A.M.McIntyrea,C.Guéguenb,⇑abEnvironmentalandLifeSciencesGraduateProgram,TrentUniversity,1600WestBankDrive,Peterborough,ON,CanadaK9J7B8ChemistryDepartment,TrentUniversity,1600WestBankDrive,Peterborough,ON,CanadaK9J7B8highlights
\"Oneprotein-andtwohumic-likecomponentsarefoundinScenedesmusacutusexudates.\"BiologicalactivitiescanproducedpeaksCandM.\"Visiblephotodegradationandmicrobialdegradationaffectalgalexudatecomposition.\"UnlikeCd,PbandZn,CustronglybindstoalgogenicDOM.\"SignificantdifferencesinlogKvaluesarefoundbetweenhumic-likecomponents.articleinfoabstract
Thenatureandcompositionofdissolvedorganicmatter(DOM)stronglyinfluencesitsbindingpropertiestoheavymetalsandthustheirfate,mobilityandtoxicityinaquaticenvironments.Fluorescencespectros-copywithparallelfactoranalysis(PARAFAC)wasusedtocharacterizeDOMexudedbythecosmopolitanfreshwatergreenalgaeScenedesmusacutusduringearlyexponentialgrowthphase.Oneprotein-like(peakT;C2)andtwohumic-likecomponents(peaksA+CandA+M,C1andC3,respectively)weresplithalfvalidatedon122emission-excitationmatrices(EEMs).Ourdatashowthatbothhumic-likecouldbeassociatedwithbiologicalactivities.UnlikeCd,PbandZn,CustronglybindstoalgogenicDOMwithcon-ditionalstabilityconstants(logK)averaging5.26±0.29(from4.85to5.36).SignificantdifferencesinlogKvalueswerefoundbetweenhumic-likePARAFACcomponents,indicatingcleardifferencesinthebindingpropertiesofhumic-likecomponentswithcopper.Ó2012ElsevierLtd.Allrightsreserved.Articlehistory:Received1March2012Receivedinrevisedform27August2012Accepted28August2012Availableonline27September2012Keywords:PARAFACScenedesmusFluorescencequenchingExudatesBindingconstantCopper1.IntroductionDissolvedorganicmatter(DOM)isaheterogeneousmixtureofdissolvedmaterialfoundubiquitouslyinaquaticsystems.AquaticDOMfallsintotwomaincategories:autochthonous(i.e.producedinsitusuchasalgalexudates)andallochthonousDOM(i.e.pedo-genic).Intheauthochthonouspool,algogenicDOMarisesextracel-lularlyviametabolicexcretionorintracellularlyduetoautolysisofcells.Theformerisdominantduringtheexponentialgrowthperi-odwhereasthedominanceofthelatterincreasesduringthesta-tionaryphaseofthealgaesystem.Thedeterminationofthechemicalpropertiesofalgal-derivedDOMisimportantforunder-standingaquaticecosystemfunctions.DOMplaysalargeroleinaquaticecosystemsincludinglightattenuationandmobility,transportandfateofchemicalspecies(Williamsonetal.,1999;Mylonetal.,2003).DOM,andparticularly⇑Correspondingauthor.Tel.:+17057481011;fax:+17057481625.E-mailaddress:celinegueguen@trentu.ca(C.Guéguen).0045-6535/$-seefrontmatterÓ2012ElsevierLtd.Allrightsreserved.http://dx.doi.org/10.1016/j.chemosphere.2012.08.057humicandfulvicacids,influencesgreatlythefate,mobilityandtransportofmetalsinnaturalwaters(TessierandTurner,1995).Dependingonthenatureofthechelatingligands,metaltoxicitymaybedifferent.Forexample,Cuassociatedwithlipophiliccom-plexesismoretoxictoalgae(StauberandFlorence,1987;Camp-bell,1995;Crootetal.,2000)thanboundwithhydrophilicmoieties(Koukaletal.,2007).AlgogenicDOMandparticularlyextracellularpolymericsubstances(EPS;Koukaletal.,2007)havebeenfoundtoincreasethecoagulationandsedimentationratesofcolloidalmaterialandassociatedmetals(Wilkinsonetal.,1997).Theproductsofexcretionbythealgaebloomsmaycontrib-utesignificantlythedissolvedorganiccarbonpoolandbeanexcel-lentsourceofligandsformetalcomplexation(Moffett,1995).ForexampleVasconcelosetal.