Comparison of batch and fed-batch fermentations using corncob hydrolysate for bioethanol production

玉米芯分批补料发酵工艺优化

Comparisonofbatchandfed-batchfermentationsusingcorncobhydrolysateforbioethanolproduction

Yi-HuangChanga,Ku-ShangChanga,Cheng-WeiHuangb,Chuan-LiangHsuc, ,Hung-DerJanga,

a

DepartmentofFoodScience,YuanpeiUniversity,Hsinchu300,TaiwanInstituteofFoodScience,YuanpeiUniversity,Hsinchu300,Taiwanc

DepartmentofFoodScience,TunghaiUniversity,Taichung407,Taiwan

b

articleinfoabstract

Theoptimalconditionsforthemaximumproductionofethanolfromcellulosichydrolysateinbatchandfed-batchcultureswereinvestigatedandcompared.Thepretreatedcorncobcouldbeconvertedintoreducingsugarwithmaximalyieldsaftertheenzymemixtureswerefed.After48hofhydrolyticreaction,overallreducingsugarandglucoseconcentrationsreached0.61and0.36g/gdriedsubstrate,respectively.FurtherbatchfermentationofcellulosichydrolysatewasperformedusingbatchculturesofSaccharomy-cescerevisiaeBCRC21812,23.3–41.1g/lbiomassand6.9–23.0g/lethanolwasobtained.Forthefed-batchfermentation,theeffectsoffeedingglucoseconcentrationsonethanolfermentationwerestudied.Thefeedingglucoseconcentrationof30g/lresultedinahigherethanolyieldthanthatof20g/land10g/ldid.Thecellbiomass,ethanolyields,andethanolconversionrateforthefed-batchfermentation,feedingat30g/lglucoseconcentration,were44.5g/l,32.3g/land0.64gethanol/gglucose,respectively.Theresultsindicatethatthefed-batchfermentationhadahigherethanolyieldthanthatofthebatchfermentation.

Ó2012ElsevierLtd.Allrightsreserved.

Articlehistory:

Received31January2011

Receivedinrevisedform5February2012Accepted6February2012

Availableonline18February2012Keywords:

CellulosichydrolysatesBatchfermentation

Fed-batchfermentationBioethanol

1.Introduction

Tolessentheworld’sdependenceonnon-renewableresources,useofagriculturalbiomassfortheproductionofbiofuelssuchasbioethanolhasdrawnmuchattentiontomanyresearchersinthepastfewdecades.Cellulosicbiomassisanidealsourceofenergybecauseitisbothrenewableandavailableinlargequantitiesaroundtheworld.However,theprocessfortheproductionofethanolfromcellulosicmaterialsismorecomplicatedthanitsproductionfromsugarorstarch-basedones.Speci cally,therearetechnicalandeconomicalimpedimentsinregardstothedevel-opmentofcommercialprocessesthatutilizecellulosicbiomass.Technologiesthatwillallowforthecost-effectiveconversionofcellulosicbiomassintofuelsandotherchemicalsarebeingdevel-oped.Thesetechnologiesincludelow-costthermoorchemicalpre-treatment,highlyeffectivecellulasesandhemicellulases,andef cientandrobustfermentativemicroorganisms,havemadethecommercializationofbiofuelproductionmuchmorepromising[1–3].

Forproductionofbioethanol,alowerrawmaterialprice,togetherwithahighethanolyieldandef cientenzymes,willde-creasetheproductioncostsigni cantly.Severaldifferentpretreat-mentmethodshavebeenusedtofacilitatetheenzymaticCorrespondingauthors.Tel.:+886423590121x37306;fax:+886423599059

(C.-L.Hsu),tel.:+88635381183x8482;fax:+88636102342(H.-D.Jang).

E-mailaddress:hungder@mail.ypu.edu.tw(H.-D.Jang).

