反硝化除磷系统的驯化及反硝化聚磷菌的筛选刘艳萍

*225127//A2OBTB、PHBβ-16SrDNA。80%。DPB-A511、DPB-A9DPB-A103、50%。3DPB-A511、DPB-A9Dechloromonasaromatica、Candidatusaccumulibacterphosphatis97%DPB-A10Bacilluspumilus99%。16SrDNAX172A0517－6611201118－11040－03AcclimationofDenitrifyingPhosphorusRemovalSystemandScreeningofDenitrifyingPhosphateAccumulatingBacteriaLIUYan-pingetalDepartmentofEnvironmentScienceandEngineeringYangzhouUniversityYangzhouJiangsu225127AbstractUsingtheurbanriversedimentsdenitrifyingphosphorusremovalsystemwassuccessfullyacclimatedbyanaerobic/anoxic/aerobicA2Oprocess．BasedonthesystemdenitrifyingphosphorusaccumulationbacteriawerescreenedwithBTBmetachromaticgranulesandPHBpoly-β-hydroxybutyratestainingexperimentsandtheirgeneticbackgroundswerefoundoutby16SrDNAsequenceanalysistechnology．Undertheexperimentalconditionsdenitrificationphosphorusremovalsystemhadmorethan80%removalefficiencyofnitrogenandphosphorusandthreedenitrifyingphosphorusbacteriawereisolatedfromthesystemwhichwerenamedDPB-A511DPB-A9andDPB-A10separatelyandhadmorethan50%nitrogenandphosphorusremovalefficiency．DPB-A511andDPB-A9had97%similaritywithDechloromonasaromaticaCandida-tusaccumulibacterphosphatisandDPB-A10had99%similantywithBacilluspumilus．KeywordsDenitrifyingphosphorusremovalsystemDenitrifyingphosphorusaccumulationbacteriascreening16SrDNAsequenceanalysis226770472008ZX07317-007-1。1986－。*[e-mail]。2011-03-28DenitrifyingPhosphorusRemovingBacteriaDPB1－2。β-PHBPHBNOx－3－4。DPB。、5－7。。//A2OBTB、PHBβ-、。11．118LSBR25cm40cm1。。1．2。MLSS3000mg/LSBR。//A2O。300CODmg/LNH4Cl14Nmg/LK2HPO4·3H2O10Pmg/LMgSO445mg/LKCl18mg/LCaCO340mg/LKNO350Nmg/L0．5ml。0．25h12L3．0h0．25h12LKNO3K2HPO4·3H2O3．0h2．0h0．5h0．5h。pH7．0～8．520℃。－200～－250－100～－150mVDO2～3mg/L。、EDTA1．5g/LCoCl2·6H2O0．15g/LCuSO4·5H2O0．25g/LMnCl2·4H2O0．99g/LZnSO4·7H2O0．43g/LNaMoO4·2H2O0．22g/LH3BO30．014g/L1g/LFeCl3·6H2O0．15g/LKI0．35g/L。KNO3。、80%DPB8－10。1．33g10gNaCl5g15g1000mlpH7．0。BTBKNO31gKH2PO41gMgSO4·7H2O1g8．5gFeCl2·6H2O0．5g。CaCl2·7H2O0．2gBTB1%1ml20gH2O1000mlpH7．0～7．3。KNO31gNa2HPO42g1g1g20gH2O1000mlpH7．2。CH3COONa1gKNO30．361gNH4Cl0．038gKH2PO4·3H2O0．151gMgSO4·7H2O0．184gCaCl2·2H2O13mg0．5mlH2O1000mlpH7．2。CH3COONa3．32gNH4Cl153mgJournalofAnhuiAgri．Sci．2011391811040－1104211060DOI:10.13989/j.cnki.0517-6611.2011.18.008MgSO4·7H2O82mgK2SO418mgCaCl2·2H2O71mg0．5mlH2O1000mlpH7．2。1．4。BTB、、PHB11－12。24h30℃24h。230ml30℃140r/min48h－NO3－-NPO43－-P13。1．516SrDNA16SrDNA27F5’-AGAGTTTGATCCTGGCTCAG-3’/1492R5’-GGTTACCTTGTTACGACTT-3’DNA。1500bpMarker50～1500bp。PCR1450μl10×Buffer5μlBSA5μl5mmol/LdNTPs25mmol/LMgCl24μl20μmol/L27f1μl20μmol/L1492r1μl37．