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Valorizationofoliveoilmillwastewaterfortheproductionofβ-glucansfromselectedBasidiomycetesstrains

A.Zerva1,G.Mousavi1,L.-M.Papaspyridi1,P.Christakopoulos2andE.Topakas1*

1BiotechnologyLaboratory,SchoolofChemicalEngineering,NationalTechnicalUniversityofAthens,5IroonPolytechniouStr.,ZografouCampus,Athens15780,Greece2BiochemicalandChemicalProcessEngineering,DivisionofSustainableProcessEngineering,DepartmentofCivil,EnvironmentalandNaturalResourcesEngineering,LuleåUniversityofTechnology,SE-97187Luleå,Sweden

*Correspondenceto:E.Topakas.Tel:+30-210-7723264;fax:+30-210-7723163;e-mail:vtopakas@chemeng.ntua.gr

Abstract

Purpose

TheaimofthepresentstudywastoinvestigatethefeasibilityofpolysaccharidesproductionbyselectedBasidiomycetesinsubmergedculture.Oliveoilmillwastewater(OOMW)wasalsotestedasapotentialsubstrateforpolysaccharidesproductionbymushroomstrains,focusingonthesimultaneousdegradationandvalorizationofthewastematerial.

Methods

Thetestedstrainsweregrownintwodifferentsubstrates,andafterbiomassharvesting,polysaccharideswereisolatedusingtwodifferentmethods.Theextracellularpolysaccharideswereisolatedfromtheculturebroth,withethanolprecipitation.TheisolatedfractionswerepartiallycharacterizedwithFT-IRspectroscopy.

Results

Allthreestrainsperformedwellinbothsubstrates.MaximumdegradationperformanceofOOMWwasachievedbyG.lucidum.Extracellularpolysaccharides(EPS)productionwasobservedduringgrowthindefinedmediumonly,exceptfromP.ostreatus,whereEPSwerealsoisolatedfromOOMWcultures.Inregardtobiomasspolysaccharides,P.citrinopileatusbiomassgrowninOOMWwasfoundtobethericheringlucans,with14.1%(w/w)totalglucancontent.Afterpurificationofbiomasspolysaccharideswithtwomethods,thefractionwiththehighestglucancontentwasfoundtobetheonefromG.lucidumaftergrowthindefinedmediumcultures,with49.1%(w/w)totalglucans.FT-IRspectraoftheisolatedsamplesrevealedthebandscorrespondingtoα-andβ-glucosidicbonds,butalsotheexistenceofproteincontamination.

Conclusions

Purificationofbiomasspolysaccharideswithtwodistinctmethodsrevealedthatα-amylaseandSevagtreatmentsfailedtoremovecompletelyα-glucansandproteinsrespectively,leadingtothe

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suggestionthatthesetwostepscouldbeomittedwithoutsignificantimpact.Moreover,theresultsimplythatthevalorizationofOOMWmightbefeasiblewiththeuseofmushroomstrains,leadingtotheproductionofimportantproducts,suchasglucans.

Keywords:β-glucan,oliveoilmillwastevalorization,Basidiomycetessubmergedculture,polysaccharideisolation

1.Introduction

Glucansarepolysaccharidesconsistingsolelyofglucoseunits.Thenumberofdifferentglucosidicbondswithwhichtheglucosemonomerscanbeconnected,togetherwiththeoccurrenceofbranchedchains,resultsinavarietyofdifferentpolysaccharidemoleculeswithdistinctproperties.Amongthem,β-1,3-D-glucansorβ-1,4-D-glucanscanbefoundinthecellwallsofhigherplantsandcerealseeds,whileβ-1,3-D-glucansandβ-1,6-D-glucansareusuallyfoundinthefungalcellwalls[1],togetherwithα-1,3-Dglucans,frequentlyincomplexwithchitinorproteins[2].β-D-glucansareusuallybranchedchains,withvaryingmolecularweight,whileα-D-glucansarelinearpolymers.

Researchinterestinglucansissteadilygrowingthelast20years,duetotheirimportanteffectsinthehumanhealth,includingantitumor,immunostimulating,(activatingphagocytosis)[3],antidiabetic[4],apoptosis-inducing[4],andantioxidant[5]activities.Activityagainstbacteria,virusesandfungihasalsobeenreported[5].Moreover,theyarefoundtoenhancepatientrecoveryfromthetoxiceffectsofchemotherapy,lowerbloodcholesterolconcentration,andparticipateinbloodpressurecontrol[3].

