Separation And Purification Techniques In BiotechnologyDechow生物化学生物技术教案 1

Separation And Purification Techniques In Biotechnology Dechow生物化学生物技术教案 1 (1)as thelast hurdleto beovere inbringing biotechnologyfrom thelaboratory tomercial status.This textwill attemptto describehow adsorptivematerials,ion exchangeresins,column chromatographyand affinitychromatography can be utilizedin theserecovery andpurification operations.This chapterwill examihe natureof the fermentation broth and willserve toput theserecovery operationsin perspectivewith otherpurification techniquesnot coveredby thistext.It isessential tounderstand therelative advantagesof eachand theirinterrelationships sincemost purificationswill requirebinations ofdifferent techniques.1.l FERMENTATIONBROTH Thefermentation broth is thebinationofinsoluble,gelatinous biomass,the nutrientfluid,and thesoluble metabolitesresulting from the fermentationoperation.When the fermentation iscarried outwithout anyform ofinert supportfor the biomass,the limitof fluidityfor stirringor aerationis approximately3to7%WV dryweight ofbiomass.Physically,biomass isa pressible,gelatinous solidwith surfacelayers ofpolysaharide materialwhich makeit cohereand adhere.Downstream processing,therefore,has todeal with a viscous,highly non-Newtonian slurryas itsfeedstock.12Separation andPurification Techniquesin BiotechnologyFor example,a bacterialfermentation forsingle cellprotein willproduce abroth of3%WV in which theslurry isabout60%(by volume)wet biomassand40%interparticle fluid.When biomasssupports areused,somewhat higheroperating biomassconcentrations arepossible and,in wastedisposal fermentations,the concentrationhasbeenraised from2to5g/liter to10to40g/liter.Compared with the feedstreams torecovery processesin conventionalchemical processing,fermentation broths are verydilute aqueoussystems(see Table1.1).Therefore,it will be particularlyimportant to avoid energyintensive thermaloperations and to selectprocesseswhichgive largeconcentration increasesin thefirst stageor stages.Table1.1:Typical ProductConcentrations LeavingFermenters ProductGrams perLiter Antibiotics(e.g.,Penicillin G)10-30Enzyme protein(serum protease)2-5Ethanol70-120Lipids10-30Organic acids(citric,lactic)40-100Riboflavin10-15Vitamin B120.02The fluidvolume inmicrobiological processesmust bereduced byat leastan orderof magnitudebetween the broth and the finalfluid stagesof the recovery processesand,in somecases,by verymuch more.For vitaminB-12the reductionratio isover1OOO:l.Consequently,the plantdesign and the range of aeptableunit operationschanges significantlyas the fluid progressesfrom the broth handlingstages to the finalisolation stage.Many fermentation brothsareunstable.Once anybroth leavesthe controlled,aseptic environmentof thefermenter,it isexposed toa drastihange ofconditions.An activelygrowing biomassfrom anaerated culturewillbesuddenly deprivedof oxygenand willexperience arapidly fallingconcentration ofnutrients.This frequentlyproduces rapidchanges inphysical properties,leading todestruction ofdesired product.Lipids may be consumedas analternative energysource forcontinuing metabolicactivity.Enzymes maybe destroyedby proteasesreleased from the deterioratingcells.The brothalso beessusceptible toIntroduction3contamination fromforeign organismswhich canhave the same effects.Similar problemscan ourif therecovery operationsof abatch fermentationare delayed.The problemscan bereduced bychilling toaround5OC.This ismonly donefor enzymes and otherrelatively smalloutput processes.However,this isto beavoided,if possible,with largerfluid volumessince chillingfrom a typical fermentationtemperature of35OC to5OC requiresrefrigeration energyof about40kWh/m3of brothand considerablecapital expenditure.The timefor appreciableproduct lossto ourcan beas littleas20minutes atfermentation