Biochemicalcharacterizationof

BiochemicalcharacterizationofextracellularpolymericsubstancesextractedfromanintertidalmudflatusingacationexchangeresinGuillaumePierrea,MarianneGrabera,FrancisOrvainb,ChristineDupuya,ThierryMaugarda,*aUMR6250CNRS-ULRLIENSs,UniversitédeLaRochelle,UFRSciences,BatimentMarieCurie,AvenueMichelCrépeau,17042LaRochelle,FrancebUMR100IFREMER-UCBNLBBM,UniversitédeCaenBasse-Normandie,esplanadedelaPaix,14032Caen,FrancearticleinfoArticlehistory:Received22April2010Accepted26September2010Availableonline16October2010Keywords:ExtracellularcompoundsBiochemicalcharacterizationBiofilminsituquantificationBenthicecologyabstractThebiochemicalcharacterizationofExtracellularPolymericSubstances(EPS)excretedinaEuropeanintertidalmudflat(Marennes–OléronBay)wasperformed.Experimentswerecarriedoutforthefirsttimeinsitu,byusinganimprovedextractionrecentlydeveloped.Thisinnovativeprocedure,usingacationexchangeresin(Dowex),allowsseparatingpreciselydifferentfractionsofEPS,especiallypureboundEPS.Moreover,itavoidsthecontaminationofEPSfractionsbyresidualandintracellularpolymers,enablingtoproperlyestimatepolymericcontentsineachfraction.Theresultswerepartlysimilartoconven-tionalresultsdescribedintheliteratureandtheamountofcolloidalcarbohydrates(146mg/gofdrysediment)extractedbytheDowexmethodfittedwellwithdifferentEPSestimationinEuropeanmudflats.Colloidalcarbohydrateswereessentiallycomposedofglucose(50%),acarbonsourcerapidlyconsumedbythevariouscommunitiesinthesediment.Pureboundcarbohydrateswerecomposedofspecificcarbohydrates(28%rhamnose,22%xylose).Residualfractions,consideredascontainingsomerefractoryboundEPSandmostlyotherinternalpolymericsubstances,presentedamorevariedcompositionrichincarbohydrates:galacturonicacid(20%),mannose(19.5%),glucose(19%),arabinose(15%),xylose(8%),galactose(7%).
2010ElsevierLtd.Allrightsreserved.1.IntroductionThebenthicbiofilmswhichdevelopedduringtheemergedperiodsinintertidalmudflatsarewidelystudiedforvariousreasons.Consideringanecologicalperspective,themainreasonistounderstandtheinfluenceofbenthicbiofilmsinintertidalecosystems,byanalyzingtheircompositionsandtheirchanges,dependingonenvironmentalparameters.Numerousstudiesofthelastdecadeshavealloweddeterminingtheircompositionandhighlightedthepresenceofmicroalgae(micro-phytobenthos),bacteriaandfungi,tangledinacomplexmixtureofpolymericcompoundsthattheyproduce(Frølundetal.,1996).TheseExtracellularPolymericSubstances(EPS)arerichinpolysaccharides,proteins,proteoglycans,lipidsandmanyothercompoundsexpressedatdifferentlevels(Stoodleyetal.,2002;Stal,2003),relatedtothelocationorenvironmentalconditionswhichaffectbothfoodwebandprimaryproductionofthisecosystem(UnderwoodandPaterson,2003).EPSareinvolvedinthemobilitysystemofepipelicdiatoms(StalandDéfarge,2005)andcanbeusedascarbonsourcesbythebacterialcommunity(vanDuyletal.,1999;Hofmannetal.,2009).EPSalsoaffectthemicroenvironmentofbiofilmsbyvaryingphysico-chemicalparameterslikeporosityormechanicalstabilityofthesediment(Orvainetal.,2003;Perkinsetal.,2004;Spearsetal.,2008).Ontheotherhand,benthicEPScanpresentinterestingstructuresandfunctions,whichcanbeusedin*Correspondingauthor.Tel.:þ33546458277;fax:þ33546458265.E-mailaddress:[e-mail](T.Maugard).ContentslistsavailableatScienceDirectBiochemicalSystematicsandEcologyjournalhomepage:–seefrontmatter
2010ElsevierLtd.Allrightsreserved.doi:10.1016/j.bse.2010.09.014BiochemicalSystematicsandEcology38(2010)917–923manybiochemicalfields.Theextractionofsulfatedpolysaccharidesformedicinalapplicationsisoneexample(WitvrouwandDeClercq,1997).ManyworkshavealreadyproposedextractionprotocolsallowingthecollectionofparticularEPS,havingspecificbiochemicalproperties(Staatsetal.,1999;deBrouwerandStal,2001;Azeradoetal.,2003;Bellingeretal.,2005;Comteetal.,2006).AllthedataobtainedhavebeeneachtimecriticizedandauthorshaveagreedthattherewasnouniversalextractionmethodforEPS.Recently,Takahashietal.(2009)haveoptimizedaprotocolforEPSextractionandproposedaninnovativemethod,usingacationexchangeresin,toextractcultureddiatomsEPSwithoutanycontaminationbyinternalcompounds.Furthermore,themethodallowedtheextractionofboundEPS,afractionpoorlystudiedandheavilycontaminedinotherpreviousstudies(deBrouwerandStal,2004;Chiovittietal.,2004).TheaimofthepresentinvestigationwastocharacterizethebiochemicalcompositionofEPScollectedfrombenthicbiofilmsduringemergedperiodsonamacrotidalbay(Marennes–OléronBay,France),usingforthefirsttimeinsitutheDowex-resinmethod(Takahashietal.,2009).2.Methods2.1.IntertidalmudflatsamplesThemudsamplesusedinthisstudywerecollectedfromMarennes–OléronBay(AtlanticCoastofFrance)inFebruary2008(winter)atlowtide(Fig.1).Twohoursafterthebeginningoftheemersion,sedimentcoresweresampledforthreedifferentsquares,totakeintoaccountspatialheterogeneity.Sedimentsampleswerecollectedusingcorediameterof20cm,andthetop1cmwascollectedthreetimesandpooledtogiveamainsedimentcore,foreachsquare.Aftersampling,sedimentwasbroughtbackonfromthefieldbyusingawatercraftforanimmediateEPSextractiononfreshsedimentsontheupper