisisae, Saccharomyces carlsbergenesiae, Saccharomyces saki, Saccharomyces oviformis, Candida utilis, Mucur sp.
        
        Material used:
        
        Sugar - molasses, sweet potato, whey, glucose, sucrose, lactose
        
        Starch - cereals, roots, corn, wheat, potato, maize, barley, rice
        
        Cellulose - wood, paper waste, agriculture waste
        
        Nutrients medias:
        
        Carbon - molasses, starch
        
        Nitrogen - corn, steep liquor, soybean meal, pure ammonia/ammonia salt, urea, nitrate salt
        
        Vit and grow factors - bioin, yeast extract, beef extract, corn steep liquor, wheat germ meal
        
        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
    - - Cobalt complex - active material from liver, crystallized as vit B12, treatment of pernicious anemia
        
        Recovered active crystalline B12 from Streptomyces griseus culture which also produce antibiotic grisein
        
        Fermentation process - 2 alternative routes:
        
        Aerobic pathway - bacteria
        
        Anaerobic pathway - archea
        
        Fermentation - Pseudomonas denitrificans, Propionibacterium shermnaii, Sinorhizobium meliloti
        
        Disadvantages:
        
        Long fermentation cycles
        
        Complex and expensive systems for strain engineering
        
        Lack of suitable genetic system for strain engineering
        
        Research focus on: random mutagenesis & fermentation process optimization
        
        New finding: Escherica coli as platform for vit B12 production
        
        well-studied cell level
        
        metabolic engineering and synthetic biology strategies hv extensively well-applied
        
        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
    - - Extracellular protein
        
        Extracted from beta hemolytic streptococcus
        
        MOA: Combined with plasminogen to form activator complex, then convert plasminogen to plasmin, which is then able to degrade clot-bound firbin
        
        therapeutic action:
        
        Lysis of thrombi in coronary arteries, with prevention of ventricular function
        
        Lysis of pulmonary emboli and subsequent restoration of blood flow
        
        Restoration of cannula patency and function
        
        MO: Streptooccus equisimillis, Streptococcus mutans, Streptococcus faecalis, Streptococcus uberis
        
        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
  - - - First discovery of killing Staphylococcus culture
        
        Inhibits synthesis of peptidoglycan in cell walls
        
        Penicillin produced by fungus Penicillin sp.
        
        Production by fed-batch fermentation process using bioreactor of 30k-250k capacity
        
        Requires lactose, other sugars and a source of nitrogen
        
        Batch fermenter normally used to prolong the stationary period and increase production
        
        Strain improvement process:
        
        Penicillum nonatum - low yield, poor response to submerged cultures
        
        Penicillum chrysogenum - strains isolated form moldy fruit, give high % yield
        
        Strain are treated with UV, X-rays, nitrogen mustard to induce mutagenesis
        
        Repeated treatments to get improved strains of the species - higher yield
        
        Older strains - 200 units/mL :arrow_right: Newer strains - 1000 units/mL
        
        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
      - Semi-synthetic penicillin
        
        B-lactam ring makes penicillin molecule susceptible to metabolism
        
        Not orally active & must be injected
        
        The present drug development focuses on:
        
        To increase chemical stability for oral administration
        
        To increase resistance to B-lactamses
        
        To increase the range of activity
        
        Analogues of penicillin are produced by varying the carboxylic acid
        
        These analogues have increased stability & effective against wide range of pathogens
        
        They are broad-spectrum antibiotics & can be taken oraly
        
        Natural penicillin is treated to yield 6-APA which modified chemically by the addition of side chain
    - - Broad-spectrum, Aminoglycoside antibiotics
        
        Antimycobacterium
        
        Active against Gram+ve - Eg. Staphyloccocus aureus
        
        Ysed against the pathogenic bacteria resistant to penicillin
        
        Active against plague, brucellosis, tularemia diseases
        
        Specifically active against Gram-ve Mycobacterium tuberculi & Mycobacterium leprae
        
        Discovered from Streptomyces griseus isolated from soil
        
        A protein synthesis inhibitor
        
        Streptomycin & dihydrosreptomycin are the basic compounds
        
        Prepared as salts
        
        Available as crystalline hydrochloride, phosphate, sulfate
        
        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