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3/21/2017 Aquarium Nitrication Revisited: Thaumarchaeota Are the Dominant Ammonia Oxidizers in Freshwater Aquarium Biolters

Aquarium Nitri cation Revisited: Thaumarchaeota Are the


Dominant Ammonia Oxidizers in Freshwater Aquarium
Bio lters
LauraA.Sauder, KatjaEngel, JenniferC.Stearns, AndreP.Masella, RichardPawliszyn, JoshD.Neufeld

Published:August16,2011 http://dx.doi.org/10.1371/journal.pone.0023281

Abstract
Ammoniaoxidizingarchaea(AOA)outnumberammoniaoxidizingbacteria(AOB)inmanyterrestrialandaquaticenvironments.
Althoughnitrificationistheprimaryfunctionofaquariumbiofilters,veryfewstudieshaveinvestigatedthemicroorganisms
responsibleforthisprocessinaquaria.ThisstudyusedquantitativerealtimePCR(qPCR)toquantifytheammonia
monooxygenase(amoA)and16SrRNAgenesofBacteriaandThaumarchaeotainfreshwateraquariumbiofilters,inadditionto
assessingthediversityofAOAamoAgenesbydenaturinggradientgelelectrophoresis(DGGE)andclonelibraries.AOAwere
numericallydominantin23of27freshwaterbiofilters,andin12ofthesebiofiltersAOAcontributedalldetectableamoAgenes.
Eightsaltwateraquariaandtwocommercialaquariumnitrifiersupplementswereincludedforcomparison.Boththaumarchaealand
bacterialamoAgenesweredetectedinallsaltwatersamples,withAOAgenesoutnumberingAOBgenesinfiveofeightbiofilters.
BacterialamoAgeneswereabundantinbothsupplements,butthaumarchaealamoAand16SrRNAgenescouldnotbedetected.
Forfreshwateraquaria,theproportionofamoAgenesfromAOArelativetoAOBwasinverselycorrelatedwithammonium
concentration.DGGEofAOAamoAgenesrevealedvariablediversityacrosssamples,withnonmetricmultidimensionalscaling
(NMDS)indicatingseparationoffreshwaterandsaltwaterfingerprints.CompositeclonelibrariesofAOAamoAgenesrevealed
distinctfreshwaterandsaltwaterclusters,aswellasmixedclusterscontainingbothfreshwaterandsaltwateramoAgene
sequences.Theseresultsrevealinsightintocommonplaceresidentialbiofiltersandsuggestthataquariumbiofiltersmayrepresent
valuablebiofilmmicrocosmsforfuturestudiesofAOAecology.

Citation:SauderLA,EngelK,StearnsJC,MasellaAP,PawliszynR,NeufeldJD(2011)AquariumNitrificationRevisited:
ThaumarchaeotaAretheDominantAmmoniaOxidizersinFreshwaterAquariumBiofilters.PLoSONE6(8):e23281.
doi:10.1371/journal.pone.0023281

Editor:JackAnthonyGilbert,ArgonneNationalLaboratory,UnitedStatesofAmerica

Received:June30,2011Accepted:July11,2011Published:August16,2011

Copyright:2011Sauderetal.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthor
andsourcearecredited.

Funding:ThisresearchwasfundedbytheNaturalSciencesandEngineeringResearchCouncilofCanada(NSERC).The
fundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.

Competinginterests:Theauthorshavedeclaredthatnocompetinginterestsexist.

Introduction
Ammoniaisatoxicmetabolicwasteproductexcretedbyfishandotheraquaticorganisms.Ammoniatoxicitycanthreatenaquatic
ecosystemhealthandisaparticularconcernforrelativelyclosedecosystems,suchasaquacultureoperationsandhomeaquaria,
inwhichammoniacanquicklyaccumulatetolethalconcentrationsintheabsenceofactivenitrification.Theunionizedformof
ammonia(NH3)isparticularlytoxictofishstress,disease,anddeathmaybeassociatedwithconcentrationsthatexceed0.1mg
L1inaquariumandaquaculturesystems[1],[2].Inordertoconvertammoniatonitrate,aquariumbiofiltersaredesignedto
promotethegrowthandactivityofnitrifyingpopulationsduetothehighsurfaceareaoffiltersupportmaterial(e.g.sponge,ceramic
orpolymer)andrapidflowratesofaeratedwater.Despitetheirimportancetofishhealthandidenticalfunctionwithinmany
industrialbiofilters,includingaquacultureandwastewatertreatment,littleisknownofthemicroorganismscatalyzingnitrificationin
associationwithaquariumbiofiltersupportmaterial.
Beforethediscoveryofammoniaoxidizingarchaea(AOA),belongingtothenewlyproposedphylumThaumarchaeota[3],[4],
molecularapproacheswereusedtoinvestigateammoniaoxidizingbacteria(AOB)andnitriteoxidizingbacteriainfreshwaterand
marineaquaria[5],[6].Inparticular,HovanecandDeLongusedoligonucleotideprobestotargetbacterialnitrifiersinfreshwaterand
saltwateraquariumbiofilterDNAextracts.AlthoughNitrosomonaslikebacteriafromtheBetaproteobacteriawereassociatedwith
thesaltwateraquariaintheirstudy,theydidnotdetectthesebacteriainmostofthefreshwateraquariumbiofilterextractstested.
Theyconcludedthatthebacterialspeciesresponsiblefornitrificationinsimplefreshwatersystemsremainunknown[5].
SubsequentstudiesdeterminedthatNitrosomonasspp.couldindeedbeenrichedfromfreshwateraquariumbiofilters[7],
suggestingtheirpotentialinvolvementinammoniaoxidationunderinsituconditions.SincethediscoveryofAOA,twostudieshave
investigatedthepresenceanddiversityofbothAOAandAOBinmarinebiofiltrationsystems[8],[9].Foeselandcolleaguesfound
thatNitrosomonaslikeAOBwerenumericallydominantinamarineaquaculturebiofilm.Urakawaandcolleaguesidentifiedthe
presenceofamoAgenesfromAOBandAOAinselectedmarineaquariumbiofiltersfromapublicaquariuminJapan(i.e.sunfish
tank,coldwatertank,andacoastalfishtank),andsuggestedthatthediversityofAOAandAOBwasdecreasedinlowtemperature

