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ProcessBiochemistry44(2009)516–520ContentslistsavailableatScienceDirect

ProcessBiochemistry

journalhomepage:www.elsevier.com/locate/procbio

SpeciﬁcactivityandviabilityofNitrosomonaseuropaeaduringdiscontinuousandcontinuousfermentation¨vena,*,IngoSchmidtbDidemGuab¨yu¨kcFatihUniversity,FacultyofEngineering,EnvironmentalEngineeringDepartment,34500Bu¸ekmece,Istanbul,TurkeyIngoSchmidtUniversityofBayreuth,DepartmentMicrobiology,Universitaetsstrasse30,95447Bayreuth,GermanyARTICLEINFOABSTRACTArticlehistory:

Received19August2008Receivedinrevisedform5November2008Accepted13January2009Keywords:NitrosomonaseuropaeaSpeciﬁcactivityViabilityCultivationGrowthphaseFermentationTheenergyconservationandnumberofviablecellsofNitrosomonaseuropaeaﬂuctuatedramaticallyduringcultivation.Indiscontinuousculturethespeciﬁcactivity(SA)reachesitsmaximumafter9hwithabout2700nmolO2(mgprotein)À1minÀ1,wherethehighestnumberofviableN.europaeacellsisdetectableafter21hwith2Â108cellmlÀ1.Afterwards,bothSAandviablecellnumberimmediatelystarttodecrease.Accordingly,theexponentialgrowthturnsintoalineargrowth,wherebythenumberofviablecellspermanentlydecreases.Theexponentialgrowthphasecanbeextendedfromabout21to38hbyincreasingtheconcentrationofCO2ortraceelements.IncontinuousfermentationofN.europaea,SAofabout2500nmolO2(mgprotein)À1minÀ1andviablecellnumberof2.5Â108cellmlÀ1isdetectableatdilutionratesbetween1and1.8dayÀ1.Atdilutionratesbelow1dayÀ1,SAandnumberofviablecellsarereduced.Theminimaldoublingtimeis13and15hduringcontinuousanddiscontinuousfermentation,respectively.Consequently,cellproductionofN.europaeashouldbeperformedincontinuousfermentation.Whenbacteriaaregrownindiscontinuoussystems,theyshouldbeharvestedintheearlyexponentialgrowthphase.ß2009ElsevierLtd.Allrightsreserved.1.IntroductionThenitriﬁerN.europaeaobtainsenergyfromtheaerobicoranaerobicoxidationofammoniatonitriteandCO2servesasitscarbonsource[1–6].Theinitialreaction,theformationofhydroxylamine,iscatalyzedbytheammoniamonooxygenase(AMO)[2,3,7,8].Inthesecondstep,hydroxylamineisoxidizedtonitritebythehydroxylamineoxidoreductase(HAO)[6].TwoofthefourelectronsreleasedfromtheoxidationofhydroxylaminearerequiredfortheinitialAMOreaction,whiletheothertwoareemployedinthegenerationofaprotonmotiveforceinordertoregenerateATPand,viaareverseelectrontransport,NADH[6].Ammoniaoxidizersplayanimportantroleintheglobalnitrogencycleandarekeyorganismsinthetreatmentofindustrialanddomesticwastewater[9–12].Theyhavebeenfoundinmanyecosystemslikefreshwater,saltwater,sewagesystems,andsoils[13,14],aswellasinanoxicenvironments[15,16].Asigniﬁcantamountoftheenergyobtainedfromoxidationofhydroxylaminehastobeinvestedinammoniaoxidation(AMO),reverseelectrontransporttogenerateNADH,andCO2ﬁxation.Asaconsequence,growthratesofN.europaeaarerelativelylow.Growthratesof0.94and0.41dayÀ1werereportedforcontinuous*Correspondingauthor.Tel.:+902128663300x5613;fax:+902128663412.¨ven).E-mailaddress:dguven@fatih.edu.tr(D.Gu1359-5113/$–seefrontmatterß2009ElsevierLtd.Allrightsreserved.doi:10.1016/j.procbio.2009.01.005anddiscontinuouscultures,respectively[17].Inotherstudiesgrowthratesof0.43[18],0.58[19],and3dayÀ1[20],anddoublingtimesof8[21]or10–24h[17]weremeasured.Ammoniumconcentrationsbetween2and50mM,pHvaluesbetween7.2and7.6,oxygenconcentrationsbetween1and6mglÀ1,andtempera-turesbetween28and358CwerereportedtobeoptimaltogrowN.europaea[17,21–28].Indifferentstudieseitherthespeciﬁcammoniaoxidationoroxygenconsumptionactivitywererecorded.Thespeciﬁcactivity(SA)ofN.europaeaindiscontinuousculturedifferedbetween300and1670nmolO2(mgprotein)À1minÀ1[17],infed–batchsystemsbetween600and1500nmolO2(mgprotein)À1minÀ1,andincontinuousfermentationwithbiomassretentionbetween240and900nmolO2(mgprotein)À1minÀ1atdilutionratesbetween0.05and0.1hÀ1[29,30].Interestingly,celldensityofNitrosomonasculturesseemstobelimited.Indiscontinuousculturesthetotalcellnumberhardlyincreasedabove5Â108cellsmlÀ1[30]andthemaximumcellnumberincontinuouscultureswithcompletebiomassretentionwasapproximately2Â1010cellsmlÀ1inthepresenceof50ppmNO2[30].TheaccumulationoftheendproductnitritewassuggestedtoinhibitgrowthandtolimitthecellnumberofN.europaea[31].Thelimitedcelldensitymakesitdifﬁculttoproducesigniﬁcantamountsofbiomass[17,29,32,33].ThecultivationtechniquesforbiomassproductionofthemodelbacteriumN.europaeadiffersigniﬁcantlywithregardtoappliedfermentorsystems(discontinuous,continuous),ammo-¨ven,I.Schmidt/ProcessBiochemistry44(2009)516–520D.Gu517

