BiopulpingofwoodchipswithPhlebiabrevisporaBAFC633reduceslignincontentand

improvespulpquality
MaraIsabelFonsecaa,
*,JuliaInsFariab,MaraLorenaCastrilloa,MaraDanielaRodrgueza,
CarlosEduardoNuezc,LauraLidiaVillalbaa,PedroDaroZapataa
a
LaboratoriodeBiotecnologaMolecular,MdulodeBioqumicayFarmacia,FacultaddeCienciasExactasQumicasy
Naturales,UNaM,Posadas,Misiones,Argentinab
DepartamentodeBiotecnologaFngica,PROIMICONICET,T4001MVB,Tucumn,Argentinac
ProgramadeCelulosayPapel,FacultaddeCienciasExactasQumicasyNaturales,UNaM,Argentina
articleinfo
Articlehistory:Received22April2013Receivedinrevisedform15November2013Accepted16November2013Available
online28February2014
Keywords:PhlebiabrevisporaBAFC633PinustaedaLaccaseBiopulping
abstract
ThewhiterotfungusPhlebiabrevisporaBAFC633produceslaccasesinlargeproportions.InthisworkP.brevisporaBAFC633
wasgrownonPinustaedawoodchipsin10Lbioreactors.Toselectthebiopulpingexperimentalconditions,weanalyzedthe
variablesaffectingenzymaticlaccaseactivityintheculturesupernatants,indicatingthatthesuitableincubationtemperaturewas
30Cinordertopromoteenzymestability.PhlebiabrevisporaBAFC633secreted744U/goflaccase,selectivelyremoving
ligninduringbiotreatmentofwoodchips,causingareductioninKappanumberand10%weightloss,andcreatingamoreopen
structure and better access to the pulping liquor, which would require less chemical con sumption, thus diminishing the
environmentalimpactofthechemicalpulpingprocess.
TheseresultssupportthebiotechnologicalpotentialofP.brevisporaBAFC633forbiopulpingprocessesandenhancethe
potentialforbioprospectingnativeisolatesofthemicrofloraofourcountrysnaturalenvironment.
2014ElsevierLtd.Allrightsreserved.
1.Introduction

PulpandpaperconstituteoneofthemajormanufacturingactivitiesinMisiones,Argentina.Thestrongdemandforpaperasa
commodityleadstoasteadyexpansionofpaperindustriesinourcountry.Themainobjectiveofthepapermanufacturing
processesistheremovalofligninfromwood;thisisthefirststepinchemicalpulping.Kraftpulpingisthemostwidespreadpro
cess(DaReetal.,2010).Kraftpulpingandbleachingstagesuselargeamountsofchlorineandchloridechemicals(Polainaand
MacCabe,2007;Selvametal.,2011).Thederivedproductsofthesechemicalsarechlorinatedorganicsubstances,someofwhich
aretoxic,mutagenic,persistentandcancausenumerousharmfuldisturbancesinbiologicalsystems(BajpaiandBajpai,1997).
InthissensefilamentousfungiofthephylumBasidiomycotahavebeenwidelystudiedfortheirabilitytodegradewood(Kirk
andCullen,1998);thesefungiareuniqueintheirabilitytodegrademostcomponentsofwoodduetotheirabilitytosynthesize
therelevanthydrolyticandoxidativeextracellularenzymes(Poojaryetal.,2012).Withinthebasidiomycetes,whiterotfungi
(WRF)havereceivedspecialattentionbecausetheyaretheonlyorganismscapableofmineralizinglignintoCO
2
andH
2
O(Martinez,2002)bysecretingoxidativeenzymes,suchasperoxidases
andlaccases,whichhaveabroadrangeofsubstrates(Fieldetal.,1993).Somewhiterotfungihavetheabilitytoselectively
removeextensiveamountsofligninwithonlyinsignificantlossesofcelluloseandmoderatetolowlossesofhemicellulose
(Blanchetteetal.,1985;OtjenandBlanchette,1986).Oneofthelessharmfulandmorepromisingalternativestoimprove
conventionalpulpandpaperprocessesistheuseofmicroorganisms(suchasWRF)andenzymesasatreatmentforwoodchipsto
reducelignincontent.
