处理水产养殖污水潜流湿地中的厌氧氨氧化菌群特征

37220162ENVIＲONMENTALSCIENCEVol．37No．2Feb．2016122*2221．2013062．200092anaerobicammoniumoxidationANAMMOX．16SrＲNAPCＲANAMMOX、．CandidatusbrocadiaCandidatuskuenenia2ANAMMOX3ANAMMOXCandidatusbrocadiaANAMMOXH'1.21H'0.64ANAMMOX8.0×104～9.4×106copies·g－17.3×109～9.1×1010copies·g－1ANAMMOXANAMMOX．ANAMMOX．．X172A0250-3301201602-0615-07DOI10．13227/j．hjkx．2016．02．0292015-05-142015-09-2231372570“”2012BAD25B012012030831990～[e-mail]*[e-mail]csofANAMMOXBacteriainSubsurfaceFlowConstructedWetlandSSFCWforProcessingofAquacultureWasterWaterZENGXian-lei12LIUXing-guo2*WUZong-fan2SHIXu2LUShi-min21．CollegeofFisheriesandLifeScienceShanghaiOceanUniversityShanghai201306China2．FisheryMachineryandInstrumentＲesearchInstituteChineseAcademyofFisherySciencesShanghai200092ChinaAbstractAnaerobicammoniumoxidationANAMMOXisoneoftheimportantfunctionsinwastewatertreatmentbysubsurfaceflowconstructedwetlandSSFCWhowevertherearefewstudiesonANAMMOXinSSFCWenvironmentatpresent．ThecommunitycharacteristicsofANAMMOXintheSSFCWofprocessingaquaculturewastewaterwereexploredinthisstudy．InordertoanalyzethestructurediversityandabundanceofANAMMOXbacteriaseveral16SrＲNAclonelibrarieswereconstructedandreal-timePCＲtargetingspecific16SrＲNAgenestogetherwithdiversityanalysiswasadopted．TheobtainedresultsshowedthattheSSFCWidentifiedatotalofthreeunknownclustersandtwoknownclustersincludingCandidatusbrocadiaandCandidatuskuenenia．ThedominantclusterwasCandidatusbrocadia．ThehighestdiversitylevelsofANAMMOXbacteriaoccurredinautumnH'1.21whilethelowestinspringH'0.64．TheabundanceofANAMMOXbacteriainSSFCWenvironmentrangedfrom8.0×104to9.4×106copies·g－1offreshweightandthecopynumberoftotalbacterial16SrＲNAgenesrangedfrom7.3×109to9.1×1010copies·g－1offreshweightduringculturecycle．ThereweresignificantdifferencesintheANAMMOXbacteriaabundancesofdifferentstratumandseasonsinSSFCWenvironmentbutthedifferencesintotalbacterialabundanceswerenotobvious．InadditionthedifferencesinANAMMOXbacteriaabundancesindifferentstratumandseasonsinSSFCWenvironmentwereirregularindifferentculturecycle．AccordingtothedistributioncharacteristicsofANAMMOXbacteriainthewetlandthedenitrificationeffectofSSFCWcouldbeimprovedbychangingthesupplyingmannersofaquaculturewastewaterandadjustingthestructureofwetland．TheresearchresultswillprovidereferenceforfurtheroptimizingtheSSFCWandimprovingtheefficiencyofpurification．Keywordssubsurfaceflowconstructedwetlandanaerobicammoniumoxidationbacteriamicrobialcommunitystructureabundancediversity、、、12．、、34．