Manyfungalstrainsproducepolysaccharides,eitherasasecretedmetabolite,orasaconstituentoftheircellwall.Amongthem,theBasidiomycetesspeciesbelongingtothegeneraPleurotusandAgaricus,withgreatsignificanceinhumannutrition,andsomeofpharmaceuticalimportance,suchasGanoderma,arethemostpromisingcandidatesforpolysaccharidesproduction,andthusthefocusofmostresearchstudies[4,6,7,8,9,10].Therearealsosomestudiesreportingthedetectionoftheenzymesresponsibleforthesynthesisofpolysaccharides,suchasβ-D-glucansynthases,andtheircorrespondinggenes[5,11].

Apartfromtheirabilitytoproduceglucanswithdietaryvalue,manyBasidiomycetestrainsarealsoveryefficientlignindegraders,duetotheirpotentoxidativeenzymesystem,comprisingmainlyoflaccases(Lac,EC1.10.3.2),manganeseperoxidases(MnP,EC1.11.1.13)andmanganese-independentperoxidases,suchasligninperoxidase(LiP,EC1.11.1.14),togetherwithavarietyofotheraccessoryenzymes.Duetotheproductionofthisbiocatalyticfactory,Basidiomycetesareabletodegradecomplexphenolicsubstratesandusethemasacarbonsourcetosustaintheirgrowth.Takingthistraitintoadvantage,suitableBasidiomycetesstrainscouldbegrowninlignin-containingwastes,andwhiledegradationofphenolsisunderway,thesecretedpolysaccharidescouldbeisolatedfromtheextracellularfluid,andthebiomasscouldbeharvestedfortheisolationofcellwallpolysaccharides.Thisway,acombinedbioremediationandvalorizationofthewastecanbeachieved[12,13].Apromisingphenolicwasteforthispurposeisoliveoilmillwastewater(OOMW),acommoneffluentfromtheoliveoilproductionprocess.OOMWishighlytoxicagainstplants,soilmicroorganismsandmarineorganisms,duetoitshighorganicloadandphenoliccontent.Basidiomycetesareabletogrowusingitscomponentsascarbonandenergysource,seeingthat,

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apartfromphenolcompoundsthatcanbeoxidizedbytheirligninolyticenzymesystem,OOMWalsocontainssugars,lipidsandorganicacidsthatpromotefungalgrowth[13,14].

Inthepresentwork,wereporttheproductionandisolationofβ-D-glucansfromthreeendogenousGreekstrains,Pleurotusostreatus,PleurotuscitrinopileatusandGanodermalucidum.Theproductionofbiomasspolysaccharidesandexo-polysaccharideswastestedduringgrowthintwodifferentsubstrates,adefinedsugarmedium,andacomplexwastewatermedium,containingOOMW.Theisolationofbiomasspolysaccharideswastestedwithtwodifferentmethods.TheisolatedfractionswerefurtheranalyzedwithFT-IRspectroscopy.

2.MaterialsandMethods

2.1Oliveoilmillwastewater

OOMWwasobtainedfromanoliveoilmillwithathree-phasedecanterinKalamata(Peloponnese,S.W.Greece),pretreatedandmaintainedat-20oC.ThecompositionandphysicochemicalpropertiesofOMWWwaspreviouslyassessed[15].Priortouse,pHwasadjustedto6with3NNaOH,andtheOOMWwascentrifugedat8000rpm,4oCfor20minandsubsequentlyfilteredthroughWhatmanNo.1,toremoveanysuspendedsolids.