*Correspondingauthor.Av.MarianoMoreno1375,3300Posadas,Misiones,Argentina.Tel./fax:543752427687.
Emailaddress:biotecmol2010@gmail.com(M.I.Fonseca).
Thisalternativeprocessisknownasbiopulping(OtjenandBlanchette,1987;BlanchetteandBurnes,1988;Villalbaetal.,2006).
Thisprocesscansavesubstantialamountsofenergy,
http://dx.doi.org/10.1016/j.ibiod.2013.11.01809648305/2014ElsevierLtd.Allrightsreserved.
ContentslistsavailableatScienceDirect

InternationalBiodeterioration&Biodegradation
journalhomepage:www.elsevier.com/locate/ibiod
InternationalBiodeterioration&Biodegradation90(2014)29e35
improvespaperquality,reducestheenvironmentalimpactofpulping,andenhanceseconomiccompetitiveness.
TheobjectiveofthisstudywastoevaluatetheWRFPhlebiabrevisporaBAFC633forbiotechnologicalapplicationinthe
biopulpingofPinustaedachips.
2.Materialsandmethods

2.1.Fungalstrainandwoodchips
PhlebiabrevisporaBAFC633wasprovidedbytheMycologicalCultureCollectionoftheDepartmentofBiologicalSciences,
FacultyofExactandNaturalSciences,UniversityofBuenosAires,Argentina.Stockculturesweremaintainedonmaltagar(12.7
gl
1
),agar(2%w/v)(MEA)slantsat4Cwithperiodictransfer.
WoodchipsofPinustaedawereselectedforthisstudybecausetheyhaveregionalimportanceintheproductionofcellulose,
andtheyarefastgrowingtrees.Therearepreviousstudiesusingwhiterotfungiforthetreatmentofthissubstrate(Otjenand
Blanchette,1987;BlanchetteandBurnes,1988;Villalbaetal.,2006).
2.2.PreliminarystudiesoflaccasetemperatureandpHoptimum,andenzymethermostabilityonculturesupernatants
Preliminarytestswereperformedusingculturesupernatantsfrom50mlofMEmedium(12.7gl
1
maltextractand5gl
1
corn steepliquor) at10 daysof incubation, pH4.5, instatic
conditionstoestablishoptimalconditionsforlaccaseactivity.Theoptimumtemperatureoflaccasewasdeterminedbetween30
and80C.TheoptimumpHforlaccasewasdeterminedusingsodiumacetatebuffer0.1MatapHrangeof3.6e5.6.
Forthermostabilityevaluationduringtheenzymaticbioprocess,culturesupernatantswereincubatedcontinuouslyfor7hat
optimumpHandtemperature.Theeffectoftemperatureonstabilityoflaccaseonculturesupernatantswasdeterminedfor7hat
20,30,40,50,and60C.
2.3.Preparationoffungalinocula
Oneagarplug(0.5mm
2
)offungusgrownonanMEAplateat29Cfor5dayswasaddedto50mLofMEmediumand
incubatedat29Cstaticallyfor10days.Fortheinoculationexperiments,themyceliumwasremovedunderasepticconditions,
washedinsteriledistilledwaterthreetimes,mashedfor5stoachieveahomogenousmixture,andfinallyaddedtothesubstrate.
2.4.Woodpreparation
Idealchipsizeforcommercialpulpingis12.50.3cm,soinordertoachievethesedimensions,P.taedachipsfromthe
sawmillwerecarefullyselectedandseparatedmanually.Afterclassification,theywerethoroughlymixedandplacedindishes.
Threealiquotsoftheinitialchipsweretakentodeterminethemoisturecontent.Woodchipswereautoclavedfor30minat121C
topreventcontaminationbymicroorganismsthatcanantagonizeorinhibitthegrowthoffungus.Subsequently,400g(onadry
weightbasis)understerileconditionswerepouredintothe10Lbioreactors.