temperatures.The necessaryand practicalrecovery operationsemployed andthe orderin which they areused indownstream processingcan bereduced todeceptively simplelooking recoverysequences.Figure1.1 (2)shows aschematic of therecoverysequence andthe techniquesassociated witheach process.Producl inCIIII.Product inbroth or.queous phaseSedimentation Filtrationfrom wholecdls solvsnf/dlrrgent rend&-q membranes,rtc,“leaky!Solvent extractionIon exchanpeAdsorption Gelfilfrstion Affinitymethod9Distillation MembranesElectrophoresir Differentialfreezing EvaporationMembrsner PmcipitalionAdsorption.ion exchange,clf%ily.erc Frocra.ThDwing-cryslalliralion Figure1.1.Schematic of the processeswhichmaybe involvedin theseparation andfractionation offermentation products(Reference2).4Separation andPurification Techniquesin BiotechnologyIf the desired product is extracellular,it isonly necessary to filterthe biomassfrom thebrothand isolate the product fromthe fluid.If aproduct isintracellular,a celldisruption stepmust firstbe employed.If the productisalso watersoluble,this disruptionshould beperformed whilethe biomass is stillin aslurry form.The chemicalstability andsolubility of theproductwill dictatethe mostsuitable recovery techniques.Most microbiologicalproducts havelimited chemicalstability.This putssevere restrictionson thetemperatures,the reactants,and pHlevels which can be used.An importantconsideration indetermining theappropriateness ofa recoverytechnique is the actualpurity requirementfor theproduct.Many products,such asenzymesandvaines,will notneed to be isolatedas purepounds.An appropriateproduct inthese caseswould bea plexmixture havingthedesiredproperties.However,removal ofspecific materials,such aspyrogens ininjectable medicinals,would benecessary.The majorrecovery difficultiesarise whenit isnecessaryto separate specifiompounds fromother chemicallyand physicallysimilar materials.The isolation of enhancedpurity enzymeprotein fromother proteinrequires highlyspecific physico-chemical effectsto be used.Today it is theexception ratherthan thenorm for the separatedpounds tobe thatsimilar.Normally thereisarangeofalternative recoverytechniquesand a selectioncan bemade on the basisof cost,familiarity,and reliabilitycriteria.1.2RECOVERY UNITOPERATIONS Thevarious methodsfor treatingthefermentation broth can be dividedinto mechanicalor chemicalunit operations.Table1.2lists theprocesses normallyincluded ineach category.Filtration andcentrifugation areunit operationsin which the suspended solids areseparated fromthe fluidphase.Drying is theremoval of moisture or solvent from solid particles,while evaporationis theremoval ofmoistureorsolventfroma solution.In crystallization,the conditionsofasolution areadjusted tochange thesolubility ofone ofIntroduction5Table1.2:Separation andRecovery TechniquesMechanical FiltrationCentrifugation EvaporationCrystallization DryingReverse osmosisUltrafiltration ChemicalAdsorption Ion exchange Column chromatography Affinitychromatography Solventextraction ElectrophoresisElectrodialysis thedissolved poundsso thatit leavesthe solutionasa solid.Microfiltration,ultrafiltration,and reverse osmosis aremembrane processesinwhichseparation isbased ondifferences inability toflow through a thinbarrier that separates twofluids.Microfiltration isa hydraulicallydriven processusing amembrane witha poresize inthe100to3000A range.For ultrafiltration,the poresize isfrom10to125A,while forreverseosmosis,the poresize isfrom3to loA.Adsorption,ion exchange,column chromatography,and affinitychromatography can be groupedas recoverytechniquesinwhichthe removed poundor soluteestablishes an equilibrium betweensites ona solidphase materialandthesolution.In adsorption,the removedspecies isbonded to the solidphase materialby polarityor weakchemical bonds.Ionexchangerecovers materialbytheinterchange