BioresourceTechnologyjournalhomepage:www.elsevier.com/locate/biortechModificationofwheatstrawligninbysolidstatefermentationwithwhite-rotfungi
MariaJ.Dinisa,RuiM.F.Bezerrab,FernandoNunesc,AlbinoA.Diasb,CristinaV.Guedesa,LuísM.M.Ferreiraa,JohnW.Coned,GuilherminaS.M.Marquese,AnaR.N.BarroscMiguelA.M.Rodriguesa,*aCECAV–UniversidadedeTrás-os-MonteseAltoDouro,DepartmentofAnimalScience,Apartado1013,5001-801VilaReal,PortugalCITAB–UniversidadedeTrás-os-MonteseAltoDouro,DepartmentofBiologicalandEnvironmentalEngineering,Apartado1013,5001-801VilaReal,PortugalcCQ–UniversidadedeTrás-os-MonteseAltoDouro,DepartmentofChemistry,Apartado1013,5001-801VilaReal,PortugaldAnimalNutritionGroup,DepartmentofAnimalSciences,WageningenUniversity,TheNetherlandseUniversidadedeTrás-os-MonteseAltoDouro,DepartmentofAgronomy,Apartado1013,5001-801VilaReal,Portugalbarticleinfoabstract
Thepotentialofcrudeenzymeextracts,obtainedfromsolidstatecultivationoffourwhite-rotfungi(Tra-metesversicolor,Bjerkanderaadusta,GanodermaapplanatumandPhlebiarufa),wasexploitedtomodifywheatstrawcellwall.Atdifferentfermentationtimes,manganese-dependentperoxidase(MnP),ligninperoxidase(LiP),laccase,carboxymethylcellulase(CMCase),avicelase,xylanaseandferuloylesteraseactivitieswerescreenedandthecontentofligninaswellashydroxycinnamicacidsinfermentedstrawweredetermined.AllfungisecretedferuloylesterasewhileLiPwasonlydetectedincrudeextractsfromB.adusta.Sincenosignificantdifferences(P>0.05)wereobservedinremaininglignincontentoffer-mentedstraw,LiPactivitywasnotalimitingfactorofenzymaticligninremovalprocess.Thelevelsofesterifiedhydroxycinnamicacidsdegradationwereconsiderablyhigherthanpreviousreportswithligno-cellulosicbiomass.ThedatashowthatP.rufa,maybeconsideredformorespecificstudiesashigherferu-licandp-coumaricacidsdegradationwasobservedforearlierincubationtimes.Ó2009ElsevierLtd.Allrightsreserved.Articlehistory:Received3February2009Receivedinrevisedform8April2009Accepted19April2009Availableonline17May2009Keywords:WheatstrawWhite-rotfungiBiodegradation1.IntroductionWheatstrawisoneofthemostabundantcropresiduesintheworld,representingaround149milliontonsperyearonEuropeaccordingtoFAO(2004).Thishugeamountofresiduesmayconsti-tuteapromisingrawmaterialthatcouldpotentiallybetrans-formedintoamoreediblefeedforruminants(Rodriguesetal.,2008)oralternativelyitcouldalsobeusedfortheproductionofethanol(Fangetal.,2002).Ineitherofthesepossibilitiesthemainconstrainttoimprovehydrolysisofthislignocellulosicmaterialisthecomplexityofthecellwallstructure.Thedegradationoftheplantcellwallisofteninefficientbe-causemostpolymersofcelluloseandhemicelluloseareeitherinsolubleortoocloselyassociatedwiththeinsolublematrix(Pan-agiotouetal.,2007).Furthermore,hydroxycinnamicacids,particu-larlyferulicandp-coumaricacid,arecovalentlyboundtocellwallpectinsandpolysaccharides(arabynoxylans,xyloglucans)through*Correspondingauthor.Tel.:+351259350422;fax:+351259350482.E-mailaddress:mrodrigu@utad.pt(M.A.M.Rodrigues).0960-8524/$-seefrontmatterÓ2009ElsevierLtd.Allrightsreserved.doi:10.1016/j.biortech.2009.04.036esterlinkagesandtolignin,mainlybyetherbonds,thusinfluenc-ingcellwallpropertiesanditsbiodegradability.Theutilizationofwhite-rotfungienzymecomplexesmaybeconsideredanalternativeresearchfieldtoincreasetheaccessibil-ityofcellwallstructure.Researchhasshownthatligninisoxidisedanddegradedbyaligninolyticsystem(Elisashvilietal.,2008;Rodriguesetal.,2008)composedbyligninperoxidase(LiP),man-ganeseperoxidase(MnP)andlaccase.Inaddition,cellulases,hemi-cellulasesandesterasesarealsoconsideredtobeextremelyimportantinthedegradationprocessoflignocellulosicbiomass(Tabkaetal.,2006;Panagiotouetal.,2007).Inthisway,wecon-siderthattheseenzymesactingtogethershouldperformacomple-mentaryactioninordertofacilitatethecompletedegradationofcellwalls.Theaimofthepresentworkwasto(i)studytheproductionofenzymecomplexesbyfourdifferentfungi–Trametesversicolor(TV),Bjerkanderaadusta(BA),Ganodermaapplanatum(GA)andPhlebiarufa(PR)–atdifferenttimesofincubationundersolidstatefermentation(SSF)ofwheatstraw;(ii)toevaluateitsinfluenceinthedegradationofesterifiedhydroxycinnamicacidsand(iii)intheremovaloflignin,themostrecalcitrantbiopolymerofplantcellwall.4830M.J.Dinisetal./BioresourceTechnology100(2009)4829–48352.Methods2.1.FungalstrainsFourfungalstrains,T.versicolor,B.adusta,G.applanatumandP.rufa,wereusedtoobtaintheenzymaticextracts.Fungiwerecol-lectedonthenorthofPortugalandweremaintainedonpotatodextroseagar(PDA)platesat4°Candperiodicallysubcultured.2.2.EnzymeproductionEnzymaticextractswereobtainedfromasolidculturemediumcontaining15gofwheatstrawwith0.05gofglucosein45mlofdeionizedwater.Incubationswerein250mlErlenmeyerflaskscontainingtheculturemediaandtwo10mmagarplugsremovedfromeachisolatedfungus.Flaskswereincubatedat27°Candfer-mentedstrawfromfourflasksofeachfunguswasharvestedevery7daysuntil28daysafterinoculation.Afterharvesting,contentsofthecultureflasksweresuspendedin150mlofdeionizedwaterandincubatedonarotaryshaker(100rpm)for3h(Hofrichteretal.,1999).Extractswerefiltered,centrifugedandaliquotswereusedtodetermineenzymeactivities.2.3.ExperimentaldesignandstatisticalanalysisAlltreatmentcombinationswerecompletedinquadruplicate.Themainandinteractioneffectsoftimeofincubationandfungalspeciesfactors(4Â4factorialdesign),enzymetimecoursepro-duction,hydroxycinnamicacidsandligninamountwereevaluatedusingPROCGLMinSAS9.1software(SASÒInc.,Cary,NC)foranal-ysisofvarianceandsignificancetests.Whensignificant(i.e.P<0.05)differenceswereobtainedusingleastsquaremeanspro-ceduresofSAStocomparemeansatthe5%levelofconfidenceusingamultiplecomparisont-test.2.4.EnzymeassaysEnzymaticactivitiesweredeterminedat25°CusingaHeliosUV–visspectrophotometer(ThermoFischerScientific,Waltham,MA,USA).Manganeseperoxidase(MnP)activitywasdeterminedaccordingtothemodifiedmethodofHeinflingetal.(1998)bytheformationofMn3+–tartrate(e238=6.5mMÀ1cmÀ1)from0.10mMMnSO4using100mMtartratebuffer(pH5)and0.10mMH2O2.LigninperoxidaseactivitywasmonitoredatpH3.0accordingtoTienandKirk(1988),andtheformationofveratr-aldehydewasmonitoredat310nm(e310=9.3mMÀ1cmÀ1).Lac-caseactivitywasdeterminedaccordingtoDiasetal.(2003)bymeasuringtheoxidationof2mM2,20-azino-bis-3-ethylbenzo-thiazoline-6-sulfonate(ABTS)bufferedwith100mMcitrate–phos-phate(pH4.0)andformationofABTScationradicalwasmonitoredat420nm(e420=36.0mMÀ1cmÀ1).AssayofferuloylesteraseswasperformedasreportedbyMastihubaetal.