2.5.Inoculationprocedure
Crushedfungusmyceliumwasusedasinoculum(0.5mgmycelium/gchips)andpouredoverthewoodchipswithextensive
mixing.Sodiumacetatebuffer0.1MpH3.6wasaddedtothesystemtoreach65%moisturecontent.Theincubationstepwas
carried
M.I.Fonsecaetal./InternationalBiodeterioration&Biodegradation90(2014)29e3530
outatbioreactorsmaintainedat29Cinanenvironmentallycontrolledchamber.Theexperimentswereconductedinduplicate.
 After30days,theincubationperiodwascompleted.Thechipswereairdriedandstoredat20Cinplasticbagstostopfungal
activitybeforepulping.
2.6.Weightlossmeasurement
Beforeincubation,thewoodchipsweredriedtoconstantweightat80C,andtheinitialweightwascalculated.Afterthe
incubation,thewoodchipswerewashedwithsteriledistilledwaterandfilteredtoremoveresidualmycelium.Thewashedchips
weredriedat80Ctoconstantweight,andtheweightlosswascalculatedbasedontheinitialandfinaldryweights.
2.7.Microscopiccharacteristics
Opticalmicroscopywasusedtovisualizemorphologicalchangesinthewoodasaresultoffungaldegradation.Toobtainthin
slices(0.1mmthickness)formicroscopicanalysis,thecommercialchipsamplesweretakenfromeachofthetreatmentsand
controls,boiledfor3hwithdistilledwater,andthenslicedbyhandusingascalpel.Thewoodsectionswerestainedaccordingto
thetechniquedescribedbyIsenberg(1967).
2.8.Enzymeassays
Laccase(EC1.10.3.2)activitywasmeasuredat30Cusing5mMof2,6dimethoxyphenol(DMP)in0.1Msodiumacetate
bufferpH3.6.Absorbancewasmonitoredat469nm(E
469
1427.5mM
1
cm
1
)inaShimadzuUV3600spectrophotometer.Onelaccase
activityunitwasdefinedastheamountofenzymerequiredtooxidize1mmolofDMPperminuteat30CandexpressedasUml
1
(Moreiraetal.,2004).
Endob1,4glucanase (EC 3.2.1.4) activity was determined by measuring the liberation of reducing sugar with the 3,5
dinitrosalicylicacid(DNS)method(Miller,1959)using0.5%carboxymethylcellulose(w/v)assubstratein0.05Msodiumcitrate
bufferpH5.Reactionswereincubatedat50Cfor30min.Absorbancewasmeasuredat540nminaShimadzuUV3600
spectrophotometer.Thecarbohydratefractionwasextractedfromtheculturesupernatant,andtheamountofsugarliberatedwas
calculatedusingaglucosestandardcurve.Oneendob1,4glucanaseactivityunitwasdefinedastheamountofenzymethat
releases1mmolofreducingsugarperminuteat50C.
Endoxylanaseactivitywasdeterminedindirectlythroughreducingsugarsreleasedbyhydrolysisofsolublexylanbeechwood
(SigmaeAldrich,USA)andsubsequentdetectionbythe3,5dinitrosalicylicacidmethod(DNS)previouslydescribedbyMiller
(1959).
Theenzymereactionwascarriedoutat50Cfor60mininsodiumacetatebuffer0.05M(pH4.8)containing1%beechwood
solublexylan(w/v).Oneendoxylanaseunitwasdefinedastheamountofenzymethatreleases1mmolofreducingsugarper
minuteunderassayconditions.
EnzymeactivitieswerecalculatedonachipweightbasisandreportedinUg
1.
2.9.Analysisofwoodcomposition
Thechemicalcompositionofnontreatedandbiotreatedwoodchipswasdeterminedaccordingtothelaboratoryanalytical
pro cedure (LAP) and biomass analysis of the National Renewable En ergy Laboratory (NREL). Sample preparation for
compositionalanalysiswasdoneaccordingtoNREL/TP51042620(2008).Ethanol
extractives(42619;2008),ash(NREL/TP51042622,2008),andlignin(NREL/TP51042618,2008)werealsodetermined.
CarbohydratesdeterminationbyHPLCwasperformedwithneutralizedsamplesonanSHODEXSP810columnunderthe
followingconditions:purewateraseluentataflowrateof0.6ml/min,85C,andrefractiveindexdetector.Theacetylcontent
wasdeterminedbyHPLCusinganAMINEXHPX87Hcolumnunderthefollowingconditions:4mM$H
2
SO
4
aseluent,flowrate0.6ml/min,35C,andrefractiveindexdetector.Allresultsareexpressed
onadrywoodbasis(OD).