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marineaquaria.CandidatusNitrosopumilusmaritimusSCM1,thefirstAOArepresentativeisolatedinpureculture,wasobtained
fromsaltwateraquariumgravel[10].Despitetheseinitialstudies,noresearchhasyetinvestigatedtheabundanceofAOBandAOA
infreshwateraquariumbiofilters.
BasedontheubiquityandhighabundanceofAOAinnaturalenvironments,theinabilityofHovanecandDeLong(1996)todetect
AOBinfreshwateraquaria,andtheisolationofthefirstammoniaoxidizingarchaeonfromaquariumsubstrate[10],we
hypothesizedthatAOAdominatefreshwateraquariumbiofiltersandplayanimportantroleinaquariumnitrification.Inadditionto
determiningtheabundancesofAOAandAOBinaquaria,theobjectivesofthisstudyweretoassessthediversityofAOAamoA
genesinaquaria,andtodeterminehowthesegenesclusteredwithsequencesderivedfromenvironmentalsourcesandcultured
AOArepresentatives.Theresultsofthisstudyrevealedthat,basedonamoAgeneabundances,AOAwerethedominantputative
ammoniaoxidizersinthemajorityoffreshwaterandsaltwateraquaria.Theseresultsprovidefirstevidencefortheimportantroleof
AOAinfreshwateraquariumfiltrationandsuggestpossiblenicheadaptationofAOAtoconditionsassociatedwithfreshwater
aquariumbiofilters.

Results
Aquariumsamples

Twentysevenfreshwaterandeightsaltwateraquariumfiltersampleswerecollectedfromretailandresidentiallocationsinthree
cities(TableS1).Allbiofilterssampledinthisstudywerederivedfromstandaloneaquariainhomes(oroffices),orfromdisplay
tanksinretailoutlets,reflectingconditionscommontomostresidentialorretailaquaria,respectively.Inadditiontoaquarium
biofilters,weincludedtwoaquariumsupplementsintheanalysis.TheaquariaselectedforsamplingrangedinpHfrom7.6to9.2
andvariedintheirfishandliveplantcomposition.Theaquariacontainedavarietyoffishincludingmixedtropical,goldfish,South
AmericancichlidsandAfricancichlids.Threeaquariahadreceivedantibiotictreatmentintheprevioussixmonths(SW4,SW5,
FW8)andseveralwereknowntohavereceiveddosesofbacterialfiltersupplementwhenfirstestablished(e.g.FW12,FW13,
FW19,FW25).Ammoniumconcentrationsofaquariarangedfrombelowdetectiontoapproximately0.5mgL1,withthemajorityof
aquariabelow100gL1.In28ofthe32aquariastudied,nitrite(NO2)wasbelowdetection.Asexpected,significantpositive
correlationswereobservedbetweenammoniumandnitriteconcentrations(r=0.48,p<0.05TableS2)andnitriteandnitrate
concentrations(r=0.52,p<0.05TableS2).Aquariarangedinsizefrom5gallonstogreaterthan400gallons(forlargeretailshow
tanks)andapproximatefishnumbersrangedfromzero(inaplanttankFW3)upto300(FW11).Althoughnotaperfectmeasureof
fishbiomass,theapproximatenumberoffishpergallonwaspositivelycorrelatedwithammoniumconcentration(r=0.60,p<0.001
TableS2).Waterhardnessoffreshwateraquariawasaslowas25ppm(inFW12,abreedingtankusingsoftenedwater),however,
thewaterhardnessof25of27aquariawas>150ppm.Hardnessofsaltwatersamplescouldnotbedeterminedusingthekit
utilized.Alkalinity(i.e.carbonatealkalinity/hardness)rangedfrombelowdetection(e.g.FW24,FW17)to300ppm(e.g.SW1,FW3).
Neitherhardnessnoralkalinitycorrelatedsignificantlywithanyotherwaterchemistryparameters(TablesS2andS3).

AOAandAOBabundances

RealtimePCRresultsdemonstratedthatthaumarchaealamoAgenesweredominantin23ofthe27sampledfreshwaterfilters
(Fig.1TableS1).For12ofthefreshwaterbiofilters,thaumarchaealamoAgenesrepresentedtheentiredetectedamoAgene
signal,includingFW13,FW15,FW16,andFW25,aquariathatreceivedbacterialaquariumsupplementswhenfirstestablished.For
saltwateraquaria,amoAgenesfrombothAOAandAOBweredetectedinallsamples,withAOAdominatingfiveofeightsamples.
Forbothcommerciallyavailableaquariumsupplements,AOBamoAgeneswereabundant,andbothAOAamoAand16SrRNA
geneswerebelowdetectionlimits.