(2002)showedthatthenatureandconcentrationofmarinephytoplanktonexudatesinfluencedmetaluptake.TheproductionofDOMfollowingalgalbloomsanditsimpactonmetalbindinginrivers,streamsanddrinkingreservoirsisrelativelylessstudiedcomparedtoothertypesofDOM.Excitation–emissionmatrix(EEM)fluorescencespectroscopyisahighlysensitivetechniquecommonlyusedtostudyDOMinA.M.McIntyre,C.Guéguen/Chemosphere90(2013)620–626621freshwaterandmarineecosystems(Coble,2007;Guéguenetal.,2011).ThistechniqueprovidesimportantinformationonDOMcomposition.Forexample,itispossibletodistinguishbetweenla-bileprotein-likeandhumic-likeDOM,twofluorescentgroupsre-portedinalgalcultures(Hendersonetal.,2008;Lietal.,2008;Romera-Castilloetal.,2010),dependingontheirexcitationandemissionwavelengths(Ex/Em).AsanEEMcancontainseveralthousandsdatapoints,itmaybedifficulttoassessthedynamicsoffluorescentDOMbasedonthetraditionalEEMpeak-pickingtechniquesastheycanbetimeconsumingandmaynotbereliable.However,thecharacterizationoffluorescentDOMhasimprovedwiththeapplicationofamultivariatemodelingapproachcalledparallelfactoranalysis(PARAFAC)(Stedmonetal.,2003;StedmonandBro,2008),whichallowsEEMstobedecomposedintoindivid-ualfluorescentcomponents.Thisnewapproachhasbeensuccess-fullyappliedtostudyvariabilityinDOMcompositioninnaturalsystems(e.g.StedmonandMarkager,2005;YamashitaandJaffé,2008;Guéguenetal.,2011).FluorescencespectroscopyhasalsobeensuccessfullyappliedtoinvestigatetheDOMinteractionswithparamagnetic(Cu)anddia-magneticmetals(Cd,PbandZn)(RyanandWeber,1982;Dudaletal.,2006;YamashitaandJaffé,2008;Wuetal.,2011,2012).ThechangeinDOMfluorescenceintensityisusedtodetectthecomplexationwithmetal.RecentworkusingEEM-PARAFACre-vealedthatdifferenttypesofDOMcomponents(e.g.humic-likeandprotein-like)wereassociatedwithmetalbinding(YamashitaandJaffé,2008;Wuetal.,2011).Forexample,Cu(II)hasbeenre-portedtoquenchhumic-andprotein-likefluorescence(YamashitaandJaffé,2008;Wuetal.,2011)whereastheadditionofPb(II)quenchedintheprotein-andfulvic-likeregionofmunicipalsolidwasteleachate(Wuetal.,2011).TitrationofextractedhumicacidswithCd(II),Cu(II),Pb(II)andZn(II)atpH6resultedinamarkedde-creaseofhumic-likefluorescence(Plazaetal.,2006).Wuetal.(2012)showedthatCd2+complexationwasonlyassociatedwithfulvic-likecomponent(220/428nm)atpH6whereasCu2+bindingwasfoundwithallsixPARAFACcomponents(fulvic-,humic-andprotein-like).TheapplicationofEEM-PARAFACmodelingcom-binedwithfluorescencequenchinghasnotyetbeenusedtostudytheinteractionsofmetalswithDOMderivedexclusivelyfromfreshwateralgae,despiteitspotentialtoprovideadditionalinsightintothebiogeochemicalcyclingofmetals.Theobjectivesofthisstudywere(1)tocharacterizetheDOMproducedduringtheexponentialphaseofthegreenalgaeScene-desmusacutus,and(2)toinvestigatethebindingpropertiesofalgalDOMforCd,Cu,PbandZnusingfluorescencequenchingandPARAFAC.2.Materialsandmethods2.1.AlgalculturingS.acutus(strain282)wasobtainedasaxenicculturefromtheCanadianPhycologicalResearchCentre(UniversityofWaterloo,Canada).TheinoculatedmediumwastransferredtoaConviron,A-1000environmentalchamber(23°C;16:8hlight:dark).Anegli-gibleamountofUVlight(below400nm)penetratedtheculturingflasks,minimizingUVphotobleachingoccurringduringtheculturegrowth.ThealgaeweregrowninCOMBOmedium(250–300lScmÀ1,pH=7.8;Andersen,2005)withtheanimaltracecomponentre-movedfor72h.Thealgalcellswerethenseparatedfromthemed-iumbycentrifugationat2500rpmfor10min.Thealgalpellet(2Â105cellsmLÀ1;ReichertBright-Linehaemocytometer)wasthenre-suspendedinsterileEDTA-freeCOMBOmediumforanadditional72h.EDTAwasremovedfromthemediaduringtheexperimentalphasetoavoidbindingcompetitionwithDOM(Guéguenetal.,2003).GrowthwasnotimpairedbytheuseofEDTA-freeCOMBOmediaovertheperiodofstudy.Finally,thealgalculturewasharvestedduringitsearlyexponentialperiod(day6)andfilteredthroughapre-combusted0.7lmglassfiberfilter(GFFWhatman).Threealgalcultureswerealso0.02lmfiltered(WhatmanAnotop)toinvestigatetheinfluenceofalgogenicDOMsizeonCubinding.Theculturemediaandglasswarewereauto-clavedtominimizebacteriaactivityatthebeginningoftheculture.Unfortunatelynobacterialabundancewasavailableinthestudy.2.2.QuenchingtitrationTheexponentiallygrowing0.7lmalgalfiltrateisolatedfromseparatealgalcultureswastreatedwiththeselectedconcentra-tionsofCd2+,Cu2+,Pb2+andZn2+usingstocksolutionsprepareddailyfromtheiranalyticalgradesaltsCd(NO3)2,Cu(NO3)2,Pb(NO3)2,Zn(NO3)2.Cu(II)quenchingtitrationwasalsoperformedon0.02lmalgalfiltrate(Anotop10,Whatman)isolatedfromsep-aratealgalcultures.Themetalconcentrationsinthefinalsolutions(pH7.8;0.1MNaNO3)rangedfrom2to100lM.Allsolutionswerekeptatroomtemperatureinthedarkfor5mintoensurecomplex-ationequilibriumpriortofluorescencescanning.Nosignificantdif-ferenceinbindingpropertieswasfoundwhensampleswereallowedtoequilibratefor5minor24h(p<0.05),confirmingthatthereactionofDOMandmetalcomplexationwasfairlyrapidandpseudo-equilibriumconditionscanbeachievedwithin10–20s(Linetal.,1995;Wuetal.,2004).Fluorescencequenchingexperimentswerealsoconductedwithtwoaminoacids(tyrosineandtryptophan;FisherScientific)andtwofulvicacidsPonyLakeandSuwanneeRiverfulvicacid(PLFAandSRFA,respectively;InternationalHumicSubstancesSociety,USA).Theseextractedful-vicacidsarecommonlyusedasmicrobially-derivedandterrestrialfulvicacids,respectively(Coryetal.,2010).FluorescenceofthesamplesbeforeandaftermetaltitrationwasrecordedusingaFluoromax-4JobinYvonspectrofluorometer(Fig.1).TheEEMspectrawerecollectedatexcitationwavelengths250–500nmandemissionwavelengths300–600nm(Guéguenetal.,2011).SpectrumofMilli-Qwater(Millipore,18.2MXcmÀ1)wasrecordedbetweeneachalgalsampletoensurenocontamina-tionandsubtractedfromthesamplespectratoremovemostofthefirstandsecondorderscatterpeak.FluorescenceintensitieswerenormalizedtotheMilli-QwaterRamanareaatexcitation350nmmeasureddaily(LawaetzandStedmon,2009)andreportedinRamanunit(r.u.).InadditiontoEEMspectra,emissionspectraof1.21.00.8ferI0.6/I0.40.20.0020406080100120Cu [µM]Fig.1.ExampleoffluorescencequenchingcurvesofC1titratedwith0.01MCu(NO3)2basedonthreeseparatealgalcultures.622A.M.McIntyre,C.Guéguen/Chemosphere90(2013)620–626thealgalexudatewereacquiredatthemaintwofluorescentcom-ponents(i.e.C1340/455nmandC2Ex/Em270/340nm;seebelowfordetails).ThequenchingmodeldevelopedbyRyanandWeber(1982)wasusedtodeterminethebindingconstantsandligandconcentra-tionoftheexudatesasfollows:II¼1þIMLÀ1=2KCqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiLðKCLþKþCMþ1ÞÀðKCLþKCMþ1Þ2À4K2CLCMrefIrefð1ÞwhereIistheoverallfluorescenceintensity,Irefisthereferencefluorescenceintensityofsample(i.e.withoutmetaladded),IMListhefluorescenceintensityoftheligand:metalcomplexwhenthecomplexingcapacityhasbeenreached,Kisthebindingconstant,CLandCMaretheconcentrationsofligandandaddedmetal,respec-tively.Thisquenchingmodelassumesa1:1ligand:metalbindingratio.2.3.PARAFACmodelingParallelfactoranalysis(PARAFAC;Stedmonetal.,2003)decom-posestheEEMintoindependentcomponentsthatexplainthemax-imumamountofvariationbetweenthesamples.TheanalysiswascarriedoutinMatlabR2011b(MathWorks)usingtheDOMFluortoolbox(StedmonandBro,2008).Althoughmostofthefirstandsecondorderscatterpeakswereremovedafterblanksubtraction,theregionoftheEEMspectrummaystillbeinfluencedbyremain-ingscatterpeakswhichhavetobecutandreplacedwithmissingvaluespriortorunPARAFACanalysisasrecommendedbyStedmonandBro(2008).Splithalfanalysis,randominitializationandTuck-ercongruencecoefficientswereusedtovalidatetheidentifiedcomponents(Stedmonetal.,2003).Themodelwasconstrainedtononnegativevalues.Nooutlierswereidentifiedduringtheanal-ysis.ThePARAFACmodelwasvalidatedon122EEMs(Table1)fromthe0.02and0.7lmfiltrateofalgalexudates(n=27and41,respectively),tyrosine(n=11),tryptophan(n=17),SRFA(n=9)andPLFA(n=9).EachtitrationseriesusedinthePARAFACmodelincludedtheoriginalsampleandupto8titratedsampleswithmetalconcentrationsrangingfrom2to100lM.2.4.DissolvedorganiccarbonDissolvedorganiccarbon(DOC)concentrationsweredeter-minedforallfiltrateexudatesamplesasinGuéguenetal.(2002).Briefly,DOCismeasuredasnon-purgeableorganiccarbonusingaShimadzuHighTemperatureTotalOrganicCarbonAna-lyzer(TOC-V).Inorganiccarbonwasremovedbyacidificationwith2MHClandspargingwithhighpurityairfor7min.Thesamplethenflowedthroughacatalyst-packedcombustiontubeat680°Cwherecarbondioxidewasformedandmeasuredusingnon-disper-siveinfrareddetection.UnfortunatelynoexternalstandardfortheDOCmeasurementswasusedasthematrixoftheCRMssuppliedTable1
PARAFACcomponentsforalgalexudatesarecomparedtotheprobablecomponentsidentifiedinCoble(1996).ComponentEx/Emwavelength(nm)atCobleDescriptionmaximumfluorescenceintensity(1996)basedonEEMlocationCl(255)340/455PeakUVC+UVAA+Chumic-likeC2275/340PeakTProtein-like,tryptophan-likeC3<250(300)/465PeakUVC+UVAA+Mhumic-likebyDr.Hansell(UniversityofMiami)doesnotmatchsamplesmea-suredinthisstudy(seawatervs.freshwater).2.5.StatisticalanalysisNon-linearregressionforquenchingexperiments(Eq.(1))wascarriedoutusingtheSolverapplicationinMicrosoftExcel2003andverifiedwithSigmaPlot10.0.StatisticalanalyseswerecarriedoutusingSTATISTICA8.Significancewasestablishedfromrepli-catesobtainedfromexperimentalflasksculturedseparately.3.ResultsanddiscussionTheconcentrationofDOCinS.acutusfiltrateswas195±92lM($2.106cellsmLÀ1).TheconcentrationofDOCintheexudate-freemedia(i.e.EDTA-freeCOMBO),was$80timeslowerthanintheexudatesolutions(2.5vs.195lM-C),meaningthatDOCinthefil-tratesamplesderivedfrombiologicalactivity.ThealgaeintheKoukaletal.study(2007)hadacomparableDOCproductionpercellthanalgaeinthepresentstudy(17–27vs.52–143fmolcellÀ1,respectively).HowevertheDOCproductiononapercellbasiswastwoordersofmagnitudehigherintheLevyetal.study(2011)(1.2–5.6pmolcellÀ1).3.1.FluorescencecharacteristicsofalgalexudatesFig.2showstheEEMsofalgalexudates,tyrosine,tryptophan,PLFAandSRFAintheabsenceofmetals.Algal-derivedDOMshowedfluorescencefeaturesinboththeprotein-like(Em<400nm)andhumic-like(Em>400nm)regions.MillerandMcKnight(2010)alsofoundthatDOMproducedduringsummerplanktonbloominsubalpinelakesincludedbothhumic-andpro-tein-likecomponents.Asexpected,tryptophanandtyrosinehaddiscretedominantpeaksatEx/Em=275/350and275/310nm,respectively,whichcorrespondswellwiththeliterature(Coble,2007;Hudsonetal.,2007andreferencestherein).SRFAandPLFAbothpresentedtwomainhumic-likepeaksinsimilarregionsoftheEEM(Em>400nm).Athree-componentPARAFACmodelwassplit-halfvalidatedon122EEMs(Table1).Component1(C1),whichaccountedforthegreatestamountofvariationinEEMsacrosssamples,hadadomi-nantpeakwithmaximumexcitationandemissionvaluesof340/455nmandasecondsmallerpeakatEx/Em=255/455nm.Thisred-shiftedpeakresembledmorehumic-likepeakC(Ex/Em320–360/420–460nm;Coble,2007)thanhumic-likepeakM(Ex/Em290–310/370–410nm;Coble,2007).Therefore,attendingtothepositionofpeakmaximum,humic-likeC1wasclassifiedaspeakA+C.ThispeakhasbeenpreviouslydescribedasUVC/UVAhu-mic-like(peakA+C;Coble,1996;Stedmonetal.,2003;Guéguenetal.,2011).ComponentC1hasbeenreportedinalgalculture(Lietal.,2008;Romera-Castilloetal.,2011),suggestingthatbiologicalactivitiesareabletoproducematerialfluorescingatpeakA+C.Component2(C2)hadapeakatEx/Em=275/340nm,whichresembledtotryptophan(peakT;Coble,1996).ThepredominanceofC2inpuretyrosineandtryptophan(85%and98%,respectively;Fig.2)wasconsistentwithitsprotein-likeassignment.Thepres-enceoftheprotein-likematerial(C2)wasexpectedasalgalmate-rialiscomposedmostlyofproteins(HaasandWild,2010).ThiscomponentalsohadsimilarfeaturestoacomponentfoundintheStedmonandMarkager(2005)study(theirComponent6),whichtheyattributedtoprotein-likederivedfromalgaeexuda-tion.Again,thiscomponentwasreportedinmarinephytoplanktoncultures(Romera-Castilloetal.,2010,2011)andbacterialextracel-lularpolymericsubstances(EPS)(Zhangetal.,2010;2012).A.M.McIntyre,C.Guéguen/Chemosphere90(2013)620–626623Fig.2.EEMsof(A)Scenedesmusa.exudates,(B)tryptophan,(C)tyrosine,(D)SRFAand(E)PLFA.Fluorescenceintensityisinramanunit(r.u.).Component3(C3)hadaprimarypeak(Ex/Em<250/465nm)andasecondarypeak(Ex/Em300/465nm).Thisred-shiftedpeakresembledmorehumic-likepeakM(Ex/Em290–310/370–410nm;Coble,2007)thanhumic-likepeakC(Ex/Em320–360/420–460nm;Coble,2007).Attendingtothepositionofpeakmax-imum,humic-likeC3resembledmorehumic-likepeakM(Ex/Em290–310/370–410nm;Coble,2007)andthusclassifiedaspeakA+M.InareviewpaperonDOMcompositionbasedonPARAFACcomponent,IshiiandBoyer(2012)foundapeakA+Mwithinthesamerange(Ex/Em<240–260(295–380)/374–450nm).Thiscom-ponentaccountedfortheleastamountofvariationinEEMsacrosssamplesandoftenappearedasashoulderoftheEEMpeaks(e.g.Fig.2BandC).Component3hasbeenfoundinawiderangeofaquaticandterrestrialenvironments(OhnoandBro,2006;IshiiandBoyer,2012andreferencetherein).Althoughmoststudiesfoundthedominanceofprotein-likefluorescenceinalgogenicDOM(Hendersonetal.,2008;Lietal.,2008;Romera-Castilloetal.,2010,2011),themainpeakproducedbyS.acutuswaspeakA+M(49.8±1.9and49.9±15.9%in0.02and0.7lmfiltrates,respectively).Thisworkprovidesfurtherevidencethatphyto-planktonisabletoproducesubstancesfluorescingatpeakM.Pro-ductionofpeakMwasalsoreportedinfreshwaterandmarinealgalcultureduringtheexponentialgrowthphase(Hendersonetal.,2008;Romera-Castilloetal.,2010,2011).Therelativeabun-danceofC3washigherinalgalexudatesandPLFAthaninaminoacid(i.e.tyrosineandtryptophan)andSRFA,suggestingthatC3isassociatedwithbiologicalactivity.120C1 