Comparativeanalysisofthebacterialdiversityin

Comparativeanalysisofthebacterialdiversityinalab-scalemovingbedbiofilmreactor(MBBR)appliedtotreaturbanwastewaterunderdifferentoperationalconditionsKadiyaCalderóna,⇑,JaimeMartín-Pascualb,JoséManuelPoyatosb,BelénRodelasa,AlejandroGonzález-Martínezb,JesúsGonzález-LópezaaDepartmentofMicrobiology,UniversityofGranada,Granada18071,SpainbDepartmentofCivilEngineering,UniversityofGranada,Granada18071,SpainhighlightsCarrierFRisthemajoroperationalparameteronthebiofilmformationinalab-scaleMBBR.HRTandcarriertypenotinfluencestatisticallyonthebacterialdiversityofthebiofilm.CarrierFRisthecriticalparameterinthestart-upofMBBR-basedWWTP.articleinfoArticlehistory:Received4May2012Receivedinrevisedform26June2012Accepted27June2012Availableonline5July2012Keywords:BiofilmMBBRTGGECarrier-FRHRTabstractDifferenttypesofcarriersweretestedassupportmaterialinalab-scalemovingbedbiofilmreactor(MBBR)usedtotreaturbanwastewaterunderthreedifferentconditionsofhydraulicretentiontime(HRT)andcarrierfillingratios(FR).Thebacterialdiversitydevelopedonthebiofilmsresponsibleofthetreatmentwasstudiedusingacultivation-independentapproachbasedonthepolymerasechainreaction-temperaturegradientgelelectrophoresistechnique(PCR-TGGE).ClusteranalysisofTGGEfin-gerprintsshowedsignificantdifferencesofcommunitystructuredependentuponthedifferentopera-tionalconditionsapplied.Redundancyanalysis(RDA)wasusedtodeterminetherelationshipbetweentheoperationalconditions(typeofcarrier,HRT,FR)andbacterialbiofilmdiversity,demonstratingasignificanteffectofFR=50%.PhylogeneticanalysisofPCR-reamplifiedandsequencedTGGEbandsrevealedthattheprevalentBacteriapopulationsinthebiofilmwererelatedtoBetaproteobacteria(46%),Firmicutes(34%),Alphaproteobacteria(14%)andGammaproteobacteria(9%).2012ElsevierLtd.Allrightsreserved.1.IntroductionUrbanandindustrialwastewaterreclamationisnowadaysoneofthemajorresearchtopicsresourcedbypeopleandgovernments.Untreatedorinsufficientlytreatedwastewaterdischargedtotheenvironmentcausesseveralproblems,suchaseutrophication(Luostarinenetal.,2006;Plattesetal.,2007).Inordertoimprovethequalityoftreatedwastewaterandmeetthedemandsofenvironmentalregulations,implementationofadvancedtechnolo-giesfortreatmentisrequired(Trapanietal.,2010).Biologicalprocessesbasedonbiofilmshavebeenprovedtooffersatisfactorysolutionsfortheremovaloforganiccomponentsandnitrogenfromwastewater,avoidingsomeoftheproblemsassociatedwithacti-vatedsludgeprocess,suchaslargereactorsize,needforsettlingtanks,andbiomassrecycling(Luostarinenetal.,2006;McQuarrieandBoltz,2011).Movingbedbiofilmreactors(MBBRs)havebeenwidelyappliedtotreatbothurbanandindustrialwastewaters.ThistechnologyallowsBOD5andNremovalratessimilartothoseofactivatedsludge-basedprocesses,withtheadvantageofasmallertankvol-ume(Andreottolaetal.,2000).MBBRscanbeoperatedasanoxicoraerobicphaseswithfreelymovingbuoyantplasticbiofilmcarriers.Thesystemsincludeasubmergedbiofilmreactorandaliquid-solidseparationunit.TheobjectiveoftheMBBRsystemsistoachievethegrowthofthebiomassasabiofilmonsmallcarriers,whichhavelowerdensitythanwater,arecontinuouslykeptinthetankandareabletomovefreelyinthereactorwithoutsludgerecycling(Ødegaard,2008).Biofilmcarriersmadeoutofdifferentmaterialsanddesignshavebeendevelopedandarecommerciallyaccessible.0960-8524/$-seefrontmatter2012ElsevierLtd.Allrightsreserved.⇑Correspondingauthor.Address:DepartamentodeMicrobiología,FacultaddeFarmacia,UniversidaddeGranada,18071Granada,Spain.Tel.:[phone];fax:[phone].E-mailaddress:[e-mail](K.Calderón).BioresourceTechnology121(2012)119–126ContentslistsavailableatSciVerseScienceDirectBioresourceTechnologyjournalhomepage:(McQuarrieandBoltz,2011).KnowledgeonthemicrobiotacompositioninvolvedinbiofilmprocessesandthemechanismsbywhichoperationalvariationsmayinfluencetheirstructureisregardedascruciallyimportantfortheoptimizationofnutrientremovalratesonMBBRsystemsandtoimplementcontrolstrategies(Boltzetal.,2011;Ciesielskietal.,2010).However,todate,littleresearchisavailablerelatedtothecharacteristicsofthemicrobialbiofilmsgrownonMBBRs,inparticular,informationconcerningtheircommunitycomposi-tion(McQuarrieandBoltz,2011).Theaimofthisworkwastofindtherelationshipsbetweendifferentoperationalconditions(typeofcarrier,carrierfillingratio,andhydraulicretentiontime)andthecommunitystructureofbacterialbiofilmsdevelopedonMBBRs.Forthispurpose,scanningelectronmicroscopy(SEM)andmolecu-larfingerprintingtoolssuchaspolymerasechainreaction(PCR)coupledtotemperaturegradientgelelectrophoresis(TGGE)wereused.IdentificationofthedominantbacteriainhabitingthebiofilmwasachievedbyDNAsequencingoftheprevalentTGGEbands.2.Methods2.1.Descriptionofthelab-scaleMBBRsandoperatingconditionsThelab-scaleplantconsistedofthreereactors(eachonewitha3-Loperatingvolume),operatedinparallelandfedfromacommonfeedtankbyamultichannelperistalticpump.Thefeedtankwasfilledperiodicallywithwaterfromtheoutletoftheprimarysettlerfromthewastewatertreatm