Figure1.RelativegeneabundancesofBacteriaandThaumarchaetoainaquaria.
Relative16SrRNA(A)andamoA(B)geneabundancesforbacteriaandthaumarchaeainsaltwater(SW1SW8)and
freshwater(FW1F27)aquariaandinaquariumsupplements(SP1,SP2).ThesedatawerecalculatedfromduplicateqPCR
amplifications.
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Bothbacterialandthaumarchaeal16SrRNAgenesweredetectedinallaquariumDNAextractshowever,forthemajorityof
samples,bacterial16SrRNAgenesgreatlyoutnumberedthaumarchaeal16SrRNAgenes(Fig.1TableS1).Inoneaquarium
biofilter(FW11),thaumarchaealandbacterial16SrRNAgenecopynumberswereapproximatelyequal.Forallaquaria,bacterial
amoAgenecopynumberswereatleastthreeordersofmagnitudelessthanbacterial16SrRNAgenes(TableS1).Insomeaquaria
(e.g.FW2,FW27,SW4),thaumarchaealamoAand16SrRNAgenecopynumberswereapproximatelyequal.Inothercases(e.g.
FW11,FW12,SW8),thaumarchaealamoAgenecopieswereordersofmagnitudelessabundantthanthaumarchaeal16SrRNA
genes.
Wealsoexaminedaspectsofwaterchemistry(TableS1)toassesscorrelationsthatmightprovideanexplanationfordifferential
amoAgeneabundances.Regressionanalysesfocusedonfreshwateraquariumsamplesbecausetheseweretheprimaryfocusof
thisstudy,andbecausetoofewsaltwatersampleswereavailabletoyieldstatisticallysignificantcorrelations(datanotshown).
Correlationswerealsocalculatedthatincludedbothfreshandsaltwatersamples,whichyieldedsimilarresultstofreshwater
correlations(TablesS2andS3).Forfreshwateraquaria,weobservedasignificantnegativecorrelationbetweenammonium
concentrationsandtheproportionofamoAgenesbelongingtoAOAratherthanAOB(r=0.85,p<0.001, R2=0.72Fig.2andTable
S3).LowammoniaconcentrationsweretypicallyassociatedwithhighAOAamoAgeneabundances,althoughonesample(FW20)
1

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hadhighproportionsofAOBamoAgenes,despiteanammoniaconcentrationoflessthan20gL1.Inallcases,higher
concentrationsofammoniumwereassociatedwithhigherrelativeabundancesofAOBamoAgenes.Nootherfactorsrelatedto
waterchemistryoraquariumsetup(e.g.pH,hardness,alkalinity)yieldedsignificantcorrelationswithamoAgeneabundances
(TableS2).AmmoniaconcentrationsinaquariumFW27fluctuatedverylittle,bothondailyandmonthlyscales(Fig.S1A).Thehigh
proportion(>85%)ofAOAinthisfilterwasalsoconsistentoveratwoyearsamplingperiod(Fig.S1B).Together,theseresults
suggesttemporalconsistencyinindividualfreshwateraquariumfilterenvironmentalconditionsandAOAcommunities.

Figure2.FreshwateraquariaammoniumconcentrationsandrelativethaumarchaealamoAgeneabundances.
AOAamoAgenecopiesareexpressedasapercentageofthetotalamoAgenecopies(perngDNA).Thecoefficientof
determination(R2)forthelinearregressionis0.7249.TheassociatedPearsoncorrelationcoefficient(r)is0.8518,withan
associatedpvalueof<0.001.SeeTableS1forallsampledata.
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AOAgenediversity

InordertoassessthesampletosamplevariabilityofthaumarchaealpopulationspossessingamoAgenes,allbiofilterDNAextracts
weresubjectedtoDGGEanalysis.BasedonthephysicalseparationofPCRproductsbysequenceheterogeneityandG+Ccontent
[11],thepatternsgeneratedbyamoAgeneampliconsvariedbetweensamplesfromverysimplewithfewbands(e.g.FW8,FW20,
SW4)torelativelycomplexwithmanybands(e.g.FW10,FW15,SW1Fig.3).DGGEpatternsrevealedsharedbandsbetween
aquariumbiofiltersfromthesamelocation(e.g.FW8andFW9,FW25andFW26,andSW1andSW2),indicatingthatlocation
specificfactorslikelyinfluencedthespecificcompositionofAOA.Despitetheselocationspecificsimilarities,distinctclusteringof
fingerprintswasnotobservedbasedonlocation,andmanybandsweresharedbetweenmultiplesamplesfromvariouslocations
(Fig.3AandB,TableS1).Basedonvisualinspectionofalignedfingerprints,apossibleshiftinG+Ccontentwasobservedbetween
saltandfreshwaterfingerprints,withsaltwaterbandstypicallymeltingatlowerdenaturantconcentrationsthanthemajorityof
freshwatersequences(Fig.3AandB).ThisapparentdifferenceinG+Ccontentwassupportedbysequencesfrombothclone
librariesandsequencedDGGEbands.FreshwateramoAgenesequencesfromclonelibrarieshadanaverageG+Ccontentof
45.4%,versus43.8%insaltwaterclones.SequencesofDGGEbandsshowedasimilarpattern,withaverageG+Ccontentsof
45.5%and44.0%forfreshwaterandsaltwaterbands,respectively.Althoughrelativelysmall(1.5%),thisdifferenceinaverage
G+Ccontentbetweenfreshwaterandsaltwatersequenceswasstatisticallysignificant(p<0.0001)forbothclonesandDGGE
sequences,asdeterminedbyunpairedttests.Nonmetricmultidimensionalscaling(NMDS)usingdensitometriccurvesgenerated
fromDGGEfingerprintsrevealedthatfreshwaterandsaltwaterfingerprintswerelargelyseparatedintwodimensionalspace.
However,somefreshwatersamples(e.g.FW1,FW12,FW15)wereintermediatetofreshwaterandsaltwaterclusters(Fig.3C).

