CANON系统中4种脱氮相关菌丰度和多样性高景峰

1005-0930201605-0863-015X703.1Adoi10.16058/j．issn．1005-0930.2016.05.0012015-01-212015-09-15510780075137802751578015“”2013-JH-L061974—[e-mail]41111121．1001242．100022CompletelyAutotrophicNitrogenremovalOverNitriteCANON、．PCＲ、、PCＲLab-scaleLPilot-scaleP2CANON4Ammonia-OxidizingBacteriaAOB、Nitrite-OxidizingBacteriaNOB、DeNitrifyingBacteriaDNBANaerobicAMMoniumOxidationAnammox4．1LPAOB、NOB、DNBAnammox1010、109、1081010copies/g．AOBAnammoxCANONNOBDNB．2AOB、NOB、DNBAnammoxDNB105AnammoxCandidatusBrocadiaCandidatusKuenenia2AOBNitrosomonasN.europaeaN.eutropha2NOBNitrospiramarine．34AOBDNBAnammoxNOB．CANON．CANONCANONCompletelyAutotrophicNitrogenremovalOverNitrite-ANaerobicAMMoniumOxidationAnammox．DissolvedOxygenDO、、、．CANONAmmonia-245201610JOUＲNALOFBASICSCIENCEANDENGINEEＲINGVol．24No．5October2016OxidizingBacteriaAOBNH+4-NNO－2-NAnammoxNO－2-NNH+4-NN2NO－3-N1AOBNH+4-NNO－2-NDeNitrifyingBacteriaDNBNO－2-NN2≤20%2．CANONAnammoxAOBDNB．Nitrite-OxidizingBacteriaNOBAOBO2AnammoxNO－2-NCANONCANON．3AnammoxThird45AOBAnammox2CANONNOBDNB4．4CANON6CANON4CANONCANON．2CANONAOB、NOB、DNBAnammox4CANON．11.12CANONLLab-scalePPilot-scaleLSBＲ+SBBＲSBＲSBBＲ0.12m3SBＲ0.1m3SBＲSludgeＲetentionTimeSＲTSBＲSBBＲSBBＲ0.06m3-0.012m3AnammoxDO0.2～0.5mg/L-AnammoxAPA/OCANON500m310t100m3AnammoxDO0.5mg/LTN、NH+4-NTP80、153mg/L．L-SBBＲPCOD50%68%TN77.5%85%NH+4-N83.7%97%TP15%82%．1．1.273L-SBBＲ4P．L-SBBＲ3L1～L3L120～25℃10d1cmL214℃45d1cmL320～25℃10d．P4P1～P4P1P210cm468Vol．241cm8mmP31cmP4．－20℃．12CANONTable1CharacteristicofwastewaterintwoCANONsystems/m3/mg/L/mg/LCODTNNH+4-NTPCODTNNH+4-NTPMLSS/mg/LSＲT/dHＲT/hDO/mg/LL-SBＲ0．1220752444．18049430．1766004～53～43～5L-SBBＲ0．128049430．2401170．173000456～100．2～0．5P500516500～600450～50014．6164＜80＜152．653000～5000810＜0．5MLSSMixedLiquidSuspendedSolidsHＲTHydraulicＲetentionTime．1.31.3.1DNAFast-DNASpinkitforsoilQIAGENCAUSADNANanodrop-1000ThermoFisherScientificUSADNA．1.3.2PCＲqPCＲ2AOB、NOB、DNBAnammoxqPCＲ274．2PCＲqPCＲTable2PrimersusedforPCＲandqPCＲamplificationinthisstudy5'-3'/bpPCＲAOBamoA-1FGGGGTTTCTACTGGTGGT491PCＲ7-8amoA-2ＲCCCCTCKGSAAAGCCTTCTTCqPCＲ9NOBNsr1113FCCTGCTTTCAGTTGCTACCG151PCＲNsr1264ＲGTTTGCAGCGCTTTGTACCGqPCＲ10DNBFlaCuATCATGGTSCTGCCGCG473PCＲ11Ｒ3CuGCCTCGATCAGＲTTGTGGTTnirK876ATYGGCGGVCAYGGCGA165qPCＲ12nirK1040GCCTCGATCAGＲTTＲTGGTTAnammox-hzsB_396FAＲGGHTGGGGHAGYTGGAAG385PCＲ13BacteriahzsB_742ＲGTYCCHACＲTCATGVGTCTGqPCＲ1.3.3PCＲ、、L-SBBＲAnammoxSBＲP20%A/O．L1P4LP．2AOB、NOB、DNBAnammoxPCＲ2．PromegaAgaroseGelDNAPromegaMadisonUSAZeroBackgroundTATOPOCloningKitCloneSmarterHoustonUSApCloneEZ-TA-AMPTOPO568No．5CANON4．