生物工程專業(yè)畢業(yè)設(shè)計英文翻譯生物工程

High-RateContinuousProductionofLacticAcidbyLactobacillusrhamnosusinaTwo-StageMembraneCell-RecycleBioreactorSunhoonKwon,Ik-KeunYoo,WooGiLee,HoNamChang,YongKeunChangDepartmentofChemicalEngineeringandBioProcessEngineeringResearchCenter,KoreaAdvancedInstituteofScienceandTechnology,373-1Kusong-dong,Yusong-gu,Taejon305-701,SouthKorea;E-mail:hnchang@kaist.ac.krAbstractItisimportanttoproduceL(+)-lacticacidatthelowestcostpossibleforlacticacidtobecomeacandidatemonomermaterialforpromisingbiodegradablepolylacticacid.Inanefforttodevelopahigh-ratebioreactorthatprovideshighproductivityalongwithahighconcentrationoflacticacid,theperformanceofmembranecellrecyclebioreactor(MCRB)wasinvestigatedviaexperimentalstudiesandsimulationoptimization.Duetogreatlyincreasedcelldensity,highlacticacidproductivity,21.6gL?1h?1,wasobtainedinthereactor.Thelacticacidconcentration,however,couldnotbeincreasedhigherthan83g/L.Whenanadditionalcontinuousstirredtankreactor(CSTR)wasattachednexttotheMCRBahigherlacticacidconcentrationof87g/Lwasproducedatsignificantproductivityexpense.WhenthetwoMCRBswereconnectedinseries,92g/Llacticacidcouldbeproducedwithaproductivityof57gL?1h?1,thehighestproductivityamongthereportsofL(+)-lacticacidthatobtainedlacticacidconcentrationhigherthan85g/Lusingglucosesubstrate.Additionally,theinvestigationoflacticacidfermentationkineticsresultedinasuccessfulmodelthatrepresentsthecharacteristicsoflacticacidfermentationbyLactobacillusrhamnosus.ThemodelwasfoundtobeapplicabletomostoftheexistingdatawithMCRBsandwasingoodagreementwithLevenspiel’sproduct-inhibitionmodel,andtheLuedeking-Piretequationforproduct-formationkineticsappearedtobeeffectiveinrepresentingthefermentationkinetics.Therewasadistinctivedifferenceintheproductionpotentialofcells(cell-density-relatedparameterinLuedeking-Piretequation)aslacticacidconcentrationincreasesover55g/L,andthisfindingledtoamorepreciseestimationofbioreactorperformance.?2001JohnWiley&Sons,Inc.BiotechnolBioeng73:25–34,2001.Keywords:Lactobacillusrhamnosus;lacticacid;highproductivity;cellrecycle;membranebioreactor