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Figure3.DenaturinggradientgelelectrophoresisofthaumarchaealamoAgeneamplicons.
Saltwater(A)andfreshwater(B)fingerprintshavebeennormalizedandaligned.Bandschosenforsequencingareindicated
withtriangles:whitetrianglescorrespondtobandsappearinginFigure4.Blacktrianglesrepresentfailedsequencing
reactions.Clusteringoffreshwaterandsaltwaterfingerprints(C)isbasedonnonmetricmultidimensionalscaling(NMDS)
usingPearsoncorrelationsofbackgroundsubtracteddensitometriccurves.
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InadditiontoDGGE,wegeneratedclonelibrariesof261and84sequencesforfreshwaterandsaltwatercompositethaumarchaeal
amoAgenePCRamplicons,respectively.MultidimensionalscalingoftranslatedandalignedamoAgenesequences(fromDGGE
bands,clonelibraries,andreferencesequences)revealedfourdistinctclusters,eachofwhichcontainedbothclonesandDGGE
bandsequences(Fig.4).Thefirstcluster(cluster1Fig.4)containedsaltwaterandfreshwaterclonelibrarysequencesin
approximatelyequalproportions,aswellasbothsaltwaterandfreshwaterDGGEbandsequences.ThefreshwaterDGGEband
sequencesthatfellintothisclusterweregeneratedfromsampleswhichhadfingerprintsthatfellneartheperipheryofthe
freshwatercluster,closetosaltwatersequences(Fig.3C).

Figure4.NMDSordinationoftranslatedthaumarchaealamoAgenesequences.

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SequenceswereobtainedfromclonelibrariesandDGGEbandsderivedfromfreshwaterandsaltwateraquariumfilter
samples.Theinsetpanelprovidesasummaryofthenumberoffreshwaterandsaltwaterclonelibrarysequencescontained
withineachcluster.SequencesobtainedfromDGGEbandscorrespondtothewhitetrianglesinFigure3,andarelabelled
withsampleandbandnumbers.SelectedGenbanksequencesfromuncultivatedclonesandreferencestrainsareincluded
forcomparison.ThesamplingenvironmentandGenbankaccessionnumbersforallreferencesequencesareincludedwithin
parentheses.
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Boththesecondandthirdcluster(clusters2and3Fig.4)weredominatedbysaltwaterclonesequences.Cluster2wasadistinctly
salineclusterthatcontainedamoAgenesequencesfromAOAoriginatingfromsalineenvironments,includingCa.N.maritimusand
CandidatusCenarchaeumsymbiosumA[YP_87534212],aswellasanenvironmentalcloneobtainedfromasaltwateraquarium
biofilter[AB3732359].ThemajorityofsaltwaterDGGEbandsequencesalsofellintothiscluster,includingSW23,SW12,SW82
andSW62,sequencesthatrepresentabandsharedacrossthemajorityofsaltwaterfingerprints(Fig.3A).Cluster3wasvariable
incomposition,andcontainedbothfreshwaterandsaltwaterDGGEbandsequences.ReferenceamoAgenesequencesinthis
clusterwerefromsoilfosmid54d9[AJ62742213]andhotspringorganismsCandidatusNitrososphaeragargensis[ABY7759514]
andCandidatusNitrosocaldusyellowstonii[ABY8378815].
Cluster4wasadistinctfreshwaterclusteritwasthelargestoftheclustersandcontainedthevastmajorityofthefreshwaterclone
libraryandDGGEbandsequencesgeneratedinthisstudy.ThisclustercontainedallsequencesgeneratedfromaparticularDGGE
bandthatwassharedoverapproximatelyhalfofthefreshwaterfingerprints(i.e.FW24,611,13,14,17,19,27Fig.3B).In
addition,referencesequencesfromavarietyoflowsalinityenvironmentsfallintothiscluster,includingrivers,lakes,sediments,
andwastewater,aswellastherecentlyenrichedCandidatusNitrosoarchaeumlimnia[16].