2.10.Kraftcooking:yieldandkappanumber
Kraftcookingrunswereconductedusing200gofdrychips.Thechipswerepreviouslyclassifiedbysquaremeshsieves
throughthefractionpassingthe25mmsieveandretainedonthe5mmsieve.ThekraftcookingwasconductedinanM/K
digesterof7Lcapacity,withliquorrecirculationunderconditionsshowninTable1.
Attheendofthecookingperiod,thepulpwaswashedinthedigesterbyrecirculationofwaterat70Cfor10min.Defibration
wasthenperformedusingastandarddisintegratortoaconsistencyof2%.Thepulpobtainedwasdischargedontoa270mesh
sieveandwashedthoroughlywithwater.ThepulpwasrefinedusingaSomervilleequipmentwithslotsof0.15mm.Total,
refined(accepts),andrejectscontentweredetermined.Thepercentageofacceptsandrejectsfractionsprovidesthetotalyield.
ThekappanumberdeterminationswereperformedaccordingtoTAPPIT236om99.
2.11.Physicalandmechanicalpropertiesofthepulp
Physicalandmechanicalpropertiesofrefinedpulpsweredeterminedat4000PFIrevolutions.Thestandardsusedwere:
refining, TAPPI T248 sp00;drain resistance(SchopperRiegler method), ISO52671:1979; formationsheets forphysical
testing,TAPPIT205sp95;physicaltesting,TAPPIT220sp96.
2.12.Statisticsanalysis
TwowayANOVAwithBonferroniposttestwasperformedusingGraphPadPrism4.00forWindows(GraphPadSoftware,
SanDiego,CA,USA).
3.Results

3.1.PreliminarystudiesoflaccasetemperatureandpHoptimal,andenzymethermostabilityinculturesupernatantsofP.
brevisporaBAFC633
Manyvariablesaffectenzymaticactivityduringthebioprocess.Preliminarystudieswerecarriedoutonculturesupernatantto
determinethebestconditionstoadaptfungalgrowthonwoodchipsandlaccaseactivityduringthebiopulpingprocess.
M.I.Fonsecaetal./InternationalBiodeterioration&Biodegradation90(2014)29e3531
EstimatedtemperatureandpHoptimumforlaccaseactivityonculturesupernatantswere55Cand3.6,respectively(Fig.1A
andB).
Regardingenzymestabilityatdifferenttemperatures,lowertemperatures(20and30C)didnotaffecttheenzymeactivity,
whilehighertemperaturescausedareductionofactivity(Fig.1C).At50Cthehalflifewasapproximately2honculture
supernatants,whileat60Cthehalflifewaslessthan1h.
3.2.EffectofP.brevisporaBAFC633growthonPinustaedawoodchipsinbioreactors
Theexperimentsatthebioreactorswereconductedat30CandapHof3.6.Thistemperaturedidnotmatchtheoptimumtem
peraturefoundinthepreliminaryexperiments;nevertheless,70%ofthemaximumlaccaseactivitywasachieved.Theenzyme
activityatthebioreactortemperature(30C)showedahalflifeof50honculturesupernatants(Fig.1D).
Attheendoftheincubationstep,thefungussecreted744U/goflaccaseand0.55U/gofendob1,4glucanase,while
endoxylanaseactivitywasnotdetected.After4wkofcolonization,thefungusgrewintheformofathinlayerofvelvety
appearanceleavingareddishcoloronthechipssurface.
 Attheendofthefungaltreatmenttheweightofchipsdecreased10%comparedwiththecontrolwithouttreatment(p<0.05).
LongitudinalsectionsofwoodchipsinoculatedwithP.brevisporaBAFC633clearlyshowedfungalhyphaepenetratingthe
lumens,vessels,andcellsthroughpits(Fig.2).
3.3.Chemicalanalysis
Woodextractivesarethosecomponentssolubleinneutralorganicsolventsconsistingofwax,fats,resins,phytosterols,and
volatilehydrocarbons.TheextractivescontentofwoodchipstreatedwithP.brevisporaBAFC633increasedsignificantly(p<
0.001)(Table2).
Oneofthemainobjectivesofchemicalpulpingprocessesistoremoveligninselectively.Thegoalofwoodchiptreatment
withthefungusprevioustochemicalpulpingistoreducethelignincontent,thusobtainingahigherdelignificationinthepulping
process.Itwaspossibletoachievealossof4.22%ofthetotalligninintreatedwoodchips(p<0.01)(Table2).
Thecarbohydrateandligninanalysisprovidesinformationabouttheselectivityofthewooddegradingfungus. Arabans
decreasedsignificantlyonlyinthetreatedsamples(p<0.01),whereastheothercarbohydratesdidnotshowsignificantdiffer
ences(p>0.05).Thesedatasupportthefungusselectivitytowardligninselecteddegradation,preservingpolysaccharides,a
fundamentalfeaturerequiredforpulpingprocesses.
3.4.Pulpyield,kappanumber,andopticalproperties
Theyieldintreatedsamplesdecreased1.73points,buttherewasalsoadecreaseof6.3%kappanumber,whichindicates
significantligninreduction(p<0.01).Usuallyitisdesirabletoachievethehighestdegreeofdelignificationwiththeproduction
preservation,withthebonusthatligninremovalfacilitatestheaccessibilityofthepulpingliquor.Itisconvenienttoevaluate
thebenefitofthereductionintheligninduetofungaltreatmentagainstlossofcarbohydrateyield,sinceitisimportanttokeep
thiscontentashighaspossible.
Fungaltreatmentcauseda7.1%increaseinpaperbrightness(p<0.05).Thispropertyiscritical,sinceitaffectstheamountof
bleachingchemicalsused.Reducinguseofthesechemicalsisbeneficialforbotheconomicandenvironmentalreasons(Table3).
Table1Kraftcookingconditions.
ParameterCondition
AA/wood(%)24Sulfidity(%)30Liquor/woodrate5Max.temperature.(C)170Impregnationtime(min)60HFactor2000
3.5.Physicalandmechanicalproperties
Thechangesintroducedbyfungaltreatmentofwoodchipsandsubsequentpulpingprocessgenerallyhaveanimpactonthe
structuralfeaturesofthepaper,whichinturnwillaffectthepapersproperties(Table4).
Fig.1.LaccaseactivityinculturesupernatantsofP.brevisporaBAFC633,dependingonpH(A)andtemperature(B).Enzymatic
stabilityoflaccaseactivityatdifferenttemperatures(C):20C(),30C(:),40C(;),50C(A),and60C(C),andafterprolonged
incubationat30C(D).
M.I.Fonsecaetal./InternationalBiodeterioration&Biodegradation90(2014)29e3532
Fig.2.Microscopiclongitudinalsections(40Xbarmarkers1450mm,and100Xbarmarkers1415mm)fromPinustaedawood
chipstreated(A,B,E,F,G,H,I,J)anduntreated(C,D)withP.brevisporaBAFC633.Arrowsindicatethebluestainedhyphae
andthereddyedwood.
Table2ChemicalanalysisofwoodchipswithandwithoutP.brevisporaBAFC633treatment.
%OvendrywoodTreatedchipsControlchips
Glucan42.150.942.660.52Xylan6.100.036.190.09Araban0.980.00**1.010.04Galactan1.590.072
0.09Mannan7.440.198.570.39Acetyl1.600.081.790.15Insolubleacidlignin26.720.0828.790.01**
Solubleacidlignin2.730.19**1.960.15Totallignin29.4530.75Alcoholextractives3.260.11***1.290.03Ash
0.270.36
(*)p<0.05;(**)p<0.01;(***)p<0.001.
Table3Pulpingoftreatedanduntreatedwoodchips.
PulpsRefined
yield%
KappaNBrightness
%ISO
Withfungal
treatment
Rejects%Total
yield%
43.64043.6420.80.21**31.0*
Withoutfungaltreatment
45.370,0245.3922.20.1628.8
(*)p<0.05;(**)p<0.01;(***)p<0.001.