0.6～0.7mCOD5．70%637anaerobicammoniumoxidationANAMMOXANAMMOX24%7．ANAMMOX、、、、、8ANAMMOX1.0%～67.0%9101.0%～42.0%1140.0%～50.0%12．Long13ANAMMOX32.1%～77.9%．Zhu14ANAMMOX2.4%～35.0%．ANAMMOXANAMMOXANAMMOX．16SrＲNA16SrＲNA、hzo、nirS、hzsCANAMMOX15ANAMMOX．ANAMMOX．16SrＲNA、PCＲANAMMOX、、．11.1N30°57'1.89″E121°08'52.21″．1500m240m37.5m370cm0.5mmHDPE．、1.5m、50～80mm34.5m330cm、50～80mm30cm、20～50mm10cm、10～20mm0.5%～2%0.30～1.0．、、4．1．3110.5～4dBOD80～120kg·hm2·d－1．1Fig．1Cross-sectiondrawofthewetland1.22014311115．320、620、920、、．Wi2～3m、Wo2～3m0～5cm、20～25cm、40～45cm6．．6162500mL200mL180r·min－130min3000r·min－110min0.22μmDNA．1.3DNAPCＲFastDNASPINKitforSoilMpbioDNA．DNA1%182、．6DNAPCＲANAMMOX16SrＲNA．Pla46f-630rAmx368f-Amx820r16．PCＲPremixExTaq12.5μLForwardprimer1μLＲeverseprimer1μL1μLddH2O9.5μL．1.4PCＲGeneJETGelExtractionKitThermopEASY-T1CloningKitTrans1-T1．LB1hLB20．PCＲ181mLLB37℃10hInvitrogen．1.5DNAStarMothurOTU．OTUNCBI-BLAST．MEGA5.05Neighbor-Joining．MothurShannonindex、Simpsonindex、chaocoveragerate．1.6PCＲANAMMOX16SrＲNAAMX-808-FAMX-1040-Ｒ18ANAMMOX16SrＲNA16SrＲNA1055F-1392Ｒ16SrＲNA．PCＲiCycleriQ5Bio-ＲadCaliforniaUSAPCＲ16．22.1ANAMMOX320、620、920Mar、Jun、Sep69．Mothur3%OTU6OTU．Mar、Jun、SepOTU3、3、4ANAMMOXOTU．OTU2OTUANAMMOX．OTUMarJunOTUSep2OTUOTUANAMMOX16SrＲNAANAMMOX2．2ANAMMOXFig．2MicrobialcommunitystructureofANAMMOXbacteriainSSFCWenvironmentindifferentseasonsANAMMOX16SrＲNA3．Candidatusbrocadia、CandidatuskueneniaANAMMOX．OTU1#OTU2#Candidatusbrocadiafulgida16SrＲNA98.2%～99.8%97%～98.2%CandidatusbrocadiaANAMMOX．OTU1#ANAMMOX98.4%～100%OTU2#ANAMMOX97.7%～100%17．3OTU4#71637JunCandidatuskuenenia16SrＲNA98.4%～98.9%CandidatuskueneniaANAMMOX98.9%～99.8%．3OTU3#、OTU5#、OTU6#ANAMMOX16SrＲNA97%．OTU5#OTU6#SepANAMMOXOTU5#Candidatuskueneniastuttgartiensis16SrＲNA93.4%～93.6%．OTU6#Candidatusbrocadiacaroliniensis16SrＲNA93.1%～93.4%ANAMMOX97.5%～98.4%．OTU3#MarCandidatusanammoximicrobium16SrＲNA76.4%99.5%．3ANAMMOXFig．3PhylogenetictreeconstructedbyANAMMOXsequencesinSSFCWenvironmentandsimilarsequences2.2ANAMMOXMothurANAMMOX．ANAMMOX16SrＲNA95%1ANAMMOX．1ANAMMOXH'SepH'1.21MarH'0.64ANAMMOX．1ANAMMOXTable1DiversitylevelofANAMMOXbacteriainSSFCWenvironment/%H'D3Mar96.70.640.6236Jun1000.930.4239Sep1001.210.30481622.3ANAMMOXPCＲ18．ANAMMOX8.0×104～9.4×106copies·g－1106copies·g－1．ANAMMOXMarJun1024．WiWo4Fig．4AbundancecomparisonofANAMMOXindifferentstratumandseasonsANAMMOXMar、、ANAMMOX、、．Jun、、ANAMMOX、、．SepANAMMOXANAMMOX4．MarSepANAMMOX、JunANAMMOX＞＞5．ANAMMOX45．2.45Fig．5VerticaldistributionofANAMMOXabundanceindifferentseasons．7.3×109～9.1×1010copies·g－1MarJun6．ANAMM