GC-MS

Copyright©1999,AmericanSocietyforMicrobiology.AllRightsReserved.

Vol.65,No.4

FungalDegradationofLipophilicExtractivesin

EucalyptusglobulusWood

´RREZ,1*JOSE´C.DELRI´O,1MARI´AJESU´SMARTI´NEZ,2ANAGUTIE

2´NEZANDANGELT.MARTIInstitutodeRecursosNaturalesyAgrobiologı´adeSevilla,ConsejoSuperiordeInvestigaciones

1Cientı´ficas,E-41080Seville,andCentrodeInvestigacionesBiolo´gicas,Consejo

SuperiordeInvestigacionesCientı´ficas,E-28006Madrid,2Spain

Received2October1998/Accepted5January1999

Solid-statefermentationofeucalyptwoodwithseveralfungalstrainswasinvestigatedasapossiblebiologi-calpretreatmentfordecreasingthecontentofcompoundsresponsibleforpitchdepositionduringCl2-freemanu-factureofpaperpulp.First,differentpitchdepositswerecharacterizedbygaschromatography(GC)and

GC-massspectrometry(MS).Thechemicalspeciesidentifiedarosefromlipophilicwoodextractivesthatsurvivedthepulpingandbleachingprocesses.Second,adetailedGC-MSanalysisofthelipophilicfractionafterfungaltreatmentofwoodwascarriedout,anddifferentdegradationpatternswereobserved.Theresultsshowedthatsomebasidiomycetesthatdecreasedthelipophilicfractionalsoreleasedsignificantamountsofpolarextractives,whichwereidentifiedbythermochemolysisasoriginatingfromlignindepolymerization.Therefore,theabilitiesoffungitocontrolpitchshouldbeevaluatedafteranalysisofcompoundsinvolvedindepositformationandnotsimplybyestimatingthedecreaseinthetotalextractivecontent.Inthisway,Phlebiaradiata,Funaliatrogii,Bjerkanderaadusta,andPoriasubvermisporastrainswereidentifiedasthemostprom-isingorganismsforpitchbiocontrol,sincetheydegraded75to100%ofbothfreeandesterifiedsterols,aswellasotherlipophiliccomponentsoftheeucalyptwoodextractives.Ophiostomapiliferum,afungususedcommer-ciallyforpitchcontrol,hydrolyzedthesterolestersandtriglycerides,butitdidnotappeartobesuitableforeucalyptwoodtreatmentbecauseitincreasedthecontentoffreesitosterol,amajorcompoundinpitchdeposits.

Downloaded from aem.asm.org by on March 29, 2010 Biotechnologyhasbeenintroducedintopulpandpapermanufacturing,thefirstnonfoodindustrialuseofplantbio-mass(7).Xylanase-aidedelementarychlorine-free(ECF)bleachingofpaperpulpisthebestexampleoftheapplicationsdevelopedinrecentyears(22).However,otheraspectsofpaperpulpmanufacturingalsoofferpromisingavenuesforusingmicroorganismsandenzymes;oneoftheseisbiologicalcontroloftheso-calledpitchdeposits.Accumulationofwoodextractivesinpulpandpapermills(pitchdeposits)resultsinlow-qualitypulpandblockagesthatcauseshutdownsofoper-ationsandimportanteconomiclosses(15).Theincreasingneedforrecirculatingwaterinpulpmillsandtheneedtore-duceeffluentsinordertomeetenvironmentalprotectionre-quirementsareleadingtoincreasesintheconcentrationsofpitchcompoundsintheproductionprocess.Thissituationandtheuseoftotallychlorine-free(TCF)andECFbleachingpro-cessesbasedonthereplacementofCl2bymilderchemicaloxidantsorenzymesresultingreaterpitchdepositionprob-lemsinmills.Traditionally,pitchdepositsduringpulpingpro-cesseshavebeenreducedbydebarkingandseasoninglogsandwoodchipsandbyaddingpitchcontrolagents(1,5).However,oftentheresultsarefarfromsatisfactory.Alternatively,bio-logicalcontrolofpitchdepositsbytreatmentofpulpwithenzymes(9–11)andtreatmentofwoodwithdifferentmicro-organisms(2,6,8,12),havebeensuggestedinrecentyears.Thesebiotechnologicalapproacheshaveinvolvedmainlytreat-mentofpinewoodwithOphiostomapiliferumandrelatedspecies,aswellassomebasidiomycetes.Thestudiesofpitch

*Correspondingauthor.Mailingaddress:IRNAS,CSIC,P.O.Box1052,E-41080,Seville,Spain.Phone:349624711.Fax:349624002.E-mail:anagu@irnase.csic.es.