(2002)throughaspec-trophotometricmethodbymeasuringtheproductionof4-nitro-phenol(4NP)from4-nitrophenylferulate(4NPF),whichwasobtainedfromtheInstituteofChemistryoftheSlovakAcademyofSciences(Slovakia).After30minincubationat50°C,4NPre-leasedfrom1mMsubstrate(finalconcentration)bufferedwith100mMphosphatepH6.5wasdeterminedbyabsorbancereadingsat410nm.Absorbancewasconvertedintoconcentrationthroughastandardcurvepreparedwith4NP(0.05–0.5mM).Forcellulolyticenzymeassays,activitiesofcarboxymethylcel-lulase(CMCase)andAvicelldigestingcellulase(avicelase)weremeasuredaccordingtotheIUPAC(WoodandBhat,1988).Thereducingsugarsreleasedweredeterminedbythedinitrosalicylicacid(DNS),usingglucoseasastandard(BezerraandDias,2004).Xylanaseactivitywasdeterminedundersimilarconditionsasde-scribedabove,exceptthat1%xylansolutionwasusedasthesub-strate(Mandelsetal.,1974;Tabkaetal.,2006).2.5.DeterminationofligninandofesterifiedhydroxycinnamicacidsinwheatstrawsamplesTheacetylbromidesolublelignin(ABSL)wasusedtodeterminelignincontentofsamplesasdescribedbyFukushimaandHatfield(2001).ForthedeterminationofhydroxycinnamicacidsanadaptationoftheproceduresdescribedbyChien(1992)wasused.Samplesweremilledtoaparticlesizeof1mmandfurtherdriedat55°Cfor48handdewaxedwithtoluene/80%ethanol(2:1,v/v)inaSox-tecapparatusfor3h.Afterthis,sampleswereincubatedwith30mlofethanol(80%)at85°Cfor1hwithcontinuousagitation.Followingcentrifugation(14,000rpm)at5°Cfor15min,theresi-duewasdriedat60°Covernight.A25mgportionofthedewaxedsampleswassaponifiedwith5mlof1MNaOHfor18hunderN2at25°C.Aninternalstandardsolution(100ll)ofß-resorcylicacidinasolution(1:1,v/v)ofmethanolandwaterwasadded.Thesam-pleswereneutralizedtopH2.0with6MHCl.Theacidifiedsolu-tionwasextractedwith3Â2mlofethylether.Theextractwasdriedundernitrogenat25°C.Thefinalresiduewasredissolvedin1mlofwater:methanolandstoredinthedarkpriortoanalysisbyhigh-performanceliquidchromatography(HPLC).FortheHPLCanalysisaDionexUltimate3000withaPDAdetectorwasused.Thereversephasecolumn(Kromasil,250Â4mm,particlesize5lm,Teknokroma,Spain)wasmaintainedat30°Cduringtheruns.Thesamples(50ll)wereanalysedbygradientelutionof5%formicacid(SolventA)andmethanol(SolventB)(0min,B=5%;2min,B=5%;and65min,B=65%)ataflowrateof1ml/minandmoni-toredbetween200and400nm.PhenolicacidswereidentifiedbycomparisonofretentiontimeandUV-Spectrumofpurestandards.Calibrationcurveswereestablishedwithappropriatemixturesofferulic,caffeic,syringicandcoumaricacids.Resultsforlignin(g)andhydroxycinnamicacids(mg)areexpressedinrelationtowheatstrawinitialweight(15g).3.Resultsanddiscussion3.1.EnzymeactivitiesEnzymeproductionduringincubationoffourdifferentfungiispresentedinFig.1.MnPactivitydevelopedgraduallyovertheincubationperiodshowingmaximumvaluesforP.rufaandB.adu-sta(2.95and1.95U/ml,respectively)onday28,butnosignificantdifferenceswereobservedbetween21and28daysofincubation(Table1).Onthecontrary,MnPproductionwaslowerforG.applanatumandT.versicolorforallincubationtimes(Table1).B.adustashowedthelowestlaccaseactivityforallincubationperiods(Table1).Foralltheremainingfungilaccaseshowedmaximumactivityatday7(Fig.1)withoutanydifferencesbetweentheotherincubationperi-ods(Table1).