ofions betweenthe liquidand solidphases.Columnchromatographymay useadsorptive,ion exchangeor molecularsieve materialstoseparatesolutes whichare firstloaded ontoa columnof theseparation materialand theneluted insuch amanner thatthe individualsolutes arecollected inseparate fractions.In affinitychromatography,the removedspecies isbound witha highlevel ofselectivity toligands covalentlyattachedtoasolidmatrix.In solventextraction,theremovedpound establishesanequilibriumdistribution betweenimmiscible solvents,usually waterand anorganic liquid.Electrophoresis andelectrodialysis areseparation techniquesthatseparatecharged moleculesor ionsusing an6Separation andPurification Techniquesin Biotechnologyelectric field.Electrophoresis separatescharged ponentsby aentuatingsmall differencesin ionicmobility inan electricfield usinga movingcarrier fluid.Electrodialysis concentratesponents onthe basisof electromigrationthrough ionicmembranes.1.2.1Mechanical OperationsFiltration:Filtration istypically thefirst stepintheisolationof any productfromthefermentation broth.This processseparates the biomass cells,the celldebris,and anyprecipitates fromthebrothfluid.The mathematicalrepresentation of the incrementaltime dtto filteran additionalincremental volumedV aftera volumeV hasbeen filteredis given by:dt/dV=atbV(1.1)The rightside of the equationcontains twoponents,a andb.The aterm isr)r,/AP andthebterm,which dependson V,is equaltorr,/A2PW,where nis the liquid viscosity,rs is the specifiake resistance of the filter material,A isthe filterarea,P isthe constantapplied pressuredifference,rC isthe specifiake resistance,and Wisthe cake dryweight perunit volume of filtrate.Normally the resistanceofthe filtermaterial termincludes the resistance contributionof anyfiltration aid,pipes,and valves.Aording tothis equation,theresistanceto filtrationis dueinitially toonly theconstant terma.In theory,as filtrationproceeds andthebiomass cake beesthicker,theresistancewould beexpected toincrease linearlyaording tothebterm withthis dependenceon V.Although manypractical considerationsmust betaken intoaount whenapplying thisequation,its simplicity,the plexityofamore exactdescription,andtheuniqueness ofmany industrialapplications resultin Equation1.l beingthe mostuseful filtrationrepresentation.The specificpractical limitationsthat mustbe consideredare theblockage orblinding ofthe filter,the pressibilityofthebiomasscake,andthevariable porestructure ofthe cake.Blinding ofthe filtermaybeprevented bystarting the filtration ata lowhydraulic pressureby partiallyby-passing thepump.This willavoid Introduction7driving thefirst solidsinto the filter support.The biomasscakes pressibilitywill usuallybe proportionaltotheapplied hydraulicpressure uptoacertain pressure.Beyond thatpressure,the cakewill collapsetoanew pressedform so that throughputis reducedwiththeincremental pressureincrease.Table1.3shows filtrationdesign andoperation fordifferent fermentation broths.In twoofthecases notedin Table1.3a precoatwas used.The filterwill oftenbe precoatedwithafiltration aidsuch asdiatomaceous earthto reduceblinding ofthe filterandtoincrease filtration rates.The filtration aid mightbe addedtothebroth butthen thequantity offiltrationaidrequired ismore thandouble theprecoat amount (3).Table1.3:Representative Designand OperatingResults forFermentation Broth(Vacuum0.68-0.85Bar)Bacillus StreptomycesPenicillium licheniformisery threuschrysogenum filtertype Vacuumprecoat Vacuumdrum Vacuumdrum orprecoat Designfiltrationrate(9_/hr-m2)160-3204001,400-1,800Solids inslurry(wt%)8252-8Cycle time(min/rev)0.533Filter mediumPrecoat NylonPolypropylene Cakedischarge mechanismPrecoat StringString orprecoat Theequipment inthis operationcan beas simpleas anenlarged laboratoryvacuum filterto moreelaborate rotaryvacuum filters.These latterfilters