Biosorptioncharacteristicsofextracellularpolymericsubstance(EPS)producedbyRhizobiumradiobacterforremovalofCu(II)andMn(II)ionsformaqueoussolutionsLi-LiWanga,b,FangMaa,b*,Shan-ShanFenga,b,De-ZhiSunb,c,BingYua,b,JieXinga,baStateKeyLaboratoryofUrbanWaterResourceandEnvironment,Harbin,150090,ChinabDepartmentofMunicipalandEnvironmentalEngineering,HarbinInstituteofTechnologycCollegeofEnvironmentalScienceandEngineering,BeijingForestryUniversity,Beijing100083,Chinae-mail:AbstractThispaperdealswiththeextracellularpolymericsubstance(EPS)ofRhizobiumradiobacterF2usedasanovelbiosorbenttoremoveCu(II)andMn(II)ionsfromaqueoussolutions.TheCu(II)andMn(II)ionsbiosorptioncharacteristicsoftheEPSwereexaminedasafunctionofinitialpH,contacttimeandEPSconcentration.TheproperpHoftheaqueoussolutionwasfoundtobe6.0forboththebiosorptionofCu(II)andMn(II).BiosorptionequilibriumtimesofCu(II)andMn(II)wereachievedin1.0h.ThemaximumbiosorptioncapacitiesunderoptimumconditionsofCu(II)andMn(II)ontheEPSweredeterminedtobe45.301.62mg/gand28.861.28mg/g,respectively.ThefitnessofthebiosorptiondataforLangmuirandFreundlichisothermmodelswasinvestigated.ItwasfoundthatbiosorptionofCu(II)andMn(II)ionsontotheEPSwerebettersuitabletoLangmuirthanFreundlichisothermmodel.CompetitivebiosorptionofMn(II)andCu(II)ionswasalsoinvestigatedtodeterminetheselectivityofthebioflocculant.Keywords-biosorption；extracellularpolymericsubstance(EPS)；Cu(II)；Mn(II)；competitivebiosorptionI.INTRODUCTION(HEADING1)Waterandwastewaterscontaminatedbyheavymetalshaveresultedinadverseimpactonpublichealthandeconomy1,andexposuretoheavymetalsevenattracelevelsisbelievedtobeariskforhumanbeings2.Inthepastdecadesvariousprocesseshavebeendevelopedtotreatsuchwatersincludingchemicalprecipitation,ion-exchange,adsorptionandreverseosmosis3.However,thesemethodsaresometimesrestrictedbecauseoftechnicaland/oreconomicalconstraints4.Biosorptionisconsideredasanalternativeprocessfortheremovalofheavymetals,metalloidspecies,compoundsandparticlesfromaqueoussolutionbybiologicalmaterials5.Comparedwithconventionalmethodsfortheremovaloftoxicmetalsfromwastewater,thebiosorptionprocessofferspotentialadvantagessuchaslowoperatingcost,minimizationofthevolumeofchemicaland/orbiologicalsludgetobedisposedof,andhighefficiencyindetoxifyingverydiluteeffluents6.ThispaperwascarriedouttoinvestigatetheEPSproducedbycultureofRhizobiumradiobacterF2asabiosorbentfortheremovalofMn(II)andCu(II)fromaqueoussolutions.TheoptimumbiosorptionconditionsweredeterminedsuchasinitialpH,contacttimeanddosageoftheEPS.Thesuitablebiosorptionisothermwasputforwardbasedonthebiosorptiondata.Inaddition,thebiosorptionperformanceoftheEPSinbinarymetalsolutionsystemswasalsoexamined.Thechoiceofmetalswasmaderegardingtheirindustrialuseandpotentialpollutionimpact.Cuisnotacutelytoxictohumansbutitsextensiveuseandincreasinglevelsintheenvironmentmaycauseserioushealthproblems7.MnremovalisoneoftheproblemsinusinggroundwatersinceMnisacommonelementingroundwater.ElevatedlevelsofMnindrinkingwaterareconsideredundesirable.Whenwaterisexposedtoair,Mn(II)isoxidizedtoMn(IV).TheMn(IV)precipitatecanstainhouseholdutensilsandclothesandmayimpartametallic,bitter,astringentormedicinaltastetothewater8.II.METHODSA.MicroorganismsStrainF2wasoriginallyscreenedfromsoilbyStateKeyLaboratoryofUrbanWaterResourceandEnvironment(SKLUWRE)andidentifiedasRhizobiumradiobacter.