INTRODUCTIONTheefficiencyofthemembranecell-recyclebioreactor(MCRB)wassuccessfullydemonstratedinanumberofpreviousstudiesofthehigh-volumetricproductivityoflacticacid.Withgreatlyincreaseddensityofbiocatalysts,i.e.,microbialcells,thevolumetricproductivityoflacticacidcouldgoupto160gL?1h?1asreportedinthestudyofOhleyeretal.(1985),whichismorethan20timeshigherthanthatoftheconventionalbatchandchemostatprocesses.However,thehighproductivityisnottheonlyrequirementfortheeconomicfeasibilityoftheprocess.TimmerandKromkamp(1994)foundthattheprocessmightbeprimarilyinfluencedbyproductioncapacityandproductconcentrationandtoalesserextentbythevolumetricproductivitywhenannuallacticacidproductioncapacityrosetoashighas4540metrictons.Incaselacticacidconcentrationissignificantlylow,theenergycostforwaterremovalinthedownstreamprocessoffsetsthebenefitsoftheincreasedproductivity.Fromthispoint,MCRBhasanimportantproblemtobetackled:Theconcentrationsoflacticacidaresignificantlylowwhencomparedwithbatchprocesseswherethelacticacidconcentrationabove120g/Liseasilyattainable.Exceptforareportshowing117g/LD(?)-lacticacidwithavolumetricproductivityof84gL?1h?1(MehaiaandCheryan,1987),allotherMCRBoperationsresultedinlacticacidconcentrationsoflessthan90g/Land,moreover,mostofthemhadconcentrationsbelow60g/L(Cheryan,1998;Litchfield,1996;Ohleyeretal.,1985).ThemicroorganismscannotgrowaboveacertainrangeoflacticacidconcentrationandtheMCRBsarerunundercontinuousmannerwithcontinuousbleedingofcellstopreventthelossoffluiditythatoccurswhencellconcentrationgoestoohigh.Thus,toenhancetheeconomicaladvantageoftheMCRBprocess,methodsthatincreasethelacticacidconcentrationalongwiththehigh-celldensityarerequired.Someauthors,whoconsideredthispersistentproblemoflow-productconcentration,conductedstudiestoobtainhigherlacticacidconcentrationinMCRB.Xavieretal.(1995)reportedalacticacidconcentrationof90g/Lwithaproductivityof36gL?1h?1,whileTejayadiandCheryan(1995)achieved89g/Land22gL?1h?1oflacticacidconcentationandproductivity,respectively.Atypicalapproachtoovercometheabove-mentionedproblem,alow-productconcentrationduetosevereproductinhibition,istheuseofaplug-flowreactor,whichcanbeapproximatedbyseveralcontinuous-stirred-tankreceptors(CSTRs)inseries(deGooijeretal.,1996;KellerandGerhardt,1975;LuedekingandPiret,1959b;Levenspiel,1984).TheadvantagesoftheCSTRs-in-seriesagainstasingleCSTRespeciallyinlacticacidproductionwererevealedbyothersintwo-andthree-stageCSTRs(Aeschlimannetal.,1990;Bruno-Ba′rcenaetal.,1999;Mulliganetal.,1991):increasedproductivityandconcentrationoflacticacidviapartlyseparatingcellgrowthandlacticacidproductionphases;increasedlacticacidyieldattheexpenseofbiomassformationatalatterstage;highpurityofthelacticacidisomer,L(+)-lacticacidviaincreasedpopulationoffreshcells;andreducedusageofacostlynutrient,yeastextract.Inanefforttocombinetheadvantageofboththebioreactorconfigurations—MCRBandmulti-stagedbioreactor—Kuloziketal.