城市污水处理厂Candidatu省略bacter的菌群结构及定量分析张丽敏

36420164ActaScientiaeCircumstantiaeVol．36No．4Apr．2016No．51278007No．2012AA063406SupportedbytheNationalNaturalScienceFoundationofChinaNo．51278007andtheNationalHighTechnologyＲesearchandDevelopmentProgramofChinaNo．2012AA0634061987—[e-mail]*[e-mail]1987—[e-mail]*[e-mail]10．13671/j．hjkxxb．2015．0594．2016．CandidatusAccumulibacterJ．3641226-1235ZhangLMZengWWangAQetal．2016．CommunitystructuresandquantitativeanalysesofCandidatusAccumulibacterinmunicipalwastewatertreatmentplantsJ．ActaScientiaeCircumstantiae3641226-1235CandidatusAccumulibacter*1001242015-06-142015-07-142015-07-30CandidatusAccumulibacter．polyphosphatekinase1geneppk1912Accumulibacter．PCＲQuantitativePCＲQPCＲAccumulibacter6IA、IIA、IIB、IIC、IID、IIF12．A2OAccumulibacter22．77%．AccumulibacterType-IIIIC、IID108cells·g－1MLSSAccumulibacter75．34%14．87%．IAAccumulibacter0．32%．AccumulibacterIIA、IIC、IID．QPCＲAccumulibacterIIDAccumulibacter．CandidatusAccumulibacterppk1EBPＲ0253-2468201604-1226-10X172ACommunitystructuresandquantitativeanalysesofCandidatusAccumulibacterinmunicipalwastewatertreatmentplantsZHANGLiminZENGWei*WANGAnqiZHANGJiePENGYongzhenCollegeofEnvironmentalandEnergyEngineeringBeijingUniversityofTechnologyBeijing100124Ｒeceived14June2015receivedinrevisedform14July2015accepted30July2015AbstractCandidatusAccumulibacterhasbeenidentifiedasdominantpolyphosphate-accumulatingorganismsPAOsinenhancedbiologicalphosphorusPremovalEBPＲsludges．Thisstudyinvestigatedtheclade-levelcommunitystructuresandpopulationdynamicsofAccumulibacterusingpolyphosphatekinase1geneppk1genesin12sludgesamplesfromninefull-scalewastewatertreatmentplantsWWTPs．ＲesultsofQuantitativePCＲindicatethatsixcladesIAIIAIIBIICIIDandIIFofAccumulibacterwerefoundinthe12sludgesamples．AbundancesofAccumulibacterintheanaerobic-anoxic-oxicA2Oandimprovedprocesseswerethehighestupto22．77%oftotalbacteria．AbundanceofIICandIIDreached108cells·g－1MLSSaccountingfor75．34%and14．87%oftotalAccumulibacterrespectively．TheywerethedominantspeciesofAccumulibacter．CladeIAabundancewasthelowestwithanaverage0．32%oftotalAccumulibacter．CladesIIAIICandIIDwerefoundinallsludgesamplesindicatingthecladesdiversityinthesludgesamples．QuantitativePCＲandphylogeneticanalysisconfirmedEBPＲperformancewascloselyrelatedtotheclade-levelcommunitystructuresofAccumulibacter．PoorEBPＲperformancewaspossiblyrelatedtoahighproportionofIIDtototalAccumulibacter．