G.applanatumshowedhigherlaccaseactivityforallincubationperiods(Table1).RegardingLiP,enzymeactivitywasonlydetectedforB.adusta,showingthesametrend(Fig.1)alreadyobservedforMnPwithincreasingvaluesuntiltheendoftheexperiment.Theproductionofligninolyticenzymesduringwheatstrawdegradationbyfungihasalreadybeenreportedbyseveralauthors(Aroraetal.,2002;Rodriguesetal.,2008;Zhangetal.,2008).How-ever,thedataregardingP.rufaandG.applanatumisbeingpre-sentedforthefirsttimeusingaculturemediumcontainingwheatstraw.LaccaseandMnPareconsideredtobethemostcom-monligninolyticenzymeswithinwhite-rotfungi(NerudandMisurcová,1996).Inseveralstudieswithdifferentwhite-rotfungiM.J.Dinisetal./BioresourceTechnology100(2009)4829–483548313.503.00G. applanatumT. versicolorP)12.50B. adusta. rufa-lm 2.00U( P1.50nM1.000.500.0007142128Time (days)0.400.35G. applanatumT. versicolor)P-1l0.30mB. adusta. rufa U0.25( e0.20sac0.15caL0.100.050.0007142128Time (days)0.600.50G. applanatum)10.40T. versicolor-lmP. rufa U0.30B. adusta( PiL0.200.100.0007142128Time (days)Fig.1.Ligninolyticenzymeactivitiesofwhite-rotfungiduringtheincubationperiod.strainsactivitiesofMnPandlaccasearepredominant(Vyasetal.,1994;Hofrichteretal.,1999;Tekereetal.,2001;Aroraetal.,2002)andourresultsalsoshowageneralpredominanceontheactivityofthesetwoenzymes.OurdataalsoshowthatMnPactiv-ityismuchhigherthanthatoflaccase.ThisisinaccordancetowhatwasreportedbyVyasetal.(1994)andHofrichteretal.(1999)whorefertomaximumlevelsofMnP10timeshigherthanthemaximumleveloflaccaseactivity.ProductionofLiPwasonlyobservedforB.adustawithageneralincreasealongtheincubationtime.WhileLiPisnotalwaysdetected(Tekereetal.,2001),Aroraetal.(2002)andVyasetal.(1994)detectedLiPforP.chrysosporiumandT.versicolorinSSFwheatstrawmedia.Onepossibleexplana-tionforthismayresideonthenatureofLiPactivitydetectionwhenusingtheveratrylalcoholoxidationassay(Aroraetal.,2002)duetothepresenceofinhibitorsorcolorinterferencebyaromaticcom-pounds(Hofrichteretal.,1999).Whileallfungitestedproducedavicelase(Fig.2)itsactivitywasverylow,comparativelywithCMCase.Therewerenosignificantdifferencesfordays7,14and21(Table1)andthemaximumvalueobserved(Fig.2)wasonT.versicoloronday28(0.03U/ml).InthecaseofCMCaseactivitythemostactiveproducerappearedtobeG.applanatum,withamaximumvalue(0.13U/ml)onday14,whileinT.versicolor(0.07U/ml),P.rufa(0.06U/ml)andB.adusta(0.02U/ml)thepeakwasobservedonday7(Fig.2).Thexylanaseactivity(Table1)wasquitedifferentforthefourfungi(P<0.05).G.applan-atumshowsafirstphaseuptothesecondharvesttime(day14)whereithadamaximumvalue(0.22U/ml),thendeclined,butitstillexistedwithalowervalueonday28(Fig.2).TheactivityofP.rufabetweenday7and14showedvaluesfairlystable(Fig.2).Finally,B.adustaandT.versicolorshowedavariableproductionforxylanasewithnoappreciablechangeoverthe28daysofthetrial.Inlinewithourfindings,CMCaseactivityusuallypresentsvaluesgreaterthanavicelaseindependentlyofculturemediumandfungalstrains(ValáškováandBaldrian,2006;Rodriguesetal.,2008).Xylanaseandcellulolyticactivitiespresentedinthisstudyarenotwithinahighrangeofvalues.However,theserela-tivelylowvaluesmustbeinterpretedinrelationtotheoverallobjectives.Infact,aspreviouslyreported(Rodriguesetal.,2008)oneofthemaingoalsinenzymeproductionforruminantfeedsshouldbethedevelopmentofenzymecomplexesthatcouldpro-motemaximumlignindegradationwithoutanextensivedegrada-tionofcelluloseandhemicelluloseThesecellwallcarbohydratescouldthenbeusedasamoreefficientenergysourcebytheanimals.Feruloylesteraseactivitywasquitesimilar(Table1)forallfungi(P<0.05)withtheexceptionofG.applanatum.Therewerenodif-ferencesbetweenday7and28(Table1).Feruloylesterasepeakactivitywasdetectedonday7inallfungiwiththeexceptionofT.versicolorwithahigheractivityonday14andP.rufawhichpre-sentedasecondpeakat21daysofincubation(Fig.2).Dataonferuloylesteraseactivityforthesefungigrownonwheatstrawisscarceoratleastitisnotavailable.GenerallythemainfungiinvolvedonferuloylesteraseproductionareAspergillusandPenicilliumanditsactivityisdependentonthesubstrateused,morespecificallywiththecarbonsource(Pana-giotouetal.,2007).Nevertheless,ourdataseemtofallwithintherangeofvaluesreportedbytheseauthorswhostudiedtheproductionofferuloylesterasebyP.brasilianumgrownonsev-eralsubstrates.ItshouldbepointedoutthatG.applanatumshowedthehighest(P<0.05)xylanaseandferuloylesteraseactivities(Table1)whicharekeyenzymesinthedegradationoflignin–hemicellulosematrix.Thepossibilityofacomplemen-taryactionbetweentheseenzymes,directlyinterrelatedtothestructuralconfigurationofplantcellwall,hasbeenevidencedbyotherauthors(Ferreiraetal.,2007;Panagiotouetal.,2007)anditwillbediscussedfurtheron.Fromthedatawehaveobtained,andsimilarlytowhatwasal-readyreportedforsomeofthesefungi(Rodriguesetal.,2008),thedifferencesbetweenenzymeactivitiesarequitehighandmustbeinterpretedastheresultofdifferentfungalstrainscultivateddur-ingdifferentperiodsofincubationand/orinthepresenceofdiffer-entgrowthmediacomposition.3.2.LigninandesterifiedhydroxycinnamicacidsPhenoliccompositionofwheatstrawcellwallispresentedinTable2.Asexpectedresultsshowedthatferulicandp-coumaricacidswerethedominantesterifiedhydroxycinnamicacidsandlig-ninconcentrationsarewithintherangeofvaluesnormallyre-portedforwheatstraw.Inspiteofusingdifferentextractionmethods,similarresultshavebeenreportedbyYosefandBen-Ghe-dalia(2000)andSunetal.(2001).4832M.J.Dinisetal./BioresourceTechnology100(2009)4829–4835Table1
Enzymeactivitiesfromfourwhite-rotfungiincubatedwithwheatstrawduring28daysinsolidstatefermentation.SourceEnzymeactivities(U/ml)MnPFungiABAGAPRTVTime(days)7142128EffectsBFungiTimeTime*fungi1.363b0.271a1.928c0.144a0.307a0.831b1.242c1.326c*********LiP0.259b0.000a0.000a0.000a0.000a0.058b0.094c0.108c*********Laccase0.004a0.216c0.049b0.062b0.157b0.049a0.061a0.062a*********Avicelase0.006a0.003a0.010b0.014c0.008a0.008a0.007a0.011b*********CMCase0.006a0.067c0.028b0.054c0.020a0.016a0.054b0.065a*********Xylanase0.041a0.132d0.063b0.112c0.025a0.106b0.109b0.107b*********Feruloyl0.018ab0.032c0.016a0.021b0.027c0.022b0.022b0.016a*********Valueswithinacolumnbearingthesamesuperscriptarenotsignificantlydifferent(P>0.05)accordingtoTukey’stest.ABA,Bjerkanderaadusta;GA,Ganodermaapplanatum;PR,Phlebiarufa;andTV,Trametesversicolor.B**P<0.01and***P<0.001.0.0450.040.0350.030.0250.020.0150.010.0050071421280.35Avicelase (U ml)Xylanase (U ml-1)G. applanatumT. versicolorP. rufaB. adusta0.30.250.20.150.10.0500714G. applanatumT. versicolorP. rufaB. adusta-1Time (days)0.182128Time (days)G. applanatumT. versicolorP. rufaB. adustaG. applanatumT.versicolorP.rufaB. adustaFeruloyl esterase (mU ml-1)0.160.1445.040.035.030.025.020.015.010.05.00.00714CMCase (U ml)-10.120.10.080.060.040.020071421282128Time (days)Time (days)Fig.2.Cellulolytic,hemicellulolyticandferuloylesteraseenzymeactivitiesofwhite-rotfungiduringtheincubationperiod.Table2