(1992)investigatedtheperformanceofaseven-stagedcascadereactorwithcellrecycle.Cellsintheoutflowofthelastreactorwerefivefoldconcentratedbyamicrofilterandrecycledbacktothefirstreactor.Incomparisonwithasingle-stageMCRB,thecascadereactorshowed4timeshigherproductivity,28gL?1h?1,withcompleteutilizationof100g/Llactose,inwhichthecellconcentrationsweremaintainedat20g/Landthelacticacidconcentrationswerearound72g/L.Inthisstudy,theperformanceofanewbioreactorconfiguration,twoMCRBsinseries,wasinvestigatedaimingatthehighestvolumetricproductivityeverobtainedalongwiththelacticacidconcentrationashighaspossible.Moreover,asimulationstudywasconductedtoestimatetheperformancelimitofMCRBwithanunstructuredkineticmodel,whichisvalidatedbytheexperimentresults.MATERIALSANDMETHODSMicroorganismandCultureConditionsLactobacillusrhamnosus(ATCC10863),anobligatoryanaerobichomofermentativeL(+)-lacticacidproducer,wasobtainedfromAmericanTypeCultureCollection(Rockville,MD).One-mLstockcultureswerestoredat?76°CinLactobacilliMRSmedium(Difco,Detroit,MI)with25%(v/v)glycerol.Precultureswerepreparedbytransferringastockcultureto200mLofMRSmediumandincubatedat42°Cfor12handtransferredtothemainculture.Theculturetemperaturewas42°CandtheculturepHwascontrolledat6.0withammoniawater(ca.8N).ForMCRBoperationsthebasalmediumhadthefollowingcomponentsperliter:0.2gNa3-Citrate·2H2O,1.0gKH2PO4,0.2gMgSO4·7H2O,0.03gMnSO4·H2O,0.03gFeSO4·7H2O,and0.015mLsulfuricacid.TheconcentrationsofglucoseandyeastextractwillbedescribedintheResultssection.Allthemediacomponentswereheatsterilizedtogetherat121°Cfor100minexceptforyeastextractthatwasseparatelysterilizedfor15min.Theculturevolumenotedintheresultsincludesthebrothvolumeintherecyclestream.AnalyticalMethodsCellgrowthwasmeasuredbyaspectrophotometer(PharmaciaUltrospec3000,Cambridge,UK)atawavelengthof620nm.Drycellconcentrationwascalculatedfromtheopticaldensity(OD620)withalinearcorrelationfactor(oneOD62040.32g-drycellweightperliter).Concentrationsoflacticacidandglucoseweredeterminedbyahighperformanceliquidchromatography(HPLC)systemequippedwitharefractive-indexdetector(HitachiL-6000,Tokyo,Japan).AnHPLCcolumn(Aminex87H,Bio-Rad,Richmond,CA)wasusedwith0.005Msulfuricacidasthemobilephaseatanelutionspeedof0.6mL/minwhilethecolumntemperaturewasmaintainedat50°C.Theconcentrationstandardsof1.0Mlacticacid(Fluka,Buchs,Switzerland)and10g/Lglucose(Sigma,St.Louis,MO)wereusedfortheHPLCanalysis.MembraneCell-RecycleBioreactor(MCRB)Intheexperimentsofasingle-stageMCRB,a400-mLwater-jacketedglassreactorwasemployed,thatwasequippedwithahollow-fiberfiltrationunitUFP-100-H-4X2TCA(100kNMWC,0.065m2filtrationarea;A/GTechnologyCorporation,MA).Aperistalticpump,07090-40(Cole-Parmer,IL)wasusedtocirculatetheculturebroththroughthemembraneunitwithaflowrateofca.100mL/min.Forthetwo-stageoperations,twoidenticalMCRBswereseriallyconnected.EachMCRBconsistedofa1-Lglassreactorattachedwithaplate-and-framefiltrationunit,Pellicon2BIOMAX100V(100kNMWC,0.1m2filtrationarea,Millipore,Bedford,MA),andadiaphragmpump,P-07090-40(Cole-Parmer)forcellrecyclewithaflowrateofca.600mL/min.TheMCRBwassterilizedwith50%(v/v)ethanolandwashedthoroughlywithsterilewaterbeforeinoculation.Inoperation,afreshmediumwasfedcontinuouslyintothereactorwhilefilterpermeatewassimultaneouslypumpedout.Topreventthecelldensityfromgoingoveracertainlimit,whichcausesthelossoffilterfunctionality,smallamountsofculturebrothwerecontinuouslydrawnoutfromthereactor(bleedflow).Inthetwostageoperation,thebleedandpermeateflowfromthefirstreactorwerefedtogetherintothesecond.NumericalMethodsTheleastsquaresregressionwasusedtoestimatetheparametersofthefermentationkinetics.Numericalintegrationtofindsteady-statevaluesandconstrainedmultivariableoptimizationtofindtheoptimaloperationvariableswereperformedwiththehelpofasoftwarepackage,Matlab5.0(TheMathworks,Inc.,USA).Theconstraintsutilizedintheoptimizationwerethemaximumcelldensity(Xm)andthemaximumremainingglucoseconcentration(S).DISCUSSIONToincreasethebioreactorperformancefortheproductionoflacticacid,acontinuouslacticacidfermentationsystemcoupledwithmembranecell-separationtechnique(MCRB)hasbeenstudied.Bygreatlyincreasedcelldensityinthereactorvolumetricproductivitycouldbeincreasedover10timesthantheconventionalbatchandcontinuousfermentation.However,theconcentrationoflacticacidproduced,amajorfactorforeconomicfeasibility,couldnotbeincreasedhigherthan95g/Lbeyondwhichcellgrowthisinhibitedalmostcompletely.InthepreliminaryexperimentswithsingleMCRB,lowlacticacidconcentrationsaround51g/Lwereobtainedevenwhenthecelldensitywasmaintainedathigherthan90g/L(Fig.4).WhenaCSTRwith9timeslargervolumethantheMCRBwasattached,intendingforalongerreactiontimeforthecellsintheMCRBoutflow,87g/Llacticacidcouldbeobtainedwithagreatsacrificeintheproductivity(Fig.6).Itwasconcludedthathigherlacticacidconcentrationwithhighproductivitycouldbeobtainableifthesecondreactor,CSTR,hadbeenreplacedwithanotherMCRB,whichmadeupthetwo-stagebioreactorwithcell-recycleatbothstages.WiththetwoMCRBsinseries,92g/Loflacticacidwasobtainedatahighproductivityof57gL?1h?1(Fig.12).Inconclusion,asystematicapproachwithMCRBswithmultistagedoperationcanbecarriedouttopredictoptimalperformancesoflacticacidproduction,whichexperimentallyprovedthattwostageMCRBscanproducelacticacidinahighconcentrationwithgreatlyincreasedvolumetricproductivity(typeA).References[1]AeschlimannA,StasiLD,vonStockarU.1990.ContinuousproductionoflacticacidfromwheypermeatebyLactobacillushelveticusintwochemostatsinseries.EnzymeMicrobTechnol12:926–932.[2]AmraneA,PrigentY.1999.AnalysisofgrowthandproductioncouplingforbatchculturesofLactobacillushelveticuswiththehelpofanunstructuredmodel.ProcBiochem34:1–10.[3]BerryAR,FrancoCMM,ZhangW,MiddelbergAPJ.1999.GrowthandlacticacidproductioninbatchcultureofLactobacillusrhamnosusinadefinedmedium.BiotechnolLett21:163–167.[4]BibalB,KappC,GomaG,PareilleuxA.1989.ContinuouscultureofStreptococcuscremorisonlactoseusingvariousmediumconditions.ApplMicrobiolBiotechnol32:155–159.[5]BibalB,VayssierY,GomaG,PareilleuxA.1991.HighconcentrationcultivationofLactococcuscremorisinacell-recyclereactor.BiotechnolBioeng37:746–754.[6]Bo¨rgardtsP,KrischkeW,Tro¨schW,BrunnerH.1998.Integratedbioprocessforthesimultaneousproductionoflacticacidanddairysewagetreatment.BioprocessEng19:321–329.[7]Bruno-Ba′rcenaJM,RagoutAL,CordobaPR,Sin?erizF.1999.ContinuousproductionofL(+)-lacticacidbyLactobacilluscaseiintwo-stagesystems.ApplMicrobiolBiotechnol51:316–324.[8]CheryanM.1998.Ultrafiltrationandmicrofiltrationhandbook.Lancaster,PA:TechnomicPublishingCompany.467p.[9]deGooijerCD,BakkerWAM,BeeftinkHH,TramperJ.1996.Bioreactorsinseries:Anoverviewofdesignproceduresandpracticalapplications.EnzymeMicrobTechnol18:202–219.[10]DuttaSK,MukherjeeA,ChakrabortyP.1996.Effectofproductinhibitiononlacticacidfermentation:Simulationandmodelling.ApplMicrobiolBiotechnol46:410–413.[11]Gonc?alvesLMD,XavierAMRB,AlmeidaJS,CarrondoMJT.1991.Concomitantsubstrateandproductinhibitionkineticsinlacticacidproduction.EnzymeMicrobTechnol13:314–319.[12]KellerAK,GerhardtP.1975.Continuouslacticacidfermentationofwheytoproducearuminantfeedsupplementhighincrudeprotein.BiotechnolBioeng17:997–1018.