AppliedandEnvironmentalMicrobiology1991DeBoer3600f

APPLIEDANDENVIRONMENTALMICROBIOLOGY,Dec.1991,p.3600-3604Vol.57,No.120099-2240/91/123600-05$02.00/0Copyright©1991,AmericanSocietyforMicrobiologyNitrificationatLowpHbyAggregatedChemolithotrophicBacteriaW.DEBOER,'*P.J.A.KLEINGUNNEWIEK,lM.VEENHUIS,2E.BOCK,3ANDH.J.LAANBROEK'InstituteforEcologicalResearch,P.O.Box40,6666ZGHeteren,'andLaboratoryforElectronMicroscopy,BiologicalCentre,UniversityofGroningen,9751NNHaren,2TheNetherlands,andAbteilungfurMikrobiologie,InstitutfurAllgemeineBotanikderUniversitatHamburg,D-2000Hamburg52,Germany3Received23July1991/Accepted28September1991Astudywasperformedtogaininsightintothemechanismofacid-tolerant,chemolithotrophicnitrification.MicroorganismsthatnitrifiedatpH4wereenrichedfromtwoDutchacidsoils.Nitrateproductionintheenrichmentcultureswasindicatedtobeofachemolithoautotrophicnatureasitwas(i)completelyinhibitedbyacetyleneataconcentrationaslowas1,umol/literand(ii)stronglyretardedunderconditionsofcarbondioxidelimitation.Electronmicroscopyoftheenrichmentculturesshowedthepresenceofbacteriathatweremorphologicallysimilartostrainsofknownchemolithotrophicnitrifyinggenera.Manyoftheenrichedbacteria,inparticularthosethatwereidentifiedasammoniumoxidizers,wereaggregated.FiltrationexperimentsindicatedthataggregatedcellswereabletonitrifyatlowpH,whereassinglecellswerenot.Itishypothesizedthatcellsinsidetheaggregatesareprotectedagainstthetoxicityofnitrousacid.NitrificationbyaggregatedchemolithoautotrophicbacteriamaybethedominatingprocessofnitrateformationinmanyacidsoilsasitdoesnotappeartodependontheexistenceofmicrositesofhighpH(acid-sensitiveautotrophicnitrification)orontheavailabilityoforganiccarbon(heterotrophicnitrification).Itisestablishedthatnitrificationinacidforestsoilsincreasesafterdisturbanceoftheforestecosystem,e.g.,byclearcuttingorahighdepositionofammonium(14,20).Thismayresultinundesirableleachingofnitrateandaluminumintothegroundwater(16,21).Dependingonthetypeofacidsoilstudied,nitrateproduc-tionisbelievedtobemainlycausedbyfungi(13,19)orchemolithoautotrophicbacteria(5,25).Autotrophicnitrifi-cationinacidsoilsisthoughttodependonthepresenceofmicrositesofrelativelyhighpHbecauseallchemo-lithotrophic,ammonium-oxidizingstrainsthathavebeenisolatedfromsuchsoilsappeartobeacidsensitive(10,17).Recently,evidencehasbeenpresented(6)fortheexistenceofchemolithotrophicbacteriathatareabletooxidizeammo-niumatpH4.Acetylene-sensitive,acid-tolerantnitrateproductionwasgenerallyfoundtooccurinDutchheathlandandforestsoilswithahighavailabilityofammonium,indicatingthatacid-tolerantammonium-oxidizingchemo-lithotrophsmaycommonlybepresentinsuchsoils(5,8).Nevertheless,chemolithotrophicoxidationofammoniumatlowpHremainsaratherobscureprocessas,thusfar,theresponsiblebacteriacouldnotbeisolated.Toobtainmoreinformationonthemorphologyandgrowthcharacteristicsofacid-tolerantammonium-oxidizingstrains,wedidelectronmicroscopicinvestigationsofenrichmentculturesnitrifyingatlowpH.MATERIALSANDMETHODSEnrichmentprocedures.Acid-tolerantnitrifyingbacteriawereenrichedatpH4inaliquidmineralmedium[2.5mM(NH4)2SO4,0.2mMKH2PO4,0.2mMMgSO4,0.2mMCaCl2,traceelements(2)]thatwasinoculated(0.1%,wt/wt)withorganicmaterial(FHlayer)ofeitheraheathlandsoil(pH3.6)oraDouglasfirforestsoil(pH3.7).Characteristicsofbothsoilsarepresentedelsewhere(6,8).Previously,ithadbeenshownthatnitrateaccumulationinbothsoilswas*Correspondingauthor.completelyinhibitedby60-Paacetylene(6,8),indicatingthatnitrificationmaybeofachemolithotrophicnature(12).ThepHofthe0.1%suspensionswasmeasureddailyandadjustedto4withNa2CO3.Incubationsweredoneat20°Conarotaryshaker(100rpm).Afteranincubationof5to10weeks,morethan90%oftheammoniumwasconvertedtonitrate.Subsequently,freshmineralmediumcontaining1mM(NH4)2SO4wasinoculated(1:10)withnonfilteredor5-p.m-filtered(nylon)0.1%suspension.The5-p.mfiltrationtreatmentwasperformedtoremovemostoftheparticulateorganicmatter.ThepHofthesubcultureswasmeasureddailyandadjustedto4or6withNa2CO3.Incubationconditionswerethesameasdescribedabove.Samplesfordeterminationofammoniumandnitrateweretakenweekly.Inasimilarway,the5-p.m-filteredsubculturesthathadbeenadaptedtonitrifyatpH4weresubcultured.Testsforchemolithoautotrophy.Nitrateproductioninsub-cultureswastestedforitssensitivityto(i)acetyleneand(ii)limitationofcarbondioxide.Fortheseexperiments,subcul-tureswereusedthathadbeeninoculatedwithnonfiltered0.1%suspensionsandadjustedtopH4.Intheacetyleneinhibitionexperiment,25-mlportionsofthesesubcultures(0.01%suspensions)weretransferredintoscrew-capbottles(315ml).Subsequently,thebottleswereclosedwitharubberseptum.Acetylene,whichwasmadefreeofacetoneandCObypassingthegasthroughasolutionofCuCI2inconcentratedHClandthenwater(22),wasaddedthroughtheseptumtogiveequilibriumconcentrationsof0,1,10,and100p.M,respectively.SincenopHadjustmentswerepossi-bleinthescrew-capbottles,incubationswerestartedatpH4.5insteadofpH4.0.Thebottleswerekeptclosedfor10dayswhilebeingincubatedonarotaryshaker.Attheendoftheincubationperiod,thegasphasewasanalyzedforthepresenceofoxygenbyusingagaschromatographequippedwithanelectron