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Development of industrial fermentation processesMoney makingCompetitionEconomically feasible on large scale basisRecovery of product ready for open marketCompetitive advantageCriteria for being important in choice of organismNutritional characteristics of the organism when grown on a cheap medium

Optimum temp of the organism

Reaction of the organism with the equipment and suitability for the type of process

Stability of the organism and its amenability for genetic manipulation

Productivity of the organism i.e. ability to convert substrate into product per unit time

Ease of product recovery from the culture

What are the R&D approaches for finding of a MO of economic value, and large scale fermentation process?Micro-organism

Source Environment (soil)Stock culture collectionsScreening Primary screeningSecondary screeningPrimary screeningHighly selective procedures for detection and isolation of MO of interest

Few steps will allow elimination of valueless MO

Eg. Crowded plate technique for Ab screening, serial dilution, acid base indicator dyes, CaCO3, sole source carbon or nitrogen, enrichment tech

Does not give too much information on detail ability of the micro-organisms

May yield only a few organisms and few of them may have commercial valueCommon techniquesDirect wipe or sponge of the soilSoil dilution (10-1 to 10-10)Gradient plate method (streak, pour)Aerosol dilutionFlotationCentrifugation I.II.Enrichment, screening for metabolites or microbial products III. Unusual environmentsSecondary screeningSorting of MO that have real commercial value for industrial processes and discarding those which lack potential

Conducted on agar plates (not sensitive), small flasks or small fermentors (more sensitive) containing liquid media or combination of these approaches.

Liquid culture provide better info on nutritional, physical and production responses.

Can be qualitative or quantitativePreservation of Industrially important MOViable and Free from contamination

Stored in such a way so as to eliminate genetic change and retain viability

Viable by repeated sub-culture (avoid mutations by keeping stocks and strain degeneration and contaminations)

Preservation of Industrially important MOStorage at reduced temperatureAgar slopes at 50C or in -200C freezer: viable for 6 months

Liquid nitrogen (-1960C): problems of refilling, advantagesStorage at dehydrated formDried cultures

LyophillizationQuality control of preserved stock: batch system, single colony, typical pattern, large number, purity, viability and productivityIf sample fails entire batch is destroyedMICROBIAL METABOLIC PRODUCTS OR METABOLITESWide range of products having commercial valueAlgae SCP

Bacteriaacetic acidbactracingramicidinendotoxinglutamic acidvitamin B12Actinomycetesantibiotics (tetracycline, streptomycin, neomycin, rifamycin, gentamycin)

Fungicitric acid, amylase, cellulase, SCP, lipase, pencillin, ethanol, wine, steroids, gibberllinSUBSTRATEPrimary metabolitesSecondary metabolitesBioconversionsEssential metabolitesAmino acidsNucleosidesvitaminsMetabolic end products

Ethanol, acetone, lactic acid, butanolAntibiotics

Alkaloids

Gibberlins

Pigments Steroids

Amino acids

Ascorbic acidTYPES OF LOW MOLECULAR WEIGHT COMPOUNDS BY MOTrophophase Idiophase Limiting nutrientSecondary metaboliteCell MassPrimary metabolismSecondary metabolismTime Concentration PRIMARY METABOLITESFormed in trophophase (log phase)

Balanced growth of MOOccurs when all nutrients are provided in the mediumIts is essential for survival and existence of the organism and reproductionCells have optimum concentration of all macromolecules (proteins, DNA, RNA etc.)

Exponential growth

PRIMARY METABOLITESPrimary essential metabolites:

Produced in adequate amount to sustain cell growthVitamins, amino acids, nucleosidesThese are not overproduced, wastefulOverproduction can be genetically manipulated

Primary essential end products:Normal end products of fermentation process of primary metabolismNot have a significant function in MO but have industrial applicationsEthanol, acetone, lactic acid, CO2LIMITATIONS:growth rate slows down due to limited supply of any other nutrient. Metabolism does not stop but product formation stops.OVERPRODUCTION OF PRIMARY METABOLITESManipulation of feedback inhibitionAuxotrophic mutants having a block in steps of a biosynthetic pathway for the formation of primary metabolite (intermediate not final end prod).End product formation is blocked and no feedback inhibition

Mutant MO with defective metabolite productionA ---- > B ----> C -----> D ------> EFinal end prodRequired metaboliteStartingsubstrateintermediateBlocked reactionUnbranched pathwaySECONDARY METABOLITESCharacterized by secondary metabolism and secondary metabolites (idolites)

Produced in abundance, industrially importantCharacteristics:

Specifically producedNon essential for growthInfluenced by environmental factorsSome produce a group of compds eg a strain of Streptomyces produced 35 anthracyclinesBiosynthetic pathways are not establishedRegulation of formation is more complex

Functions:May or may not contribute for existence or survival of the MOidiophaseOVERPRODUCTION OF SECONDARY METABOLITESMore complexSeveral genes are involved eg may be 300 to 2000 genesRegulatory systems are more complexSome regulatory mechanisms

Induction: eg tryptophan for ergot production etc

End product regulation: some metabolite inhibit their own biosysnthesis

Catabolite regulation: key enzyme inactivated, inhibited or repressedeg. Glucose can inhibit several antibioticsammonia as inhibitor for antibiotic prod.Phosphate regulation: Pi for growth and multiplication in pro and eukaryotes. Increase in pi conc can increase secondary metabolites but excess harmfulAutoregulation: self regulation mechanism for production like hormonesBIOCONVERSIONS OR BIOTRANSFORMATIONSUsed for chemical transformation of unusual substrates for desired prods

Conversion of ethanol to acetic acid, sorbitol to sorbose, synthesis of steroid hormones and certain amino acids

Structurally related compounds in one or few enzymatic reactions

Can use resting cells, spores or even killed cells.