Phenoliccompositionofwheatstrawbeforeincubation.PhenoliccompositionAEsterifiedhydroycinnamicacids(mg)SyrA15.4ALignin(g)FerA101.21.76CafA3.6p-CoumA136.3SyrA,syringicacid;CafA,caffeicacid;p-CoumA,p-coumaricacid;andFerA,ferulicacid.TheeffectofSSFinphenoliccompositionofwheatstrawispre-sentedinTable3.Whilenosignificanteffect(P>0.05)wasob-servedinesterifiedp-coumaricacidcontentamongthefourfungitreatments,B.adustagavethelowestdecrease(P<0.05)inesterifiedferulicacidconcentration.ForbothesterifiedsyringicandcaffeicacidsP.rufatreatmentresultedinthehighestvalues(P<0.05)inwheatcellwallafter28daysincubation.However,alongthetimeofincubationtherewasasignificantdecrease(P<0.001)onthecontentofesterifiedhydroxycinnamicacidswithM.J.Dinisetal./BioresourceTechnology100(2009)4829–48354833Table3
Phenoliccompositionofwheatstrawincubatedwithfourwhite-rotfungiinsolidstatefermentation.SourcePhenoliccompositionAEsterifiedhydroxycinnamicacids(mg)Lignin(g)SyrACafAp-CoumAFerAFungiBBA11.0a1.5a59.2a41.0b1.3aGA13.5b1.7a54.8a35.9ba1.4aPR17.8c2.2b50.7a32.34a1.2aTV12.3ab1.4a50.2a31.0a1.2aTime(days)715.6b2.6c95.1c66.4c1.5c1414.2b1.6b51.4b32.7b1.3b2113.9b1.5b38.1a23.1a1.0a2810.9a1.0a30.2a18.0a1.0aEffectsCFungi*****–*–Time**************Time*fungi–*–***–Valueswithinacolumnbearingthesamesuperscriptarenotsignificantlydifferent(P>0.05)accordingtoTukey’stest.ASyrA,syringicacid;CafA,caffeicacid;p-CoumA,p-coumaricacid;FerA,ferulicacid;G,guaiacyl,S,syringyl;andH,p-hydroxyphenyl.BBA,Bjerkanderaadusta;GA,Ganodermaapplanatum;PR,Phlebiarufa;andTV,Trametesversicolor.C*P<0.05;**P<0.01and***P<0.001.nosignificantdifferencesbetween21and28daysofincubation(P>0.05)foresterifiedp-coumaricandferulicacidsconcentra-tions.Alreadyat7daysofincubation(Fig.3)T.versicolorandP.rufatreatedstrawshowedlowerconcentrationsofesterifiedp-couma-ricandferulicacid,indicatingthatdegradationwasmoreeffectivewiththesetwofungiinthebeginningofincubation.Thelevelofdecreaseinesterifiedp-coumaricandferulicacidongrasslignocellulosefromBermudagrasstreatedwithtwowhite-rotfungi(CeriporiopsissubvermisporaandCyathusstercoreus)re-portedbyAkinetal.(1996)wasaround50%and65%,respectively.Morerecently,Topakasetal.(2007)inareviewonmicrobialpro-duction,characterizationandapplicationsofferuloylesterases(FAE)reportedthereleaseofferulicacidinseveralagroindustrialby-productsandonlyonepaperisreferredforwheatstrawdata.Init,Benoitetal.(2006)refertoquitelowerlevelsofreleaseofp-coumaricandferulicacids(16%and58%,respectively)fromsteamexplodedwheatstraw.Nevertheless,theseauthorsconsid-eredtheseresultsgoodduetothehighlyresistantcellwallstruc-tureofwheatstraw.Tapinetal.