C2 C3 10080Abundance [%]6040200anateatexudxudtopheep yrmTum0.7u0.02sineTyroASRFAPLFFig.3.Relativeabundanceoffluorescencecomponentsinalgalexudates,aminoacids(i.e.tryptophanandtyrosine)andextractedfulvicacids(i.e.SRFAandPLFA).TherelativeabundanceofthethreecomponentsisshowninFig.3.Thealgalexudatecompositionbasedon0.02and0.7lmal-galfiltrateisolatedfromseparatealgalcultures(n=3and6,respectively)wasdominatedbyhumic-likematerial(C1+C3)with87.0±8.5and71.2±17.3%respectively.Protein-likematerialC2,aproxyforfreshlyproducedDOM(YamashitaandTanoue,2003),624A.M.McIntyre,C.Guéguen/Chemosphere90(2013)620–626represented13.0±8.5and28.8±17.3%offluorescingDOMin0.02and0.7lmalgalfiltratesrespectively,whichiscomparabletoalgalexudates(37–45%ofDOMexudates;seeTable1inRomera-Castilloetal.,2011).NosignificantdifferenceinDOMcompositionwasob-servedbetween0.02and0.7lmalgalfiltrates(n=3and6,respec-tively;p<0.05),suggestingthatprotein-likefluorescenceduetobacterialbiomass(Determannetal.,1998)wasnotsignificantinthisstudy.Theproductionofhumic-likematerialhastraditionallybeenassociatedwithterrestrialorigin(C1)andinsituproduction(C3).Inthisstudy,DOMwasderivedexclusivelyfromalgalactivities,confirmingthatpeakCcanhaveanautochthonoussource.ThisagreeswithRomera-Castilloetal.(2011)whoshowedthatpeakCwasassociatedwithbacterialdegradationofpeakMproducedbyphytoplankton.StedmonandMarkager(2005)foundthatfluo-rescenceofbothhumic-likepeakscanbemicrobiallygenerated.Inadditiontomicrobialdegradation,photodegradationcanalsoaf-fectDOMcomposition.AlthoughUVexposureduringgrowthphasewaslimitedinthisstudy,visiblephotodegradationcanbeasinkforbothhumic-likecompoundswithpeakCbeingthemostsensitive(StedmonandMarkager,2005).Thusvisiblephotodegra-dationandmicrobialtransformationcouldhaveresultedinpro-ductionandtransformationofhumic-likematerial.ThealgalexudatesandPLFAshowedsimilarDOMcomposition.TheexudateswereexpectedtoshowresemblancetoPLFAduetotheircommonautochthonousnature(McKnightetal.,1994).Ontheotherhand,fluorescencecompositioninSRFAwasdispropor-tionatelydominatedbyC1($80%ofthetotalfluorescence;Fig.3),confirmingthepedogenicoriginofSRFA.3.2.Bindingpropertiesofalgal-derivedDOM3.2.1.CopperquenchingTheadditionofCu(II)inducedamarkeddecreaseinC1(Fig.1)andC3fluorescenceintensityinbothalgalexudate,pureaminoacidandfulvicacidsolutionswhereasC2intensitywasreducedonlyinalgalexudateandpureaminoacids;noconsistenttrendwasobservedinC2ofSRFAandPLFA.Thequenchingdegreeran-gedfrom14%to67%inhumic-likeC1,from6%to86%inpro-tein-likeC2,andfrom30%to38%inhumic-likeC3withtheadditionofCu(II).Theseresultsrevealdifferencesinquenchingef-fectamongvariousfluorescentcomponentswithC1andC2inten-sitiesbeingthemostreduced.Thefluorescencequenchingmethodwassuccessfullyappliedtoprotein-likealgalexudate(thisstudy)andEPSfluorescenceintensity(Zhangetal.,2012)suggestingthatfluorescencequenchingcanbeusedtoevaluatethebindingchar-acteristicsofprotein