2.2Microorganismsandcultureprocedures

TheP.citrinopileatusLGAM28684,G.lucidumLGAM9720andP.ostreatusLGAM1123strainsusedforthisstudy,wereobtainedfromtheculturecollectionoftheLaboratoryofGeneralandAgriculturalMicrobiology(AgriculturalUniversityofAthens),andwereselectedafterascreeningevaluationincludingnumerouswhite-rotbasidiomycetes[16].ThestrainsweremaintainedinPotatoDextroseAgarplates(PDA-Applichem,Germany)at4oC.Liquidcultureswerepreparedasreportedpreviously[17].Briefly,the100mLliquidmediumcontainedxylose57gL-1,cornsteepliquor37gL-1,K2HPO41gL-1,andMgSO4(H2O)70.2gL-1.Alternatively,forthepreparationofliquidcultures,OMWWwasdilutedatafinalconcentrationof50%(v/v)withtheappropriatebuffersolutiontoafinalvolumeof100mLin250mLErlenmeyerflaskswithcottonstops,supplementedwith30gL-1yeastextract,andautoclavedat121oCfor20min.Batchfermentationswerealsocarriedoutin2.5LBio-Flo310bioreactors(NewBrunswickScientific,US),usingeithertwo-folddilutedOOMWwith0.1MpotassiumphosphatebufferpH6,orthedefinedmediumdescribedpreviously.Agitationspeedwassetto100rpm,andthemediumwassupplementedwith30gL-1yeastextractpriortosterilization.Theinoculationof1.5Lofmediumwascarriedoutwith200mLoffullygrownprecultures,resultingatastartingconcentrationof1.5-2.5gL-1ofdrybiomass.Thefermentationwasmaintainedat26oC,100rpm,unlessotherwisestated.Samplesweretakenatselectedtimeintervals,centrifuged(1520g,10min),andthesupernatantwasusedforanalysis.Attheendofthebioreactorfermentation,biomasswasseparatedfromtheculturemediumbycentrifugationatthesameconditions,freeze-driedandweighed.

2.3DeterminationofOMWWtotalphenoliccontentanddecolorization

Totalphenolscontentwasdetermined,asdescribedbyWaterhouse[18].Phenolsconcentrationwasexpressedinppmofgallicacidequivalents,usingtheappropriatecalibrationcurve.OMWW

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decolorizationwasestimatedspectrophotometricallybymeasuringtheabsorbanceat525nm,aspreviouslydescribed[19].

2.4Assessmentofglucancontentinfungalbiomass

Thecontentintotalglucansandα-glucansweredeterminedinthefreeze-driedmyceliumofalltestedspecies,obtainedfromculturesgrowninOMWW-basedmedia,accordingtotheMushroomandYeastβ-glucanAssayProcedureK-YBGL10/2005(MegazymeInternationalIreland,Bray,Ireland).Thecontentinβ-glucanswascalculatedasthedifferencebetweentotalglucansandα-glucans.

2.5Isolationandpartialpurificationofβ-glucans

Thepurificationofβ-glucansfromthemycelialbiomasswastestedwithtwomethods:ThefirstwasamodifiedmethodofSynytsyaetal.,2009(Fig.1,ProtocolA),andthesecondthemethoddescribedbyWeietal.,2008[32](Fig.2,ProtocolB).

Extracellularpolysaccharides(EPS)wereisolatedasfollows:Theliquidculturesupernatantwasconcentrated10-foldinarotaryevaporator(RotavaporBuchiRE111,Buchi,Switzerland).Intheconcentratedsupernatant,polysaccharideswereprecipitatedwiththeadditionof4volumescoldethanol(96%)andovernightincubationat4oC.Themixturewascentrifugedandtheprecipitatewasfreeze-driedandweighed.

2.6FT-IRspectroscopy

FT-IRspectraofthedriedglucanpreparationswererecordedwithaNicoletMagna-IR560Spectrophotometer(ThermoFisherScientificInc.)inthe4000-400cm-1rangeintheformofKBrpellets.

2.7Statisticalanalyses

DataanalysiswasperformedwiththeuseofSigmaPlotsoftware(SystatSoftware,Inc.,SanJose,CA,USA).Errorbarsrepresentthestandarderrorofthemeanvalue.

3.Results

3.1Fungalgrowthundersubmergedfermentation

Allthreestrainsshowedasatisfactorygrowthinsubmergedcultureinbothmediatested.P.ostreatusyielded4.87mgmL-1biomassafter13daysofgrowthinthe2-Lbioreactorindefinedmedium,and8.38mgmL-1biomassafter13daysunderthesameconditionsinOOMW-basedmedium.Inthelattercase,totalphenolicsreductionreached43.6%,andmediumdecolorization11.1%.BiomassproductionforG.lucidumreached89.5mgmL-1indefinedmedium,butonly4.2mgmL-1inOOMW-basedmedium,after6daysofgrowth.ForOOMW-growncultures,takingintoaccountthelowbiomassproduced,thereductionoftotalphenolicsreachedonly19.4%,butdecolorizationofthemediumwas47.56%attheendoftheculture.