Measurementofthedrainabilityoftherefinedpulp,anindexofthedegreeofrefinement,wasconductedintheSchopper
RieglerapparatusandexpressedinSR.Determiningthevalueofthisindexisoneofthemostimportantstagesinthepaper
productionprocessandstronglyinfluencestheconformationofthesheetanditsphysicalproperties.Aslightincreaseof1SRof
therefinedpulpsisconsideredanimprovementofthepulpcharacteristicscomparedtounrefinedsamples.Forthecorrect
formationofthepulpinthepapermachine,higherSRisrequired.
Thegrammage(gm
2
)ofthetreatedsamplesincreased0.49pointscomparedtothecontrolsamples,whilethethickness,
the
distancebetweenbothsidesofthepaper, remained constant. Thedensityofthepaperdecreasedin0.01 points. Bulkand
percentageelongationdidnotshowsignificantdifferences(p>0.05).
Theairpermeabilityofthepaperisofgreatimportanceinthematerialspecificationsforthepackagingindustryandprinting.
Permeabilityistheairflowthroughtheunitareaofunityunderthepressuredifferenceintheunitoftimeunderstandard
conditionsofthetest.Inourcasethepaperpermeabilitydecreasedslightly(0.03mm/Pas,p<0.05),sothattherewasaminor
effectofcompactionofthefiberstructure.Theairresistance,whichistheresistancetothepassageofairofferedbythepaper
structure,increased24.45points,indicatingthattheresultingfibroussheetismoreclosed.
Regardingtheremainingmechanicalproperties,nosignificantdifferencewasfoundinstrength,explosion,ortearresistance
(p>0.05),norinthetensileenergyabsorption(TEA)(p>0.05).
4.Discussion

Theenzymaticactivityandthelignindegradationareinfluencedbyanumberoffactors,includingthefungalstrain,the
nutrientcomposition(e.g.,Mn
2
andCu
2
),moisturecontent,pH,andtemperature(Fonsecaetal.,2010;Kaaletal.,1995;Zhaoet
al.,1996;Fuetal.,1997;Doradoetal.,2001;SnajdrandBaldrian,2007;Pateletal.,2009).Tocontrolthesefactors,optimal
conditionsforthepretreatmentprocessshouldbeachieved,whichwouldresultinanoptimalgrowthofwhiterotfungionthe
selectedsubstrates.Knowingthebiochemicalcharacteristicsoflaccaseinthesupernatantalsocouldservetoreducethecostof
enzymepurification.TheliteraturedisclosesthatthefungallaccasehavehigheractivitiesatmoreacidicpHlevels(Xu,1997)
andthattheoptimaltemperatureisbetween50and60C(Baldrian,2006).Inourcase,theoptimumpHforlaccaseactivity
estimatedinthesupernatantwas3.6,whileoptimumtemperaturewasestimatedat55C.However,conditionsforfungalgrowth
andenzymeproductioncouldbedifferent,andthesemustbeconsideredduringbioprocessingwithlivingmicroorganismssuch
asfungi.Moreover,enzymestabilityiscrucialduringthebiotechnologicalprocess.Thesupernatantscontaininglaccaseactivity
producedbyP.brevisporaBAFC633reachedahalflifeofw50hat30C,beingfurtherextendedtolowertemperatures,which
wouldbeveryimportantforproductioninbioreactorswithoutthenecessityofrefrigeration.Addressingthesefundamentals,we
decidedtoconductthebiopulpingexperimentsat29CandpH3.6toensurefungalgrowthandhigherlaccasestability.
Whiterotfungiarecapableofdegradingallcomponentsofthecellwall,butthereiswidevariationinthetypesof
degradation
M.I.Fonsecaetal./InternationalBiodeterioration&Biodegradation90(2014)29e3533
produced:Cellulose,hemicellulose,andligninmaybedegradedsimultaneouslyoreachcomponentmaybedegradedatdifferent
rates,andalsothepreferentialattacktoligninandhemicellulosemightoccur(Blanchetteetal.,1987).Inthisstudy,thelevelsof
ligninolytic enzymes indicated a predominance of laccase activity associated with very low endocellulase activity and no
evidenceofendoxylanaseactivity.