-likesubstancesandmetalions.Thiscontrastswithpreviousstudiesonmunicipalsludgeandsurfacewaterswherelargefluctuationsinthequenchingcurveofprotein-likecomponentswerefound(YamashitaandJaffé,2008;Wuetal.,2011).Inbothcases,DOMexhibitedsimilarfluorescencecharac-teristicstothoseofprotein-likebuttheintrinsiccompositionmaybevariablebetweensamplesaffectingtheirquenchingprop-erties.Alternatively,thestabilityofprotein-metalcomplexmaychangebetweensamples(Wuetal.,2011),metalconcentration(YamashitaandJaffé,2008)orboth.Furtherstudiesarerequiredtoclarifytheroleofprotein-likeinmetalbinding.ThelogKvaluesforCu-algalDOMrangedfrom4.59to6.23withanaverageof5.26±0.39(n=29;Table2).NosignificantdifferenceinlogKwasfoundbetween0.7and0.02lmalgalDOMsamples(p<0.05)whichagreewithsimilarfluorescentcomposition(Fig.3).Stabilityconstantsofalgalexudateswerecomparablewiththoseofextractedfulvicacids(SmithandKramer,2000;Table2).Overall,thebindingpropertiesofalgogenicDOMwithCu(II)agreewithpreviousfluorescencequenchingstudiesusingvarioussourcesofDOMincludingwetlandhalophytes,naturallakeandTable2
CopperbindingparameterscalculatedbytheRyan–Webermodelforalgalexudates(C1,C2,C3),SRFAandPLFA.Standarddeviationappearsinbracketswhereavailable.TypeComponentCl(lM)LogKAlgalDOMC16.90(9.70)5.35(0.39)C222.5(16.6)5.29(0.43)C30.24(0.24)4.85(0.06)SRFAC11.265.06C2N/AN/AC30.644.97PLFAC10.515.44C2N/AN/AC30.325.37ASVISEFQFQ (this study)23456789Log K Fig.4.Stabilityconstantsofcoppercomplexes(logK)usingfluorescencequenching(FQ;WuandTanoue,2001;YamashitaandJaffé,2008;HurandLee,2011;Mounieretal.,2011;Panetal.,2011;Wuetal.,2011),ISE(McKnightandMorel,1979;LombardiandVieira,1999;Lombardietal.,2005;LombardiandVieira,2000;Brooksetal.,2007a,b)andASV(Mylonetal.,2003;Lorenzoetal.,2007;Andradeetal.,2010;Sánchez-Marínetal.,2010a,b).municipalsolidwasteleachate(Fig.4).LogKvalueswerealsocom-paredwiththoseobtainedbyionselectiveelectrode(ISE),anodicstrippingvoltammetry(ASV)andcathodicstrippingvoltammetry(CSV)(Fig.4andreferencetherein).Thesetechniquesrevealbind-ingcharacteristicsofbulkDOMasopposedtoonlythebindingofDOMfluorophoresinfluorescencequenching.ThelogKvaluesspan4–5ordersofmagnitude(Fig.4).ThismaybeduetodifferentDOMsourcesandmethodsused.Thedifferencesinmetalconcen-trationrangeusedinthetitrationleadtothedetectionofdifferentligands.Thestrongersitesaregenerallydetectedatlowmetalcon-centrationwhereasthemoreabundantbutweaksitesarefoundathighmetalconcentration.ThisunderscorestheimportanceofonlycomparinglogKvaluesderivedusingthesameanalyticalwindow(i.e.metal/ligandratio)(FilellaandHummel,2011).Consideringsimilarmetalconcentrationrange,thebindingstrengthsfoundinthisstudywerecomparablewiththosedeterminedbyISEinalgalexudatestudies(logK=5.0–6.3;LombardiandVieira,1999,2000;Lombardietal.,2005).ThecalculatedlogKvaluesforthethreePARAFACcomponentswere5.35±0.39(n=12),5.29±0.43(n=13)and4.85±0.06(n=4)forC1,C2andC3,respectively(Table2).Itcanbenotedthattheprotein-likeEPShadabindingaffinityrangingfrom2.63to3.90forCu(II)atpH8(Zhangetal.,2012),whichis$2-foldlowerthanforalgalexudates(5.29±0.43;thisstudy).Theweight-aver-agemolarmass(Mw)ofalgalexudatesdeterminedbyfieldflowfractionationwas<1.3kDa(unpublisheddata)whereasMwofEPSrangedfrom81to689kDa(AlasonatiandSlaveykova,2011,2012