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3.2Isolationofextracellularpolysaccharides

ForP.ostreatuscultures,theEPSproductionreached15.9mgmL-1inthedefinedmediumand18.8mgmL-1intheOOMW-basedcultures.ThecrudeEPScorrespondingtothecultureswiththedefinedmediumwasfoundtocontain4.57%totalglucans,consistedof0.27%α-glucansand4.3%β-glucans.LoweryieldwasobtainedintheEPSfractioncorrespondingtotheculturesgrownonOOMWmedium,containing1.91%totalglucansconsistedof0.46%α-glucansand1.45%β-glucans.

InthecaseofG.lucidum,EPSwereonlyproducedatanidentifiablequantityinthecaseofdefinedmediumcultures.Inthiscase,EPSconcentrationwasestimatedonlyat2.53mgmL-1.Nonetheless,EPSexctractedwerefoundtobehighlyrichintotalglucans,reachingthepercentageof37.9%,withonly0.34%accountingforα-glucansand37.6%forβ-glucans.Theseresultsareinaccordancewithpreviousstudies,makingG.lucidumapromisingcandidateforlarge-scaleproductionofvaluableβ-glucans.

ForP.citrinopileatuscultures,EPSsamplescontained6.6%oftotalglucans,ofwhich0.46%wereα-glucansand6.17%wereβ-glucansduringgrowthindefinedmedium,withafinalconcentrationof1.2mgmL-1.However,EPSwerenotdetectedinculturesgrowninOOMWmedium.Forallthreestrains,itseemsthatthemajorityoftheexo-polysaccharidesisolatedaccountsforβ-glucans.

3.3Isolationofmycelialbiomass-derivedglucans

P.ostreatuscrudebiomasssampleswereanalyzedfortotalglucancontentinbothfermentationusingdefinedmediumorOOMWasgrowthsubstrate(Table1).Inthedefined-mediumcultures,totalglucancontentwasfoundat8.68%(w/w),with3.36%(w/w)accountingforα-glucansand5.32%(w/w)accountingforβ-glucans.ConcerningOOMW-basedcultures,thecorrespondingnumberswere7.58%(w/w)fortotalglucans,1.1%(w/w)forα-glucans,and6.48%(w/w)forβ-glucans.IsolationofbiomassderivedpolysaccharidesfromP.ostreatus,wascarriedoutfollowingProtocolA,includingtwoisolationsteps,oneforwatersolublepolysaccharidemoleculesandoneforalkali-solublepolysaccharidemolecules(Table1).ForP.ostreatusgrownindefinedmedium,polysaccharidefractionswereobtainedwherethewatersolublefractioncontained20.45%(w/w)oftotalglucans,ofwhich11.05%(w/w)wereα-glucans,and9.4%(w/w)wereβ-glucans.Additionally,thealkali-solublefractioncontained16.39%(w/w)totalglucans,composedof11.02%(w/w)α-glucansand5.37%(w/w)β-glucans.InthecaseofOOMW-grownbiomass,thealkali-solublepolysaccharidefractionwasabsent,whilethewater-solublefractioncontained34.76%(w/w)totalglucans,with3.02%(w/w)α-glucans,and31.74%(w/w)β-glucans.Fromtheseresultsitseemsthatthetypeofmoleculesproducedmaydependonthegrowthconditions,atleastconcerningtheirphysicochemicalproperties.

TheisolationofpolysaccharidesfromP.citrinopileatusbiomassaftergrowthindefinedmediumwascarriedoutfollowingProtocolB.Theobtainedpolysaccharidefractionwasonly37mg,andthusthedeterminationofitsglucancompositionwasnotpossible.Ontheotherhand,P.citrinopileatusbiomassgrowninOOMWcultureswastreatedwithbothProtocolsAandB,allowingthecomparisonofbothmethodologies.IsolationofpolysaccharidesfollowingProtocolB(Table2),resultedinonlyonepolysaccharidefractionwiththecompositionof31.71%(w/w)totalglucans,15.72%(w/w)α-glucansand16%(w/w)β-glucans.Ontheotherhand,theisolationofpolysaccharidesfollowingPotocolA,yieldedawatersolublefractionwith13.13%(w/w)totalglucans,composedof6.33%(w/w)α-glucansand6.8%(w/w)β-glucans,andanalkali-solublefractionwith23.76%(w/w)totalglucansconsistedof2.21%(w/w)α-glucansand21.55%(w/w)β-glucans.