niumconcentrations,pHvalues,oxygenconcentrations,andtemperatures.N.europaeaisusuallygrownindiscontinuousfermentorsystems(oftenErlenmeyerﬂasks)andbacteriaareharvestedbetweenlategrowthandearlystationaryphase.Asaresultofcultivationdifferentqualitiesofbiomassareproduced.Thecatabolicactivity(ammoniumoroxygenconsumption)andviabilityﬂuctuatesigniﬁcantly,inﬂuencingtheresultsofexperi-mentsperformedwiththesebacteria.Togeneratereproducibleandcomparableresults,ahomogenousbiomasswithknownSAandviability,preferentiallywithmaximumSAandviability,isrequiredinsufﬁcientquantities.Thegoalofthisstudywastoverifythecorrelationofgrowthconditionsduringbiomassproductionandactivity/viabilityofthebacteria.DiscontinuousandcontinuousfermentorsystemswereappliedandSAandviabilityofN.europaeaweremeasuredinallgrowthphases.Theinﬂuenceofgrowthfactorslikeammonium,oxygen,CO2,andtraceelementsonactivityandviabilitywereexamined.Theresultsopenthepossibilitytoproduceahomo-genousN.europaeabiomasswithoptimalanddeﬁnedactivityandviability.2.Materialsandmethods2.1.MicroorganismandgrowthconditionsStockculturesofN.europaea(ATCC19718)weregrownaerobicallyin1-l-Erlenmeyerﬂaskscontaining400mlmineralmedium[4].Theculturesweregrowninthedarkat288Cshakingat50rpm.Theywereharvestedduringthelateexponentialgrowthphaseandwereusedasinoculum(20%ofthetotalvolume)forcontinuousanddiscontinuousfermentations.2.2.Experimentaldesign2.2.1.DiscontinuousfermentationN.europaeawasgrowninafermentorof30ltotalvolume(modelBiostat,Braun)suppliedwith20lmineralmediumcontaining75mMammoniumunderdiscontinuousconditions.Airwassuppliedataﬂowrateof0.2–5lairminÀ1tomaintainadissolvedoxygenconcentrationof5Æ1mglÀ1.Temperaturewas298CandpHwaskeptat7.3employing20%(w/v)Na2CO3.Samplesforoff-linedeterminationofSAandcellnumberaswellasammoniumandnitriteconcentrationweretakenwithinregulartimeintervals.Celldensity(bacterialgrowth)wasmonitoredat578nminaspectrophotometer.Cultureswerealwayskeptinthedark.AsubstrateinhibitionofN.europaeawasnotobservedatammoniumconcentrationsbelow150mMandnitriteasaproductwasnotinhibitoryinconcentrationsupto100mM.

