白洋淀富营养化湖泊湿地厌氧氨氧化菌的分布及对氮循环的影响

3221201211ACTAECOLOGICASINICAVol．32No．21Nov．2012http//．ecologica．cn208770869732009CB4211032008ZX07421-00120110952011-09-152012-03-26*[e-mail]10．5846/stxb201109151354．．201232216591-6598．WangSYZhuGBQuDMYinCQ．Widespreadofanaerobicammoniaoxidationbacteriainaneutrophicfreshwaterlakewetlandanditsimpactonnitrogencycle．ActaEcologicaSinica201232216591-6598．121*311．1000852．1000833．100085。15N、、。。16SrRNA2%305OTUsGenebankCandidatusBrocadiafulgidaCandidatusBrocadiaanammoxidans97%96%。0．19—7．78nmolNg－1h－10．64%—20．65%。1．8—78gNm－2a－1。。WidespreadofanaerobicammoniaoxidationbacteriainaneutrophicfreshwaterlakewetlandanditsimpactonnitrogencycleWANGShanyun12ZHUGuibing1*QUDongmei3YINChengqing11StateKeyLaboratoryofEnvironmentalAquaticQualityResearchCenterforEco-EnvironmentalSciencesChineseAcademyofSciencesBeijing100085China2Civil＆EnvironmentalEngineeringSchoolUniversityofScience＆TechnologyBeijingBeijing100083China3KeyLaboratoryofTibetanEnvironmentChangesandLandSurfaceProcessesChineseAcademyofSciencesBeijing100085ChinaAbstractAnaerobicammoniaoxidationanammoxisoneofthemostinnovativediscoveriesinfieldofmicrobialnitrogencyclewithitsdirectoxidationofammoniatonitrogengaswithnoN2Oproduction．Atpresenttheanammoxresearchesarelimitedtotwoareasbiologicalwastewatertreatmentandmarineecosystems．EvidenceforanammoxwasobtainedfromBaiyangdianthelargestfreshwaterlakeinNorthChinausinganisotopepairingtechniqueandmolecularanalysis．Thislakeischaracterizedbyhyper-eutrophicconditionswithagreatamountofaccumulatedammonianitrogen95—336mg/kginsurfacesediments0—5cm．Theanammoxprocessesweredetectedonanextensivespatialscalen=10inthelakesedimentsbyPCRand16SrRNAgeneclonelibrariesusingprimerscombinationpla46f-630randAmx368f-Amx820r．Thehttp//．ecologica．cn30sequencessharing98%similarityweregroupedintoonlyfiveoperationaltaxonomicunitOTUusingsoftwareDOTURbythefurthestneighborapproach．Thebiodiversityindicator“Shannon1．4andChao1estimator5”werealsocalculatedwithDOTURsoftware．Theresultofbiodiversityanalysisshowedthatanammoxbacteriabiodiversityinthelakesedimentswaslow．Phylogenetictreeswereconstructedbyneighbor-joiningNJwiththeJukes-CantorcorrectioninMEGA4package．The16SrRNAgenesequencesofanammoxbacteriainthesurfacesedimentswerecloselyrelatedtotheknownanammoxbacteriaCandidatusBrocadiafulgida97%andCandidatusBrocadiaanammoxidans96%anditrevealedmostofanammoxbacteriafromBaiyangdianLakemaybelongto“Brocadia”．Isotopeanalyseswerebasedon15Nlabeledammoniumandnitrate．