BiodiversityofNcyclebacteriainnitrogenremov

BiodiversityofN-cyclebacteriainnitrogenremovingmovingbedbiofiltersforfreshwaterrecirculatingaquaculturesystemsMaartjeA.H.J.vanKessela,b,HarryR.Harhangia,KatinkavandePas-Schoonena,JackvandeVossenbergc,GertFlikb,MikeS.M.Jettena,PeterH.M.Klarenb,HuubJ.M.OpdenCampa,⁎aDepartmentofMicrobiology,IWWR,RadboudUniversityNijmegen,Heyendaalseweg135,NL-6525AJNijmegen,theNetherlandsbDepartmentofOrganismalAnimalPhysiology,IWWR,RadboudUniversityNijmegen,Heyendaalseweg135,NL-6525AJNijmegen,theNetherlandscKWRWatercycleResearchInstitute,Groningenhaven7,NL-3433PENieuwegein,TheNetherlandsabstractarticleinfoArticlehistory:Received12January2010Receivedinrevisedform19May2010Accepted19May2010Keywords:N-cyclebacteriaMovingbedbiofilmreactorFreshwateraquaculturesystemNitrificationDenitrificationAnammoxMaintenanceofoptimalwaterqualityandremovalofnitrogencompoundsposechallengestoaquacultureworldwide.Presenceandactivityofdifferentbacteriainvolvedinnitrogencyclinginthebiofilmofamovingbedbiofilmreactor(MBBR)connectedtoaKoicarptankwereinvestigatedexperimentally.ForthisMBBRsystem,anitrogenremovingrateof3.5gnitrogenperdaywasfoundinactivityassays,whereasthepredictednitrogenremovalratewascalculatedtobe58gnitrogenperday.Thepresenceofammonium-oxidizingandnitrite-oxidizingbacteriainthebiofilmwasdemonstratedbysituhybridization(FISH)analysis.Alargenumberofplanctomyceteswereshowntobepresentinthebiofilmofthereactor,buttheydidnothybridizetooligonucleotideprobesofknowngeneraofanammoxbacteria.Molecular16SrRNAgeneanalysisoftheMBBRbiofilmrevealedanovelgroupofplanctomycetecellswaspresentinthisbiofilter,whichmayexplainthedifferenceinexperimentallyfoundandcalculatednitrogenremovalofthesystem.Thenovel16SrRNAgenesequencesshowedalowsimilaritytodescribedanammoxbacteria.Thisandotherstudiesmayimplicatethattheanammoxreactioncanbeincorporatedinbiofiltersystemsforaquaculturalfreshwatersystems,whichmakesitpossibletoremovenitrogencompoundsfromthesesystemsinarelativelyinexpensiveandsustainablemanner.©2010ElsevierB.V.Allrightsreserved.1.IntroductionIntheNetherlands,fishaquacultureiscarriedoutinclosed,recirculatingsystems.Amajorprobleminthesesystemsisthemaintenanceofaconstantandoptimalwaterquality.Eliminationofwasteisgenerallymanagedthroughmechanicalfiltrationtoremovesolids,andbiofiltrationfortheconversionoftoxicnitrogenmetabolitesintolesstoxicforms(Crabetal.,2007).Theidentityofthemicroorganismsresponsibleforthisconversionofnitrogenmetabolitesislargelyunknown(Taletal.,2003).Giventhelowammoniumandnitriteconcentrationsinthesesystems,K-specialists(bacteriawithhighsubstrateaffinity)suchasNitrosospiraandNitrospiramaybelikelycandidatesforaerobicammoniumandnitriteoxidation.Further,therecentlydiscoveredammonium-oxidizingcrenarchaea(Könnekeetal.,2005)couldplayarole.Nitrateaccumulationinaquaculturesystemscanbepreventedbyadailyreplacementofavolumeofpondwater(vanRijn,1996;Hargreaves,1998),butthisisexpensiveandinefficient.Furthermore,strictenviron-mentallegislationonnitratelevelsinthedischargewater(vanRijn,1996)promptstheaquacultureindustrytoinvestinmoreefficientnitrogenremovalsystems.Thisshouldresultintheremovalofnitrateinaquaculturewastewater,forexamplebydenitrification,bysomeconsideredtobethebestcandidatetoremovenitratefromaquaculturesystems.Denitrificationiscarriedoutbyfacultativeanaerobicbacteriathatutilizeeitherorganic(heterotrophicdenitrification)orinorganic(autotrophicdenitrification)compoundsaselectronsourcestoreducenitratetoformnitrogengas(N2).Difficultiesassociatedwiththedenitrificationprocess,suchasreleaseofnitrousoxideaswellastherequirementforanexternalelectronsource,havethusfarpreventeditsapplicationinfull-scalecommercialrecirculationsystems.Anotherattractiveoptionforcompletingthenitrogencycleinaquaculturesystemsistheintegrationoftheanaerobicammonium-oxidation(anammox)processinthesesystems.Asanimportantpathwayofthenitrogencycle,anammoxallowsammoniumtobeoxidizedbynitriteunderanoxicconditionsyieldingmolecularnitrogen.Theimportanceofthisprocessintheglobalnitrogencycleisshownbytheestimationthatanammoxbacteriaareresponsiblefor30%–50%oftheglobalpresent-daymarinenitrogenloss(Brandesetal.,2007;Lametal.,2009).Theapplicationofanammoxisattractiveforthecompletenitrogenremovalfromaquaculturesystemssinceitiseconomicallyadvantageous;itrequiresatleast50%lessoxygencomparedwiththeconventionalnitrification–denitrificationprocess(Jettenetal.,2001).Furthermore,noadditional(in)organicelectrondonorisrequiredforthisautotrophicnitrogenremovalcycleinwhichammoniumisoxidizedviahydrazine(N2H4)asanintermediateAquaculture306(2010)177–184⁎Correspondingauthor.Tel.:[phone];fax:[phone].E-mailaddress:[e-mail](H.J.M.OpdenCamp).0044-8486/$–seefrontmatter©2010ElsevierB.V.Allrightsreserved.doi:10.1016/j.aquaculture.2010.05.019ContentslistsavailableatScienceDirectAquaculturejournalhomepage:(Jettenetal.,2009).Sincetheirdiscoveryinthelate1990sanammoxbacteria,whichbelongtotheorderoftheBrocadialesandareaffiliatedtothePlanctomycetales(Jettenetal.,inpress),havebeenfoundinwastewatertr