2.2.2.ContinuousfermentationN.europaeawasgrowninafermentorof12ltotalvolumesuppliedwith10lmineralmediumundercontinuousconditions.Airwassuppliedataﬂowrateof0.1–3lairminÀ1tomaintainadissolvedoxygenconcentrationof5Æ1mglÀ1.Temperaturewas298CandpHwaskeptat7.3employing20%(w/v)Na2CO3.Thedilutionratewasvariedbetween0.1and2.2dayÀ1andfreshmineralmedium(55mMammonium)wascontinuouslyaddedviaaperistalticpump.Inthefermentortheammoniumconcentrationwaskeptbelow5mM.

2.2.3.AnalyticalproceduresAmmoniumwasmeasuredaccordingtoSchmidtandBock[4],nitriteandnitrateaccordingtoVandeGraafetal.[34],andproteinaccordingtoBradford[35].TheoxygenconsumptionwasmeasuredandtheSAofthebacteriawascalculatedasoxygenconsumptionpermgproteinandminute.OxygenconcentrationwasmeasuredinaClarkCellTypeOxygenMembranePolarographicDetector(O2-MPD,RankBrothersLtd.,Cambridge,England).TotalcellnumberwasdeterminedbylightmicroscopyusingaHelberchamber(S.D.10%).Todetermineviablecellnumber,MPNassayswereperformedbypreparingserial10-folddilution(1to10À10)andtransferringdilution10À5to10À10eachinﬁveglasstubeswithmineralmedium[4].Thetubeswereincubatedonarotaryshaker(100rpm)at308C.After3and6weeksthetubesweretestedfornitriteproduction.NitritewastakenasindicationforthepresenceofviablecellsofN.europaeaandtheresultswereusedtocalculatetheviablecellnumber(S.D.22%).TheMPNmethodtendstounderestimatethecellnumber,especiallywhenbacteriaformaggregates.Inthisstudy,cellsuspensionswithplanktonicsinglebacteriawereanalyzed.ThemaximumcellnumbermeasuredviaMPNwasalmostashighasthetotalcellnumber(Helberchamber),givingevidencethatanunderestimationofviablecellnumberdidnotoccur.3.Resultsanddiscussion3.1.ActivityandviabilityofN.europaeaduringdiscontinuousfermentationInitialexperimentsweredesignedtoinvestigateSAandviabilityofN.europaeaduringdiscontinuousfermentation.Thefermentorwasinoculatedwith1.2Â108N.europaeacellsmlÀ1.AfterinoculationtheSAincreasedsigniﬁcantlyreachingitsmaximumof2700nmolO2(mgprotein)À1minÀ1after9h(Fig.1a).Thetotalcellnumberstartedtoincreaseafterinoculation,butthenumberofviablecellsincreasedsigniﬁcantlyfasterandtheratioofviabletototalcellschangedfromabout0.02(0h)to0.95(18h)(Fig.1a).At9htheSAandat18hthenumberofviablecellsreachedtheirrespectivemaximumandstartedtodecreaseafterwards.Therefore,‘‘ﬁttest’’bacteriawithregardtoSAandviability(generationtimeofabout15h)werepresentinearlyexponentialgrowthphase.Inlategrowthphaseandearlystationaryphase,SAandviabilityofthebacteriaweresigniﬁ-cantlyreduced(Fig.1a).Inotherstudies,SAofabout1500nmolO2(mgprotein)À1minÀ1[29]and1670nmolO2(mgprotein)À1minÀ1[17]werereported.Thedataﬁtsresultspresentedinthisstudy,whereaSAofabout1500nmolO2(mgprotein)À1minÀ1wasdetectableinearlystationaryphase(Fig.1a).N.europaeareachedstationaryphasewithin50h(Fig.1).Ammonium(76mM)wasmainlyconvertedintonitrite(about60mMnitritewereproduced)(Fig.1b).Nitratewasnotdetectable.Onlyabout1%oftheconsumedammoniumisincorporatedintobiomass[36].Therefore,thedifferencebetweenconsumedammoniumandproducednitritewasmainlyconvertedintogaseousN-compounds(N-loss;[37])andN-losswasapproxi-mately20%.Fig.1.N.europaeagrowninafed–batchfermentationfor48hina30-l-laboratoryscalefermentor.(a)Totalcellnumber(~),viablecellnumber(~),andspeciﬁcactivity(*);(b)ammonium(&)andnitrite(^)concentrationinthefermentor.518¨ven,I.Schmidt/ProcessBiochemistry44(2009)516–520D.GuProductionofN.europaeaindiscontinuousfermentationhastwomajordisadvantages.First,highestSAandviabilityofthebacteriawereobservedinearlyexponentialgrowthphase(Fig.1a).Atthismomentoffermentationtheamountofbiomasswaslowandharvestingbacteriaforfurtherexperimentsorapplicationswouldonlyprovidesmallamountsofbiomass.Anincreasedfermentorvolumewouldberequiredtocompensateforthelowbiomassconcentration.Second,highestSAwasalwaysdetectablebeforethenumberofviablecellsreacheditsmaximum.Asaconsequence,itisnotpossibletogenerateN.europaeabiomasswhichhasoptimalSAandviability.3.2.GrowthlimitingfactorsforN.europaeaduringdiscontinuousfermentationExponentialgrowthphaseofN.europaeaindiscontinuousculturewasshort,becausethenumberofviablecellsdecreased(Fig.1a),andwasfollowedbyanon-exponential(linear)growthphase.Itwasassumedthatagrowthlimitingfactorcausedtheendofexponentialgrowth.Aseriesofdiscontinuousfermentation’swereperformedtoidentifygrowthlimitingfactorsandtoverifywhethergrowthcanbeimproved(extendedexponentialgrowthphase)bychangingthesegrowthfactors.Ammoniumandoxygen(energyconservation),CO2(assimilation),traceelements(saltsandmetals),andnitrite(toxicproduct)concentrationswerevariedandtheeffectonlagandexponentialgrowthphaseaswellasontheratioofviabletototalcellnumberwasdetermined(Table1).FromdatapresentedinTable1,itisevidentthatthedurationoflagphasewassimilarunderstandardconditionsaswellasinthepresenceofincreasedCO2,traceelements,orammoniumconcentration.Lagphasewasextendedwhennitritewasremovedfromthemediumoroxygenconcentrationwasincreased.Thelongestexponentialgrowthphasewasobtainedwhenthecellsuspensionwasaeratedwithairsupplementedwith3%CO2.Alsoaerationwithaircontaining0.5%CO2orapplicationofmediumwithanincreasedconcentrationoftraceelementsledtoaprolongedexponentialgrowth.Highestviablecellnumber(4.5Â108cellmlÀ1)andhighestratioofviabletototalcells(0.92Æ0.03)wereobtainedattheendofexponentialgrowthphasewhenthefermentorwasaeratedwithanair–CO2(97:3%)mixture(Table1).Increasedammoniumortraceelementconcentrationsledtoanextendedexponentialgrowthphase,aswell.Increasing