Threetreatments115NH+4negativecontrol215NH+4and14NO－3positivecontrol314NH+4and15NO－3testgroupwaspreparedandexperimentswereoperatedinhermeticanaerobiccontainersat19℃．OutcomegaseousN2wasdeliveredtoGaschromatography-isotoperatiomassspectrometryforseparationof28N229N2and30N2．Thentheratesofanammoxanddenitrificationactivitieswerecalculated．Theanammoxratesrangedbetween0．19to7.78nmolNg－1h－1withsignificantspatialheterogeneitywhichcontributed0．64to20．65precenttosedimentsN2productionwiththeremainderbeingduetodenitrification．ItwasestimatedthatthetotalremovedN1．8—78gNm－2a－1waslinkedtoanammoxinthiseutrophicfreshwaterlake．About56to9426t/aofNwasremovedbywayofanammoxprocesswiththewaterlevelchangingfrom5．5to9．5mofBaiyangdianLake．Thisresearchprovidedsomenewknowledgeonanammoxinfreshwaterecosystems．KeyWordsanammoxabundanceactivityeutrophicationlakesedimentsnitrogencycle。AOBamoA。/Anammox1-2。103。24%—67%4。。5-67-9。PCR、、、15N。30。11．138°43'—39°02'115°38'—116°07'366km285%。、、、9。37001431A。36%、1∶3．5。。295632http//．ecologica．cn101B0—5cm。4℃15N－80℃DNA。1ABFig．1ThelandscapeviewofBaiyangdianreed-bed/ditchsystemsAandgeographicaldistributionofsamplingsitesB1．2TOM、NH+4-N、NO－3-N、TN、TP、pHDO、T。4℃《》5102mol/LKCl2hSANplusSkalarAnalyticalB．V．theNetherlands。DO、TYSIYSI6600。1．3DNAPCR0．30g－50℃FastDNASPINKitforSoilBio101VistaCADNA。PCRpla46f5'-GGATTAGGCATGCAAGTC-3'630r5'-CAKAAAGGAGGTGATCC-3'PCR11-12PCRAmx368f5'-TTCGCAATGCCCGAAAGG-3'Amx820r5'-AAAACCCCTCTACTTAGTGCCC-3'16SrRNAPCR13。PCR50μL10×buffer5μLdNTP2．5mmol/L4μL10mmol/L1μLBSA0．5μLTaq2．5U0．25μLDNA2μLddH2O50μL。PCR14。1．4、PromegaAgaroseGelDNAPromegaMadisonWIPCRpGEM-TeasyPromegaMadisonWIJM109Luria-BertaniLB。100PCRT7、SP6。HhaⅠ1—2。DNASTARNCBI-BLAST。16SrRNAMEGA4．1。operationaltaxonomicunitOTUDOTUR。GenbankHQ847787-HQ847799、HQ847808-HQ847824。1．515N2．5g12．0mLLabcoUK395621http//．ecologica．cn30hNO－3。15N99．29%Na15NO315N98．2%15NH42SO43①15NH+4②15NH+414NO－3③14NH+415NO－3。19℃0h、1h、2h、4h、8h200μL7MZnCl2。-Gasbench-MAT25328N229N230N215-16。①、②③。。③。NH+4NO－3。③28N229N2NO－315NO－3A28=Atotal×1－FNA29=Atotal×FN1AmFNNO－315N。NO－3。③28N2、29N230N2D28=Dtotal×1－FN2D29=Dtotal×2×1－FN×FND30=Dtotal×FN22Dm。Pm-12D30=P30Dtotal=P30×FN－23D29=P30×2×1－FN×FN－14A29=P29－D29=P29P30×2×1－FN×FN－15A28=A29×FN－1×1－FN656Atotal=FN－1×P29+2×1－FN－1×P307DtotalAtotal。VDVA。。。VAVA'。VA'=VA×MN×ρs×HS×T8MNρsHS0—5cmT。18SW9Q。Q=VA″×SW9VA″VA'。22．11。。17。495632http//．ecologica．cn1Table1CharacteristicsofthesedimentsamplesinBaiyangdianLakeSamplingsitesGeographiccoordinatesOverlyingwater/℃DO/mg/LSedimentsNH+4-N/mg/kgNO－3-N/mg/kgTN/g/kgTP/g/kgpHTOM/g/kg154'27．1