).ThedifferenceinbindingconstantandMwsuggestthatA.M.McIntyre,C.Guéguen/Chemosphere90(2013)620–626625EPSwerenotpredominantinthisstudy.NosignificantdifferencewasfoundbetweenlogKvaluesforC1andC2(p=0.99).ThelogKvalueforC3was,however,significantlylowerthanC1andC2(p<0.01).Humic-likecomponentsinSRFAandPLFAwerefoundtohavesimilarlogKvaluestothoseinalgalexudates(Table2),indicatingthatfreshly-producedalgalmaterialandisolatedfulvicacidshavesimilarcopper-bindingaffinity.Inadditiontothebindingconstants,aconsiderablerangeofthecomplexationcapacities(CL)wasobservedwithintheDOMrang-ingfrom0.24±0.24lMforC3to6.90±9.70lMforC1to22.5±16.6lMforC2(Table2).Thisrangeagreeswellwithpub-lishedcomplexationcapacityvalues(FilellaandHummel,2011;andreferencestherein).Protein-likeC2hassignificantlyhigherCLvaluesthanbothhumic-likecomponents(C1andC3)(p<0.05).Nosignificantdifferencewasfoundbetweenhumic-likecomponents(p=0.27).3.2.2.Cd,PbandZnquenchingNosignificantquenchingofthealgal-derivedPARAFACcompo-nentsinourstudyindicatesthatthediamagneticCd2+,Pb2+andZn2+cationsdidnotinteractstronglywithalgal-derivedDOM.Sim-ilarresultswerefoundinthesolidwasteleachatestudywherehu-mic-likeandprotein-likematerial(C1andC3;Wuetal.,2011)didnotquenchsignificantlyCd,PbandZn.OnlyonePARAFACcompo-nent(C4220/432nm;Wuetal.,2011)boundstronglytobothmet-alsbutthiscomponentwasnotfoundinouralgalstudy.NochangeinfluorescenceintensitywasalsoobservedinZnandCdassaysusingsurfacesoilDOM(Dudaletal.,2006).Ontheotherhand,hu-micacidsisolatedfromsewagesludgeshowedsignificantfluores-cencereductioninthepresenceofCd,PbandZn(Plazaetal.,2006;Wuetal.,2012).Wuetal.(2012)showedthatCd2+complexationwasonlyassociatedwithfulvic-likecomponent(220/428nm),acomponentnotfoundinalgalexudates.4.ConclusionThebindingcharacteristicsofDOM,releasedbyS.acutusduringtheexponentialperiodofgrowth,withCu,Cd,PbandZnwerestudiedusingfluorescencequenchingtitrationscombinedwithEEMandPARAFAC.Threefluorescentcomponents(twohumic-like:peakA+CandpeakA+M;andoneprotein-like:peakT)werefoundintheexudatematerial.Basedonthisstudyandpreviouswork,peakCwasassociatedwithmicrobialdegradationofalgo-genicmaterialwhereaspeakMwasassociatedwithalgalproduc-tion.StrongquenchingeffectsforCu(II)whilenegligiblequenchingeffectsforCd(II),Pb(II)andZn(II)wereobserved.Thelowestbind-ingconstantwasfoundforC3,suggestingthatmicrobialactivitieshavesignificantimpactsoncopperbindingcharacteristics.AcknowledgmentsThisstudywasfundedbytheNaturalSciencesandEngineeringResearchCouncilofCanadaandtheCanadaResearchChairpro-gram.TheauthorsthankA.PerroudandB.MarcereforDOCanaly-sisandW.Chen,C.W.CussandC.Wylieforcommentsonearlierdrafts.Threeanonymousreviewersarethankedfortheirthought-fulreviewsandcommentswhichsubstantiallyimprovedanearlierversionofthismanuscript.ReferencesAlasonati,E.,Slaveykova,V.I.,2011.Compositionandmolarmasscharacterisationofbacterialextracellularpolymericsubstancesbyusingchemical,spectroscopicandfractionationtechniques.Environ.Chem.8,155–162.Alasonati,E.,Slaveykova,V.I.,2012.EffectsofextractionmethodsonthecompositionandmolarmassdistributionsofexopolymericsubstancesofthebacteriumSinorhizobiummeliloti.Biores.Technol.114,603–609.Andersen,R.A.,2005.AlgalCulturingTec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