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APPLIEDANDENVIRONMENTALMICROBIOLOGY,Apr.1995,p.1246–1251Vol.61,No.40099-2240/95/$04.0010Copyrightq1995,AmericanSocietyforMicrobiologyAnaerobicOxidationofAmmoniumIsaBiologicallyMediatedProcessASTRIDA.VANDEGRAAF,1†ARNOLDMULDER,2PETERDEBRUIJN,1MIKES.M.JETTEN,1LESLEYA.ROBERTSON,1ANDJ.GIJSKUENEN1*KluyverLaboratoryofBiotechnology,DepartmentofMicrobiologyandEnzymology,DelftUniversityofTechnology,2628BCDelft,1andTNO-IMW,2600JADelft,2TheNetherlandsReceived25July1994/Accepted9January1995Anewlydiscoveredprocessbywhichammoniumisconvertedtodinitrogengasunderanaerobicconditions(theAnammoxprocess)hasnowbeenexaminedindetail.Inordertoconfirmthebiologicalnatureofthisprocess,anaerobicbatchcultureexperimentswereused.Alloftheammoniumprovidedinthemediumwasoxidizedwithin9days.Incontrolexperimentswithautoclavedorrawwastewater,withoutaddedsludgeorwithaddedsterilized(eitherautoclavedorgammairradiated)sludge,nochangesintheammoniumandnitrateconcentrationswereobserved.Chemicalreactionscouldthereforenotberesponsiblefortheammoniumconversion.Theadditionofchloramphenicol,ampicillin,2,4-dinitrophenol,carbonylcyanidem-chlorophenyl-hydrazone(CCCP),andmercuricchloride(HgIICl2)completelyinhibitedtheactivityoftheammonium-oxidizingsludge.Furthermore,therateofammoniumoxidationwasproportionaltotheinitialamountofsludgeused.Itwasthereforeconcludedthatanaerobicammoniumoxidationwasamicrobiologicalprocess.Astheexperimentswerecarriedoutinanoxygen-freeatmosphere,theconversionofammoniumtodinitrogengasdidnotevenrequireatraceofO2.Thattheendproductofthereactionwasnitrogengashasbeenconfirmedbyusing15NH41and14NO32.Thedominantproductwas14-15N2.Only1.7%ofthetotallabellednitrogengasproducedwas15-15N2.ItisthereforeproposedthattheN2producedbytheAnammoxprocessisformedfromequimolaramountsofNH41andNO32.Theremovalofammoniumisanimportantprobleminmod-ernwastewatertreatmentsystems.Itisgenerallyachievedbyacombinationoftwoprocesses,nitrificationanddenitrification.Ammoniumisoxidizedfirsttonitriteandthentonitratebynitrifyingbacteria.NitrificationisanO2-requiringprocessandthereforerequiresanaerobicenvironment(1,7).Duringthesubsequentdenitrificationstep,nitrateornitriteisconvertedtodinitrogengas(11).Denitrificationiscarriedoutbyawidespectrumofrespiratorybacteriarepresentingmostgeneraandphysiologicaltypes(27).Mostdenitrifyingbacteriacarryoutthesereactionsonlyunderanaerobicconditions(20).Veryrecently,however,ammoniumlossesunderanaerobicconditionswerediscoveredtooccurinalaboratory-scaledeni-trificationreactor(12).Increasedremovalofammoniumwasparalleledbyincreaseddisappearanceofnitrate.Thissug-gestedthatthefollowingreactionwastakingplace(4):5NH4113NO3234N219H2O12H1(1)DG89521,483.5kJperreactionTheoverallreactionforthisanaerobicammoniumoxidation(Anammox)processisexergonicandthuscould,intheory,supplyenergyforgrowth.Itwasthereforepostulatedthattheremovalofammoniumobservedtooccurinthedenitrifyingreactorwascarriedoutbybacteriausingammoniumasanelectrondonorfornitratereduction(12).Theaimofthere-searchdescribedherewasthreefold:first,toconfirmthattheobservedammoniumremoval(12)wasmediatedby(micro)bi-ologicalactivity;second,toestablishthatoxidationofammo-niumtookplaceunderfullyanaerobicconditions;andfinally,todemonstratethatammoniumwasconvertedtodinitrogengasby[15N]ammonium.MATERIALSANDMETHODSOriginofbiomassandpreparation.Sludgefromthedenitrifyingfluidizedbedreactorinwhichanaerobicammoniumoxidationoccurredwasusedasasourceforbiomass(12).Thesludgefromthereactorwaseitherusedimmediatelyorelsestoredat48Cuntilneeded.Beforeuse,thesludgewashomogenizedbypassingitseveraltimesthrougha60-mlsyringe.Forexperimentswith[15N]am-monium,thesludgeinthereactorwasused.Medium.Effluentfromthedenitrifyingfluidizedbedreactor,supplementedwithvariousconcentrationsofammoniumandnitrate,wasusedasthemediumforallbatchexperiments.Theeffluentcontainedthefollowing(perliter):totalorganiccarbon,130to155mg;SO422-S,80to130mg;NH41-N,10to70mg;NO32-N,40to120mg.Detailsofthedenitrifyingreactor,whichwasbeingfedwitheffluentfromamethanogenicreactor,wereasdescribedbyMulderetal.(12).Anaerobicbatchcultureexperiments.Serumbottles(500-mlvolume)werestaticallyincubatedinthedarkat378C.Eachbottlecontained460mloftreatedwastewater(pH7.0)and40ml(approximately60mg[dryweight]perml)ofsludgeinoculum.Theinitialammoniumandnitrateconcentrationsweread-justedbyaddingNH4NO3,(NH4)2SO4,orNaNO3.TheinitialNH41-NandNO32-Nconcentrationswere75to115and360to430mg/liter,respectively.Thesulfate-Sconcentrationwasbetween80and130mg/liter,dependingontheperformanceofthewastewatertreatmentsystem.TopreventO2contamination,thebottleswerefirmlyclosedwith4-mm-thickbutylrubbersepta.Anaerobicconditionswereestablishedineachbottlebyflushingwithpurenitrogengasfor15minormore.Samplesweretakendailythroughinsitulongsamplingneedles.Duringsampling,N2wassuppliedbyusingaseparatesampleport.Anadditionalsyringeservedtoindicateandcontroloverpressure.Beforebeingsampled,thebottleswereshakenvigorously.Afterthesludgehadsettled,thesamplewasdrawnoffandcentrifuged.Ammonium,nitrite,nitrate,sulfate,andpHweredetermined.Allexperim