1株异养硝化好氧反硝化细菌DK1的分离鉴定及其脱氮特性牟东阳

3811201711ENVIＲONMENTALSCIENCEVol．38No．11Nov．20171-DK1111*1121．1001242．1175761Pseudomonassp．DK1．C/N5NaNO3NaNO24.09mg·L·h－14.43mg·L·h－1．100%DK1NH+4-N2.32mg·L·h－1．NO－2-NDK1NO－2-N100～300mg·L－136h0．NO－3-NNO－2-NDK1NO－3-N．SNDNH+4-N、NO－2-NNH+4-N、NO－3-N30h95.06%94.69%．NH+4-N、NO－2-NNO－3-N100%．DK1SND．/X172A0250-3301201711-4763-11DOI10．13227/j．hjkx．2017041312017-04-132017-05-312016YFC04011030050005221061992～[e-mail]*[e-mail]genＲemovalCharacteristicsoftheHeterotrophicNitrificationandAerobicDenitrificationBacterialStrainDK1MUDong-yang1JINPeng-fei1PENGYong-zhen1*LIXi-yao1ZHANGQiong1HEJian-zhong21．NationalEngineeringLaboratoryforAdvancedMunicipalWastewaterTreatmentandＲeuseTechnologyEngineeringＲesearchCenterofBeijingBeijingUniversityofTechnologyBeijing100124China2．DepartmentofCivil＆EnvironmentalEngineeringNationalUniversityofSingaporeSingapore117576SingaporeAbstractNitrogenremovalbyanewlydiscoveredPseudomonssp．strainDK1isolatedfromactivatedsludgewasinvestigated．UsingglucoseasacarbonsourceandanC/nNratiooffivebatchexperimentsshowedthattheaerobicdenitrificationremovalratewas4.09mg·L·h－1and4.43mg·L·h－1withNaNO3orNaNO2respectively．Completelynitrogenremovalwasachievedwhenusingthesetwonitrogensources．DK1wasalsofoundtoheterotrophicallyremoveNH+4-Natarateof2.32mg·L·h－1andtocarryoutanoxicdenitrificationofarangeofconcentrationsofNO－2-Nfromabout100to300mg·L－1withinamaximumof36hoursofinoculation．InthepresenceofbothNO－3-NandNO－2-NDK1wasfoundtopreferentiallydenitrifyNO－3-N．SimultaneousnitrificationanddenitrificationSNDcapacityoftheDK1strainwasobservedwhenusingammoniumandnitrateorammoniumandnitriteandthecorrespondingnitrogenremovalratesreachedashighas95.06%and94.69%within30hoursofinoculationrespectively．Ammoniumwithbothnitrateandnitriteresultedina100%nitrogenremovalwithinthesametimeframe．TheabilitytoachieveSNDandtodenitrifybothNO－3-NandNO－2-NmakestheDK1strainpotentiallyusefulforfutureapplicationinnitrogenouswastewatertreatment．Keywordsheterotrophicnitrificationaerobic/anoxicdenitrificationsimultaneousnitrificationanddenitrificationmixednitrogensourcesnitrogenremovalbacterialstrain．NH+4-NNO－2-NNO－3-NN2-．-1～4．NH+4-NNH2OH、NO－2-N、NO－3-N5NO－2-NNO－3-N638NH+4-N．-、pH、C/N7～9．1--．11.1g·L－1NaCl1.0MgCl2·6H2O0.5KH2PO40.2KCl0.3CaCl2·2H2O0.015TES2.292Na2S·9H2O0.012NaHCO32.52．1mLSe/W1mLVitamin5mL．g·L－1HCl1∶410mLFeCl2·4H2O1.5CoCl2·6H2O0.19MnCl2·4H2O0.1ZnCl20.07H3BO30.006Na2MoO4·2H2O0.036NiCl2·6H2O0.024CuCl2·2H2O0.002Se/Wg·L－1Na2SeO3·5H2O0.006Na2WO4·2H2O0.008NaOH0.5．