0016-2361/$-seefrontmatterÓ2012ElsevierLtd.Allrightsreserved.doi:10.1016/j.fuel.2012.02.006

hydrolysisoflignocellulosicmaterial[4].Anef cientprocessforobtainingreducingsugarsfromlignocellulosicmaterialistousechemical/physicalpretreatment,followedbyenzymatichydroly-sis.Thehydrolysisofnaturallignocellulosetoglucosedependsonthesynergyofenzymessystem,i.e.,b-1,4-endoglucanase,b-1,4-exoglucanaseandb-glucosidase[5],andb-1,4-endoxylanase.Thesecellulolyticenzymeshavebeenappliedtoincreasethehydrolysisef ciencyofcellulosicmaterials[6].

Manydifferenttypesofprocessesforethanolfermentationhavebeenproposed,includingbatchfermentation,continuousfermen-tation,continuousfermentationwithcellrecycling,fed-batchcul-turesandrepeated-batchcultures[7].Batchfermentationprocessisusedextensivelytoconvertsugarstoethanolfortheproductionofbeveragesandbiofuels.Asforfed-batchfermentation,theinter-mittentadditionofglucose,withouttheremovalofthefermenta-tionbroth,intothefed-batchcultureisoneofthemostcommonmethodsfortheproductionofethanolintheindustry.Theadvan-tagesofthisprocessincludethereductionofsubstrateandend-productinhibition,higherproductivityofethanol,higherdissolvedoxygenrate,decreasedfermentationtime,andhighersacchari ca-tionrate[8].Fed-batchfermentationhasbeenreportedasagoodprocessforethanolproductionwhenperformedondifferentrawmaterialssuchascornstover[9]andrecycledpaper-derivedmaterial[10].

Evenconversionofcorncobhydrolysatetobioethanoleitherbybatchfermentation[1,11]orbyfed-batchfermentation[12,13]separatelyhasbeenstudiedinthepast,thesimultaneous

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comparisonoftheef cienciesofbothbatchandfed-fermentationsbythesamecellulosichydrolysatewererare.Additionally,theim-pactofdifferentfermentationtreatments,i.e.batchandfed-batch,ofcellulosichydrolysateonthedynamicsofmicrobialgrowthandethanolproductionrateintherespectivefermentorswereseldomevaluated.Inthiswork,thehydrolysisprocessofthecorncobsub-strateusingpretreatmentwithacid,autoclavingandthenhydroly-siswiththeenzymemixtureswasexamined.Thefermentationprocessesandkineticsofthecorncobhydrolysateinthebatchandfed-batchculturesofSaccharomycescerevisiaeBCRC21812werealsocompared.

Acidpretreatmentwasperformedwith1%(v/v)sulfuricacidfor30minatasolid-to-liquidratioof1:10.Themixturewas ltered.Thenthe ltratewasfurtherhydrolyzedbyautoclavingat121°Cfor60min,accordingtotheproceduresdescribedinourpreviousreport[14].Afterthepretreatment,thecellulosicresiduewassoakedindistilledwaterandincubatedinwaterbathat50°Cfor30min,andthen ltered.

2.2.Microorganismsandcultivation

ThestrainofS.cerevisiaeBCRC21812,purchasedfromBiore-sourcesCollectionandResearchCenter,FIRDI(Hsinchu,Taiwan),wasusedasaninoculumforethanolfermentation.S.cerevisiaeBCRC21812istraditionallyusedforalcoholicbeverageandbioeth-anolproduction.IthadbeenfoundthatS.cerevisiaeBCRC21812couldgrowwellinYPDmediainthepresenceof8%(v/v)ethanolaccordingtoourpreviouspreliminarystudy.YeastculturesweremaintainedinaYPDmediumcontaining2%(w/v)dextrose,1%(w/v)peptoneand0.5%(w/v)yeastextractat25°Cfor48h.TheinitialpHwasadjustedto6.5either1NHClorNaOHpriortoster-ilizationat121°Cfor20min.