(2006)studiedthepotentialofwheatstrawasrawmaterialforpapermakingandrecoveryofsomephenoliccompoundswithFAEfromA.nigerandxylanaseandreferredthattreatmentwithFAEreleased7.5%and12%offerulicandp-coumaricacid,respectively.Howeverifthistreat-mentwasassociatedwithalightalkalineextractiontherecoveryincreasedto20%and25%,respectively.Takingintoaccountthemeasurementsobtainedduringtheexperimentalworkourresultsshowadecreasearound80%forbothesterifiedp-coumaricandferulicacidsafter28daysofincu-bation(Table3).However,inrelationtotheinitialconcentrationsofthesehydroxycinnamicacidsinwheatstraw,adecreaseof72%and77%wasmeasuredat21daysofincubationforp-coumaricandferulicacids,respectively,whilefortheperiodbetween21and28daysofincubationthedecreasewasonlyaround20%forbothesterifiedhydroxycinnamicacids.Inthiswayitseemsthatthereisafirstphaseuptothe14–21daysofgrowthonwhichthereisarapiddecreaseofesterifiedp-coumaricandferulicacids,andasecondphaseinwhichvaluesremainfairlystableindicatingthatprobablyfungiproducedallthenecessaryenzymestoreleasethesecompoundsinthefirst21daysofincubation(Fig.3).160.00G. applanatum140.00T. versicolorP. rufa)g120.00mB.adusta( c100.00iram80.00uoc60.00-p40.0020.000.0007142128Time (days)120.00100.00G. applanatumT. versicolor)g80.00P. rufam(B. adusta cil60.00ureF40.0020.000.0007142128Time (days)2G. applanatumT. versicolor1.5P. rufa)B. adustag( anih1neL0.5007142128Time (days)Fig.3.Timecourseofhydroxycinnamicacidsandlignindegradationduringtheincubationperiod.Theseresultsindicatethatalltestedfungalisolateswereabletoreducethecontentofesterifiedp-coumaricandferulicacidsinhigherextentthantheapplicationofcommercialenzymes,proba-blyduetoasortofcomplementaryactionbetweentheconstitu-entsoffungalenzymaticcomplexesaswehavementionedinapreviouswork(Rodriguesetal.,2008).Thisassumptionisalsova-lidtoexplainwhyT.versicolorandP.rufatreatmentsshowedamorepronounceddegradationofthesehydroxycinnamicacidsinthefirst7daysofincubation(Fig.3).Thelignincontentdecreasedidnotdifferwidelybetweenthedifferentfungaltreatments(Table3).Howevertherewasasignif-icantdecrease(P<0.001)inthetotalamountofligninfromday74834M.J.Dinisetal./BioresourceTechnology100(2009)4829–4835untiltheendoftheincubationperiod(Table3)reachingavalueof33%.Whenanalysingthecontentofwheatstrawligninwithoutanyfungaltreatment(Table2)itispossibletoseethatthisde-creaseisaround43%,indicatingthatligninlosswasquitelowwithinthefirst7daysofincubation(13%).Differenceswereob-servedamongthefungalisolatesduringtheincubationperiod(Fig.3)withT.versicolorandP.rufatreatmentsleadingtoahigherdecreaseinlignincontentbetween14and21daysofincubation.Severalauthorshavereportedthepotentialforwhite-rotfungitodegradeligninandpresentedvaluesthatarequitevariabledependingonthestrain,typeoffermentationaswellasontheincubationperiod.Forinstance,Jalcˇ(2002)onareviewpaperana-lysingtheresultsofseveralauthorspresentedvaluesofwheatstrawlignindegradationthatvariedbetween2%and65%.Aroraetal.(2002)showedpercentagelossofwheatstrawligninforP.radiataandT.versicolorof18.5%and12.5%,whileotherPhlebiaspp.showedligninlossesthatreached25%.Morerecently,Zhangetal.(2008)whenstudyingtheeffectofsteamexplosionpre-treat-mentonligninbiodegradationofwheatstrawbyT.versicolorre-portedadecreasearound30%.Asstatedbefore,ligninlossesonthefirst7daysofincubationwerequitelowwithamorepronounceddecreaseafterwards.ThesamehasbeenobservedbyAroraetal.