B.PreparationoftheEPSofF2Thecompositionofproductionmediumwasasfollows(perliter):10gglucose,5gK2HPO4,2gKH2PO4,0.5gpeptone,0.5gurea,0.2gMgSO47H2O,and0.2gNaCl.CorrelativedetailsweredescribedinapreviouspaperbyZhuetal.9.National863program(SQ2009AA06XK1482412)978-1-4244-4713-8/10/$25.002010IEEEThefermentationbrothwascentrifugedat16,000gfor20mintoremovecells.Twovolumesof4coldethanolwereaddedtothesupernatantandstoredat4for24h.Theprecipitatewasobtainedbycentrifugationat10,000gfor5minandwashedseveraltimeswithethanol.Thentheprecipitatewaslyophilizedandthecrudecompoundbioflocculantwasobtained.C.BiosorptionexperimentsMn(II)andCu(II)solutionswerepreparedfrom1000mg/lstocksolutionsofCu(II)andMn(II)obtainedbydissolvingCuSO45H2OandMnSO4H2Oindoubledistilledwater,respectively.ThelyophilizedEPSwasdissolvedindoubledistilledwastogivea2g/lsolution.Theadsorptionstudieswerecarriedoutin250mlErlenmeyerflasks.Inthefirstgroupofexperiments,biosorptionofmanganese(II)andcopper(II)fromaqueoussolutionscontainingasinglemetalionwasinvestigated.TheeffectofpHonthebiosorptionofcopper(II)andmanganese(II)ontheEPSwasinvestigatedinthepHrangeof2.0-7.0.100mlofsolutionscontaining100mg/lmanganese(II)and100mg/lCopper(II)ionswerepreparedusingthestocksolutions,respectively.TheinitialpHofeachmetalsolutionwasadjustedtotherequiredpHvalue.Then3mloftheEPSsolutionwasaddedtotheeachmetalsolutionandthemixturewasshakenonanorbitalshakerat125r/minand250.1for2h.Similarly,metalsolutionswith100mg/lmetalionswereusedtoinvestigatethepropercontacttimerangingfrom0.5to3hwithatimeintervalof0.5h.Inthelatterexperiments,thedosageoftheEPSvariedfrom1to5mlusingsolutionscontaining100mg/lmetalions.Theircontentswerecentrifugedat3600gfor5minandthesupernatantwasanalyzedforresidualmetalionconcentration.Inthesecondgroupofexperiments,competitivebiosorptionofMn(II)andCu(II)ionsfromsolutionscontainingthesetwometalswasinvestigated.Themedium(100ml)containing100mg/lofeachmetalionwasincubatedwith3mloftheEPSsolutionat250.1.Afterbiosorption,supernatantwasseparatedfromtheEPSbycentrifugationasdescribedabove.D.AnalyticalproceduresTheconcentrationsofremainedMn(II)andCu(II)ionsinthesupernatantweredeterminedbyICP-AES(Optima5300DV,PE,USA)withworkingwavelengthsforCu(II)andMn(II)at327.393.3nmand257.610nm,respectively.TheamountofadsorbedmetalionsonpergramoftheEPSwasobtainedbyusingthegeneralequation:()MVeCiCeq/=(1)whereqeistheamountofmetalionsadsorbedontheEPS(mg/g)；Ciistheinitialmetalionconcentrationinsolution(mg/l)；Ceistheequilibriummetalionconcentrationinsolution(mg/l)；Visthevolumeofthesolution(l)；MistheamountoftheEPSusedinthereactionmixture(g).Alltheexperimentswerecarriedoutintriplicateandthearithmeticalmeanvalueswereusedincalculations.Allthechemicalsusedinthisstudywereofanalyticalgrade.III.RESULTSANDDISCUSSIONA.EffectofpHonbiosorptioncapacityHydrogenionconcentrationisconsideredasoneofthemostimportantparametersthatinfluencetheadsorptionbehaviourofmetalionsinaqueoussolutions10.TheeffectofinitialpHonthebiosorptionofCu(II)andMn(II)ionsontotheEPSwereevaluatedwithinthepHrangeof2.0-7.0at100mg/linitialmetalionconcentrations,respectively(Fig.1).StudiesbeyondpH7werenotattemptedbecauseprecipitationwouldbelikelyinthepresenceofhigherconcentrationofhydroxylionsinthebiosorptionmedium.FromFig.1,itwasobservedthatboththeadsorptionofCu(II)andMn(II)werehighlypHdependentandincreasedwiththeincreaseofpHoftheaqueoussolutionuptopH6.ThebiosorptionofmetalionsreachedthemaximumatpH6anddecreasedthereafter.ThispHdependencyofbiosorptioncapacitycouldbeexplainedbythefunctionalgroupsinvolvedinmetaluptakeandmetalchemistry.BecauseofthecompetitionofhighprotonconcentrationatlowpHconditions,heavymetalswerepartiallyreleasedtoaqueousphase11.WhenthepHincreases,themetalionsthencompetemoreeffectivelyforavailablebindingsites,whichincreasesbiosorption12.B.EffectofcontacttimeonbiosorptioncapacityTheeffectofthecontacttimeonthebiosorptionofCu(II)andMn(II)ontheEPSwasdepictedinFig.2.InthecaseofCu(II)biosorptionontotheEPS,thebiosorptionprocessreachedequilibriumat1.0handabiosorptioncapacityof42.071.22mg/gwasachieved.ThemaximumbiosorptionofMnoccurredat1.0h.AfterthisequilibriumperiodtheamountofadsorbedmetalionsdidnotchangesignificantlywithanincreaseincontacttimeforbothCu(II)andMn(II).ThussubsequentbiosorptionexperimentswereperformedusingtheoptimumpHandcontacttimerequiredtoreachequilibriumofeachmetalion.Thisrapiduptakeofmealsmaybeanimportantparameterforapracticalapplicationofbiosorptioninindustrialwastewatertreatment.23456701020304050copper(II)mangansese(II)qe(mg/g)pHFig.1.EffectofpHontheCu(II)andMn(II)biosorptionontheEPS.0.51.01.52.02.53.001020304050copper(II)manganese(II)qe(mg/g)Time(h)Fig.2.EffectofcontacttimeontheCu(II)andMn(II)biosorptionontheEPSC.EffectoftheEPSdosageonbiosorptioncapacityThenumberofavailablesitesandexchangingionsforadsorptiondependsupontheamountofadsorbentinthebiosorptionprocess.TheeffectofadsorbentconcentrationonthemetalremovalefficiencywaspresentedinFig.3.ItwasfoundthattheCuuptakeofpergramEPSreacheditsmaximumattheEPSdoseof10mginto100mlsolutionofmetals.Similarlyinthecaseoflead,thebiosorptioncapacitywasfoundtoincreasewithincreaseinEPSdosageupto30mg.However,thebiosorptioncapacitydecreasewithincreasingdosageathighEPSconcentrationandthismaybebecausethatanincreaseinpolysaccharideconcentrationleadstointerferencebetweenbindingsites.Thedecreaseofspecificuptakemightbeattributedtometalconcentrationshortageinsolution13.ThebiosorptioncapacitiesoftheEPSreached45.301.62mg/gforCu(II)ionsand28.861.28mg/gforMn(II)ions；thesearecomparablewiththevaluesreportedinpreviousstudies.ThebiosorptioncapacitiesofGeobacillustoebiisub.sp.decanicusweredeterminedas41.5and23.2mg/gbiosorbentforCu(II)andMn(II)14.ThemaximumbiosorptionvaluesofCu(II)ontheAspergillusflavusfungalbiomasswasfoundtobe13.460.99mg/g3.102030405001020304050copper(II)mangannese(II)qe(mg/g)DosageoftheEPS(mg)Fig.3.EffectofEPSdosageontheCu(II)andMn(II)biosorptionontheEPSTABLEI.ISOTHERMPARAMETERSFORCU(II)ANDMN(II)BIOSORPTIONMetalsLangmuirparametersFreundlichparametersQ0(mg/g)b(1/mg)R2nKfR2Cu(II)51.2300.1560.9894.83920.4170.882Mn(II)28.1691.3080.9908.13318.0480.513D.BiosorptionisothermsLangmuirandFreundlichmodelsarethemostwidelyusedmodelsinthecaseoftheadsorptionofmetalions.Theseisothermequationsareasfollows:FreundlichneCFKeq/1=(2)eCnFKeqlog/1loglog+=(3)Langmuir)1/(0ebCebCQeq+=(4)01011QebCQeq+=(5)10203040500.00.51.01.52.0copper(II)manganese(II)Ce/qe(g/l)Ce(mg/l)Fig.4.LangmuirisothermsforbiosorptionofCu(II)andMn(II).1.52.02.53.03.54.04.53.03.84.0copper(II)manganese(II)lnqelnCeFig.5.FreundlichisothermsforbiosorptionofCu(II)andMn(II).01020304050binarymetalmixturesinglemetalionqe(mg/g)copper(II)manganese(II)Fig.6.BiosorptionofCu(II)andMn(II)ontheEPSinbinarymetalionmixtureIntheaboveequations,KFandnareFreundlichconstants,whichaffecttheadsorptionprocess,suchasadsorptioncapacityandintensityofadsorption,respectively.Q0(mg/g)andbareLangmuirconstantsrelatedtomonolayeradsorptioncapacityandenergyofadsorptionrespectively.Thevaluesoftheconstantsaswellasthecorrespondingregressioncoefficients,whichwereobtainedbyfittingtheexperimentaldataatpH6andacontacttimeof1.0h,werelistedinTable1.Figs.4and5indicatedtheLangmuirandFreundlichisothermsforbiosorptionofCu(II)andMn(II)ontotheEPS,respectively.Withregardtothevaluesofregressioncoefficients(R2)giveninTable1,itwasnotedthattheLangmuirisothermmodelexhibitedbetterfitnesstothebiosorptiondataofbothCu(II)andMn(II)ontotheEPSthantheFreundlichisothermmodel,whichcouldalsobeconfirmedfromFigs.4and5.Langmuirmodelsuggestsmonolayersorptiononahomogeneoussurfacewithoutinteractionbetweenadsorbedmolecules.Inaddition,themodelassumesuniformenergiesofsorptionontothesurfaceandnotransmigrationofthesorbate15.Therefore,biosorptionprocessinthisstudymaybeinterpretedasmonolayeradsorption.E.BiosorptionfrombinarymetalsolutionSimultaneousbiosorptionofCu(II)andMn(II)ionsbytheEPSwasstudiedusinganaqueoussolutioncontaining100mg/lofeachmetalion.Thebiosorptioncapacitieswere36.261.52mg/gforCu(II)and16.231.09mg/gforMn(II),whichwerelowerthanthatofnoncompetitiveconditions(Fig.6).Theorderofthebiosorptioncapacityforcompetitiveconditionswasasfollows(basedonmg/gaccumulation):Cu(II)Mn(II),whichwasinagreementwiththatinthenoncompetitiveconditions.IV.CONCLUSIONSResultspresentedinthispaperindicatedthatthebiosorptionprocessofCu(II)andMn(II)ontotheEPSproducedformRhizobiumradiobacterF2wasdependentonexperimentalconditionssuchaspH,contacttimeanddosageoftheEPS.TheequilibriumdatawereappliedtotheLangmuirandFreundlichisothermmodelsanditwasfoundthattheLangmuirisothermdescribedthebiosorptionofCu(II)andMn(II)ionsontotheEPSverywell.Inthecaseofabinarymetalmixture,thebiosorptioncapacityoftheEPSdecreasedforeachmetalion,Cu(II)ionswerefoundtobeadsorbedmoreeffectivelythanMn(II)ionsontotheEPSinbothsingleandbinarymetalsolutions.ThepresentdatasuggestthatEPSproducedformRhizobiumradiobacterF2maybeusedasefficientbiosorbentfortheremovalofCu(II)andMn(II)ionsfromaqueoussolution.ACKNOWLEDGMENTTheauthorswishtoexpresstheirgratitudetotheStateKeyLaboratoryofUrbanWaterResourceandEnvironment(SKLUWRE)andtheNational863program(SQ2009AA06XK1482412)forfinancialsupport.REFERENCES1J.O.Nriagu,J.M.Pacyna,“Quantitativeassessmentofworldwidecontaminationofair,waterandsoilsbytracemetals,”Nature,vol.333,pp.134-139,1988.2X.Bosch,“Cadmiumcutstwoways:TheheavymetalmutatesDNA,andthenpreventscellsfromrepairingthedamage,”Science,vol.609,pp.1-1,2003.3T.Akar,S.Tunali,“BiosorptioncharacteristicsofAspergillusflavusbiomassforremovalofPb(II)andCu(II)ionsfromanaqueoussolution,”BioresourceTechnology,vol.97,pp.1780-1787,2006.4F.Pagnanelli,M.Trifoni,F.Beolchini,A.Esposito,L.Toro,F.Vegli,“Equilibriumbiosorptionstudiesinsingleandmulti-metalsystems,”ProcessBiochemistry,vol.37,pp.115-124,2001.5A.Artola,M.D.Balaguer,M.Rigola,“Heavymetalbindingtoanaerobicsludge,”WaterResearch,vol.31,pp.997-1004,1997.6D.Kratochvil,B.Volesky,“Advancesinthebiosorptionofheavymetals,”TrendsinBiotechnology,vol.16,pp.291-300,1998.7A.Snchez,A.Ballester,M.L.Blzquez,F.Gonzlez,J.Muoz,A.Hammaini,“BiosorptionofCuandz