1367

removalperformedwithbasidiomycetes,suchasPhanero-chaetechrysosporium,Poriasubvermispora(synonym,Ceripori-opsissubvermispora),andPhlebiopsisgigantea,areinthepre-liminarystagesandareoftenassociatedwithstudiesoftheuseofthesefungiforso-calledwoodbiopulping(i.e.,biologicalremovalofligninforpaperpulpmanufacturing).AwhitestrainofO.piliferum(CartapipfromClariant)hasbeenusedcommerciallytodepitchsometypesofwoodpriortopulping.However,successfuluseofCartapiptocontrolpitchdeposi-tioninKraftpulpobtainedfromeucalyptwood(whichisextensivelyusedasarawmaterialforpaperpulpmanufac-turinginSpain,Portugal,Brazil,andothercountries)hasnotbeenreported.Moreover,noinformationaboutbiologicalde-pitchingofthistypeofwoodwithotherfungalspeciesisavail-able.

Designingeffectivebiotechnologicalsolutionsforwoodex-tractiveremovalrequiresthoroughcharacterizationofthecompoundsresponsibleforpitchdeposition.Inthiscontext,thefirstaimofthisworkwastoidentifythespecificconstitu-entsofpitchdepositsduringKraftpulpingofEucalyptusglobu-luswoodcomparedwithwoodextractivesobtainedfromthiseucalyptspecies.Then,themainaimwastoevaluatethevia-bilityofbiotechnologicalsolutionsforeliminatingtheseprob-lematicpitchcompoundsbyanalyzingindetailthepatternsofremovalofthemainlipophiliccompoundspresentineucalyptwoodbyaselectionoffungalstrains.

MATERIALSANDMETHODS

Samples.PitchdepositsafterTCFandECFbleachingwereobtainedfromeucalyptKraftpulpmillsinHuelvaandPontevedra,Spain.TheTCFsequenceusedincludedtwooxygendelignificationstages,achelationstage,anH2O2stage

1368

GUTIE

´RREZETAL.TABLEesters,1.Hydrocarbons,andandpitchtriglyceridesfattydepositsafterinextractsacids,waxes,sterols,ketones,sterol

ECFandfromTCFE.bleachingglobuluswood

aCompound(s)a%in:

Wood

ECF

TCF

Alkanes

Steroidhydrocarbons(compoundVI)b3.05.3Ͻ20.20.10.59.6SaturatedUnsaturatedfattyfattyacidsacids7.65.8ND13.3c19.76.3Waxes(C20–C40)

3.0Ͻ0.11.6SitosterolStigmastanol(compoundI)

24.6Fucosterol(compoundIII)3.0Ͻ0.121.0Othersterols

(compoundV)1.23.2ND20.85.4Ͻ0.11.21.4AliphaticStigmastan-3-oneketones

NDStigmast-4-en-3-one(compoundIX)0.6Stigmasta-3,5-dien-7-one(compoundVII)

4.810.71.10.11.0Stigmastane-3,6-dione(compound(compoundX)VIII)4.41.012.67.91.27.93.70.5SitosterolStigmastanolesters17.3Ͻ0.1Othersterolestersesters(compoundd(compoundII)dIV)d3.55.24.511.01.011.05.0Triglycerides

6.5

ND

ND

abSeeFig.1forthechemicalstructuresofsteroidcompounds.cStigmasta-3,5-diene(compoundVI)isthemaincompound.dND,notdetected.Estersofsterolsandseveralunidentifiedfattyacids(includinglinoleicacid).

underpressure(withoxygen),andafinalH2Odelignificationstage,followed2stage.TheECFsequenceusedincludedanoxygenbytwoClOintermediatealkalineextractionstage.E.globuluswoodchips2stageswithanweregroundtosawdust.ThedifferentpitchdepositsandsawdustwereSoxhletextractedwithacetonefor6h(21).Theacetoneextractswereevaporatedtodrynessandredissolvedinchloroformbeforetheywereanalyzedbygaschromatography(GC)andGC-massspectrometry(MS).