2.3.Hydrolysisofcellulosicresiduebytreatmentwiththeenzymemixture

Themixtureofprehydrolysateobtainedfromtheacidandauto-clavingpretreatmentswascollectedand lteredwithWhatman

2.Materialsandmethods

2.1.Cellulosicmaterialandpretreatment

Thecorncobmaterials,purchasedfromalocalmarket,wereoven-driedfor24hat50°C,groundedintoparticles(diameter2–10mm)andstoredinpillvialsat25°C.Thecorncobmaterialsconsistedmainlyof42%(w/w)celluloseand28%(w/w)hemicellu-lose,whichcouldbehydrolyzedtoreducingsugar.Inaddition,therewas20%oflignininthecorncobmaterials.Therestwereashesandminorcomponents.Thestructuralcarbohydrateandlig-nininbiomassweredeterminedaccordingtoStandardBiomassAnalyticalProceduresofNationalRenewableEnergyLaboratory(NREL).

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No.4 lterpaper.Acommercialcellulasemixture,1.5ml(1000IU/ml)Cellulase(Sigma,StLouis,MO,USA)supplementedwith0.52ml(250IU/ml)Novozyme188(Sigma,StLouis,MO,USA),wasusedtohydrolyzethecellulosicresidue.Enzymatichydrolysiswasperformedwitha100mlprehydrolysateandthecommercialcellulasesolution.Theprotocolsofenzymatichydrolysisofprehy-drolysatetoproducereducingsugarwerealsoaccordingtoourpreviousreport[14].Themixtureswereincubatedat50°Cinanorbitalshakerwithaspeedof160rpmfor72h.Sampleswerewithdrawnandanalyzedforlevelsoftotalreducingsugar,glucose,xylose,andcellobioseconcentration.

2.4.Batchandfed-batchfermentationofcellulosichydrolysateInoculumwaspreparedbytransferring5%(v/v)ofthecells(108/ml)ofS.cerevisiaeBCRC21812intofermentationmedia.Themediuminthebatchethanolfermentationwas(%,w/v):cellu-losichydrolysate,1–4;peptone0.5;yeastextract0.25atpH6.0.Themediumforfed-batchfermentationwas2%cellulosichydroly-sate,0.5%peptoneand0.25%yeastextract.After24h,thecellulosichydrolysatecontaining1–3%(w/w)glucosewasfed.Thecultureswereshakenat150rpmfor2d,thenadjustedto100rpmat25°C.Sampleswerecollectedregularlyand lteredthrougha0.45lmMilliporemembrane.Glucose,xylose,cellobioseandeth-anolconcentrationswereanalyzedbyHPLC(WatersCo.,MA,USA).2.5.Analysismethods

Thedryweightcontentoftherawmaterialswasdeterminedbydryingsamplesfor24hat110°C.Sampleswerewithdrawnfromthefermentationbroth,andyeastbiomasswasdeterminedbymeasuringcellopticaldensityrecordedwithaUltrospec2100

prospectrophotometersetat600nm(GEHealthcareCo.,IL,USA).Thereducingsugarsliberatedbythesereactionsweremea-suredusingthe3,5-dinitrosalicylicacidmethod[15],withglucoseasstandard.Reducingsugarwascalculatedasg/gdriedsubstrate(DS).

Glucose,xylose,cellobioseandethanolwereanalyzedbyHPLC(WatersCo.,MA,USA)withacationexchangerSugarpakcolumn(300Â6.5mmi.d.).Secondaryde-ionizedwater,ata owrateof0.5ml/min,wasusedasthemobilephase.Theinjectionvolumewas20llandthecolumntemperaturewasmaintainedat90°C.Allsampleswere lteredthrougha0.22lm lterbeforeundergo-ingHPLCanalysis.TheeluateoutofHPLCwasdetectedbyarefrac-tiveindexdetectorat50°C.