Discussion
Thepresentstudyhasgenerateddatathatchallengedecadesofcommonknowledgeregardingnitrogencyclingwithinaquarium
filtrationsystemsandsolvesoutstandingquestionsremainingsinceNitrosomonasspp.wereundetectedinfreshwateraquaria[5].
Fromthesamplingof27standalonefreshwateraquariafromhomesandretailoutlets,ourqPCRresultshaverevealedadominant
AOApopulationinmostofthefreshwaterfilterssampled(Fig.1).Indeed,12oftheaquariumfilterswereassociatedonlywithan
AOAamoAgenesignal,despiteusing40cyclesforthePCR.Theseresultsareimportantbecausetheyprovidethefirstqualitative
evidencethatAOAmayactaloneincatalyzingammoniaoxidation.ThoughalsogenerallydominatedbyAOAamoA,saltwater
aquariaweremorevariableintherelativeabundancesofAOAandAOB,withbothgroupsdetectedineachbiofilter.Basedon
amoAgeneabundances,theresultsofthisstudysuggestthatbothAOAandAOBcontributetonitrificationinsaltwateraquariaand
thatAOAarethedominantcontributorstonitrificationinmostestablishedfreshwateraquaria.
Theresultsofthisstudyalsoanswerlongstandingquestionsrelatedtonitrificationinaquariumbiofilterenvironments.Forexample,
thediscoverythatabundanceofbacterialamoAgenesmaybeordersofmagnitudelessthanbacterial16SrRNAgenes(TableS1)
providesanexplanationforpreviousstudiesthatwereunabletodetectNitrosomonasspp.withgeneprobes[5],despitetheability
todetectNitrosomonasspp.withPCRprimersformostsamples[7].Probehybridizationmethodshavelimiteddetectionforgenes
thatrepresentlessthan1%ofthetotalcommunity[17],whereasthehighsensitivityofPCRcanallowevenasinglecopyofagene
tobedetectediftheamplificationisnotinhibited.RelativeproportionsofAOBinsaltwateraquariaweregenerallygreaterthanin
freshwaterbiofilters(Fig.1),whichmayexplaintheabilityofbothprobehybridizationandPCRamplificationtodetectNitrosomonas
spp.insaltwateraquariumbiofilters[5],[7],[8].
ForavarietyofgenesinBacteria,includingboth16SrRNAandamoAgenes,copynumberswithinacellarevariableandoften
greaterthanone,andcanthereforenotbetakenasadirectindicationofpopulationsizes.Conversely,availablegenomesfrom
AOArepresentatives,includingCa.N.maritimus[18]andCa.C.symbiosum[12],suggestthatAOAcellscontainonegenecopyof
eachamoAand16SrRNA.WhetherallThaumarchaeotapossessammoniamonooxygenasegenesisunknown.However,
thaumarchaeal16SrRNAgeneshavebeendetectedthatareupto100timesmoreabundantthanarchaealamoAgenes[19],
suggestingthatsomethaumarchaeallineagesdonotgainenergybyoxidizingammonia.Someaquariumbiofiltersexaminedinthis
study(e.g.FW2,FW27,SW4TableS1)yieldedamoAgenecopynumbersapproximatelyequaltothaumarchaeal16SrRNAcopy
numbers,implyingthatallThaumarchaeotapresentintheseaquariapossessamoAgenesandpresumablyoxidizeammonia.
Interestingly,otheraquariacontained16SrRNAgenesthatwereordersofmagnitudehigherthanAOAamoAgenes(e.g.FW11,
FW12,SW8TableS1),whichmayimplytoexistenceofthaumarchaeallineagesthatdonotoxidizeammonia.
AOAamoAgenediversitywasvariableacrossthefilterscollectedinthisstudy(Fig.3AandB),withDGGEpatternsrangingfrom
relativelysimplewithfewbandstocomplexwithgreaterthantendiscerniblebands.Inaddition,manybandsweresharedacross
multipleaquariumfiltersfromavarietyoflocations.MultidimensionalscalingofDGGEfingerprintsrevealedthatfreshwaterand
saltwaterfingerprintswerelargelyseparatedintwodimensionalspace.However,fingerprintsofsomefreshwatersampleswere
intermediatetomajorsaltwaterandfreshwaterclusters(Fig.3C).DGGEbandsequencesderivedfromtheseintermediate
fingerprintsfellintoaclusterofsequencescontainingapproximatelyequalproportionsoffreshwaterandsaltwaterclones(cluster1,
Fig.4).VisualinspectionofDGGEprofiles(Fig.3AandB)suggestedapossibleshiftinamoAgeneG+Ccontentbetween
freshwaterandsaltwatersamples,andthiswaslikelyafactorintheseparationobserved.Thistrendwassupportedbyclone
libraries,whichindicatedthatfreshwaterAOAamoAsequenceshadahigheraverageG+Ccontentthantheirmarinecounterparts.
Theecologicalimplicationsofthisfindingareyettobedetermined,butthisposesaninterestinghypothesisforfutureresearch.
MultidimensionalscalingofthaumarchaealamoAclonelibrarysequences(Fig.4)revealedthatfreshwaterandsaltwaterAOA
amoAgenesequenceslargelyclusterindistinctgroups,whichsupportstheseparationobservedinordinationanalysisofDGGE
profiles.Clusterswereobservedthatpredominantlycontainedsaltwatersequences(i.e.clusters2and3Fig.4),whileonecluster
(cluster1Fig.4)containedapproximatelyequalproportionsoffreshwaterandsaltwatersequences.Basedonmultidimensional
scalingofbothAOAamoADGGEprofilesandgenesequences,salinityappearstobeamajorfactorinAOAdifferentiation.
Nonetheless,theseresultssuggestthatatleastsomesequencesaresimilarbetweenenvironments,anobservationthatmay
indicatehalotoleranceofsomephylotypes.DespiteincompleteseparationofsaltwaterandfreshwaterAOAamoAgenesequences,
themajority(>80%)offreshwatersequencesderivedfromclonelibrariesandDGGEbandsclusteredwithsequencesderivedfrom
avarietyoffreshwaterenvironments,includinglakesediments,wastewater,paddysoil,lakesandrivers(Fig.4).Thisclustering
confirmspreviousevidencefornicheadaptationofaquaticfreshwaterAOA[e.g.20],[21]andsuggeststhataquariamightprovide
valuablemicrocosmsforinvestigatingtheecologyofaquaticAOA.
AlthoughthaumarchaealamoAgenecopiesmaybeseveralthousandfoldmoreabundantthanbetaproteobacterialamoAgenesin
somemarine[22]andterrestrial[23]environments,therelativecontributionsofAOAandAOBtoenvironmentalammoniaoxidation
andthefactorsthataffecttheiractivityhavebeendifficulttoconfirm.Theconcentrationofammoniamaybeamajorfactoraffecting
bacterialandthaumarchaealammoniaoxidation.RecentstudieshaveprovidedevidencethatAOBarethedominantammonia