Mixed cultures can also be used, use of immobilized cells at low cost?BIOCONVERSIONS OR BIOTRANSFORMATIONS (BTs)When and why is biotransformation done? when production of a particular compound is difficult or costly by chemical methods

BTs are preferred over chemical reactions due to substrate specificity, stereospecificity, mixed reaction conditions (pH, temp, pressure)Environmental pollution is negligibleEasy to apply recombinant DNA technology

Easy to scale up the processes sue to limited number of reactionsTYPES OF HIGH MOLECULAR WEIGHT COMPOUNDS BY MOPolysaccharides, proteins (enzymes)

Pharmaceutical productsEnzymes naturally occurring biocatalysts; accelerate metabolic reactions

Production of primary and secondary metabolites are not possible without enzymes

Enzymes during fermentation are EXTRACELLULAR (amylase, cellulase, lipase, b-galactosidase, esterase, protease, chitinase, xylanase, glucose isomerase) and some are INTRACELLULAR (invertase, asparginase)

Extremozymes

Immobilized enzymesMicrobial BiomassMicrobes can themselves be products or main source of biomass

Microbial biomass is exploited as microbial protein or single cell protein (SCP)

METABOLIC PATHWAYS IN MICRO-ORGANISMSPROVIDES PRECURSORS FOR THE CELL COMPONENTS

ENERGY FOR ENERGY REQUIRING PROCESSESUnique feature of heterotrophic MOSecrete extracellular enzymesCatabolismAmphibolism (Intermediate metabolism requiring central metabolic pathways)AnabolismFunction of enzymes: substrate specificity, catalysisCoenzymes and prosthetic groupMethods of ATP generation: SLP, OP (respy), OP (photosyn)Uptake of substrates (diffusion, FD, AT, Gp Trans, siderophoresDegradation of carbon and energy sources (sugar breakdown)METABOLIC PATHWAYS IN MICRO-ORGANISMSThe ways in which microorganisms degrade sugars to pyruvate and similar intermediates are introduced by focusing on only three routes:

Glycolysis (Embden Meyerhof Pathway)

The pentose phosphate pathway,

(3) The Entner-Doudoroff pathwaySugars to PyruvateCarbon and energy source breakdown(1) Glycolysis: glucose to pyruvate

6-carbon phaseoxidation phase energy harvest phaseHexokinasephosphofructokinaseFructose biphosphate aldolaseGlucose Pyruvic acidGlucose 6 PhosphatePentose phosphate pathwayKDPGPathwayOr EntnerDourdoffPathwayCentre of Intermediate metabolismAcetyl CoAPrecursor for NumerousBiosynthetic pathwaysGlucose

Glucose-6-P

6 Phosphogluconolactone 6-phosphogluconate instead of Fructose 6-P

2-keto-3-deoxy-6-phosphogluconate (KDPG)

Pyruvateglyceraldehyde-3-P

ATP2ATPNADPH(2) Entner-Doudoroff pathway or KDPG pathway Pyruvate

Only in prokaryotes, many gram negative bacteria some G+veOperates when glycolytic enzymes like phosphofructokinase-1 are lacking1 net ATP is produced1 NADPH and 1 NADH is also produced1 NADH6 phosphogluconate dehydrase2-keto-3-deoxyphosphogluconate aldolaseGlucose ----> 2 pyruvic acid + ATP +NAD(P)H2 + NADH2Embden-Meyerhof pathway(3) Pentose Phosphate pathway (PPP) or HMPHeterofermenter lactobacilli

Bacteria which lack aldolase for conversion to triose phosphate

PPP takes place

Reducing equivalentsDehydrogenationhydrolysisGlucose 6 phosphateOxidative catabolism of glucose*

To glycolysis****

Microbial fermentation pathways

LAFBuDFMxAFacetaldehydeEthanolNADHCO2AFBuAcidFBuAFBuAcetoneFPropAF

METABOLIC PATHWAYS IN MICRO-ORGANISMS

2C6C6C5C4C4C4C4C4C3CCO2NADHCO2NADHCO2Precursors for biosynthesisNADHGTPMacromolecular constituentsDNA, RNAProteinsPeptidoglycansPolysaccharides (glycogen, starch, PHB)LMW constituentsPurinesPyrimidinesLipidsPhospholipidsAmino acidsGlycolysisPentose phosphate pathwayEntner Douordoff pathwayKrebs cycleGlycolysisPentose phosphate pathwayEntner Douordoff pathwayKrebs cycleGlucose

G-6-P

pyruvateOxaloacetatea keto glutaratePPPRibose 5PErythrose 4PHistidineTryptophan

Tyrosine

Phenylalanine AROMATIC AASerine Gly

CysAlaLeu

ValSERINE FAMILYPYRUVATE FAMILYATPLys

DAPAspAsn Homoserine - MetThr ----- IleASPARTATE FAMILYGlu --- Gln ProOri -- citruline -- ArgGLUTAMATE FAMILYAA syn