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EnhancedethanolproductionbyfermentationofricestrawhydrolysatewithoutdetoxificationusinganewlyadaptedstrainofPichiastipitisChiung-FangHuang,Ting-HsiangLin,Gia-LuenGuo*,Wen-SongHwangCellulosicEthanolProject,InstituteofNuclearEnergyResearch,ExecutiveYuanNo.1000,WunhuaRd.,JiaanVillage,LongtanTownship,TaoyuanCounty32546,Taiwan,ROCarticleinfoArticlehistory:Received15October2008Receivedinrevisedform19February2009Accepted20February2009Availableonline5April2009Keywords:RicestrawPichiastipitisAdaptationNaOHneutralizationOverlimingdetoxificationabstractAnenhancedinhibitor-tolerantstrainofPichiastipitiswassuccessfullydevelopedthroughadaptationtoacid-treatedricestrawhydrolysate.TheethanolproductionobtainedbyfermentationofNaOH-neutral-izedhydrolysatewithoutdetoxificationusingtheadaptedP.stipitiswascomparabletofermentationofoverliming-detoxifiedhydrolysate.TheethanolyieldusingtheadaptedP.stipitiswithbothtypesofhydrolysateatpH5.0achieved0.45gpgs1,whichisequivalentto87%ofthemaximumpossibleethanolconversion.Furthermore,thenewlyadaptedP.stipitisdemonstratedsignificantlyenhancedtolerancetosulfateandfurfuraldespitethefactthatbothinhibitorshadnotbeenremovedfromthehydrolysatebyNaOHneutralization.Finally,theethanolconversioncouldbemaintainedat60%andabovewhentheneutralizedhydrolysatecontained3.0%sulfateand1.3gL1furfural.2009ElsevierLtd.Allrightsreserved.1.IntroductionTheTaiwangovernmenthasaggressivelypromotedanE3lowethanol-blendedfuelsince2007.Atthesametime,theInstituteofNuclearEnergyResearch(INER),agovernmentalorganizationinTaiwan,hasfundedthedevelopmentofcellulosicethanolpro-ductionfromlocallignocellulosicmaterials.Ricestraw,themostabundantagricultureresidueproducedinTaiwan,hasbeense-lectedasthemostfavorablelocallyavailablefeedstockforethanolproduction(Guoetal.,2008).Priortoethanolfermentationbyamicroorganism,thefeedstockneedstobeprocessedbysaccharificationtechnologyinordertore-leasefermentablesugars.Todate,dilutesulfuricacidhydrolysisisthoughttobeoneofthepromisingpretreatmentmethodsandwasextensivelyemployedinindustry.Exceptforimprovingthecon-versionofcellulosetoglucoseinfollowingenzymatichydrolysis,thispretreatmenttechnologywasabletoproduceacceptablexy-loseyieldsof75–90%byconversionofhemicellulose(EggemanandElander,2005;Mosieretal.,2005;SunandCheng,2002).Inadditiontofermentablesugars,thedegradationproductsincludeweakacidsandfuranderivatives,whicharegeneratedaspartofthepretreatmentprocess.Thesecompoundshavebeencharacter-izedasinhibitorsofmicrobialgrowthandtheseseemtoaffectthefermentationperformance.Therefore,thedetoxificationofthelig-nocellulosichydrolysateortheuseofaninhibitor-tolerantmicro-organismisusuallyrequiredforsuccessfulhydrolysatefermentationprocess(Almeidaetal.,2007;PalmqvistandHähn-Hägerdal,2000a;SánchezandCardona,2008).Overlimingisawell-establisheddetoxificationmethodthathasbeenappliedwidelytotheacidpretreatmentofhydrolysateforethanolproduction.Thisprocesshasbeendemonstratedtohelpwiththeremovalofvolatileinhibitorycompoundssuchasfurfuralandhydroxymethylfurfural(HMF)fromthehydrolysate(Martinezetal.,2000;Ranatungaetal.,2000).However,overlimingisnotaneffectivewayofreducingtoxicitycausedbyorganicacidslikefor-micacidandaceticacid(Larssonetal.,1999;Ranatungaetal.,2000;PalmqvistandHähn-Hägerdal,2000a).Furthermore,over-limingtreatmentusuallyleadstolossofsugars,whichreducestheefficiencyofthefermentation;thislossofsugarsismostlycausedbytheirdestructionduringthestageintheprocesswherethepHissignificantlyelevated(Martinezetal.,2000;Nilvebrantetal.,2003).Afurtherproblemassociatedwithoverlimingistheneedforadditionalfacilitiesandprocesssystemstoremovethegypsumprecipitatethatisproducedasabyproduct;thissignifi-cantlyincreasesthecapitalinvestmentneededfortheethanolpro-ductionplant(CardonaandSánchez,2007).Thus,fromtheeconomicviewpoint,thedevelopmentofarobustmicroorganismthatisabletofermenthydrolysatetoethanolwithoutdetoxifica-tionwouldbehighlyimportantandveryuseful.Anumberofyeastspecies,includingPichiastipitis,Candidashe-hataeandPachysolentannophilus,havebeenfoundtobehighlyeffi-cientxylose-fermentingstrainsthatcanbeusedinethanolproduction(Agbogboetal.,2006;JeffriesandJin,2000;Sánchez0960-8524/$-seefrontmatter2009ElsevierLtd.Allrightsreserved.doi:10.1016/j.biortech.2009.02.064*Correspondingauthor.Tel.:[phone].E-mailaddress:[e-mail](G.-L.Guo).BioresourceTechnology100(2009)3914–3920ContentslistsavailableatScienceDirectBioresourceTechnologyjournalhomepage:).Amongthem,P.stipitisexhibitsthegreatpo-tentialforindustrialapplicationbecausethisspeciesproducesahighethanolyieldandhasnorequirementforaddedvitaminsdur-ingthefermentation(Agbogboetal.,2006;Nigam,2001a).How-ever,P.stipitisissensitivetoorganicacids,includingaceticacid,whicharepresentinlignocellulosichydrolysate.Thesecompoundsreducecellgrowthanddiminishedethanolproduction.Asaresult,increasingthetoleranceofthisyeasttosuchinhibitorswouldbebeneficialtothedevelopmentofP.stipitisaspartofcellulosiceth-anolproduction(Almeidaetal.,2007;Nigam,200