(2002)whosuggestedasynergisticroleinligninolysisduetothefactthatvariousenzymemaximumactivitiesoccurredpriortomaximumpeakligninloss.ThissequentialactionofenzymesinlignindegradationwasalsopointedoutbyZhangetal.(2008).AccordingtotheseauthorsthoughthemaximumofMnPandlaccaseactivitiesweredetectedduringthefirst10daysofwheatstrawincubationwithT.versi-color,lignindegradationstartedlaterandtendedtoincreaseuntilthemaximumdegradationratewasreachedonday30.Ourdataalsoshowasimilarpatterninwhichsignificantlignindegradationseemstooccurafteranincreaseintheenzymeactivitiesoflaccase.However,thesamewasnotobservedforMnPactivitiesastherewasageneralincreasealongtheincubationperiod.Synergisticeffectsarealsoattributedtotheinteractionbetweenesterasesandhemicellulases.Fungalferuloylandp-coumaroylesterasesarecapableofreleasingferuloylandp-coumaroylunitsandplayanimportantroleinbiodegradationofrecalcitrantcellwallsingrasses(Kuhadetal.,1997).Theseenzymesactinconcertwithxylanasestodisruptthehemicellulose–ligninmatrix,withoutmineralizationofligninperse(Bornemanetal.,1990).Therefore,hemicellulosedegradationseemstoberequiredbeforeefficientligninremovalcancommence,atleastforsomesubstrates.ThissynergisticeffectwasalsounderlinedbyPanagiotouetal.(2007)whosuggestedthattheproductionofferuloylesteraseandarabin-ofuranosidaseisco-regulated.4.ConclusionsOurdatashowthatenzymaticcomplexesproducedbywhite-rotfungiseemtoexerttheireffectinthecellwallstructureduetoaconcertedactionbetweenthedifferenttypesofenzymes.Infact,thehigherdegradationofesterifiedhydroxycinnamicacidsinthefirst7–14daysofincubation,whichisdirectlyrelatedtothexylanaseandferuloylesteraseactivitiesduringthisperiod,pre-cedesamoreintensivedegradationoflignin.Thepresentstudyalsoindicatedthatthefungaltreatmentswereabletoconsiderablyreducethecontentofesterifiedp-coumaricandferulicacids.Con-sideringthatwheatstrawisquiterecalcitrantandthatresultsfromtheapplicationofcommercialesterasesdonotnormallyapproachsuchhighvalues,ourresultsareofsubstantialinterest.Neverthe-less,morestudies,e.g.onthespecificityofenzymeactivitiesoffungiarestillnecessarytoimproveourknowledgeonthemecha-nismsthatregulateitsproductionandspecificinteractionwiththedifferentsubstrates.ReferencesAkin,D.E.,Morrison,W.H.,Rigsby,L.L.,Gamble,G.R.,Sethuraman,A.,Eriksson,K.E.L.,1996.BiologicaldelignificationofplantcomponentsbythewhiterotfungiCeriporiopsissubvermisporaandCyathusstercoreus.Anim.FeedSci.Technol.63,305–321.Arora,D.S.,Chander,M.,Gill,P.K.,2002.Involvementofligninperoxidase,manganeseperoxidaseandlaccaseindegradationandselectiveligninolysisofwheatstraw.Int.Biodeterior.Biodegrad.50,115–120.Benoit,I.,Navarro,D.,Marnet,N.,Rakotomanomana,N.,Lesage-Meessen,L.,Sigoillot,J.,Asther,M.,Asther,M.,2006.Feruloylesterasesasatoolforthereleaseofphenolicscompoundsfromagro-industrialby-products.Carbohydr.Res.341,1820–1827.Borneman,W.S.,Hartley,R.D.,Morrison,W.H.,Akin,D.E.,Ljungdahl,L.G.,1990.Feruloylandp-coumaroylesterasefromanaerobicfungiinrelationtoplantcellwalldegradation.Appl.Microbiol.Biotechnol.3,345–35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