3.Resultsanddiscussion

3.1.PretreatmentandenzymatichydrolysisofcellulosicmaterialThecorncobsubstratesampleswerepretreatedwithautoclaveat121°Cfor60minandeitherwith1.0%(w/v)sulfuricacidfor15min(Sample2inFig.1A)orwithoutsulfuricacid(Sample1).ForSample2,afterthepretreatment,approximately0.43g/gDSofreducingsugarswasrecovered(Fig.1A).Theresultsshowthat61.4%(w/w)ofthecellulosicsubstratewasconvertedtoreducingsugarafterpretreatmentwithautoclaveandacid.NotoxiceffectsfromfurfuralsandHMFswereobservedduringthefermentationstudiesascon rmedbySumphanwanichetal.[16].Theirresultsindicatedthecorncobwastewithacid-treatmentgeneratednon-toxiclevelsoffurfurals(0.7g/l)andHMFs(0.8g/l)inthehydroly-satesforfermentation.

TheprehydrolysatewasfurtherhydrolyzedwiththereactionofenzymemixturesatpH6.0and50°Cfor72h(Sample3).

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Thereducingsugarconcentrationreached0.61g/gDSafter48hofhydrolysis(Fig.1B).However,extendingthehydrolysistimebeyond48hdidnothelpfurtherinincreasingthereducingsu-garconcentration.Fromthereducingsugarconcentrationafterhydrolysis,itindicatesthat87.1%(w/w)ofthecellulosic/hemi-cellulosiccomponentswerehydrolyzedandconvertedtoreduc-ingsugaraftertreatmentwiththeenzymemixture.Xylosewasdetectedinthehydrolysate,showingthatthepresenceofb-1,4-endoxylanaseassistedinthehydrolysisofxylaninthesubstrate.Additionally,theb-1,4-endoglucanaseandb-1,4-exoglucanasehydrolyzecellulosechainsresultedintheformationofcellobi-ose,whichcanbefurthercleavedintoglucosebycellobiase.Itwasfoundthatasigni cantlylowamountofcellobioseexistedinthecellulosichydrolysate,indicatingthatcellobiaseincreasedthehydrolysisofcellobioseintheresultingprehydrolysate.Inaddition,ahighamountofglucoseandacomparativelyloweramountofcellobioseexistedinthecellulosichydrolysate,indi-catinggoodactivitiesofb-glucosidaseinenzymemixtures.Thereby,withtheaidofenzymatichydrolysis,higheryieldsoftotalreducingsugar(0.61g/gDS),glucose(0.36g/gDS)andxy-lose(0.17g/gDS)intheresultedhydrolysateswereachieved.These ndingsindicatethattheenzymemixtureshelpedtoin-creasethehydrolysisef ciencyofthecellulosichydrolysatesandwerenecessarytoproducethemonosaccharidesforfurtherethanolfermentation.

3.2.Batchfermentationofcellulosichydrolysateforbioethanolproduction

Batchfermentationforbioethanolproductionwasperformedinthecellulosichydrolysate-basedmediacontainingvariousconcen-trationsofglucoseasthemaincarbonsource.Duetotheconcernthatthehighconcentrationofglucoseinthehydrolysatewouldin-hibitthegrowthofyeast,themaximumconcentrationofglucose(40g/l)wasused.Todeterminetheeffectofglucoseconcentrationonthegrowthpro leofS.cerevisiaeBCRC21812,batchexperi-mentswereperformedinconical askswithglucoseconcentrationinthehydrolysatesrangingfrom10to40g/l.Fig.2showstheplotsofcellbiomassandpHvaluesagainstfermentationtime.Forthehydrolysatemedium,thepHdecreasedslowlyandremainedabove5.6throughoutthe rst vedaysofthefermentationandde-creasedrapidlyfrom5.7to5.0after6dofcultivation(Fig.2A).AsreportedbyPalmqvistandHahn-Hagerdal[17],cellgrowthincellulosichydrolysatesstronglydependedonpH,duetothelargeconcentrationofdissociatedweakacidsatlowpH.ThepH,around5.0duringtheentirefermentationprocess,didnotin uencethegrowthofyeastcellsandthusfavoredtheethanolproduction.Theyeastcellbiomassincreasedfrom23.3to41.1g/lwiththein-creasedconcentrationofglucosefrom1%to4%inthehydrolysate(Fig.2B).Inaddition,aftertheinoculationofyeastcells,themicro-bialbiomassbegantoincrease,reachedthemaximalvaluesafter