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oxidizingorganismsinammoniarichsoilandaquaticenvironments.Forexample,arecentstudyusedstableisotopelabelled
carbondioxideandsupplementedammoniainsoiltodemonstratethatthelabelledcarbonwasassimilatedprimarilyintothe
nucleicacidofbacterialnitrifiers[24].Foeselandcolleagues(2008)foundthatNitrosomonaslikeAOBwerenumericallydominant
inamarineaquaculturebiofiltrationsystemreceivinghighammoniainfluent(rangingfrom3401700gL1).Further,themetabolic
andnumericaldominanceofbacterialammoniaoxidizersinthepresenceofhighammoniumconcentrationsisconsistentwitha
previousstudy[7]investigatingtheeventualestablishmentofbacterialammoniaoxidizercolonizationofammoniasupplemented
aquariumbiofilters(560mgNH3L1).Inaddition,mosttestedactivatedsludgebioreactorswithhighinfluentammonia
concentrationshavebeendominatedbyAOBpopulations[25],[26].
PredominanceofAOAinlowammoniaconditionshasnowbeenrelativelywellestablishedinsoilenvironments[27],[28],[29].
However,theroleofammoniainregulatingammoniaoxidizingpopulationsinaquaticenvironmentshasnotyetbeenwellstudied.
ThatAOAarebetteradaptedtolowammoniaenvironmentsissupportedbythepresentstudywithaquariumbiofilters,where
ammoniumconcentrationsaremaintainedatconsistentlylowconcentrations,andcertainlywellbelow1mgNL1inallsampled
aquaria(TableS1Fig.3).Themajorityoffreshwateraquariawithverylowammoniumconcentrations(<100gL1)were
associatedwithincreasedproportionsofAOA,andasignificantinversecorrelationwasidentified(Fig.2andTableS3).For
statisticalanalyses,inclusionofadditionalsampleswithintermediatetohighammoniaconcentrationswouldhavebeenpreferable
however,establishedandmaintainedaquariaaretypicallykeptatlowammoniaconcentrationstoensurefishhealth,andasa
resultwewereunabletolocateadditionalammoniarichaquariawithinthetimeframeofthisstudy.Theresultsofthisstudysuggest
thatammoniaconcentrationinfreshwaterenvironmentsisanimportantparameterfordeterminingtherelativeabundanceofAOA
andAOB.TheseresultsareconsistentwithotherstudiesthathaveretrievedAOAamoAgenesfromlowammoniaaquatic
environmentsanddemonstratedcorrespondingactivity[30],[31],[32].Inaddition,growthkineticsofCa.N.maritimusstr.SCM1
demonstratedahalfsaturationconstant(Km)forammoniathatissubstantiallylowerthanforculturedAOBrepresentatives[33]and
similartothemeasuredammoniumconcentrationsassociatedwithmostoftheaquariasampledinthisstudy(i.e.lowgL1
ranges).However,becauseCa.N.maritimusistheonlyAOArepresentativeforwhichkineticstudieshavebeenreported,it
remainsunclearwhetherallAOAaresimilarlyadaptedtooligotrophicconditionsanddemonstratehighsubstrateaffinitiesfor
ammonia.Forexample,therecentlyisolatedNitrososphaeraviennensistoleratesammoniumconcentrationsofupto20mM[34],
whichisconsiderablyhigherthantheinhibitoryconcentrationsof2to3mMthathavebeenreportedforCa.N.maritimusandCa.
N.gargensis[14],[33].
Althoughthedetectableammoniaconcentrationsinestablishedfreshwateraquariaaretypicallylowasaresultofbiological
ammoniaoxidation,apreferenceforhighammoniaconcentrationsbyAOBsuggestsapossiblerolefortheirinvolvementinfirst
establishinganaquariumwhenammoniaconcentrationsmayapproachlevelsassociatedwithfishtoxicity.Inaddition,ammonium
concentrationwaspositivelyandsignificantlycorrelatedwiththenumberoffishpergallonofaquariumwater(TablesS2andS3),
suggestingthatAOBmayalsobeimportantforheavilystockedtanksthatexperiencechronichighammoniaconcentrations.
ThisstudyhasidentifiedthatAOAarethedominantammoniaoxidizingmicroorganismsinfreshwateraquariumbiofilters.Aquarium
ammoniumconcentrationsweresignificantlyandinverselycorrelatedwithAOAAOBratios.FreshwateraquariumAOAamoAgene
sequenceslargelyclusteredwithotherfreshwaterassociatedsequences.Thisworkprovidesafoundationforfuturestudiesof
aquariumnitrificationandAOAecology.AquariamayserveasvaluablemicrocosmstoinvestigatethefactorsaffectingAOAand
AOBdynamicsinbothnaturalandengineeredaquaticcommunities,includingwastewatertreatmentsystems,aquaculture,lakes,
riversandoceans.