[13]KulozikU,HammelehleB,PfeiferJ,KesslerHG.1992.Highreactionratecontinuousbioconversionprocessinatubularreactorwithnarrowresidencetimedistributionsfortheproductionoflacticacid.JBiotechnol22:107–116.[14]KulozikU,WildeJ.1999.RapidlacticacidproductionathighcellconcentrationsinwheyultrafiltratebyLactobacillushelveticus.EnzymeMicrobTechnol24:297–302.[15]KwonS,LeePC,LeeEG,ChangYK,ChangHN.2000.ProductionoflacticacidbyLactobacillusrhamnosuswithvitamin-supplementedsoybeanhydrolysate.EnzymeMicrobTechnol26:209–215.[16]LevenspielO.1980.TheMonodequation:Arevisitandageneralizationtoproductinhibitionsituations.BiotechnolBioeng22:1671–1687.LevenspielO.1984.Chemicalreactionengineering.NewYork:JohnWiley&Sons.p124–157.[17]LitchfieldJH.1996.Microbiologicalproductionoflacticacid.In:NeidlemanSL,LaskinAI,editors.Advancesinappliedmicrobiology.Vol42.SanDiego:AcademicPress.69p.[18]LuedekingR,PiretEL.1959a.Akineticstudyofthelacticacidfermentation.BatchprocessatcontrolledpH.JBiochemMicrobiolTechnolEng1:393–412.[19]LuedekingR,PiretEL.1959b.Transientandsteadystatesincontinuousfermentation.TheoryandExperiment.JBiochemMicrobiolTechnolEng1:431–459.[20]MajorNC,BullAT.1985.LacticacidproductivityofacontinuouscultureofLactobacillusdelbrueckii.BiotechnolLett7:401–405.

利用鼠李糖乳桿菌在兩級細胞膜循環(huán)生物反應(yīng)器中高速連續(xù)生產(chǎn)乳酸——SunhoonKwon，YongKeunChang單位：韓國高等科學技術(shù)學院，化學與生物工程研究中心，E-mail:hnchang@kaist.ac.kr摘要眾所周知，乳酸是可生物降解材料聚乳酸的主要原料，所以找到以一種以最低的成本來生產(chǎn)L（+）－乳酸的方法具有非常重大的意義。為了找到一種可以高速地生產(chǎn)高濃度乳酸的生物反應(yīng)器，我們對膜循環(huán)生物反應(yīng)器（MCRB）的性能進行了研究，并進行了實驗仿真優(yōu)化。由于大大增加了細胞濃度，這個反應(yīng)器的乳酸生產(chǎn)力可達到21.6gL-1h-1。但乳酸濃度卻不能超過83g/L，當額外增加一個連續(xù)攪拌反應(yīng)釜（CSTR）附到MCRB旁邊時，可以大幅度的提高生產(chǎn)速率，乳酸濃度也可以提高到87g/L，當兩個MCRBs串聯(lián)在一起時，乳酸的生產(chǎn)力速率達到57gL-1h-1，最終溶液中的乳酸濃度為92g/L，這比以前所報道的使用葡萄糖基生產(chǎn)L（+）乳酸濃度超過85g/L的最高的生產(chǎn)率還要高。此外，研究乳酸發(fā)酵動力學產(chǎn)生了以鼠李糖乳桿菌發(fā)酵生產(chǎn)乳酸為代表的成功典范，該模型被認為是適用于大多數(shù)現(xiàn)有的MCRBs的數(shù)據(jù)，并且很好地吻合了Levenspiel的產(chǎn)品抑制模型，Luedeking-Piret產(chǎn)品形成動力學方程似乎是有效的代表發(fā)酵動力學。然而具有生產(chǎn)潛力的細胞（細胞密度相關(guān)參數(shù)Luedeking-Piret方程）生產(chǎn)乳酸的濃度超過55g/L時，卻又有一個與眾不同的差異，而這一結(jié)果會讓我們更進一步地精確估計生物反應(yīng)器的性能。?2001JohnWiley＆Sons出版公司生物Bioeng73：25-34，2001。關(guān)鍵詞：鼠李糖乳桿菌;乳酸;高生產(chǎn)力;細胞循環(huán);膜生物反應(yīng)器