ammoniumconcentrationresultedinanextendedexponential,butmainlyinalongernon-exponentialgrowthphase.ToxiceffectsofnitriteonactivityandgrowthofN.europaeawerereported[31,33],butcouldnotbeveriﬁedinthisstudy.Incontrast,alownitriteconcentrationduringfermentationresultedinalongerlagandshorterexponentialgrowthphase(Table1).TheremovalofnitriteduringcultivationprohibitedaerobicdenitriﬁcationofN.europaea.DenitriﬁcationhadbeenshowntocontributetoenergyconservationofN.europaea[30,38]andremovalofnitritenegativelyaffectedgrowthofthebacterium(Table1;[30]).

3.3.ActivityandviabilityofN.europaeaduringcontinuousfermentationDuringdiscontinuousfermentationbacteriahadhighestSAandviabilityinearlyexponentialgrowthphase.Attheendofexponentialgrowththebiomassconcentrationwashigh,butSAandviabilitywerenotoptimal.Continuousfermentationwastestedasanoptiontosolvetheproblemsofdiscontinuousfermentation.Acontinuousfermentorwasoperatedatdilutionratesof0.3,0.65,or1.18dayÀ1,andSAandviabilityofN.europaeawereinvestigated.Atthelowestdilutionrate,SAwasonlyatabout1000nmolO2(mgprotein)À1minÀ1(Fig.2a)andtheproportionofviablecellswaslow(ratioofviabletototalcellsabout0.3).Increasingthedilutionrateto0.65dayÀ1,SAwasstronglyincreasedandwithabout2300nmolO2(mgprotein)À1minÀ1almostreachingthehighestvaluesobservedindiscontinuousfermentation.Never-theless,theratioofviabletototalcells(about0.6)wasstillsigniﬁcantlybelowthehighestvalueindiscontinuousfermentation(0.9).Furtherincreasingthedilutionrateto1.18dayÀ1,SAwasonlyslightlyincreased(2500nmolO2(mgprotein)À1minÀ1),buttheratioofviabletototalcellsreachedavalueofabout0.9(Fig.2a).Hence,adilutionrateof1.18dayÀ1weresuitabletoproducebacteriawhichhadapproximatelythesamequalitywithregardtoSAandviabilityasbacteriainearlyexponentialgrowthphaseduringdiscontinuousfermentation.Throughoutfermentationammoniawasconvertedmainlyintonitrite(Fig.2b).Nitratewasnotformed.From55mMammonium,onaverage45mMnitritewasproduced.TheN-losswasabout18%,whereaswithincreasingdilutionratethenitriteconcentrationdecreasedandconsequentlytheN-lossincreased(approximately24%atdilutionrateof1.18dayÀ1)(Fig.2b).Table1OptimizationofN.europaeagrowthinafed-batchfermentationbyvaryingtheCO2supplementation,traceelement,nitrite,ammoniumoroxygenconcentration.Thedurationofthelag,exponentialandnon-exponentialgrowthphasearegivenaswellasthemaximumtotalandviablecellnumber.Theresultsaremeanvaluesofthreereplicatedexperiments.Cellnumber:S.D.forthetotalcnwasÆ6%,forthelivingcellnumberÆ14%(MPN).GrowthconditionLagphase(h)4Æ14Æ13Æ14Æ13Æ16Æ212Æ4