MaterialsandMethods
Sampling

FreshwaterandsaltwateraquariumbiofiltersweresampledfromretailaquariumoutletsandhomesinWaterloo,Kitchenerand
Cambridge(Ontario,Canada)betweenJuneandDecember2009(TableS1).Atotalof27freshwaterand8saltwaterfilterswere
analyzedinthisstudyfiltertypesincludedsponge,floss,baffle,andliverock,andallfilterscollectedwerecomposedofcottonor
syntheticpolymericmaterial(e.g.nylon).Filtersampleswerecollectedusingflamedforcepsandscissorstocutsmallslices(e.g.1
cm1cm3cm)fromspongematerialinexternalaquariumfiltrationsystems.Allfiltersampleswereplacedinto50mlsteriletubes
andstoredoniceuntilreturnedtothelaboratorywithinafewhours.Aquariumwatersampleswerecollectedin50mlsteriletubes
andstoredonicebeforebeingfrozenat80C.Notethatadditionalsampleswerecollectedforthisstudybutwerenotincluded
eitherduetopooryieldsofnucleicacidfromthefiltermaterialorlackofamplificationforboththaumarchaealandbacterialamoA
genes.Afilterfromoneaquarium(FW27)wassampledfourtimesoverthecourseoftwoyearstoassesstemporalstabilityin
AOA/AOBratios,andwaterwassampledseveraltimes(oversixmonthsandhourlyoveroneday)toassessstabilityinammonium
concentrationswithinagivenaquarium.ThepHwasassessedforallwatersampleswithaDELTA320pHmeter(MettlerToledo,
Columbus,OH).Ammoniumconcentrationswereassessedfluorometrically,accordingtoapreviouslypublishedmethod[35]using
aTD700fluorometer(TurnerDesigns,Sunnyvale,CA)andcalculatedfromlinearstandardcurves.Otherwaterchemistry
parameterswereassessedwithasimplewatertestkitavailablecommercially(QuickDipAquariumMultiTestKit,Jungle
LaboratoriesCorporation,Cibolo,TX).AllsampleinformationiscontainedwithinTableS1.Wealsosampledfromeightsaltwater
aquariumfiltersandtwoaquariumsupplementsforcomparison(TableS1).Bacterialsupplements(typicallybottledliquid
suspensions)areintendedtoaidinpopulatingnewlyestablishedaquariawithactivenitrifyingbacteria,tohelpensurethat
ammoniaandnitriteconcentrationsremainbelowtoxiclevelsduringtheinitial12monthsofaquariumfiltercolonization.The
aquariumsupplementsincludedwereCycle(SP1RolfC.HagenInc.,Montreal,Canada),andBioSupport(SP2BigAl's
DistributionCentre,NiagaraFalls,NY).

DNAextraction

Aharshnucleicacidextractiontechnique[36]wasadaptedtoextractnucleicacidsfromthawedspongefiltermaterialthathadbeen
cutintosmallfragmentswithflamesterilizedscissors.Forsupplements,aliquots(15mL)ofliquidaquariumsupplementswere
pelletedbycentrifugationat7,000gfor30min,thensuspendedinlysisbufferforextraction.Abeadbeatingextractionwas
performedaccordingtothepublishedprotocolwithminormodifications.Nucleicacidswereextractedfromanequalvolumeoffilter
materialratherthanequivalentweightduetothevariationinfiltermediaporosity.Theporousnatureoffiltermaterialalsorequired
thephenolchloroformCTABextractionbuffertobedecantedawayfromthespongematerialaftercelllysis,priortocentrifugation
andseparationofaqueousandorganicphases.Toprecipitatepurifiednucleicacids,twovolumesofpolyethyleneglycol(PEG)
solution(30%PEG6000and1.6MNaCl)wereusedincombinationwithlinearpolyacrylamide(AppliChem,Darmstadt,Germany)
asacoprecipitanttoavoidintroducingexogenousDNAdetectedincommercialsuppliesofglycogen[37].Allextractswere
separatedona1%agarosegelwithGelRednucleicacidstain(Biotium,Hayward,CA),visualizedwithanAlphaImagerHP(Alpha
InnotechCorporation,SantaClara,CA)andquantifieddensitometricallybycomparisontodilutionsofknownquantitiesoflamba
DNA(NewEnglandBiolabs,Pickering,Canada)usingAlphaViewsoftware(AlphaInnotechCorporation).

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QuantitativerealtimePCR

QuantificationofAOAandAOB amoAgeneswasperformedusingprimersArchamoAFandArchamoAR [38]andamoA1Fand


amoA2R[39],respectively.Thaumarchaealandbacterial16SrRNAgeneswerequantifiedusingprimers771Fand957R[40]and
341Fand518R[41],respectively.AllrealtimePCRamplificationswereperformedinduplicatewithareactionvolumeof12.5l,
whichcontained2iQSYBRGreenSupermix(BioRad,Mississauga,Canada),5pmolofeachprimer,5gofbovineserum
albuminand1loftemplate.RealtimePCRwasperformedonaCFX96system(BioRad).Forboth16SrRNAgenes,PCR
conditionswere95Cfor3minfollowedby40cyclesof95Cfor20s,55Cfor30sand72Cfor30s(withfluorescencevalues
recordedaftertheextensionstep).ForamoAgenes,thePCRconditionswerethesameasabove,exceptwithanextensiontimeof
1minuteandannealingtemperaturesof60Cand58.5CforbacterialandthaumarchaealamoAgenes,respectively.Forall
amplificationreactions,meltingcurvesfrom65Cto95Cwereperformedaftereachrunwithanincrementalincreasein
temperatureof0.5C.
PCRampliconswereusedasstandardtemplateDNA,andweregeneratedusingtheprimersindicatedabovefortheirrespective
genes.WeusedgenomicDNAfromaquariumFW27togeneratestandardsforthaumarchaealandbacterialamoAand
thaumarchaeal16SrRNAgenes.GenomicDNAfromEscherichiacoligenomicDNAwasusedtogeneratebacterial16SrRNA
genestandards.StandardcurveswereconstructedusingserialdilutionsofstandardtemplateDNAplottedagainstthecycle
threshold(Ct)valuesforeachdilution.Amplificationefficienciesrangedfrom90.698.2%,andcoefficientsofdeterminations(R2)
rangedfrom0.988to0.999.MeltingcurvescalculatedforeachtargetsequenceshowedsinglepeaksandallPCRproductswere
verifiedona1%agarosegel.StartingDNAcopynumbersforeachsamplewerecalculatedfromthelinearregressionequationof
eachstandardcurve.