Vitaminmg·L－1H0.02B0.02B60.10B20.05B10.05B30.05B50.050.050.05B120.001．1．1、/mmol·L－1Table1Amountsofnitrogenandcarbonsourcesaddedtothedifferentmediums/mmol·L－1NH4ClNaNO2NaNO36——51—6—52——653—6610SND166—10SND26—610SND36661510mL0.3g·L－1NH4Cl5g．1.2NH+4-N、NO－2-N、NO－3-NDIONEXICS-2000DionexIonPacAS18-4μm30℃1.0mL·min－1AgilentHPLC1260InifintyBIO-ＲADAmenixHP76XＲID50℃80℃0.8mL·min－1DNAμg·mL－1ThermoNanodropSpectrophotometer1000．1.31mL45mL6mmol·L－1NaNO315mmol·L－1．NO－3-N05mL5．51mL10－1～10－7．．NIKONECLIPSEE200HitachiS-4300DK1．DNAQIAGENDNeasyBlood＆Tissue16SrDNAPCＲ27f5'-AGAGTTTGATCMTGGCTCAG-3'1492r5'-GGYTACCTTGTTACGACTT-3'．PCＲ100μLDNA3μL10×buffer20μLBSA1.34μLMgCl212μLdNTP2μL10pmol·L－12μL10pmol·L－12μLTaqDNA0.5μL57.16μL．PCＲ95℃130s95℃30s55℃30s72℃90s3072℃6min．PCＲQIAGENPCＲPurification1stbase．1.41.4.1DK1160mL45mL4674111-DK14mLC/N51．32℃、140r·min－130h3hNH+4-N、NO－2-N、NO－3-NDNA3．1.4.2DK160mL36mL3mL1．32℃、140r·min－1．O2N2O2N220min．NH+4-N、NO－2-N、NO－3-N3．22.1DK1DK1．1a1b2μm×0.4μm．DK11390bp16SrＲNAGenBankKY910428GenBankBLASTMEGA7Neighbor-Joining102DK1Pseudomonassp．99%．DK11DK1Fig．1MorphologicalcharacteristicsoftheDK1strain216SrＲNADK1Fig．2PhylogenetictreesoftheDK1strainandotherrelatedbacteriabasedonthepartial16SrＲNAsequencewithadjoiningneighbors567438Pseudomonassp．DK1．2.2DK1DK1NaNO33．NO－3-N87.07mg·L－10～9h9～12h15～21h21h0NO－3-N21h4.09mg·L·h－11112．DNADK13DK1NO－3-NFig．3AerobicdenitrificationabilityoftheDK1strainwithNO－3-Nusedasthesolenitrogensource12～18hNO－3-N．15hNO－3-N0NO－2-N21h28.30mg·L－130h15.68mg·L－1．13NO－2-N．30hNO－2-NDK1NaNO24NO－2-N92.90mg·L－10～9hNO－2-N9h21h0NO－2-N100%4.43mg·L·h－114Pseudomonassp．HNZhang15BacillusmethylotrophicusL7NO－3-NNH+4-N．20.02mg·L·h－121h1.08μg·mL·h－10.83μg·mL·h－1．30hNO－2-N0NO－3-NNO－2-N0.84g·L－1NO－2-N．12d6mmol·L－1NO－2-N15hNO－2-N032a．DK1NO－2-NNO－3-N5．NO－3-N385.35mg·L－121hNO－2-N94.95mg·L－118h18h109.21mg·L－124h0．DNA24hNO－2-NNO－3-N024h0.75μg·mL·h－1NO－2-N18h30h46.22mg·L·h－1．NO－2-NNO－3-NDK1NO－3-N．DK1NO－2-NNO－3-N16．2.3DK1DK16674111-DK14DK1NO－2-NFig．4AerobicdenitrificationabilityoftheDK1strainwithNO－2-Nusedasthesolenitrogensource5DK1NO－2-NNO－3-NFig．5AerobicdenitrificationabilityoftheDK1strainwithNO－2-NandNO－3-NusedasthenitrogensourcesNH4Cl6．NH+4-N0～12h96.14mg·L－191.24mg·L－112h12～21h91.23mg·L－145.63mg·L－1NH+4-N49.98%21h2.32mg·L·h－190.67mg·L·h－1171.38mg·L·h－1．NH+4-NDNA12h12～18h18h2.17μg·mL·h－1．21hNH+4-N48.00mg·L－124h25.83mg·L·h－1．12d6mmol·L－1NH+4-N20h02b．NO－2-NNO－3-N．NH+4-NAMONH2OHN2ON218．Ｒichardson197674386DK1NH+4-NFig．6HeterotrophicnitrificationabilityoftheDK1s