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168Y.-H.Changetal./Fuel97(2012)166–173

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).

玉米芯分批补料发酵工艺优化

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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

玉米芯分批补料发酵工艺优化

170Y.-H.Changetal./Fuel97(2012)166–173

2dofincubation,andthenremainedsteadythereafter.Thesere-sultsindicatedthattheyeastgrewwellonthecellulosichydro …… 此处隐藏：12739字，全部文档内容请下载后查看。喜欢就下载吧 ……