Denaturinggradientgelelectrophoresis

DGGEanalysisofAOAamoAgeneswasperformedasdescribedpreviously[42]withminormodifications.Sampleswererunon
6%acrylamidegels,whichprovidedbetterresolutionthan8%acrylamidegels(datanotshown).AOAamoAgeneswereamplified
usingprimersCrenamoA23fandCrenamoA616rwiththermalcyclingasdescribedelsewhere[43].TheDGGEsystemusedwasa
DGGEK2401(C.B.S.ScientificCompany,DelMar,CA)usingpreviouslydescribedtechnicalmodifications[11].Gelswererunfor
15hat85VandsubsequentlystainedwithSYBRgreen(Invitrogen)for1h.GelswerescannedusingtheTyphoon9400Variable
ModeImager(GEHealthcare,Piscataway,NJ).IndividualDGGEbandswereexcised,amplified(usingtheaboveprimersand
conditions)andsequenced.Fromtheoriginalgelimages,fingerprintswerenormalizedformultigelalignmentwithGelComparII
(AppliedMaths,Austin,TX)andanonmetricmultidimensionalscaling(NMDS)plotwasgeneratedbasedonPearsonproduct
momentcorrelationsofbackgroundsubtracteddensitometriccurves.

Clonelibrariesandordinationanalysis

PCRampliconsforsequencingweregeneratedusingArchamoAFandArchamoAR,withthermalcyclingasdescribedpreviously
[38].CompositesoffreshwaterandsaltwatersampleswereproducedbypoolingequalnanogramamountsofPCRproductsfrom
allfreshwaterandsaltwatersamples,respectively.CompositePCRproductswereligatedintothepGEMTEasyVector
(Promega,Madison,WI)accordingtothemanufacturer'sprotocol.SinglecolonieswerepickedrandomlyandgrownupinLuria
Burtanibrothcontainingampicillin(100gml1),followedbyaplasmidextractionandsequencingofinsertswiththeM13fprimer.A
totalof288and96clonesweresequencedforthecompositefreshwaterandsaltwaterlibraries,respectively.
Allordinationanalyseswereperformedusingtranslatedaminoacidsequences.DNAsequencesderivedfrombothDGGEbands
andclonelibrariesweretranslatedusingdna2pep[44].Afterdiscardingsequencescontainingstopcodonsintheaminoacid
translation,atotalof261freshwaterclonesand84saltwaterclonesremained.ReferencesequenceswereobtainedfromGenBank
forenvironmentalclonesaswellasisolatedorenrichedAOArepresentatives.Thecollectionofsequenceswasalignedusing
MUSCLE[45]andtheresultingalignmentwascroppedsothatallsequencesspannedthesame160aminoacidregion.All
positionsinthealignmentwereusedforordinationanalyses.Adistancematrixwasproducedusingprotdist[46]andscaledby
Kruskal'snonmetricmultidimensionalscalingusingtheMASSpackage[47].AllDNAsequencesgeneratedinthisstudywere
submittedtoGenbankwithaccessionnumbersJN183456JN183849.

Statisticalanalyses

PearsonproductmomentcorrelationcoefficientsandcoefficientsofdeterminationwerecalculatedinExcel2010(Microsoft,
Redmond,WA),andassociatedpvalueswerecalculatedusingInStat3(GraphPadInc.SanDiego,CA).Unpairedttestswere
utilizedtocomparemeansinG+CcontentbetweenfreshwaterandsaltwatersequencesandwereconductedinInStat3.

SupportingInformation

FigureS1.

AquariumFW27temporalpatterns.Ammoniumconcentrations(A)areshownoverseveralmonths(during2010),andhourlyover
a12hourperiod.ProportionsofAOA/AOBintheFW27spongefilterDNAextractareshownfromfourtimepointsoverthecourse
of2years.
doi:10.1371/journal.pone.0023281.s001
(PDF)

TableS1.

DetailsofaquariaandassociatedquantitaiverealtimePCRdata.
doi:10.1371/journal.pone.0023281.s002
(PDF)

TableS2.

PearsoncorrelationcoefficientsforaquariumchemistryparametersandAOA/AOBabundancesforallaquaria.
doi:10.1371/journal.pone.0023281.s003
(PDF)

TableS3.

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3/21/2017 Aquarium Nitrication Revisited: Thaumarchaeota Are the Dominant Ammonia Oxidizers in Freshwater Aquarium Biolters

PearsoncorrelationcoefficientsforaquariumchemistryparametersandAOA/AOBabundancesforfreshwateraquaria.
doi:10.1371/journal.pone.0023281.s004
(PDF)

Acknowledgments
ThisresearchisdedicatedtoRogerKnowles(19292009).WethanklocalaquariumretailoutletsandmembersoftheKitchener
WaterlooAquariumSociety(KWAS)fortheirenthusiasticparticipationinthisstudybyprovidingaquariumfiltersamples.

AuthorContributions
Conceivedanddesignedtheexperiments:JDNLAS.Performedtheexperiments:LASKEJCS.Analyzedthedata:LASAPMJDN.
Wrotethepaper:LASJDN.Collectedandprocessedsamples:RP.

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