Media for fermentation Technology - WELCOME aboard UG3!.pptx

mediaforindustrialfermentation,lecture6tahir,detailedinvestigationisneededtoestablishthemostsuitablemediumforanindustrialfermentationprocess,butcertainbasicrequirementsmustbemetbyanysuchmedium.allmicroorganismrequireswater,sourceofenergy,c,n,mineral,vitamins,oxygen(aerobic).onasmallscaleitseasytomaintainbutonlargescaleverydifficulttomaintainthesatisfactorygrowth.,onalargescaleonemustnormallyusesourcesofnutrientstocreateamediumwhichwillmeetasmanyaspossibleofthefollowingcriteria:,theuseofcanmolasses,beetmolasses,cerealgrains,starch,glucose,sucrose&lactoseascarbonsource&nh4salts,nitrates,cornsteepliquor,soyabeanmeal,slaughterhousewasteandfermentationresiduesasnsourcemeettheabovecriteriaforproductionmediab/ctheyarecheapmaterial.usesimplerprocedures,selectsuitablesporulationandinoculationmedia.mediumselectedwillaffectthedesignoffermentar.eg.icidevelopspecialfermentarfortheproductionofnh3&ch3oh.microbialoxidationofhydrocarbonsisahighlyaerobicandexothermicprocessthusproductionfermentarshouldhaveaveyhighotransfercapacitywithexcellentcoolingfacilities.airliftfermentardesignbyicisolvetheproblem.,theproblemofdevelopingaprocessfromthelaboratorytothepilotscale&subsequentlytotheindustrialscalemustalsobeconsidered.labmediummaynotbeidealinalargefermenterwithalowgastransferpattern.amediumwithhighviscositywillalsoneedahigherpowerinputforeffectivestirring.phvariation,foamformation,oxidationreductionpotential&themorphologicalformoftheorganism,metabolicinhibitorsthesearealsoveryimportant.mediumwillalsoaffectsproductrecovery&effluenttreatment.,historicallyundefinedcomplexnaturalmaterialsusedalthoughtheyarecheaperbutunpredictablebiomass/yieldproblems.productrecoveryverydifficult,residualcomponentsmyinterferewiththerecoveryandcontributetothebodoftheeffluent.althoughdefinedmediaismoreexpensivebutitgivemorepredictableyieldthenundefinedmedia.controlofphandfoamduringgrowthinafermenterwereindentifiedastwoimportantparameters.molasseswouldnormallybeusedasthecheapestcarbohydratetogrowyeastbiomassinalargescaleprocess.butthisisnotacceptableforrecombinantproteinproductionb/cofdifficultiesandcostforpurification.colinsuseddefinedmediumwithglucose,sucrose,animalsalts,traceelements,purevitamins&nh4asnsource&forphcontrol,alsousedantifoamingagent.,mediumformulation,mediumformulationisanessentialstageinthedesignofsuccessfullaboratoryexperiments,pilotscaledevelopmentandmanufacturingprocess.theconstituentofthemediummustsatisfytheelementalrequirementsforcellbiomass&metaboliteproductionandtheremustbeanadequatesupplyofenergyforbiosynthesis.c+n+o2+otherreq-biomass+product+co2+h2o+heatthisequationshouldbeexpressedinquantitativeterms,whichisimportantintheeconomicaldesignofmediaifcomponentwastageistobeminimal.thus,tocalculatetheminimalquantitiesofnutrientswhichwillbeneededtoproduceaspecificamountofbiomass,itshouldcalculatesubstrateconcentrationnecessarytoproducerequiredproductyields.,coonye1981somenutrientarefrequentlyaddedinsubstantialexcessofthatrequiredegp,k,sometraceeg.zn,cu.concofpisdeliberatelyraisedinmanymediatoincreasethebufferingcapacity.itdependonthemediumdesign.somemicroorganismcantsynthesizespecificnutrientsegaa,vitaminsornucleotides.onceaspecificgrowthfactorhasbeenidentifieditcanbeincorporatedintoamedium.chasadualroleinbiosynthesis&energygeneration.crequirementforbiomassproductionunderaerobicconditionmaybeestimatedthecellularyieldywhichisdefinedasquantityofcelldrymatterproduced/quantityofcsubstrateutilized.,oxygenprovidedbyaeratingtheculture,thedesignofmediumwillinfluencetheoxygendemandofacultureinthatthemorereducedcsourceswillresultinahighero2demand.water:requiredinheating,cooling,cleaning&rinsingitrequiredinlargequantities,ph,dissolvedsaltsandeffluentcontaminationmineralrequirementofwaterisveryimportantinbrewing&mostcriticalinthemashingprocess.hardwatercontainingcaso4concentrationarebetterforthe“englishburtonbitterbeer”whilewaterwithhighco3contentsarebetterforthedarkerbeersi.estouts.nowadays,watermaybetreatedbydeionization,saltaddedorphadjusted,dependuponthetypeofbreweries.reuseorefficientuseofwaterisnormallyofhighpriority.scpproduced60,000tonesbyici,usedhugeamountofwateronsingleusebases,(30gbiomass(dw)dm-3wouldrequire2700 x106dm3ofwaterperannum.eg.methylophilusmethlotrophuscouldgrownsuccessfullywith86%continuousrecyclingofsupernatantwithadditiontomakeupdepletednutrients.itresultinreducecapitalcostandoperatingcostby50%.,energysources,energycomeseitherfromoxidationofmediumcomponentsorfromlight.mostindustrialmicroorganismarechemo-organotrophs,thereforethecommonestsourceofenergywillbethecarbon(cho),lipids&protein.somemicro-organismscanalsousehydrocarbonsormethanolascarbon&energysources.,carbonsources,factorinfluencingthechoiceofcarbonsource:rateatwhichthecsourceismetabolizedcanofteninfluencetheformationofbiomassorproductionofprimaryorsecondarymetabolites.fastgrowthduetohighconcentrationsofrapidlymetabolizedsugarsisoftenassociatedwithlowproductivityofsecondarymetabolites.,someuselessreadilymetabolizedsugari.elactosebutmanyprocessneedcontinuousorsemicontinuoussupplyofsugar.mainproductoffermentationprocessdependonthechoiceofcsource.eg.ethanol,orscp-60-77%oftheproductioncostdependoncostofcsource.itisthepartofcompanydevelopmentprogramtodeterminethealternativecsourcetodeterminetheyield.thepurityofthecsourcemayalsoaffectthechoiceofsubstance.themethodofmediapreparation,particularlysterilizationmayaffectthesuitabilityofchoforindividualfermentationprocess.itsoftenbesttosterilizesugarseparatelyb/ctheymayreactwithnh4ions&aatofromblackncontainingcompoundswhichwillinhibitthegrowthofmanymicroorganism.starchwhensterilizeitbecomegelatinizesgiverisetoveryviscousliquid.thechoiceofsubstratemayalsobeinfluencedbygovtrules,eg.beetsugarandmolassesisencouraged.scotchmaltwhiskymaybemadeonlyfrombarleymalt,waterandyeast.,carbohydrates,itscommonpracticetousechoinmicrobialfermentatione.gstarchfrommaizgrainitmayalsoobtainedfrom,otherscereals,potatoes&cassava.hydrolyzedcassavastarchisusedasamajorcsourceforglutamicacidproduction.syrupsproducedbyacidhydrolysismayalsocontaintoxicproductswhichmaymakethemunsuitableforparticularprocesses.sucroseisobtainedfromsugarcane&sugarbeet.itiscommonlyusedinfermentationmediainaveryimpureformasbeetorcanemolasseswhicharetheresiduesleftaftercrystallizationofsugarsolninsugarrefining.molassesisusedintheproductionofhighvolume/lowvalueproductssuchasethanol,scp,organicandaaandsomemicrobialgums.,molassesorsucrosealsomaybeusedforproductionofhighervalue/lowbulkproductssuchasantibiotics,speciallyenzymes,vaccines&finechemicals.however,molassesbasedfermentationwilloftenneedamoreexpensive&complicatedextraction/purificationproceduretoremoveimpurities&effluentswhichmaketheprocedurecostly.cornsteepliquorisabyproductafterstarchextractionfrommaize.althoughprimarilyusedasansource,itdoescontainlacticacid,smallamountsofreducingsugars&complexpolysaccharides.