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G.lucidumbiomasswastreatedonlyfollowingProtocolB.Forthedefinedmediumgrownbiomass,theglucanscompositionwasfound6.2%(w/w)fortotalglucans,resultingin0.83%(w/w)α-glucans,and5.39%(w/w)β-glucans,whileafterpurificationtheobtainedfractioncontained49.09%(w/w)totalglucans,composedof0.51%(w/w)α-glucans,and48.58%(w/w)β-glucans(Table3).ForOOMW-grownmycelium,thecrudebiomasscompositionwas5.56%(w/w)totalglucans,0.13%(w/w)α-glucans,and5.43%(w/w)β-glucans.Afterisolation,theobtainedfractioncontained14.96%(w/w)totalglucans,0.64%(w/w)α-glucans,and14.32%(w/w)β-glucans.

3.4FT-IRanalysisofisolatedpolysaccharidefractions

Fortheidentificationoftheisolatedpolysaccharidefractions,FT-IRwasemployed.AllspectrashowninFig.3presentawidepeakaround3300-3400cm-1,correspondingtofree–OHgroupsofalargemolecule,indicatingtheexistenceofhighMWpolysaccharidemolecules[5].Thepeaksin881and893cm-1presentinEPSfromculturesgrowninbothdefinedandOOMWmediumforthesamplesderivedfromthespeciesP.citrinopileatusandG.lucidum,correspondingtoβ-glucosidicbond,andthusindicatingtheexistenceofβ-glucanmolecules[9,20,21].Thepeaksat854,862and845cm-1indicateaccordingly,theexistenceofα-glucanmoleculesinthesamplesofP.ostreatusandP.citrinopileatus[2,20,21].Moreover,thepeaksaround520-550cm-1intheP.ostreatusEPSsample,andsomeoftheothersamples,alsoindicatethepresenceofα-glucansaccordingtoseveralstudies[2,9].Peaksat1127,1153,1152,1158,1150and1143cm-1representthestretchoftheC-O-Cbondofthepyranoseringofsugarmoieties[9,21].Thepeakat1650cm-1foundinallthesamples,correspondstotheC-Nbondoftheaminoacids,andthusindicatesthepresenceofproteincontaminationinthesamples[9].Thepeaksaround2920-2930cm-1correspondto–CH2groupsoflipids,aspreviouslyreported[9].

4.Discussion

Inthepresentwork,allthestudiedBasidiomycetesstrainsgrewwellinbothmedia,yieldingsatisfactoryamountofbiomassinfermentationsindefined,aswellasinOOMWmedia.OOMWsupplementedwithnitrogensourceseemstobeabletosustainmycelialgrowthfromallstrains.However,wastedegradationinthesecaseswasnotashighasreportedpreviouslyforotherBasidiomycetesstrains[13,22,23]indicatingthatfurtheroptimizationstepsarenecessary.However,theresultssuggestthatthesubmergedfermentationmightbemoreefficientintermsofproductivity,duetothefactthatbiomassgrowthwasconsiderablyfastercomparedtosolidstateapproaches,wherecompletefruitbodyformationmighttakeuptotwomonths[24].

P.ostreatusappearstobemoreproductiveintermsofEPScomparedtotheothertwostrains.Inthiscase,aconsiderableamountofEPSwasisolatedinbothtestedmedia,albeitwithalowconcentrationofglucans.TheothertwostrainsseemtoproduceameasurableamountofEPSonlyduringgrowthinthedefinedmedium.EPSobtainedfromG.lucidumculturesinparticular,wereisolatedasagel-likesubstance,aspreviouslyreportedforBasidiomycete’sEPS[8],consistingalmostcompletelyfromβ-glucans.Theyieldsobtainedwerecomparabletothosereportedpreviouslyforotherspecies[25].Unfortunately,EPSwerenotisolatedduringgrowthinOOMW-basedmedia.ThiswasalsothecaseforP.citrinopileatus,however,inthedefinedmediumcultures,theEPSyieldexceededthoseobtainedinthepreviousworkbyWangetal.[10],bythesamespecies,whereonly0.56mgmL-1EPSwereisolated.