Exponentialgrowthphase(h)21Æ228Æ234Æ424Æ317Æ38Æ2

0Non-exponentialgrowthphase(h)26Æ310Æ18Æ124Æ254Æ1248Æ1095Æ15

Maximumviablecna(Â108cnmlÀ1)2.02.94.52.31.50.9n.d.Ratioofviabletototalcnb(cnmlÀ1)0.81Æ0.040.88Æ0.030.92Æ0.030.73Æ0.040.69Æ0.050.70Æ0.04n.d.Maximumtotalcnc(Â108cnmlÀ1)4.14.04.53.97.93.12.7Ratiooflivingtototalcnd(cnmlÀ1)0.22Æ0.040.85Æ0.07n.d.0.35Æ0.050.29Æ0.040.25Æ0.020.1Æ0.04

Standard+CO2e+CO2f+Traceelementsg+NH4+hÀNO2Ài+O2jcn:cellnumber;n.d.:notdetermined.aThemaximumlivingcellnumberwasdetectableattheendoftheexponentialgrowthphase.bTheratiobetweenlivingandtotalcellnumberwascalculatedattheendoftheexponentialgrowthphase.cThemaximumtotalcellnumberwasdetectableattheendofthenon-exponential(linear)growthphase.dTheratiobetweenlivingandtotalcellnumberwascalculatedattheendofthenon-exponential(linear)growthphase.eTheCO2concentrationintheairappliedtoaeratethefermentorcontained0.5%CO2.fTheCO2concentrationintheairappliedtoaeratethefermentorcontained3%CO2.gThetraceelementconcentrationinthemediumwasdoubled.AthreetimesincreasedtraceelementconcentrationledtolongerlagphaseandasigniﬁcantslowergrowthofN.europaea.hTheammoniumconcentrationatthestartofthefermentationwasdoubledto150mM.iNitritewasremovedbycross-ﬂowﬁltration.Nitriteconcentrationinthefermentorwasalwaysbelow0.2mM.jTheoxygenconcentrationwasadjustedat7.5Æ0.5mgmlÀ1.