(table4.7)insoyabean&pharmadedia,alongwithn,italsocontainsmallbutsignificantamountofcho.,oilsandfats,oilswerefirstusedascarriersforantifoamsinantibioticsprocesses.vegetablesoilsmayalsobeusedascsubstrates,particularlyfortheircontentofthefattyacid,oleic,linoleciandlinolenicacidb/cofcostarecompetitivewiththoseofcho.bader1984factorsfavouringtheuseofoilsinsteadofcho.atypicaloilcontainsapproximately2.4timestheenergyofglucoseonaperweightbasis.oilsalsohaveavolumeadvantageasitwouldtake1.24dm3ofsoyabeanoiltoadd10kcalenergytoafermenterwhereasitwouldtake5dm3ofglucoseorsucroseassumingthattheyarebeingadded50%w/wsolutions.ideallyinanyfermenter,maxworkingcapacityshouldbeused.oilbasedfed-batchfermenterpermitthisproceduretooperatemoresuccessfully.oilalsohasantifoampropertieswhichmaymakedownstreamprocessingsimpler.glyceroltrioleatisknowntobeusedinsomefermentationwheresubstratepurityisanimportantconsideration.methlyoleatehasbeenusedasthesolecsubstrateincephalosporinproduction,nitrogensources:mostindustriallyusedmicroorganismcanutilizeinorganicororganicsourcesofnitrogen.inorganicnmaybesuppliedasnh3gas,nh4saltsornitrates.nh3hasbeenusedforphcontrol&asthemajornsourceinadefinedmediumforthecommercialproductionofhumanserumalbuminbyyeast.nh4saltsi.e(nh4)2so4willproduceacidconditions&nh4no3normallycauseanalkalinedrift.whenthenh4ionhasbeenexhausted,thereisanalkalinedriftasthenitrateisusedasnalternativensource.inthepresenceofnitratetheassimilationofnh3isinhibitedatph2.8-3.0.nitrateassimilationcontinueuntilthephhasincreasedenoughtoallowthenh3assimilationmechanismtorestart.organicnmaybesuppliedasaa,proteinorurea.otherproteinaceousncompoundsservingassourcesofaaincludecorn-steepliquor,soyasmeal,peanutmeal,cottonseed.instoragetheseproductsmaybeaffectedbymoisture,temp,&ageing.,factorsinfluencingthechoiceofnsource,controlmechanismexistbywhichno3reductaseanenzymeinvolvedintheconversionofno3tonh4ion,isrepressedinthepresenceofnh3.inaspergilusnidulansnh3alsoregulatestheproductionofalkaline&neutralproteases.inantibioticproductionbymanymicroorganismisinfluencedbythetypeandconcentrationofthenintheculturemedium.antibioticproductionmaybeinhibitedbyarapidlyutilizednsource(nh4,no3).antibioticproductiononlybeginstoincreaseintheculturebrothaftermostofthenconsumed.e.gintheproductionofpolyeneantibiotic,soybeanhasbeenconsideredagoodnsourceb/cofthebalanceofthenutrients,lowpcontent&slowhydrolysis.thisgradualbreakdownpreventtheaccumulationofnh4ions&repressiveaa.somecomplexnmaterialcantbeutilizedbyamicro-organism&createproblemsindownstreamprocessing&effluenttreatment.thiscanbeanimportantfactorinthefinalchoiceofsubstrate.,minerals,allmicro-organismrequirescertainmineralelementsforgrowth&metabolism.inmanymedia,mg,p,k,s,ca&clareessentialcomponents,co,cu,zn,fe,mn,mb&znarealsoessentialpisusedasabuffertominimizephchangeswhenexternalphisnotbeingused.,inspecificprocessestheconcofcertainmineralsmaybeverycritical.garner1950casaltsisusedtoppttheexcessinorganicpo4soindirectlyimprovetheyieldofstreptomycin.italsoinfluencetheproductionofbactricin,citricacid,ergot,monomycin,novobiocin,oxytetracycline,polyene,ristomycin,rifamycin,streptomycin,vacomycin&viomycin,liras1990enzymeswererepressedbypo4,apo4controlsequencehasalsobeenisolated&characterizedfromthepo4regulatedpromoterthatcontrolbiosynthesisofcandicidin.wienberg1970ninetraceelementprovedtobecritical,concofmn&znarethemostcriticalinsecondarymetabolism.cldoesnotappeartoplayanutritionalruleinthemetabolismoffungibuthoweverrequiredbysomeofthehalophilicbacteria.eg.chlortetracycline&griseofulvin.otherclcontainingmetabolitesarecaldriomycin,nornidulin&mollisin.,chelators,manymediacantbepreparedorautoclavedwithouttheformationofavisiblepptofinsolublemetalpo4.theproblemofinsolublemetalpo4maybeeliminatedbyincorporatinglowconcofchelatingagentsi.eedta,citricacid,polyphosphateetcthesechelatingagentsformcomplexeswiththemetalionsinamedium.themetalionsthenmybegraduallyutilizedbythemicro-organism.chelatingagentdoesnotcauseinhibitionofgrowth.,growthfactors,vitamins,specificaa,fattyacidsorsterols.manyofthenaturalc&nsourcesusedinmediaformulationcontainallorsomeoftherequiredgrowthfactors.ifonlyonevitaminsisrequireditmaybemoveeconomicaltoaddapurevitamininsteadoflargebulkofcheapermultiplevitaminssource.e.gcapantothenatehasbeenusedinonemediumformulationforvinegarproduction.inglutamicacid,limitedconcofbiotinmustbepresentinthemedium,somerequiresthiamin.,buffers,thecontrolofphmaybeextremelyimportantifoptimalproductivityistobeachieved.manymediaarebufferedataboutph7.0bytheincorporationofcaco3,ifphdecreasestheco3isdecomposed.po4whicharethepartofmanymediaalsoplayanimportantroleinbuffering.highpo4concarecriticalintheproductionofmanysecondarymetabolites.c&nsourceswillalsoabasisforphcontrolasbufferingcapacitycanbeprovidedbytheprotein,peptides&aasuchasincornsteepliquor.thephmayalsobecontrolledexternallybyadditionofnh3ornaoh&h2so4.,theadditionofprecursors&metabolicregulatorstomedia,somecomponentsofafermentationmediumhelptoregulatetheproductionoftheproductratherthansupportthegrowthofthemicro-organism.suchadditiveinclude,precursors,inhibitors,precursors:somechemicalswhenaddedtocertainfermentationaredirectlyincorporatedintothedesiredproduct.e.gpinicillinyieldsarangeofdiffsidechaincanbeincorporatedintothepenicillinmoleucle,egcornsteepliquorincreasedtheyieldofpinicillinform20unitsto100units.cornsteepliquorcontainphenylethlaminewhenincorporated,ityieldbenzylpenicillin.phenylaceticacidiswidelyusedprecursorinpenicillinproduction.,inhibitors,whencertaininhibitorsareaddedtofermentationsmoreofspecificproductmaybeproduced.glycerolproductiondependsonmodifyingtheethanolfermentationbyremovingacetaldehyde.inhibitorshavealsobeenusedtoaffectcellwallstructureandincreasethepermeabilityforreleaseofmetabolites.thebestexampleistheuseofpenicillin&surfactantsinglutamicacidproduction.,inducers,themajorityofenzymeswhichareindustrialinterestareinducible.inducedenzymesaresynthesizedonlyinresponsetothepresenceintheenvironmentofaninducer.inducersareoftensubstratesuchasstarchordextrinsforamylase,maltoseforpullulanase&pectinforpectinase.mostinducerswhichareincludedinmicrobialenzymemediaaresubstrateorsubstrateanaloguesbutintermediatesandproductsmaysometimebeusedasinducers.e.gmaltodextrinswillinudceamylase&fattyacidsinducelipase.(usedependuponthecost)itisnotpossibletoproduceanoofheterologousprotein(hlp)inyeasts,fungi&bacteria.buthlpmayshowsomedegreeoftoxicitytothehost,itmayrestrictthegrowthandbiomass.onecommercialsystembasedonthealcapromotersinaspergillusnidul-anstoexpresshumaninterferonalpha2.thiscanbyinducedbyvolatilechemicalsi.eethylmethylketoneaddedwhenbiomassincreasetoanadequatelevel.,oxygenrequirement,veryimportantincontrollinggrowthrate&metabolicproduction.mediummayinfluence:fastmetabolism:culturemaybecomeoxygenlimitedb/csufficientoxygencantbemadeavailableinthefermeterifcertainsubstratesuchasrapidlymetabolizedsugarswhichleadtoahighoxygendemandareavailableinhighconc.rheology:theindividualcomponentsofthemediumcaninfluencetheviscosityofthefinalmedium&itssubsequentbehaviorwithrespecttoaeration&agitation.antifoam:manyoftheantifoamsinusewillactassurfaceactiveagents&reducetheoxygentrasnferrate.,fastmetabolismnutritionalfactorscanaltertheodemandoftheculture.penicilliumchrysogenumwillutilizeglucosemorerapidlythanlactoseorsucrose,&itthereforehasahigherspecificouptakerat