Enrichmentandcharacterizationofananammoxbact

AbstractAnaerobicammoniumoxidationwithnitritetoN2(anammox)isarecentlydiscoveredmicrobialreactionwithinterestingpotentialfornitrogenremovalfromwastewater.Weenrichedananammoxculturefromaro-tatingdiskcontactor(nearKölliken,Switzerland)thatwasusedtotreatammonium-richleachatewithlowor-ganiccarboncontent.Thisenrichmentledtoarelativepopulationsizeof88%anammoxbacteria.Themicroor-ganismcarryingouttheanammoxreactionwasidentifiedbyanalysisofthe16SrDNAsequenceandbyfluores-cenceinsituhybridization(FISH)with16S-rRNA-target-ingprobes.Thepercentagesequenceidentitybetweenthe16SrDNAsequencesoftheKöllikenanammoxorganismandthearchetypeanammoxstrainCandidatusBrocadiaanammoxidanswas90.9%,butbetween98.5and98.9%withCandidatusKueneniastuttgartiensis,anorganismidentifiedinbiofilmsbymolecularmethods.TheKöl-likenculturecatalyzedtheanaerobicoxidationofammo-niumwithnitriteinamannerseeminglyidenticaltothatofCandidatusB.anammoxidans,butexhibitedhighertolerancetophosphate(upto20mM)andtonitrite(upto13mM)andwasactiveatlowercelldensities.AnammoxactivitywasobservedonlybetweenpH6.5and9,withanoptimumatpH8andatemperatureoptimumat37°C.Hydroxylamineandhydrazine,whichareintermediatesoftheanammoxreactionofCandidatusB.anammoxidans,wereutilizedbytheKöllikenorganisms,andapproxi-mately15%ofthenitriteutilizedduringautotrophicgrowthwasconvertedtonitrate.Electronmicroscopyshowedaprotein-richregioninthecenterofthecellssur-roundedbyadoughnut-shapedregioncontainingribo-somesandDNA.Thisdoughnut-shaperegionwasob-servedwithFISHashavingahigherfluorescenceinten-sity.SimilartoCandidatusB.anammoxidans,theKöl-likenanammoxorganismtypicallyformedhomogenousclusterscontaininguptoseveralhundredcellswithinanextracellularmatrix.KeywordsAnaerobicammoniumoxidation·Anammox·Identification·Planctomycete·Fluorescenceinsituhybridization·Electronmicroscopy·Physiology·Wastewater·IntermediatesIntroductionNitrogenremovalisanimportantaspectofwastewatertreatmentoftenaccomplishedbymicrobialprocessessuchasnitrificationanddenitrification.Thesereactionshavebeenknownforalongtime(Winogradsky1890;KluyverandDonker1926;BeijerinckandMinkman1910)andhavebeensuccessfullyappliedinmostmodernwaste-watertreatmentplants.Recently,anewmicrobialprocessfornitrogenremovalwasobservedinafluidizedbed-re-actorinDelft(TheNetherlands)(Mulderetal.1995).Theprocess,calledanaerobicammoniumoxidation(anam-mox),involvesoxidationofammoniumtoN2withnitriteaselectronacceptorunderstrictlyanoxicconditions(VandeGraafetal.1995,1996;Strousetal.1997).15N-studieshaveshownthatoneN-atomoftheproducedN2origi-natesfromnitrite,whereastheotheroriginatesfromam-monium.Hydroxylamine(NH2OH)andhydrazine(N2H4)wereidentifiedasintermediatesoftheanammoxprocess(VandeGraaf1997).NH4++NO2-→N2+2H2O(1)KonradEgli·UrsFanger·PedroJ.J.Alvarez·HansruediSiegrist·JanR.vanderMeer·AlexanderJ.B.ZehnderEnrichmentandcharacterizationofananammoxbacteriumfromarotatingbiologicalcontactortreatingammonium-richleachateReceived:21August2000/Revised:12December2000/Accepted:10January2001/Publishedonline:22February2001ORIGINALPAPERK.Egli·U.Fanger·P.J.J.Alvarez·H.Siegrist·J.vanderMeer(✉)·A.J.B.ZehnderEidgenössischeAnstaltfürWasserversorgung,AbwasserreinigungundGewässerschutz(EAWAG),Überlandstrasse133,Postfach611,8600Dübendorf,Switzerlande-mail:[e-mail],Tel.:[phone],Fax:[phone]Presentaddress:U.FangerAndermattBiocontrolAG,Unterdorf,6146Grossdietwil,SwitzerlandPresentaddress:P.J.J.AlvarezDepartmentofCivilandEnvironmentalEngineering,UniversityofIowa,IowaCity,IA52242-1527,USAArchMicrobiol(2001)175:198–207DOI10.1007/s002030100255©Springer-Verlag2001Theexistenceofbacteriacapableofcatalyzingtheanam-moxreaction(Eq.1)hadalreadybeenpredictedin1977byBroda(1977)basedonthermodynamiccalculations.Furtherstudieshaveshownthattheanammoxprocessiscarriedoutbyanautotrophicbacterium(VandeGraafetal.1996),whichhasrecentlybeenshowntobelongtotheorderPlanctomycetalesandhasbeennamedCandidatusBrocadiaanammoxidans(Strousetal.1999a).Thisor-ganismwasalsopostulatedtocoupletheoxidationofni-tritetonitrateforgeneratingreducingequivalentsforCO2fixation(VandeGraafetal.1997).CandidatusB.anam-moxidansgrowsslowlyandhasbeenreportedtohaveadoublingtimeof11daysatpH8and40°C(Jettenetal.1999).Theadvantagesoftheanammoxprocessoverthetra-ditionalcombinationofnitrificationanddenitrificationforwastewatertreatmentareloweroxygendemand,whichisneededbynitrifiersforpartialoxidationofam-moniumtonitrite,andnorequirementforexternalcarbonsourcesbecausetheprocessisautotrophic.However,processstartupcouldbehinderedbytherelativelylowgrowthrateofanammoxbacteria.Implementingtheanammoxprocessasamanageablewastewatertreatmenttechnologywillalsorequireabetterunderstandingoftherangeofpermissiblenitrite,ammonium,andorganiccar-bonloads,andoxygenandpHregimes.Thebasicphysiologicalaspectsoftheanammoxprocesshavebeenstudiedindepthmostlybyasinglere-searchgroupinDelft(Jettenetal.1999).However,anam-moxactivityhasbeensuspectedforotherwastewatertreatmentplantswithuncharacterizedhighammoniumlosses(HelmerandKunst1998;Siegristetal.1998;Schmidetal.2