Fungalstrainsandwoodtreatmentconditions.Thefirstscreeningoffungiforremovalofeucalyptextractivesundersolid-statefermentationconditionswascarriedoutwith73strains(18).Manyofthesestrainswereisolatedfromfruitingbodiesgrowingoneucalyptwoodinforestsorinlogpilesandidentified.Addi-tionalstrainswereobtainedfromtheCentraalbureauvoorSchimmelcultures(Baarn,TheNetherlands),WageningenAgriculturalUniversity(Wageningen,TheNetherlands),andthefungalculturecollectionoftheCentrodeInvestiga-cionesBiolo´gicas(Madrid,Spain).

Treatmentofwoodwithallofthefungi(induplicate)wascarriedoutinflaskscontaining2g(dryweight)ofsmallwoodchips(1to2by10to20mm)and5mlofwater(sterilizedat120°C)thatwereinoculatedwithtwoportionsofmyceliumfromcultureson2%maltextractagar.After40daysofincubationat28°Candaconstanthumidity,thewoodwasdriedwithairat60°C,milled,andextractedwithacetoneasdescribedabove.Then,theKlasonlignincontentwasestimatedafterhot-waterextraction(21).Woodtreatmentwiththe14mostpromisingstrainswasrepeatedinquadruplicatetoconfirmtheresultsobtainedinthefirstscreening.

GCandGC-MS.TheGCanalysiswereperformedwithaHewlett-PackardmodelHP50GCequippedwithaflameionizationdetectorbyusingahigh-temperaturepolyimide-coatedfused-silicacapillarycolumn(5mby0.25mm;typeDB5-HT;filmthickness,0.1␮m;J&WScientific).Theinjectoranddetectortemperatureswere300and350°C,respectively.Theoventemperaturewasprogrammedtoincreasefrom100°C(1min)to350°C(3min)atarateof15°C/min.Sampleswereinjectedinthesplitlessmode.Heliumwasusedasthecarriergas.Amixtureofstandardcompounds(palmiticacid,sitosterol,cho-lesteryloleate,andtriheptadecanoin)wasusedtoconstructacalibrationcurveforquantitationofwoodextractivesatconcentrationsrangingfrom0.1to1mg/ml.Thecorrelationcoefficientwasgreaterthan0.99inallcases.Peakswerequantifiedbydeterminingareas.

GC-MSanalyseswereperformedwithaVarianmodelStar3400GCequippedwithaniontrapdetector(VarianmodelSaturn2000)byusingatypeDB-5HTcapillarycolumn(15mby0.25mm;filmthickness,0.1␮m;J&WScientific).

APPL.ENVIRON.MICROBIOL.

Heliumwasusedasthecarriergas.Sampleswereinjecteddirectlyintothe

columnwithanautoinjector(Varianmodel8200)byusingaseptum-equippedprogrammableinjectorsystem.Thetemperatureoftheinjectorduringinjectionwas120°C,and0.1minafterinjectionthetemperaturewasprogrammedtoincreaseto380°C(10min)atarateof200°C/min.Theoventemperaturewasprogrammedtoincreasefrom120°C(1min)to380°C(5min)atarateof10°C/min.Thetemperaturesoftheiontrapdetectorandthetransferlineweresetat200and300°C,respectively.CompoundswereidentifiedbycomparingtheirmassspectrawithmassspectraintheWileyandNistlibraries,byperform-ingmassfragmentography,and,whenpossible,byusingstandards.

Thermochemolysis.AthermochemolysisanalysiswasperformedwithaVarianmodelSaturn2000GC-MScoupledtoaCuriepointpyrolyser(HorizonInstru-ments␮Ltd.)byusingatypeDB-5column(30mapproximatelym).Afinelydivided0.5␮lofsampletetramethylammoniumwasdepositedontoby0.25mm;filmthickness,0.25hydroxideferromagnetic(25%wire,[wt/wt]mixedaqueouswithsolution)(14),insertedintotheglassliner,andthenimmediatelylocatedinthepyrolyser,andpyrolysiswascarriedoutat610°C.Thetemperatureofthechro-matographwasprogrammedtoincreasefrom40°C(1min)to300°C(20min)atarateof6°C/min.Thetemperatureoftheinjector,whichwasequippedwithaliquidcarbondioxidecryogenicunit,wasprogrammedtoincreasefrom30°C(1min)to300°Catarateof200°C/min,whiletheGC-MSinterfacewaskeptat300°C.