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2dofincubation,andthenremainedsteadythereafter.Thesere-sultsindicatedthattheyeastgrewwellonthecellulosichydroly-satewithglucoseataconcentrationupto4%.However,cellgrowthwasgreatlyrepressedwhentheglucoseconcentrationreached4%.Speci cally,whentheglucoseconcentrationincreasedfrom3to4%,theyeastcellbiomassdidnotshowobviousincrease,i.e.from40.2to41.1g/l.Thisfactshowedthattheconcentrationofglucoseat4%wouldinhibitthegrowthofyeast,duetothehaltincellbiomass.

Fig.3showsthechangeofglucose,xyloseandcellobiosecon-centration,andethanolyieldbyS.cerevisiaecultureafter6daysascomparedtotheinitialglucoseconcentrationinthehydrolysate.Inthefermentationusing1–2%glucoseinthehydrolysate,theglu-cosewasexhaustedafter2d,whereastheethanolproductionyieldincreasedrapidlyafterthe rstdayoffermentation.Thisresultindicatesthattheglucoseconsumptionwasconsistentwiththetimeperiodofethanolproduction.AsshowninFigs.3AandB,theglucosewasrapidlyusedupbytheyeastwithin2d,with1–2%glucoseinthehydrolysate.However,1.2–4.1g/lxyloseand1.7–3.3g/lcellobioseweredetectedandcouldnotbeutilizedbytheyeastcellsafter2d.Thefermentationwascompletedafter2d.Themaximalconcentrationsofethanolwere6.9and8.5g/lfortheculturesof1%and2%glucoseinthehydrolysate,respec-tively,whentheglucosewereusedup.ThefermentationresultssuggestthatS.cerevisiaecouldgrowwellinthehydrolysatemed-iumandachievevirtuallycompleteconversiontoethanolfromglucoseinthehydrolysate.However,8–10g/lofglucosewasnot

utilizedbytheyeaststrainafter6doffermentationwhentheglu-coseconcentrationsinthehydrolysatewere3–4%(Fig.3CandD).Furthermore,considerablequantitiesofxyloseandcellobiosewerealsodetected,whichcouldnotbeutilizedbytheyeastcells.Whenthesubstrateconcentrationreached4%,yeastbiomassandethanolyieldwerenotsigni cantlyincreased,suggestingthataconsider-ableinhibitoryeffecthadoccurred.Inaddition,theethanolyieldswere18.3and23.0g/lwhentheinitialglucoseconcentrationsinthehydrolysatewere3%and4%,respectively.Besides,rateoftheconversionofglucosetoethanolwas0.58–0.61gethanol/gglu-cose,using3–4%glucoseinthehydrolysate,asigni canthigheramountthanthat(0.45gethanol/gglucose)ofYuandZhang[1].TodevelopanimprovedculturemethodforethanolproductionwithS.cerevisiae,batch askcultureswere rstlycarriedouttodeterminethesuitablesubstrateconcentrationoftheinitialmedia.ItwasfoundthatS.cerevisiaegrewwithasimilarpatterninglu-coseconcentrationsupto4%,indicatingagoodabilitytodealwithosmoticstress.Thismadeitpossibletofeedconcentratedglucosesolutioninadiscontinuouswayduringthefed-batchfermentation.3.3.Fed-batchfermentationofcellulosichydrolysateforbioethanolproduction

Itrequiresahighinitialsugarconcentrationinthecellulosichydrolysatetoobtainhighconcentrationsofethanolinthefer-mentationbroth.However,highersugarconcentrationinthehydrolysateoftencausesmixingandheattransferproblems,due