essentiallyconsist ofa hollowsegmented drumcovered witha filter cloth.The drumrotates ina bathofthebroth tobe filteredwhile avacuum insidethe drumsucks liquidthrough thefiltercloth,forming acoating ofsolids onthe outsideofthedrum.Provisions areusually madeto washthefilter cake duringfiltration followedby removingthe solidfromthecloth.Instead ofvacuum,pressure can beusedto drivethe fluidthroughafiltercake.Plate andframe pressurefilters consistof wireor perforatedmetal frameswhich actas themechanical supportfor thefilter mediumwhichcanbe fine8Separation andPurification Techniquesin Biotechnologywire mesh,woven clothor cellulosicpads.With deepframe patterns,these filterscanbeused withdiatomaceous pre-coats.Example1.l Figure1.2shows theinfluence ofpH onthefiltrationrate ofS.griseus broth,1.5centipoise,using100cm2cotton clothfilter,diatomaceous earthfilter aidandaconstant pressuredifference of2bar (4).What sizeof filteris neededto filter1000L offermentation brothat pH3.8in15minutes?Figure1.2.The filtrationtime(t)divided bythe volumeof filtratecollected isplotted againstthe volumeof filtratecollected fordifferent pHvalues(Reference4).For thesake ofsimplicity,itisassumed thatthefilter:ake isinpressible sothat Equation1.1canbeintegrated10give:t nrS1()1v=-P A+arc V(12PW,x2(1.2)Introduction9The slopeand interceptfor the pH3.8curve canbeusedto obtainthe valuesfor rsand r,/W fromthe informationgiven.Using thedata points(300,0.7)and(600,1.82),rs=-55.73and r,/W=99.55.Equation1.2isaquadratic equationin(l/A),sothat:1()_(yt&gyy A=nrcV(1.3)PW WithV=1000L andt=900set,this equationsgives l/A=5.5x10w6,or A=18.18m2,The slightcurvature ofthe experimentallines inFigure1.2indicates thatthecakehas somedegree ofpressibility.This istobeexpected forcakes formedfromfermentationcells.As thevolumes,and thereforerequired times,forthesame filterarea increase,the curvaturewill beemore pronounced.While decreasingthepHwould speedup thefiltration,one mustbe carefulsothattheproductis notadversely affected.Several reviewsand texts(3,5-10)exist whichshouldbeconsulted foradditional informationon thisintegral partofanyfermentationbrothrecovery system.A listof industrialfiltration equipmentsuppliers isgiven inTable.1.2Centrifugation:Centrifugation,although widelyused forcell recovery,is notnearly asubiquitous asfiltration.This processenhances thegravitational settlingofthe suspendedsolidsinthefermentationbroth.The mathematicalrepresentation forthe centrifugationprocess isgivenby:18n St2cp=.d*(-(1.4)-PL)g2w rVIn thisequation Cpisthe time required for pleteparticle removal,disthe particlediameter,ps isthe particledensity,pL isthe fluiddensity,r7isthe fluid viscosity,sis10Separation andPurification Techniquesin BiotechnologyTable1.4:Industrial FiltrationEquipment SuppliersCompany LocationAllied FiltrationCo.Kingsley,PA AveryFilter Co.Westwood,NJ BirdMachine CompanySouth Walpole,MA CarlC.Brimmekamp&Co.Stamford,CT DenverEquipment CompanyColorado Springs,CO Dorr-Oliver,Inc.Stamford,CT EIMCO(Div.of Envirotech)Salt LakeCity,UT BC HoeschIndustries Wharton,NJ Inlay,Inc.Califon,NJ Komline-Sanderson,Inc.Peapack,NJ LenserAmerica,Inc.Lakewood,NJ PetersonFilters,Inc.Salt LakeCity,UT R&R FiltrationSystems Marietta,GA Serfilco,Ltd.Glenview,IL SparklerFilters,Inc.Conroe,TX D.R.Sperry&Co.North Aurora,IL thethickness ofthe liquidlayer inthe centrifuge,w isthe angularvelocity,risthe rotationradius,V isthe totalvolumeof liquid inthe centrifugeand gisthegravitational constant.The right-hand sideoftheequation isshown dividedinto twoterms.The firstcorresponds tothe terminalvelocity ofthe particlewith diameterd underthe forceof gravity.This termis solelyconcerned withproperties ofthefluidand suspendedparticles.The secondponent isconcerned onlywith fixedcharacteristics ofthe centrifugeoperating ata fixedspeed,w.This termis normall