RESULTSANDDISCUSSION

wood.LipophiliccompoundsinpitchdepositsandE.globulusinguseofPitchE.globulusdepositswereobtainedafterTCFandECFbleach-TwoofoftypesHwood.Theseindustrialprocessesincludethe2Oof2andorganicClOfractions2,respectively,wereasbleachingagents(20).tainedacetonedistinguishedonthebasisacidsaltsmainlysolubility.Theacetone-insolublefractionscon-(4).saltsTheofcompositionsfattyacidsandoftheminoracetone-solubleamountsofellagicfrac-

Downloaded from aem.asm.org by on March 29, 2010 FIG.1.Mainfreesterols(sitosterol[compoundI],stigmastanol[compoundIII],andfucosterol[compoundV]),esterifiedsterols(sitosterolesters[com-poundII],andstigmastanolesters[compoundIV]),steroidhydrocarbon(stig-masta-3,5-diene[compoundVI]),andketones(stigmast-4-en-3-one[compoundVII],stigmasta-3,5-dien-7-one[compoundVIII],stigmastan-3-one[compoundIX],luswoodandstigmastane-3,6-dioneandpitchdepositsduring[compoundmanufactureX])inofacetoneClextractsfromE.globu-SeeTable1forcompoundabundancevalues.

2-freeeucalyptKraftpulp.VOL.65,1999FUNGALDEGRADATIONOFEUCALYPTWOODEXTRACTIVES1369

TABLE2.andEffectspolaroffraction,twofungiandonlignintotalextractive,contents

lipophilic

Amt(g/100g)of:

VanillylPrepn

WoodLipophilicPolaracid/alde-extractivescompoundscompounds

Lignin

hyderatioaControlC.variabilis0.751.04(Ϫ26)b0.31

0.44O.valdivianum0.33(56)

0.140.27(55)(13)0.902.5

0.06((86)Ϫ104)15.8

10.816(0)(38)9.53.5

abVanillylacid/aldehyderatioafterthermochemolysisofpolarcompounds.Thevaluesinparenthesesarerelativedegradationvalues.

tionsMSextractivesareofsummarizedthedifferentinpitchTabledeposits1,whichanalyzedalsoshowsbytheGCresultsandGC-forpossiblepoundsbecauseobtainedamethodfromE.forglobulusanalysiswood.ofthisThistypeanalysiswasshowedwaswoodthatoptimizedmanyofthepreviouslychemical(13).speciesThefoundresultsobtainedofcom-sinceplestheyextractsweresurviveidentifiedtheinpulpingpitchdeposits.andbleachingineucalyptInprocesses,identified(woodlipophilic(Fig.andpitch1)accounteddeposits),allofthesam-forthedifferentsteroidcompoundsketonesbe(compoundscompoundsVIIinthemorethan70%ofthetotaltoacetoneextracts.SeveralsteroidrelativelysteroloxidationX),whichhavebeenreportedtotectedhighlevelsproductspreviously(16),werepresentatdepositsinwood.Triglyceridesinpitchdeposits,wereabsentbutfromtheywerebothalsotypesde-ofcompositionsinceingoftheythewerepitchhydrolyzeddepositsproducedduringKraftaftercooking.TCFbleach-Theextractives.wasveryveryThesimilardepositstotheproducedcompositionafterECFofE.bleachingglobuluswoodpresentdifferenttheinE.globuluscomposition.wood,wasNofoundsitosterol,inthemainhadsterolamastanol)esterifiedtanceremainedform,inwhereaseitherthefreeformortheonlyasaturatedsterol(stig-fattytheacids,tooxidation.depositsbecauseofitshigherresis-whichThesamethinghappenedwithunsaturateddetermineresultsdescribedwereabove,absentduringafterClOthe2screeningtreatment.ofBasedfungiontowoodthepriorwhethertoKrafttheycookingremovespecialextractivesemphasisfromwasE.placedglobulusonextractiveFungalbiologicaltreatmentremovalofE.ofglobulusfreeandwood.esterifiedThefirststerols.

screeningforgiformedundersterileremovalsolid-statebytreatmentfermentationofE.globulusconditionswoodwithwasfun-per-comycetes,byexaminingdifferentspeciesofbasidiomycetes,as-strainsspecies,belongingandconidialfungi(18).Theascomycetesincludedeucalyptandthreewood).twoMollisiatoeightspeciesOphiostoma(thelatterspecies,werefiveisolatedCeratocystisfromstrainsPleurotusCrepidotusofFunaliaspeciesThebasidiomycetestrogii,BjerkanderaandtwoPhlebiaincludedadustaspecies,members,P.chrysosporiumaswellofas,tervariabilis,andMelanotushepatochrous(thetwolat-strainsorganismsfromducingresinofPaecilomyceswereisolatedfromeucalyptwood).Finally,ofEucalyptussp.,Penicilliummegasporum(isolatedtigated.

