1株反硝化除磷菌的鉴定及其反硝化功能基因研究

34720137ENVIＲONMENTALSCIENCEVol．34No．7Jul．201311211231．4300702．4300723．150090denitrifyingpolyphosphate-accumulatingorganismDPAO、．ZQN2PHB．/ZQN2PHBpoly-β-hydroxybutyrateNO－3PHB．ZQN216SrＲNAGenBankBacilluscereus16SrＲNA99%．ZQN2Bacilluscereus．ZQN2nirS+nirK－ZQN2nirSnirSPseadomonasaeruginosa16SrＲNA．16SrＲNAX172A0250-3301201307-2876-062012-10-292012-12-2651208397511083602011M5012542011CDB4281983～[e-mail]rifyingPolyphosphate-accumulatingOrganismDPAOandStudyonItsDenitrifyingFunctionalGenesZHANGQian1WANGHong-yu2SANGWen-jiao1LIMeng1YANGKai2MAFang31．SchoolofCivilEngineeringandArchitectureWuhanUniversityofTechnologyWuhan430070China2．SchoolofCivilEngineeringWuhanUniversityWuhan430072China3．StateKeyLaboratoryofUrbanWaterＲesourceandEnvironmentHarbinInstituteofTechnologyHarbin150090ChinaAbstractAdenitrifyingpolyphosphate-accumulatingorganismDPAOwasisolatedandidentifiedandthedenitrifyingfunctionalgeneswereinvestigated．ThestrainZQN2wasisolatedusingdenitrifyingmedium．ItwasdistinguishedasDPAObytestsofaerobicphosphorusuptakenitratereductionmetachromaticgranulesandPHBpoly-β-hydroxybutyrategranulesdyeing．Testofanaerobicphosphorusrelease/anoxicphosphorusuptakeshowedthatstrainZQN2releasedphosphorusandsynthesizedPHBattheanaerobicphaseandusedNO－3asacceptortooxidizePHBduringtheanoxicphasewithluxuryphosphorusuptakeperformingthefunctionofsimultaneousdenitrifyingphosphorusremoval．The16SrＲNAgenesequenceofstrainZQN2wasusedforhomologyanalysisandconstructionofphylogenetictrees．Theresultssuggestedthatthe16SrＲNAgenesequenceofZQN2hadupto99%homologywiththoseofmanystrainsofBacilluscereusstrainsinGenBankdatabase．WithphysiologicalandbiochemicalreactionsthestrainZQN2wasidentifiedasBacilluscereus．TheresultofdenitrifyingfunctionalgenestudysuggestedthatstrainZQN2wasthetypeofnirS+andnirK－whichconfirmeditsdenitrifyingfunctionfrommolecularbiologypointofview．MoreoverthephylogeneticanalysisofnirSgeneofZQN2showedthatitwascloselyrelatedtomanystrainsofPseadomonasaeruginosawhichwasdifferentfromtheanalysisresultsofthe16SrＲNA．Keywordsdenitrifyingpolyphosphate-accumulatingorganismDPAO16SrＲNAdenitrificationfunctionalgenephylogeneticanalysisdenitrifierspolyphosphateaccumulatingorganismsPAOs12．Acinetobactor3Pseudomonas4．、．．PO3－4-PDOI:10.13227/j.hjkx.2013.07.05771PHApolyhydroxyalkanoatePHA．DPAOsNO－3O2PHBpoly-β-hydroxybutyratePHANO－325．Jrgensen6．7．8．16SrＲNA91011、、、412、131416SrＲNA、、15、、16、．．/16SrＲNAnirSnirK．11.1AOAanaerobic/aerobic/anoxic//SBＲ．1．