NCBIBLASTMEGAMothur97%OTUMEGANeighbor-joiningMethod．1.3.4GenbankEMBLAOBKM402198～KM402232NOBLN558519～LN558575AnammoxKM402233～KM402281DNBKM402282～KM402333．22.142.1.12CANONLPAOB、NOB、DNBAnammox2CANON7qPCＲ1．AOBamoA9.87×108～3.12×1010copies/g1.37×1010copies/g．NOB16SrＲNA5.89×107～1.81×1010copies/g4.27×109copies/g．DNBnirK1.03×108～3.53×108copies/g2.29×108copies/g．AnammoxhzsB2.70×109～3.99×1010copies/g1.46×1010copies/g．AOB、Anammox、NOBDNB1010、1010、109108copies/gAOBAnammoxNOBDNB．2AOBAnammox．12CANONAOB、NOB、DNBAnammoxFig．1QuantitativeresultsofAOBNOBDNBandAnammoxbacteriaintwoCANONsystems1AOB．LL1、L2AOB9.87×1083.4×109copies/gL3AOB1.63×1010copies/gAOB．PP1～P3AOB3.64×109、2.39×10101.62×1010copies/gP4AOB3.12×1010copies/gAOB．2668Vol．24AOBAOB．O2AOBO2O2AOB3．2NOB．LL1NOB5.89×107copies/gL32.45×108copies/gL2NOB2.48×109copies/gL32.45×108copies/gNOBPP1、P2P3NOB4.25×109、2.40×1092.34×109copies/gNOB1.81×1010copies/gNOB．LPNOBNOBNOBDO、HＲTSＲT．3DNB．LL1L2DNB2.48×1081.89×108copies/gL31.03×108copies/gLDNBPP1、P2P3DNB3.53×108、2.93×1082.23×108copies/gP4DNB1.94×108copies/gDNB．LPDNBCANONDNB．4Anammox．LL1Anammox3.99×1010copies/gL32.22×1010copies/gL21.52×1010copies/g1．L1AnammoxO2L1DOAnammoxL1AnammoxL3．L2Anammox．Anammox20～43℃14-1634℃17L214℃Anammox．PP1～P4Anammox109copies/gP1、P2P33AnammoxP41Anammox．LPLAnammox2.58×1010copies/gPAnammox6．20×109copies/g1LAnammoxP．2.1.21CANONAOBanammox．AOBAnammox1010copies/gCANON．LPAOBAOBL2AnammoxAnammox768No．5CANON4Anammox．．PAOBAnammox1cmP3AnammoxAnammox10cm8mmP2AOBAOB．．2CANONAOBAnammox．CANONAOBAnammox．NH+4-NAOBAnammoxAOBNH+4-NNO－2-NAnammoxAOBO2Anammox．CANONAOBAnammoxCANONAOBAnammox．3CANONNOBDNB．CANONAOBAnammoxAOBNH+4-NNO－2-NAnammoxNH+4-NNO－2-NN2NO－2-N．AOBNH+4-NNO－2-NDNBNO－2-NN2Anammox2．NOBAOBO2AnammoxNO－2-NNOBCANONCANONDONOBDONO－2-NNO－3-NNOBCANON．Xiao6PCＲ-DGGESBBＲAOB、NOB、DNBAnammoxNH+4-NCOD6．CANONNOBDNBNOBDNB109108copies/gNOBDNBCANON．2.242.2.1OTU2CANONAOB、NOB、DNBAnammoxLDNB75%～100%23．L1P42213AOB97%L1P442OTUL1P4L1AOBP43．OTU3aL1OTU2OTU1OTU54.55%22.73%OTU3、OTU413.64%9.10%．P4OTU1#OTU2#2OTU92.31%7.69%．868Vol．2422CANONAOB、NOB、DNBAnammoxOTUs97%Fig．2ＲarefactioncurvesindicatingtherichnessesofAOBNOBDNBandAnammoxbacteriainL1andP4clonelibrariesderivedfromtwoCANONsystemsOTUsweredefinedbasedon97%sequencessimilarity32CANONAOB、NOB、DNBAnammoxTable3TheAOANOBDNBandAnammoxsequencediversityandGood’scoverageoftheclonelibrariesintwoCANONsystemsOTUs/%Chao1OTUs/OTUs/%AOBL1224100．004100．001．16P413292．312100．000．27NOBL1301100．001100．000．00P4271100．001100．000．00DNBL1241458．332948．282．36P4281375．0017．275．582．