ThereddishpigmentationcausedbyP.brevisporaBAFC633growingonchipscouldhaveresultedinthebiosynthesisof
melanin,poweredbyphenoloxidaseactivity(Ferrazetal.,2003).Thispigmentationmaybeanindicationoffreeradicalsnearb
hyphae,indicatingtheincrementofoxidativepotentialmorethanhydrolyticpotential,whichwasobservedinquantitative
studies.Thereddishappearanceduetotheproductionofcinnabarinicacidproducedbythefungushasalsobeendescribedfor
Pycnoporuscinnabarinus(TempandEggert,1999).
Itisknownthatfungiatearlystagesofinoculationgenerallyconsumethenutrientsstoredintherays(parenchymacells),
decreasingthesugarcontentofthesecellsandcausinganinitialweightloss(Talaeipouretal.,2010).Ourresultsdidnotreveala
remarkabledecreaseofcarbohydrates(exceptforthearabans),indicatingaselectivedegradationoflignin.
BlanchetteandBurnes(1988)reportedthatwoodchipslost38%oftheirweightafter12wkoftreatmentwiththefungusP.
chrysosporiumBKMF1767.Intheirreport,46%oftheweightlossoccurredafterexposuretothefungusHHB6251,17%was
duetoFPLV1706,and38%wasduetothefungusMEPC8.Themajorchangesinthecellwallcausedbythefungusare
brokencellwalls,looseningofthetubularstructureofthefibers,separationoffiber,anddisappearanceofthemiddlelamella.In
thissense,theopticalmicroscopeobservationsshowedhowthehyphaeofP.brevisporaBAFC633wereintroducedintothe
lumenscellthroughpits(Fig.2).However,ultrastructuraldegradationcharacteristicscouldbeobservedonlywiththeelectron
microscope.Accordingtothechemicalanalysisresults,theweightlossthatoccurredinthisstudycouldbeattributedmainlyto
thelossoflignininthewoodchipstreatedsincethecarbohydratecontentremainedunchanged.
Somestudieshaveshownthatduringbiotreatment,theremovalofligninfractionsallowsbetterdiffusionofenzymesthat
causetheattackonpolysaccharides(MachucaandFerraz,2001).ThisstudyshowedthatP.brevisporaBAFC633selectively
removedtheligninwithoutcausingadecreaseofcarbohydrates,exceptforarabans.Thisnulleffectoncarbohydratesinthe
treatedsampleswasalsoobservedbyVillalbaetal.(2006),andAtesetal.(2008).Thisisbecauseligninservesasaphysical
barrierandtosomeextentachemicaloneforenzymaticdegradationofthesepolysaccharides(PewandWeyna,1962).Thelow
ornulllossofglucansisrelatedtotheselectiveremovalofligninduringthebiopulpingexperiment(Akhtaretal.,1993;Ferrazet
al.,2000).Otherresearchershaveshownthatoneofthepolysaccharidesofthewoodwassimultaneouslydegradedwithlignin
(Eriksson,1978;SetliffandEudy,1980).Ferrazetal.(2003)foundthatcelluloseremainedintactandthatthecarbonandenergy
sourcesoriginatefromhemicellulose.Thesefeaturesofwooddegradationareimportantintheprocessofbiopulping.Our
resultsshowedthatalthoughtotallignindecreased,thefractioncorrespondingtotheacidsolubleligninshowedanincreasein
thesamplestreatedwiththefungus,whichcouldbeduetodegradationproductsoflowmolecularweightandhydrophilic
derivativesoflignin(Yasudaetal.,1998).However,thehighamountofextractivespresentinthewoodduringbiopulpingwith
P.brevisporaBAFC633couldbeadisadvantageinthepulpingprocesssinceitspresenceisassociatedwithpulpyellowing
(FengelandWegener,1984).
Asmalldecreaseinyieldinthesamplestreatedwiththefunguswasobserved.Fungalpretreatmenthavebeenreportedthat
Table4Physicomechanicalpropertiesofkraftpulpsrefinedat4000PFIrevolutions,withandwithoutP.brevisporaBAFC633
treatment.