1/mg·L－1Table1Influentandeffluentindexofthereactor/mg·L－1CODNH+4-NPO3－4-PNO－3-NNO－2-NpHMLSS120.7121.712.610.020.087.4382453.570.060.140.685.191.25.0g·L－1K2HPO41.0g·L－1KH2PO41.0g·L－1MgSO4·7H2O0.2g·L－1KNO32.0g·L－1pH7.217CH3COONa·3H2O3.23g·L－1KH2PO435mg·L－1NH4Cl305.52mg·L－1MgSO4·7H2O91.26mg·L－1CaCl2·2H2O25.68mg·L－1PIPESbuffer8.5g·L－162mL·L－1pH7.2．18PHB19．1.320．1.4/ZQN2200mL20h8000r·min－18min8000r·min－18min200mL160min140r·min－130℃KNO3NO－3-N30mg·L－1NO－3-N180min140r·min－130℃．160min/180minSBＲ．30min8000r·min－18min0.22μmPO3－4-PNO－3-NPHB．PO3－4-P21NO－3-N21PHB、-22．7782341.516SrＲNADNA．16SrＲNA23、nirS24nirK252．2PCＲTable2PrimersforPCＲamplification5'-3'BSF08AGAGTTTGATCCTGGCTCAG16SrＲNA8-27BSＲ1541AAGGAGGTGATCCAGCCGCA16SrＲNA1541-1522cd3aFGTC/GAACGTC/GAAGGAA/GACC/GGGnirS916-935Ｒ3cdGAC/GTTCGGA/GTGC/GGTCTTGAnirS1322-1341nirK1FGGA/CATGGTG/TCCC/GTGGCAnirK526-542nirK2FGCC/GC/ATC/GATGGTC/GCTGCCnirK565-581nirK3ＲGAACTTGCCGGTA/C/GGC/TCCAGACnirK898-918nirK4ＲGGA/GATA/GAA/GCCAGGTTTCCnirK942-959nirK5ＲGCCTCGATCAGA/GTTA/GTGGnirK1023-1040nirKnirK1F/nirK3Ｒ、nirK1F/nirK4Ｒ、nirK1F/nirK5Ｒ、nirK2F/nirK3Ｒ、nirK2F/nirK4Ｒ、nirK2F/nirK5Ｒ．PCＲ2425．PCＲ1%．pMD18-TTOP10PCＲ．1.616SrＲNAGenBankBLASThttp//blast．ncbi．nlm．nih．gov/16SrＲNA．CLUSTALX1.826Neighbour-joining27．MEGA4Bootstrap1000NJ2816SrＲNA．22.1ZQN212hPO3－4-P90.63%PHBPHB12ZQN2．ZQN23ZQN21.6×3.8μm．ZQN23．29ZQN2Bacillussp．．1ZQN2×1000Fig．1MetachromaticgranulesphotoofstrainZQN22ZQN2PHB×1000Fig．2PHBstainingphotoofstrainZQN22.2/ZQN2/8782713ZQN2×20000Fig．3TEMphotoofstrainZQN24．/SBＲ160min/180min．．．4ZQN2/Fig．4Anaerobicphosphorusrelease/anoxicphosphorusuptakeofstrainZQN23ZQN21Table3IdentificationbybiochemicalpropertiesM．Ｒ．V．P．++++++－－+++1“+”“-”4160minPO3－4-P8mg·L－121.37mg·L－1PHB6.72mg·L－116.83mg·L－1KNO3NO－3-N30mg·L－1NO－3-N2.86mg·L－1PO3－4-P3.14mg·L－1PHB2.04mg·L－1．PHB．ZQN230ZQN2PO3－4-PPHBPHBPHBZQN2NO－3-NNO－3-NPHBPHBPO3－4-P．ZQN2NO－3-NPHB．2.316SrＲNAZQN2DNABSF08BSＲ1541PCＲZQN216SrＲNA1500bpNCBIGU384232．BLASTGenBankZQN2Bacilluscereus99%．MEGA5．ZQN2ZQN2Bacilluscereus．2.416SrＲNA9782345NJ16SrＲNAZQN2Fig．5Phylogeneticneighbor-joiningtreebasedon16SrＲNAfromstrainZQN2．Nir2Cucd1nirKnirS．6nirKnirK．nirSPCＲcd3aFＲ3cdPCＲGenBankGU563985．nirKZQN2nirS+nirK－．2．231．ZQN2．nirSMEGAnirS6．6NJnirSZQN2Fig．6Phylogeneticneighbor-joiningtreebasedonnirSfromstrainZQN26ZQN2nirSPseadomonasaeruginosa99%．ZQN216SrＲNAnirS16SrＲNA．6Pseudomonassp．GU56398416SrＲNABacillussp．ZQN2nirSPseadomonassp．nirS16SrＲNA．Heylen32．nirS16SrＲNA、nirK16SrＲNAnirK100%nirK．Nir．6ZQN2GU563985GU563984216SrＲNAnirS．31ZQN216SrＲNAZQN2Bacilluscereus．2/ZQN2PHBNO－3PHB．3PCＲZQN2nirS+nirK－ZQ