¨ven,I.Schmidt/ProcessBiochemistry44(2009)516–520D.Gu519

Fig.2.N.europaeagrowninachemostatfor1032h.0–240hadaptationofthechemostattoadilutionrateof0.3dayÀ1,240–456hsteadystate0.3dayÀ1;456–624hadaptationto0.65dayÀ1,624–792hsteadystate0.65dayÀ1;792–912hadaptationto1.18dayÀ1,912–1032hsteadystate1.18dayÀ1.(a)Totalcellnumber(~),viablecellnumber(~)andspeciﬁcactivity(*);(b)ammonium(^),nitrite(&)andO2(*)concentrationinthefermentor.Thechemostatwasstartedwithabout20mMammonium.Thenitriteconcentrationatthebeginningoftheexperimentoriginatedfromthepre-culture(20%inoculum).Thechemostatwasthenoperatedwithamediumcontaining55mMammoniumwithoutnitrite.Adifﬁcultyinthestart-upofacontinuousfermentorbecameobviouswhensystemswereimmediatelystartedatdilutionratesabove1dayÀ1(Fig.3).Intheﬁrst100h,SAincreasedtoamaximumvalueof2900nmolO2(mgprotein)À1minÀ1,butdidnotstabilizeonthislevelanddecreaseddramaticallyinthefollowing48halongwithadecreaseofthetotalcellnumber(wash-outofbacteria).DuringfermentationanN-lossupto40%wasdetectable(Fig.3).HighN-lossesareanindicationforanunbalancedgrowthandusuallyN.europaeacellsdiewithinashortperiodoftime(4–6days)([30];Fig.3).Obviously,N.europaeacellswerenotabletomaintaininasystemstartingatahighdilutionrate.Torecoverthesystem,thefermentorwasturnedintoadiscontinuousoperationfor20h(betweenhours175and195)tokeepthebiomassinthefermentorandtoverifywhetherviablecellswerestillpresentwhichisindicatedbyanincreasingcellnumber(Fig.3).Thenthefermentorwasturnedbackintocontinuousoperationwithadilutionrateof0.35dayÀ1.DuringthisperiodtheSAslowlyincreasedtoÀ1À1730nmolO2(mgprotein)min.Increasingthedilutionrateto0.6dayÀ1resultedinafurtherincreasedSAtoabout970nmolO2(mgprotein)À1minÀ1.Changingthedilutionrateto0.9dayÀ1for5daysandthento1.3dayÀ1,bacteriarecoveredactivityandviabilitycompletely.Simultaneouslywiththeactivitytheproportionofviablecellsincreasedfrom0.1(260h),0.3(413h),0.4(559h),0.7(603h),to0.9(0.9h).Therefore,recoveryofSAinthefermentorsystemwasmainlyduetotheremoval(wash-out)ofinactivebiomass.Fig.4.Chemostatsinsteadystateconditionsatdifferentdilutionrates.Totalcellnumber(~),doublingtimeofthetotalcellnumber(^),andSA(&).Calculatingtheactivityonthebasisofproteincontentofviablecells,optimalSA(about2300nmolO2(mgprotein)À1minÀ1)wasalreadyrecoveredbetween400and450hofthecontinuousfermentation(Fig.3).Anyhow,theresultsshowtheimportanceofstart-upconditionstoobtainnotonlyhighlyactive,butalsoviableandconstantlygrowingN.europaeacells.FurthercontinuousfermentationswereperformedtoevaluatesuitabledilutionratestogrowN.europaeawithoptimal(highest)SA(Fig.4).Dilutionratesbetween1and1.8dayÀ1provedtogeneratebiomasswithspeciﬁcactivitiesabove2500nmolO2(mgprotein)À1minÀ1.Aminimaldoublingtimeof0.55daywasobservedforN.europaeainthiscontinuoussystem(generationtimeofabout13h).AtlowerdilutionratestheSAwassigniﬁcantlybelowoptimumandathigherdilutionratesthebacteriacouldnotfurthergrowfasterandwerewashed-out.4.ConclusionsTheresultsofthisstudyshowthatitispossibletoproducemaximumactiveorviablecellsofN.europaeaindiscontinuousfermentation,especiallywhengrowthlimitingfactorshavebeenidentiﬁedandgrowthconditionswereoptimized.Unfortunately,optimalactivityandviabilitywereneverobservedatthesametime.Furthermore,theproducedamountofbiomasswaslimitedindiscontinuousfermentation,becauseitmustbeharvestedinearlyexponentialgrowthphase.Incontinuousfermentationbiomassofoptimalquality(speciﬁcactivityandviability)wasproducedinhighamountsandbacteriaFig.3.N.europaeagrowninachemostatfor680h.0–100hdilutionrateof1.18dayÀ1,100–175hwash-out;175–195hbatchmode,195–440hdilutionrateof0.35dayÀ1;440–560hdilutionrateof0.6dayÀ1;550–600hdilutionrate0.9dayÀ1;600–680hdilutionrateof1.3dayÀ1.Speciﬁcactivity(~),dilutionrate(À),andN-loss(*).520¨ven,I.Schmidt/ProcessBiochemistry44(2009)516–520D.Guwereavailablewheneverrequired.Modiﬁcationofthedilutionratefurtherallowedcontroloverthequality(speciﬁcactivity,viability)ofthebacteria.Adisadvantageofacontinuousfermentationisthesigniﬁcantlyhighertechnicaleffortthatisrequiredtooperatesuchfermentorsystems.Acknowledgements

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