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ForP.citrinopileatusbiomasspolysaccharides,thepurificationyieldsobtainedwiththetwomethodsweresimilar,andcomparabletorelevantstudies[27,28,29].Inthiscase,thetwomethodstesteddidnotseemtodifferintermsofpolysaccharidesyieldandrecovery,leadingtotheconclusionthatthetime-consumingandcostlystepsofProtocolA,mainlytheincubationwithα-amylaseandtheSevagtreatmentstepsmaybeomittedwithoutsignificantlossinthefinalproduct.

FollowingProtocolA,P.ostreatusbiomassglucanswerefoundmainlyinthewater-solublefraction.Theobtainedpurificationyieldsweresurprisinglyhigh,reaching30%forOOMW-grownmyceliumβ-glucans.Thisisquiteinteresting,takingintoaccountthattheseyieldsareconsiderablyhigherthanothersreportedintheliterature[6,8,30].However,comparableyieldshavebeenalsoobtainedpreviously[26].

Ontheotherhand,fortheisolationofglucansfromG.lucidumbiomassProtocolBprotocolofWeietal.[32]wasfollowed.Theprotocolyieldswereveryhighinthiscase,exceeding40%fortheβ-glucansisolatedfromthemyceliumgrownindefinedmedium,asreportedpreviouslyforotherspecies[26]butquitehigherthanmostrelevantstudies[6,8,30].

FT-IRspectraoftheisolatedfractionsrevealedproteincontamination,despitemultipletreatmentswithSevagreagent,asshowninpreviousstudies[9].Glucansoftenoccurinmushroomsincomplexwithproteinscalledproteoglucansandthuscompleteproteinremovalfromthesamplescanbechallenging[31].Intermsofproteinremoval,ProtocolAdoesnotseemtobesuperiortoProtocolB,despitetheSevagreagenttreatments,indicatingthatthisstepoftheproceduremightbeomittedwithoutsignificantchangesinthefinalproduct.

Moreover,inthecaseoftheglucansisolatedfollowingProtocolA,thetreatmentwithα-amylasefailedtoremovecompletelyα-glucansfromtheisolatedsamples.Theyieldofobtainedα-glucansvariedfrom0.63%inP.citrinopileatusalkali-solublebiomasspolysaccharideto29.29%inP.ostreatuswater-solublebiomasspolysaccharidefraction.ThecorrespondingobtainedyieldsinthecaseofthesamplesisolatedwithProtocolBvariedfrom2.86%to28.13%,indicatingthattheuseofthecommercialα-amylase,atime-consumingandcostlystepoftheprocedure,maybeomittedwithoutsignificantchangeintheα-glucanyields.

5.Conclusions

Overall,theresultsofthepresentworksupportthattheproductionandpurificationoffungalpolysaccharidesmightbefeasibleinsubmergedcultureandinlargescale,providinganalternativemethodforthevalorizationofwastewaterasmediaforfungalgrowth.ThethreeendogenousGreekBasidiomycetesstrainstestedinthisstudyperformedwellduringsubmergedcultureindefinedmedium,butalsoduringgrowthinOOMW.AlthoughproductionandpurificationofBasidiomycete’sglucanscanbeeasierandmorecost-effectiveinsolidstateculturesandinbasidiocarpform,submergedculturecouldofferaninterestingalternativeduetothreemainadvantages:Firstly,themycelialgrowthismuchmorerapid,leadingtohigherproductivity.Secondly,withthismethodtheuseofliquidmediaisrequired,leadingtoagreatervarietyofgrowthmediachoices,includingliquidwastes,suchasOOMW.BasidiomycetesareknowntoeffectivelydegradeOOMW,andafterthenecessaryoptimizationofparameters,thetwoprocessesofOOMWdegradationandpolysaccharidesproductioncouldbecombinedinasingleprocedure,leadingtothevalorizationofatoxicwaste.Finally,withthesubmergedculture,theisolationofextracellularglucanscanalsobeachieved,leadingtofurtherincreaseofthetotalpolysaccharideyieldoftheprocedure.