25μlDNA2μl5U/μlTaqDNA0．25μl。。DNAbaserNCBIBlast、NucleotideBlastCluastx。22．1163．66%46．22%1．51mg/L5．32mg/L。84．61%74．37%3．07mg/L9．72mg/L。1204．50mg/L11．84mg/L81．66%58．95mg/L98．25%。1Table1NitrogenandphosphorusremovalruleduringacclimatedstageofthesystemStagePhosphorusreleaseinanaerobicstage∥mg/LPhosphorusabsorptioninanoxicstage∥mg/LNitrationnitrogenremovalamountinanoxicstage∥mg/LPhosphorusremovalrateinanoxicstage∥%Nitrationnitrogenremovalrateinanoxicstage∥%Initialstage1．515．3238．2046．2263．66Mediumstage3．079．7250．7774．3784．61Finalstage4．5011．8458．9581．6698．252．2、PHB21。33BTBPHB3。314－16DPB-A511、DPB-A9、DPB-A102。2Table2DenitrifyingbacteriapropertyappraisalresultsBacterialstrainsNitratereductaseBTBGramMetachromaticgranulePHBCellmorphologyGemmaColonialmorphologyDPB-A511+－DPB-A9+－DPB-A10++“+”。Note“+”representtheappearanceofnitricacidreductasephenomenonorGram-positive．2．3348hTPNO3－-N50%。33DPBDPB。DPB。A2OBTB、PHB3DPBDPBDPB。1401139183Table3Nitrogenphosphorusremovaleffectofvariousstrains%BacterialstrainsRateofdenitrogenationPhosphorusremovalrateDPB-A5116265DPB-A96263DPB-A1057512．416SrDNAPCR3DNA16SrDNA-PCR1MmarkerA511DPB-A511A9DPB-A9A10DPB-A10。116SrDNAPCRFig．1PCRamplificationgelelectrophoresisof16SrDNAGenbankDPB-A511DPB-A9β-β-proteobacteriaDe-chloromonasaromaticaCandidatusaccumulibacterphosphatiscladeIIAstr．97%DPB-A10BacillusBacilluspumilus99%。DPBPseudo-monas、Bacillus、Enterobacter、Moraxella、Stenotrophomonas、AcinetobacterAeromonas。17－19。MEGAJukes-CantorNJ3。DPB-A511DPB-A9G-。22216SrDNADPB-A511DPB-A9Fig．2PhylogenictreeofsimilarsequencesaboutstrainDPB-A511andDPB-A9basedon16SrDNAsequences100%97%β-proteobacteriaβ-proteobacteria。2β-proteobacte-ria。DPB-A10G+。3BacillusBacilluspumilusBacillusamyloliquefa-ciens99%DPB-A10Bacillus。316SrDNADPB-A10Fig．3PhylogenictreeofstrainDPB-A10andsimilarsequencesbasedon16SrDNAsequences31//80%。23DPB-A511、DPB-A9、DPB-A10。3BTBPHB50%。316SrDNADPB-A511Dechloromonasaromatica97%DPB-A9CandidatusaccumulibacterphosphatiscladeIIAstr．97%DPB-A10Bacilluspumilus99%。1MERZOUKIMBERNETNDELGENESJPetal．BioloicaldenitrifyingphosphorusremovalinSBREffectofaddednitrateconcentrationandsludgeretentiontimeJ．WatSciTech2001433191－194．2DEASICHEOJJONGMP．BiologicalnitrogenremovalwithenhancedphosphateuptakeinasequencingbatchreactorusingsinglesludgesystemJ．WatRes200135163968－3976．3EINHOLDJFILIPECDMDAGGERGTetal．CharacterizationofthedenitrifyingfractionofphosphateaccumulatingorganismsinbiologicalphosphateremovalprocessJ．WatSciTech199939131－42．4KUBATSMOLDERGvanLOOSSRECHTMetal．B