PropertiesTreatedpulpsControlpulps
Drainresistance(SR)3635Paperweight[g/m2]64.89*64.40Thickness[mm]0.080.0010.080.001Bulk[cm
3
/g]1.28*1.27Density[g/cm
3
]0.780.79Burst[kPam
2
/g]7.760.287.610.3Tear[mNm
2
/g]10.160.6110.910.41Tensile[Nm/gr]100.565.396.525.1Elongation%3.30*3.45TEA[J/m
2
]146.0317.7145.8718.4IndexTEA[J/g]2.250.2732.270.258Airflowresistance[s]357.91*333.48
Permeability[mm/Pas]0.360*0.390.1
(*)p<0.05.
produceinsignificantchangesintheyieldofchemicalpulp(Giovannozzietal.,1994;Atiketal.,2006;Villalbaetal.,2006;
ImamogluandAtik,2007;Atesetal.,2008).WoodandpulplignincontentwasreducedasaresultofP.brevisporaBAFC633
pretreatment,andthisisconsistentwithresultsobtainedbyotherauthors(Akhtaretal.,1997;Bajpaietal.,2004;Villalbaetal.,
2006).Theseresultsindicatethatthefunguscausesstructuralchangesinthefiberthatwouldfacilitatethepenetrationofthe
cookingliquor,whichinturnenhancesandacceleratesthedelignification.
Thephysicalpropertiesmaybeusefulinpredictingthepotentialofpulpinpapermanufacture,aswellasinexaminingthe
efficiencyoftheprocessundercontrolledconditions,soastodescribetheperformanceofthefinalpaper(Waterhouse,1992).
Althoughthefungusactstoopenandsoftenthecellwallstructure(Kirketal.,1994),ourresultsshowednobenefitsintermsof
physicalproperties.Francoetal.(2006)foundthatthestrengthpropertiesofpulpsweresimilarinbiotreatedandcontrolpulps.
Thesequalityparametersareveryimportant,soadditionalresearchmustbeconductedtofindtheconditionstoimprovethese
properties.Moreover,fungalpretreatmentimprovedopticalproperties,allowingwhiterpulp,andtheseresultsaresimilartothose
obtainedbyotherauthorsinwheatstrawpulp(Atesetal.,2008)andbagasse(Bajpaietal.,2004),althoughweachievedhigher
brightnesswitharawmaterialwithhigherlignincontentandmoreclosedstructure.Theseincreasesarearesultofthelower
lignincontent,whichalsoleadstoshortercookingtimes,easeofbleaching,andconsequentlylowerconsumptionofbleaching
chemicals.Removingthemaximumamountofligninduringthecookingprocessisveryimportantbecausebleachingchemicals
aremuchmoreexpensivethancookingchemicals.
5.Conclusions

TheinoculationofwoodchipswiththewhiterotfungusP.brevisporaBAFC633selectivelyremovedtheligninduringbio
treatment, causing a reduction in kappa number associated with decreased levels of lignin in the pulp. The modification
introducedbythebiologicalactiononthewoodchipsleftamoreopenstructure,whichallowedbetteraccessofthepulping
liquortothecellwallcomponents.Theeffectivenessofkraftpulpingwasimproved,asdemonstratedbytheincreaseddissolution
oflignininpulpsfromwoodchipstreatedwithP.brevisporaBAFC633.Thechemicallymodifiedligninwasmorereadily
solubilizedbythecookingliquor.Consideringthatbrightnessisoneofthemostimportantparametersdefiningpulp,andthat
fungalpretreatmentwithP.brevisporaBAFC633improvedpulpbrightness,thisworkmadeanimportantcontributiontoend
product quality. While the effects of biological treatment (lignin depolymerization and enlarged pores) improved the kraft
pulpingprocess,morestudyisneededonbiopulpingwithP.brevisporaBAFC633sothatprocessingconditionscanbe
optimized.
Acknowledgments

PartoftheexperimentalworkwasfundedbytheSecretaradeCienciayTecnologadelaUniversidadNacionaldeMisiones,
Argentina,throughgrantsforinnovationprojects(16Q446and16Q486).MIF,MLC,andMDRhavefellowshipsfordoctoral
studiesofCONICET,Argentina.
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