1前言膜細胞循環(huán)生物反應(yīng)器（MCRB）的生產(chǎn)效率成功地證實了一些以往關(guān)于高容積生產(chǎn)乳酸的研究。Ohleyer等的研究報告指出，通過大量增加生物催化劑，即微生物細胞，乳酸的生產(chǎn)力可高達160gL?1h?1。（1985年），這比常規(guī)批次和恒化的生產(chǎn)工藝高出20倍以上。然而，高生產(chǎn)力并不是唯一的要求，這種工藝還必須在經(jīng)濟上有可行性。TimmerandKromkamp（1994年）發(fā)現(xiàn)，這一工藝可能主要受生產(chǎn)能力和產(chǎn)品的集中的影響，在較小程度時當年這種工藝生產(chǎn)乳酸的產(chǎn)能上升到高達4540噸。如乳酸濃度顯著低，能源成本中的水在去除抵消下游過程的好處，提高了生產(chǎn)力。從這個角度上講，MCRB有一個重要的問題有待解決：在乳酸濃度顯著低相比，間歇過程的乳酸濃度122g/L的是容易實現(xiàn)的。此外，還有一份報告顯示以84gL?1h?1的生產(chǎn)速率得到的D（+）L乳酸最終濃度為117g/L（Mehaia和Cheryan，1987年），而除部分MCRB工藝生產(chǎn)出的乳酸濃度低于90/g/L外所有其他的大多數(shù)生產(chǎn)的濃度低于60g/L的（Cheryan，1998年;里奇菲爾德，1996年;Ohleyer等，1985）。微生物無法在超過一定濃度的乳酸條件下生長，因此可以通過用MCRBs工藝進行連續(xù)放出細胞，以防止損失的流動性時所產(chǎn)生的細胞濃度不會太高。因此，為加強MCRB工藝的經(jīng)濟優(yōu)勢的方法有，隨著高密度的要求增加乳酸濃度。一些考慮到這個長期存在的低濃度產(chǎn)品問題的作者對他進行了研究，并通過MCRB工藝獲得了較高濃度的乳酸。哈維爾等人。（1995年）和Tejayadi和Cheryan（1995年）分別發(fā)表了以36gL?1h?1的生產(chǎn)速率得到濃度為90g/L的乳酸和以22gL?1h?1的生產(chǎn)速率得到濃度為89g/L的乳酸的報道。產(chǎn)品的濃度低是由于乳酸菌受到了嚴重抑制，這里有一個很好的辦法來克服上述問題，我們可以通過使用推流反應(yīng)器，它類似于很多連續(xù)化攪拌式受體（CSTRs）結(jié)合在一起（日Gooijer等。996年;Keller和戈哈德，1975年;Luedeking和Piret，1959年;Levenspiel，1984年）。CSTRs的優(yōu)勢在一系列針對單一CSTR中特別是在乳酸生產(chǎn)中所揭示的其他兩個和三個階段CSTRs（艾緒里曼等人。1990年;布魯諾-Ba'rcena等。1999年;根等。1991年）通過部分分離細胞的生長和乳酸生產(chǎn)階段提高乳酸的生產(chǎn)力和濃度，增加乳酸產(chǎn)量為代價的生物形成的后期;高純度的乳酸異構(gòu)體長，L（+）乳酸菌通過增加新鮮細胞的數(shù)量;同時減少使用昂貴的養(yǎng)分——酵母膏。為了結(jié)合雙方的優(yōu)勢，生物反應(yīng)器的配置MCRB和多階段生物反應(yīng)器Kulozik等。（1992）進行了一項七級聯(lián)反應(yīng)器與細胞循環(huán)的研究。最后一個反應(yīng)器中流出的細胞溶液通過收集器集中再生回到第一座反應(yīng)器中，相對于單級MCRB，梯級反應(yīng)器得到的生產(chǎn)率要高出4倍。達到28克L-1h-1，乳糖完整的利用率為100g/L，其中的細胞濃度保持在20g/L和的乳酸濃度約為72g/L。在這項研究中，對新型生物反應(yīng)器的配置，即兩個MCRBs串聯(lián)的性能進行了研究，旨在在最高容積生產(chǎn)力的情況下不斷得到乳酸且其濃度盡可能高。此外，對估計MCRB的性能極限與非結(jié)構(gòu)化的動力學模型，進行了模擬研究，通過這個實驗驗證了結(jié)果。

2材料與方法2.1微生物培養(yǎng)法及培養(yǎng)條件鼠李糖乳桿菌（ATCC10863），一種同型發(fā)酵的具有極強的厭氧性的L（+）乳酸生產(chǎn)菌，它是從美國特種培養(yǎng)物保藏中心獲得的（位于美國馬里蘭州羅克維爾市）。一毫升庫乳桿菌菌種與（培養(yǎng)基，底特律，MI）和的25％（V/V）的甘油混合后在-76°C的條件下保存，Precultures準備通過在MRS培養(yǎng)基中，在42°C的條件下培養(yǎng)12小時，將菌株培養(yǎng)到200毫升，并轉(zhuǎn)移到主要化?？刂婆囵B(yǎng)溫度為42℃和通過使用氨水調(diào)節(jié)pH到6.0，MCRB工藝的培養(yǎng)基要有以下組成部分每升：0.2Na3-Citrate·2H2O，1.0gKH2PO4,0.2gMgSO4·7H2O,0.03gMnSO4·H2O,0.03gFeSO4·7H2O,和0.015mL硫酸。糖的濃度和酵母提取物將在結(jié)論中指出。除酵母提取物是單獨滅菌15分鐘外，所有培養(yǎng)基一起在121°C的條件下滅菌100分鐘。在結(jié)論中談到的培養(yǎng)體積包括循環(huán)流體培養(yǎng)基的體積。2.2分析方法細胞生長可通過分光光度計在波長為620納米時測定（法瑪西亞Ultrospec3000，英國劍橋）一般可由干細胞濃度與光密度（OD620）的線性相關(guān)系數(shù)（1OD62040.32克，干重每公升）計算出來。乳酸的濃度和葡萄糖含量可由配備了折射率檢測器系統(tǒng)的高效液相色譜儀（HPLC）（日立L型6000，日本東京）測定。HPLC柱使用時（Aminex87H，酶標儀，里奇蒙，CA）以0.005M硫酸為流動相，在洗脫速度為0.6毫升/分鐘，而柱溫保持在50°C的濃度標準為1.0米乳酸（鹽，布克斯，瑞士）和10g/L的葡萄糖（六西格瑪，圣路易斯，密蘇里州）用于高效液相色譜分析中。2.3膜細胞循環(huán)生物反應(yīng)器（MCRB）在單級MCRB的實驗中，要應(yīng)用到如下實驗器材：一；400毫升水套，它采用玻璃反應(yīng)器并配備了中空纖維超微粒過濾裝置-100-H的4X2TCA（100kNMWC，0.065平方米過濾面積;阿/g技術(shù)公司，馬）。