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RESEARCHARTICLEAmmonia-oxidizingarchaeaandnitrite-oxidizingnitrospirasinthebiofilterofashrimprecirculatingaquaculturesystemMonishaN.Brown1,AurelioBriones2,JamesDiana3&LutgardeRaskin11DepartmentofCivilandEnvironmentalEngineering,UniversityofMichigan,AnnArbor,MI,USA;2Plant,Soil&EntomologicalSciencesDepartment,UniversityofIdaho,Moscow,ID,USA;and3SchoolofNaturalResourcesandtheEnvironment,UniversityofMichigan,AnnArbor,MI,USACorrespondence:LutgardeRaskin,DepartmentofCivilandEnvironmentalEngineering,UniversityofMichigan,AnnArbor,MI48109,USA.Tel.:+17346476920;fax:+17347632275;e-mail:raskin@umich.eduReceived10February2012;revised5July2012;accepted5July2012.Finalversionpublishedonline9August2012.DOI:10.1111/j.1574-6941.2012.01448.xEditor:CindyNakatsuKeywordsammoniaoxidation;nitriteoxidation;Nitrospira;Nitrosopumilusmaritimus;shrimpaquaculture;recirculatingaquaculturesystem.AbstractThisstudyanalysedthenitrifiercommunityinthebiofilterofazerodischarge,recirculatingaquaculturesystem(RAS)fortheproductionofmarineshrimpinalowdensity(lowammoniumproduction)system.Theammonia-oxidizingpopulationswereexaminedbytargeting16SrRNAandamoAgenesofammonia-oxidizingbacteria(AOB)andarchaea(AOA).Thenitrite-oxidizingbacteria(NOB)wereinvestigatedbytargetingthe16SrRNAgene.ArchaealamoAgenesweremoreabundantinallcompartmentsoftheRASthanbacterialamoAgenes.AnalysisofbacterialandarchaealamoAgenesequencesrevealedthatmostammoniaoxidizerswererelatedtoNitrosomonasmarinaandNitrosopumilusmaritimus.TheNOBdetectedwererelatedtoNitrospiramarinaandNitrospiramoscoviensis,andNitrospiramarina-typeNOBweremoreabundantthanN.mos-coviensis-typeNOB.WaterqualityandbiofilmattachmentmediaplayedaroleinthecompetitivenessofAOAoverAOBandNitrospiramarina-overN.moscovien-sis-typeNOB.IntroductionRecirculatingaquaculturesystems(RAS)fortheproduc-tionofmarineshrimpareapotentiallysustainablealter-nativetotraditionalaquaculturesystemsbecauseRASreducewaterrequirementsandlimittheconcentrationofnutrientsdischargedtoreceivingwaters.RASrequirebio-logicalfilterstooxidizetoxicammoniaandnitrite,andaerationofthewatertoremovecarbondioxideandincreaseoxygenconcentrations(Ebeling,2000).Nitrifyingbiofilterskeepammoniaandnitriteconcentrationsbelowtoxiclevels.Forthewhiteshrimp,Litopenaeusvannamei(Boone),ammoniatoxicitylevelsrangefrom2.44to3.95mgL1oftotalammonianitrogen(Lin&Chen,2001),whilenitritetoxicitylevelsvarybetween6.1and25.7mgL1ofnitrite-nitrogen(Lin&Chen,2003).Nitrificationisatwo-stepprocessinwhichammoniaisoxidizedtonitritebyammonia-oxidizingbacteria(AOB)orammonia-oxidizingarchaea(AOA)andnitriteisoxi-dizedtonitratebynitrite-oxidizingbacteria(NOB).ThesensitivityofAOBandNOBtoawidevarietyofenviron-mentalfactorsiswellknown,somuch,sothatnitrificationhasbeenregardedasthe‘Achillesheel’ofwastewatertreat-ment(Daimsetal.,2006).Inrecirculatingaquaculturesettings,thechallengesassociatedwithaccumulationofammoniaandnitritearesimilartothoseinthewastewatertreatmentfieldandalsoincludeproblemswithlowdissolvedoxygen(DO)levels,pHoutsidetheoptimalrangefornitrifyingmicroorganisms(7.5–8.6)andaccumulationoftraceamountsoftoxicsulphides(Joye&Hollibaugh,1995;Masseretal.,1999;Ling&Chen,2005).LessisknownaboutthesensitivityofAOAtoenviron-mentalparameters.ArchaealammoniamonooxygenasesubunitA(amoA)genehasbeenfoundtobeubiquitousintheenvironment,includinginmarinewaters,biofiltersofaquaria,coralreefs,estuaries,wastewatertreatmentplants,hotsprings,sedimentsandsoils(Prosser&Nicol,2008;Erguderetal.,2009;Youetal.,2009).InmoststudiesinwhichtheabundancesofarchaealandbacterialamoAgenecopieswereinvestigated,thearchaealamoAoutnumberedthebacterialamoAgenecopies(Erguderetal.,2009).Thefactorsthatinfluencethisdistributionarestillunclear,butErguderetal.(2009)proposedthatAOAmightbeimportantammoniaoxidizersinlowFEMSMicrobiolEcol83(2013)17–25ª2012FederationofEuropeanMicrobiologicalSocietiesPublishedbyBlackwellPublishingLtd.AllrightsreservedMICROBIOLOGYECOLOGYnutrient,lowpHandsulphidecontainingenvironments.Furthermore,Martens-Habbenaetal.(2009)haveshownthatAOAhaveadaptedtosurviveatlowammoniacon-centrations,forexample,0.2lM,concentrationsatwhichAOBcannotgrow.TherearefourvalidlydescribedgeneraofNOB:Ni-trobacter,Nitrospina,Nitrococcus,Nitrospira,aswellasanewlycommunicatedNOBspecies,‘CandidatusNitrotogaarctica’(Bartoschetal.,1999;Alawietal.,2007).Nitro-spiraspp.havebeenfoundtobethemainnitriteoxidiz-ersinwastewatertreatmentplants(Juretschkoetal.,1998;Burrelletal.,1999;Daimsetal.,2001,2006)andthebiofiltersofamarineRAS(Keuteretal.,2011)becausetheyarebetterscavengersfornitriteandoxygenthanNitrobacterspp.(Schrammetal.,1999;Koops&Pommerening-Roser,2001).Nitrospiraspp.havealsobeenfoundinahotspring(Lebedevaetal.,2011),amarinesponge(Offetal.,2010),andvarioussoils(Bart-oschetal.,2002).AnanalysisofthepubliclyavailablesequencesoftheNitrospiraephylum(Daimsetal.,2001)revealedthatthegenusNitrospiraconsistsofatleastfourdistinctsublineages.Nitrificationisimportanttoaquacultureingeneral,butparticularlytozerodischargeRAS,inwhichwaterquality(specificallyammoniaandnitriteconcentr