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totherheologicalpropertiesofaverydense broussuspension[7].Suchproblemscouldbeeffectivelyavoidedinthefed-batchfer-mentationprocess,wherethesubstrateisaddedgraduallyandtheviscosityofthereactionmixturecanbekeptatalowlevel.Theglucoseconcentrationinthehydrolysateincreasedfromaninitial2%to3%byadditionof1%onthe rstdayinthefed-batchprocess(Fig.4).Itwasobservedthatthecellbiomassconcentra-tionreached22.3g/lontheseconddayoffermentationandthatthepHofthefermentationbrothslightlydecreasedfrom5.8to5.5duringthe5doffermentation.Theresidualglucoseconcentra-tioninthehydrolysatewas1.8g/l,whichwasmuchlowerthanthatofthebatchculture.Aftertheadditionofthehydrolysate,theconcentrationofxyloseincreasedfrom6to15.2g/l.Thisindi-catesthatS.cerevisiaecouldreadilyfermenttheglucoseinhydro-lysatetoethanolbutcouldnotmetabolizexylose,duetothelackofxylose-degradingenzymes.Inaddition,theresultsshowthatmostoftheglucoseinthehydrolysatewasusedinfed-batchcultures,therefore,higherconcentrationsofethanol(19.0g/l)werepro-ducedthaninbatchcultureswith3%glucoseinthehydrolysate.Fed-batchculturesshortenedthereactiontimedegradinganequivalentsubstrate,thereforeenhancingtheef ciencyofutilizingthecellulosicsubstrate.

Theresultsoffed-batchculturewithinitial2%glucoseincellu-losichydrolysateandanadditionof2%glucosewereshowedinFig.5.ThecellbiomassincreasedrapidlyaftertheinoculationofS.cerevisiaeandreachedthemaximum(42.5g/l)onthethirddayoffermentation(Fig.5A).Theglucoseinthehydrolysatewasgoingtobeusedupafter1doffermentation,andtheethanolyieldswere

only15g/l(Fig.5B).Aftertheadditionofthehydrolysate,theeth-anolyieldsincreasedandreachedthemaximumontheseconddayoffermentation.Thus,theoverallethanolyieldsandglucosecon-versiontoethanolratewereestimatedtobe24.0g/land0.60gethanol/gglucose.Theresultsoffed-batchculturewithini-tial2%glucoseinhydrolysateandanadditionof3%glucoseareshowedinFig.6.Thecellbiomassincreasedrapidlyaftertheinoc-ulationofS.cerevisiaeandreachedthemaximum(44.5g/l)ontheseconddayoffermentation;moreover,thepHofthebrothde-creasedsteadilyfrom5.8to5.2duringthefermentationprocess(Fig.6A).Afterfeedingthehydrolysatetothemedia,theethanolyieldsincreasedandreached32.3g/lattheseconddayoffermen-tation.Thus,rateoftheconversionofglucosetoethanolwasesti-matedtobe0.64gethanol/gglucose.Asproposedinthisstudy,theseresultingdatawerehigherthanthosefromthebatchculturesystem.Furthermore,rateoftheconversionofglucosetoethanolfromthefed-batchfermentationinthisstudywassigni cantlyhigherthanthat(0.44gethanol/gglucose)ofthestudyusingthebatchcultureofCandidatropicalis[18].

4.Conclusions

Asaneconomicalwaytoproduceethanolfromcellulosicsub-strate,synergetichydrolysisofcellulaseandxylanasemixturescreatesafeasibleprocessthatcanbeusedintheproductionofbio-ethanol.Bioethanolproductionwithfed-batchfermentationoffersadvantagesoverthatwithbatchfermentation.Theconversionrate

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ofethanolfromglucosewashigherinfed-batchfermentationthanitwasinbatchfermentation.Moreover,thesubstrateinhibitionef-fectsoncellbiomassandyieldsofethanolwerelesspronouncedforfed-batchfermentationthanbatchfermentation.Furtherworkshouldbefocusedonscale-upoffed-batchfermentationtomaketheprocessindustriallyfeasible.Acknowledgment

TheauthorswouldliketothanktheNationalScienceCounciloftheRepublicofChina(Taiwan)for nanciallysupportingthisre-searchunderContractNo.NSC97-2313-B-264-001-MY3.References

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