Fusariumspeciesweretereticornisamong),theandconidialseveralfungilipase-pro-inves-tractiveWidedifferencesweregi,removalbythedifferentobservedfungi.intheWhileextentsomeofofwoodthefun-ex-stomasuchextractivevaldivianumasPleurotus,anderyngiiMollisia,Paecilomycesmelaleucasp.,F.trogii,Ophio-extractivecontentphoraputeanacontentby,C.variabiliswas50observedto70%,asignificant,reducedincreasetheintotalthe,P.subvermisporainwoodtreated,andotherwithConio-organ-

isms.obtainedHowever,thefromtheclosebiotreatedexaminationwoodsofrevealedtheacetonethatextractsonlyfungifractionthepolarthatdecreasedfraction,whilethetotalextractivecontentsomereducedofmainedresponsiblewhichunchanged.forpitchthedeposition,lipophilicasfractionshownabove)(themainre-obtainedincreasedthetotalTheoppositeacetoneextractoccurredcontent.withotherTheresultsfungithistreatedexperimentwithtwotherepresentativeacetoneextractsfungiareshowninTable2.Insoluble)woodsanumandpolarwere(chloroform-insoluble)fractionatedintoobtainedlipophilicfromthebio-fractions.(chloroform-decreasereducedfractioninthethetotalacetoneextractcontentduetoO.avaldivi-drasticreducedcontentpolarwasbarelyfractionmodified.content;Inhowever,contrast,theC.lipophilicvariabilisthecompoundtotaltheacetonelipophilicextractfractioncontentcontent,duetoanalthoughitincreasedpoundsthewerecontent(Table2).Theoriginsincreaseoftheinpolarthepolarcom-ligninacid/aldehydedeterminedoxidativedegradationratioofbyvanillylthermochemolysis.(14),wascompounds,higherinanWetheindicatorfoundthatpolarfrac-

ofDownloaded from aem.asm.org by on March 29, 2010 FIG.2.GaschromatogramsforlipophilicextractsfromE.globuluswoodaftertreatmentwithtwofungiandthecorrespondingcontrol.Thesamesamplevolumeforthelipophilicfractionobtainedfromthesameamountofwoodwasinjectedineachcase,andtherelativechromatographicresponsesareshown.Figure1andTable3showthechemicalstructuresandabundancevaluesforthedifferentsteroids(compoundsItoX).

1370

GUTIE

´RREZETAL.TABLEpercentage3.FungalcontentsoftotaldegradationofE.globuluswoodextractives:

andaincontrol,woodextracttreatedandmainlipophilicfraction

asdeterminedwithdifferentbyGCfungi

a%Extractivefractioncontents

Prepn

oftotal(mg/100gofwood)extract

Fattyacids

Sterols

SterolTriglyc-esters

erides

Control

Bjerkandera0.758.6

Crepidotusadusta0.4817.2

41.81.9Funaliavariabilis0.762.1(88)b9.633..9(78)Melanotustrogii

0.622.4(86)7.5(77)8.1(76)0.13..3(75)(21)49.34.6(82)()3.7()(Ϫ18)34.03.7()(0)1.4(43)3.2(84)(63)Mollisiachroushepato-Ophiostomasp.

0.46Ophiostomapiliferumpiliferum0.7410.3(40)47.276.1(Ϫ13)25.2(25)5.20.4310.09.1(47)(42)51.4((ϪϪ82)23)10.87.4(78)(68)0(39)3.0(100)(65)OphiostomaCartapip

0.3915.7(8)

.8(Ϫ55)18.5(45)

6.5(25)

Paecilomycesvianum

valdi-Phanerochaetesp.

chryso-0.630.8062.43.2((81)Ϫ263)32.211.3(23)(73)20.87.1(38)(79)14.815.8((ϪϪ72)84)Phlebiasporium

Pleurotusradiata1.011.7Pleurotuseryngii

0.425.1(90)0Poriasubvermisporapulmonariusc0.431.34

4.6(70)35.91.7(96)25.5(14)21.20(100)0.6(73)(97)

0.8(39)(98)

8.1(37)2.7(100)1.7(69)0(100)

(76)0(100)