二：蠕動泵，07090-40型（科爾-Parmer，白細胞介素）CA以100毫升/分鐘的速度推動發(fā)發(fā)酵液通過膜單元。在兩個階段的行動，兩個相同的MCRBs是串行連接。每個MCRB包括一個1一L玻璃反應(yīng)堆附有板和幀過濾單元，一個Pellicon2BIOMAX100V的（100kNMWC，0.1平方米過濾面積，超純水，貝德福德，馬）與隔膜泵，和一個P-07090-40（科爾Parmer）的細胞再生裝置，其CA流速為600毫升/分鐘。MCRB在接種前需要用含50％（V/V）乙醇的無菌水徹底清洗。在操作過程中，需不斷向發(fā)酵罐中加入新的培養(yǎng)基同時排出產(chǎn)物。為了防止細胞密度去超過一定限度，造成過濾功能下降，需要從發(fā)酵罐中不斷抽出少量的發(fā)酵液。在這兩個階段發(fā)酵過程中，從第一階段流出的發(fā)酵液用于第二階段中。2.4數(shù)值分析方法發(fā)酵動力學的參數(shù)可以用最小二乘回歸來估算。利用Matlab5.0（MathWorks公司，公司，美國）軟件進行數(shù)值積分找到穩(wěn)態(tài)值和約束多變量優(yōu)化以尋找到最佳操作變量。限制利用的優(yōu)化是最大的細胞密度（Xm）和最大其余血糖濃度（s）。

討論為了提高生物反應(yīng)器產(chǎn)乳酸的性能，我們對連續(xù)乳酸發(fā)酵系統(tǒng)加上膜細胞分離技術(shù)（MCRB）進行了研究。大大增加在固定體積發(fā)酵罐中的細胞密度，生產(chǎn)率比傳統(tǒng)的間歇和連續(xù)發(fā)酵提高了10倍以上。然而，乳酸實際生產(chǎn)中，最主要因素經(jīng)濟上的可行性，此方法乳酸濃度高于95g/L時，細胞的生長幾乎完全受到抑制。在初步單MCRB實驗中，即使在細胞密度保持在高于90g/L時，得到的乳酸濃度仍然很低，約51政/L，（圖4）。當加入一個體積比MCRB大9的CSTR時，在放出細胞液之前如果讓它在在MCRB反應(yīng)器中停留更長的時間，則乳酸濃度會明顯提升，會達到87g/L圖6）。由此我們可以得出這樣的結(jié)論：在第二個反應(yīng)器CSTR與另一MCRB相連并且兩個階段的生物反應(yīng)器與細胞循環(huán)同在這兩個階段前提下，可以高速生產(chǎn)高濃度的乳酸，如果使用兩個MCRBs系列，則可以以57gL?1h?1的生產(chǎn)速率生產(chǎn)出濃度達到92g/L的乳酸（圖12）。最后，通過優(yōu)化多步驟MCRBs反應(yīng)器，可以得到預(yù)期想得到的最優(yōu)生產(chǎn)乳酸的方法，實驗證明，利用兩階段MCRBs反應(yīng)器可以高速生產(chǎn)高濃度的乳酸，從而使固定容積反應(yīng)器的生產(chǎn)效率大大提高（A型）。

參考文獻[1]AeschlimannA,StasiLD,vonStockarU.1990.ContinuousproductionoflacticacidfromwheypermeatebyLactobacillushelveticusintwochemostatsinseries.EnzymeMicrobTechnol12:926–932.[2]AmraneA,PrigentY.1999.AnalysisofgrowthandproductioncouplingforbatchculturesofLactobacillushelveticuswiththehelpofanunstructuredmodel.ProcBiochem34:1–10.[3]BerryAR,FrancoCMM,ZhangW,MiddelbergAPJ.1999.GrowthandlacticacidproductioninbatchcultureofLactobacillusrhamnosusinadefinedmedium.BiotechnolLett21:163–167.[4]BibalB,KappC,GomaG,PareilleuxA.1989.ContinuouscultureofStreptococcuscremorisonlactoseusingvariousmediumconditions.ApplMicrobiolBiotechnol32:155–159.[5]BibalB,VayssierY,GomaG,PareilleuxA.1991.HighconcentrationcultivationofLactococcuscremorisinacell-recyclereactor.BiotechnolBioeng37:746–754.[6]Bo¨rgardtsP,KrischkeW,Tro¨schW,BrunnerH.1998.Integratedbioprocessforthesimultaneousproductionoflacticacidanddairysewagetreatment.BioprocessEng19:321–329.[7]Bruno-Ba′rcenaJM,RagoutAL,CordobaPR,Sin?erizF.1999.ContinuousproductionofL(+)-lacticacidbyLactobacilluscaseiintwo-stagesystems.ApplMicrobiolBiotechnol51:316–324.[8]CheryanM.1998.Ultrafiltrationandmicrofiltrationhandbook.Lancaster,PA:TechnomicPublishingCompany.467p.[9]deGooijerCD,BakkerWAM