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Acknowledgements

TheauthorswouldliketothankAssociateProf.GeorgeZervakisfromtheAgriculturalUniversityofAthensforkindlyprovidingtheBasidiomycetesstrainsusedinthiswork.

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Table1:GlucancompositionoftheisolatedfractionsfromtheP.ostreatuscultures.TheisolationwasperformedfollowingProtocolA.

Definedmediumcultures

Crudebiomass(%w/w)

Water-solublefraction(%w/w)

Purification(fold)

Yield(%)

Alkali-solublefraction(%w/w)

Purification(fold)

Yield(%)

EPS(%w/w)

Totalglucans

8.68 20.45 2.36 21.11 16.39 1.89 1.72 4.57

α-glucans 3.36 11.05 3.29 29.29 11.02 3.29 3 0.27

β-glucans 5.32 9.4 1.77 15.77 5.37 1.01 0.9 4.3

OOMWcultures

Totalglucans

7.58 34.76 4.59 28.57 n.d. n.d. n.d. 1.91

α-glucans 1.1 3.02 2.74 16.92 n.d. n.d. n.d. 0.46

β-glucans 6.48 31.74 4.9 30.84 n.d. n.d. n.d. 1.45

12

Table2:GlucancompositionoftheisolatedfractionsfromtheP.citrinopileatuscultures.TheisolationwasperformedfollowingbothProtocolsAandB.

Definedmediumcultures

Crudebiomass(%w/w)

Water-solublefraction(%w/w)

Purification(fold)

Yield(%)

Alkali-solublefraction(%w/w)

Purification(fold)

Yield(%)

EPS(%w/w)

Totalglucans

6.49 n.d. n.d. n.d. n.d. n.d. n.d. 6.62

α-glucans

0.56 n.d. n.d. n.d. n.d. n.d. n.d. 0.46

β-glucans

5.93 n.d. n.d. n.d. n.d. n.d. n.d. 6.16

OOMWcultures

Totalglucans

14.061

7.53213.131

31.7120.931

4.2120.61

1.79223.761 1.691 1.51 n.d.

α-glucans

3.121

1.6726.331

15.7222.031

9.4321.31

5.2222.211 0.711 0.631 n.d.

β-glucans

10.9315.862

6.791

15.9920.621

2.7321.381

1.54221.551 1.971 1.771 n.d.

1ResultsfrompurificationprotocolA,2ResultsfrompurificationprotocolB,n.d.notdetected.

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Table3:GlucancompositionoftheisolatedfractionsfromtheG.lucidumcultures.TheisolationwasperformedfollowingProtocolB.

Definedmediumcultures

Crudebiomass(%w/w)

Isolatedpolysaccharides(%w/w)

Purification(fold)

Yield(%)

EPS(%w/w)

Totalglucans 6.2 49.1 7.9 36.3 37.92

α-glucans 0.83 0.51 0.61 2.86 0.34

β-glucans 5.39 48.58 9.01 41.2 37.58

OOMWcultures

Totalglucans 5.56 14.96 2.69 14 n.d.

α-glucans 0.13 0.64 5.04 28.13 n.d.

β-glucans 5.43 14.32 2.64 13.99 n.d.

14

Figure1

Figure1.Outlineofpolysaccharidespurificationprotocol,asdescribedinSynytsyaetal.,2009(ProtocolA).

15

Figure2

Figure2.Outlineofpolysaccharidespurificationprotocol,asdescribedinWeietal.,2008(ProtocolB)[32].

16

Figure3

Figure3.FT-IRspectraofpartiallyisolatedpolysaccharidesamplesderivedfromP.ostreatus(a),G.lucidum(b)andP.citrinopileatus(c).BlackanddarkgreylinesrepresenttheFT-IRspectraofthepolysaccharidesisolatedfrommycelialbiomassandEPS,respectively,isolatedbothfromculturesgrownindefinedmedium.LightgreyanddottedlinesrepresenttheFT-IRspectraofthepolysaccharidesisolatedfrommycelialbiomassandEPS,respectively,isolatedbothfromculturesgrowninOOMWmedium.