(80)aInallcasesthestandarddeviationforreplicates(includingvariabilityfromchromatographicanalysis,acetoneextraction,fungalgrowth,andextractivedeg-radation)waslessthan15%ofthemean.Figure2showsrepresentativegaschromatogramsbcThevaluesinforparentheseslipophilicextracts.arepercentagesofdegradation.Synonym,Ceriporiopsissubvermispora.

tionthefromwoodtreatedwithC.variabilis.This,togetherwithsuggesteddecreaseinlignincontentestimatedbytheKlasonmethod,arosestudyevaluatedthatfromthatthepolarcompoundsdescribedaboveprobablythefungalabilitiesalterationofsomeoffungilignin.toWecontroldeducedpitchshouldfromthisposittotalgenerationbyspecificallyandnotanalyzingsimplyestimatingcompoundstheinvolveddecreaseininde-betheulusNext,amountlyzedwoodthetreatedcompositionsofextractives.

withtheofdifferentlipophilicextractivesfromE.glob-areinitially,shownbyGCinandFig.GC-MS.fungalspecieswereana-2.AtotalTworepresentativechromatogramstractivesandaticand/orthesignificantlyspeciesthatof73specieswereinvestigateddecreasedsignificantlythedegradedtotalex-woodlipophilicstudyweight(18),compounds,wereselectedaswellascausedcontentalimitedofproblem-lossofwereincludingobserved(Table3).withDifferentthefungalpatternsformoredetailedquantitativestrainsofused.extractivedegradationestersterols,contentO.piliferumbutsimultaneouslyandO.valdivianumSomeofthefungi,increased,reducedthesterolfattysults).acyl-sterolmainlysitosterol,esteraseactivitywhichwasthecontentoffreedetectedprobably(unpublishedrelatedtothewoodA(CartapiptreatmentsimilarpatternwithOphiostomahasbeenfoundainoaepreviouslyre-(19).O.duringpiliferumpineforwoodreducingstrainpitchfromproblemsClariant)inhasmechanicalbeenreportedpulpingtobeofusefulpinestrain(6),woodprovedaswelltoashaveinsprucelimitedsulfiteutilitypulpingin(8).However,thisreleased,becausecompoundswhich,itaswasshownnotabletodegradethecasethefreeofeucalyptsterolsmycetousofresinacidsfungiinthisfromhavetypeofabove,wood.Thisareamongfungustheandproblematicotherasco-pinealsowoodbeen(12,reported23),whereastoremovesome60basidio-

to70%APPL.ENVIRON.MICROBIOL.

mycetesofedthebasidiomycetescancompletelyassayed,degradesuchtheseascompounds(19).SomecontentbothsterolsandsterolestersbutsignificantlyP.chrysosporium,degrad-galoftriglycerides,whichwereprobablyderivedincreasedfromfun-thesporiummetabolismloidestype),whichtodegrade(24).TheabilityofliquidculturesofP.chryso-alsosterolestersfromaspen(Populustremu-Duringofwoodnumberthepresent(3),hascausepitchproblemsduringpulpingofthisstudybeendescribedwebyLeoneandBreuil(17).P.philicsubvermisporaoffungi,,includingPhlebiafoundradiataforthe,F.firsttrogiitime,B.adustathata,responsiblecompoundsandfreeforpitchthatC.depositionhavevariabilisbeen,identifiedefficientlydegradethelipo-duringmanufactureasthecompoundsofmisporapulpacids,hasfrombeeneucalyptreportedwood.tobeOneanefficientofthesedegraderfungi,P.subver-Cl2-outinpinewood(8).Furtherexperimentsarebeingofcarriedresinscaletoestablishthetimecourseofextractiveremovalandtoremoveupproblematicwoodtreatmentlipophilicwiththecompounds.

mostpromisingstrainsthatACKNOWLEDGMENTS

fungalWethankJ.M.Barrasa(UniversityofAlcala´tevedra,strains,ClariantSpain)JavierforsamplesRomeroof(CentroeucalyptdewoodInvestigacio,Madrid,andpitch´n,ENCE,Spain)Pon-forwhite(Barcelona,Spain)forasampleofCartapipdeposits,(O.andropeanThisstrain).

piliferumresearchwascarriedoutwiththefinancialsupportTechnicalproject“WoodExtractivesinPulpandPaperManufacture:oftheEu-(FAIRDownloaded from gramme.

contractandEnvironmentalCT95-560)andImplicationstheSpanishandBiologicalBiotechnologyRemoval”Pro-REFERENCES

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