KeywordsCandidatusAccumulibacterpolyphosphatekinase1geneppk1wastewatertreatmentplantsWWTPsactivatedsludgeenhancedbiologicalphosphorusremovalEBPＲ1IntroductionEnhancedBiologicalPhosphorusＲemovalEBPＲ、．β-ProteobacteriaEBPＲ4CandidatusAccumulibacterPAOsCandidatusAccumulibacterHesselmannetal．1999Crocettietal．2000．AccumulibacterPAOsZillesetal．2002．ppk1poly-PPPK116SrＲNA5Kuninetal．2008McMahonetal．2002Accumulibacter．McMahon2007ppk1TypeITypeITypeIITypeIIA、IB、IC、ID、IE5TypeIIIIA、IIB、IIC、IID、IIE、IIF、IIG7Heetal．2007Gebremariametal．2011．AccumulibacterTypeⅡ．Albertsenetal．2012．Mielczarek201328IAIIC．Peterson2008、IID、IID1．Accumulibacter．AccumulibacterIA、IIA、IIB、IIC、IID、IIF65Heetal．2007Gebremariametal．2011McMahonetal．2007Mielczareketal．2013IIF．IIF．6IB、IC、ID、IE、IIE、IIG、、McMahonetal．2007．IA、IIA、IIB、IIC、IID、IIF612．Accumulibacter6．Accumulibacter．Kim2013AccumulibacterIIC、IAIIF．Flowers2009IA、IIA、．Flowers2013CladeIIAIA．Accumulibacter．9CandidatusAccumulibacterI、IIA、IIB、IIC、IID、IIFCandidatusAccumulibacter、．2Materialsandmethods2．192211．1．A2O+CNＲ212．12A、B、C、D、E、F、G、H、I、J、KL．BCIJEFA2O+．1．1Table1Operationalparametersofwastewatertreatmentsystemsrelatedtosludgesamples/105m3·d－1/mg·L－1/mg·L－1CODPO3－4-PTPCODPO3－4-PTPMLSS/mg·L－1SＲT/dHＲT/hDO/mg·L－1AUCT+MBＲ10．06005．07．021．06800177．01．9BA2O14．06044．26．229．70．10．338961012．03．3CA2O14．06044．26．227．40．70．23946714．02．2DCASS8．03865．06．025．80．355812120．04．0722136/105m3·d－1/mg·L－1/mg·L－1CODPO3－4-PTPCODPO3－4-PTPMLSS/mg·L－1SＲT/dHＲT/hDO/mg·L－1E/FA2O+CNＲ11．03835．03．327．33100165．03．5GA2O10．04205．05．128．00．735001917．30．5H11．01152．433．01．30．447843012．30．5IA2O39．74573．05．035．30．30．55005139．54．0JAO39．74573．05．035．30．30．540001516．55．0K20．04802．84．733．60．1b3500106．62．7LA2O60．06003．85．725．00．20．330001410．71．8UCTUniversityofCapeTownA2OMBＲMembraneBio-ＲeactorCNＲCiliumNutricnlＲemovalCASSCyclicActivatedSludgeSystem“”．2．2DNA、PCＲ、1×PBS0．01mol·L－1pH=7．2～7．4314000×g2min－20℃．FastDNASpinkitforsoilMPUSADNA．DNANanodropSpectrophotometerND-1000ThermoFisherScienticUSA．ppk1-254FTCACCACCGACGGCAAGACppk1-1376ＲACGATCATCAGCATCTTGGCCandidatusAccumulibacterppk11123bp．PCＲPromegaGoTaqGreenMasterMixUSA25μL12．5μLGoTaqGreenMasterMix1μL10mmol·L－11μL10mmol·L－10．5～2μLDNA．PCＲ95℃10min95℃45s61℃1min72℃2min3572℃5min．PCＲAgaroseMS-6TaKaＲaJapanTakaraAgaroseGelDNAPuricationKitVer．2．0TakaraDalianChina．DNAZeroBackgroundTATopoisomeraseCloningKitClonesmarterUSA．10μL1μLpCloneEZ-TOPO1μL10×Enhancer0．5～8．0μLDNA．DH5a．．2．3Mothur97%OTUOTUNCBI．MEGA5．0NeighborJoiningMethodBootstrap1000．2．4PCＲQPCＲCandidatusAccumulibacter6I、IIA、IIB、IIC、IID、IIFppk116SrＲNAQPCＲHeetal．2