高通量测序技术应用于污水处理厂细菌气溶胶群落结构分析许光素

3811201811ActaScientiaeCircumstantiaeVol．38No．11Nov．2018No．201509008SupportedbythePublicWelfareProgramofMinistryofEnvironmentalProtectionofChinaNo．2015090081987—[e-mail]*[e-mail]1987—[e-mail]*[e-mail]10．13671/j．hjkxxb．2018．0208．2018．J．38114235-4242XuGSLiuJXHanYPetal．2018．Applicationofhigh-throughputsequencingtechnologytoanalyzethecommunitystructureofbacterialaerosolsinwastewatertreatmentplantsJ．ActaScientiaeCircumstantiae38114235-42421212*1121．1000852．1000492018-04-112018-05-082018-05-08．“+”“Andersen+”．、、、．．16SrDNA0253-2468201811-4235-08X172X5AApplicationofhigh-throughputsequencingtechnologytoanalyzethecommunitystructureofbacterialaerosolsinwastewatertreatmentplantsXUGuangsu12LIUJunxin12*HANYunping1LILin121．ＲesearchCenterforEco-EnvironmentalSciencesChineseAcademyofSciencesBeijing1000852．UniversityofChineseAcademyofSciencesBeijing100049Ｒeceived11April2018receivedinrevisedform8May2018accepted8May2018AbstractBacterialaerosolsinwastewatertreatmentplantsWWTPshaveaquitecomplexcommunitystructureduetotheiremissionfromwastewaterandtreatmentfacilities．Thereforeitisofgreatsignificancetoselectappropriatesamplingandanalysismethodsfortheirdiversityidentificationandriskassessment．InthisstudyparticulatemattercollectorwasselectedasthecollectionforbacterialaerosolsthroughthewholeprocessofaWWTPandhigh-throughputsequencingformicrobialanalysisrespectivelyandsimultaneouslyAndersensix-stageimpactorandcloninglibrarywereusedasacontrol．Ｒesultsshowthattheformercombinationwasnotonlysignificantlybetterintermsofanalyzingthespeciesdiversityoverallcommunitycompositionanddominantcommunitystructureofbacterialaerosolsineachsamplingpointbutalsomoreconvenientandfastertoobtainmorecomprehensivedataclosetotherealdistributionsinbacterialaerosols．TheresultsofthisstudyprovideascientificbasisfortheselectionofappropriatecollectionandanalysismethodsforbacterialaerosolsinWWTPs．Keywords16SrDNAclonelibraryhigh-throughputsequencingwastewatertreatmentplantbacterialaerosol1IntroductionDesprésetal．201220152005．．0．5～100μmBacillus、Pseudomonas、Sphingomonas、Staphylococcus2002．382013Hanetal．2013Brandietal．2000Lietal．2013．．．、、、20022003．Andersen、、Xuetal．2013．、Caoetal．2014．10%Dongetal．2016Urbanoetal．2011．2016．16SrDNAUrbanoetal．2011Hanetal．2012、-Polymerasechainreaction-denaturedgradientgelelectrophoresisPCＲ-DGGEXuetal．2013、Pecciaetal．20062016、TerminalrestrictionfragmentlengthpolymorphismT-ＲFLP2016、FluorescenceinsituhybridizationFISHLangeetal．1997、PCＲWeryetal．2008Yamamotoetal．2014Caoetal．2014Yamamotoetal．2014Dannemilleretal．2014、、．、Caoetal．2014Kumaraswamyetal．2014．．．、Andersen．2Materialsandmethods2．1SBＲ5．0×104m3·d－1．、、、SBＲ．200m200m．1．5m．2．2TH-150C90mmPALLNYU．S．99．9%100L·min－14h．．－80℃．AndersenAndersen228-9530KSKCGulfCoastInc．USA40090mm02-27528．3L·min－13min3．2．32．3．1DNADNA63241140mL1×PBS4℃200g2h．PESMO-BIOPowerSoilDNACarlsbadCAU．S．PowerBeadTubeDNAJiangetal．2015．DNAAndersen30℃48h1×PBSTanBeadDNA．2．3．2DNAV3+V4338F/806Ｒ5'-ACTCCTACGGGAGGCAGCA-3'/5'GGACTACHVGGGTWTCTAAT-3PCＲ95℃3min95℃30s55℃30s72℃45s2772℃10min10℃．2．0%AxyPrepDNAAXYGENPCＲ．DNAIlluminaMiseq97%OTUoperationaltaxonomicunitsOTU．OTUＲibosomalDatabaseProjectＲDPClassifier97%OTUphylum、class、order、family、genus．2．3．3DNAPCＲ、Hanetal．2013C1C≥75%C．C=1－n1N()×100%1N16SrDNAn116SrDNA．Shannon-Wiener2H'=∑si=1PilnPi2PiiOTU≥97%OTUOTUSOTU．3Ｒesultsandanalysis3．11．116SrDNAabFig．1Sequencinginformationofairbornebacteriaanalyzedby16SrDNAclonelibraryaandhigh-throughputsequencingb73243843～107OTUs11～3723656～42383OTUs1212～1638．、“+”“Andersen+”．2．77．50%～90．40%99%．．216SrDNAFig．2Coverageofairbornebacteriacommunitystructuresequencedby16SrDNAclonelibraryandhigh-throughputsequencing3．2Shannon-Wiener0．58～2．375．06～5．99．．316SrDNAShannon-WienerFig．3Shannon-Wienerindexforairbornebacteriaanalyzedby16SrDNAclonelibraryandhigh-throughputsequencing3．2、、、51．1Table1Taxoncomparisonofclonelibraryandhigh-throughputsequencing3711143234621122484224“+”“Andersen+”．Firmicutes、ProteobacteriaActinobacteria34a90．0%、92．2%78．9%62．1%74．4%．341≥1%11100%3．Bacteroidetes3．06%～13．48%83241172．1%～91．7%4b．416SrDNAabFig．4Communitystructureofairbornebacteriaonphylumlevelanalyzedby16SrDNAclonelibraryaandhigh-throughputsequencingtechnologyb2018、Chloroflexi、Acidobacteria4b．DNA、．5311%Bacillus87．0%74．8%6．90%15．0%Aeromonas55．2%．516SrDNAFig．5Communitystructureofairbornebacteriaanalyzedby16SrDNAclonelibraryatgenuslevel932438、7m．4221≥1%791%Others6．683．3%～98．5%．6Fig．6Communitystructureofairbornebacteriaanalyzedbyhighthroughoutsequencingatgenuslevel“+”“Andersen+”04241110．7%、12．5%Peptostreptococcaceae_unclassified15．1%、11．1%、Parkeretal．2011、Luetal．2015Jiangetal．2015Brandietal．2000Lietal．20137．1%ZoogloeaZoogloea．DNA、、、、Behzadetal．2015．、16SrDNA1500bp．4ConclusionsAndersen83．3%～98．5%、“+”、、．．ＲeferencesBehzadHGojoboriTMinetaK．2015．ChallengesandopportunitiesofairbornemetagenomicsJ．GenomeBiologyandEvolution751216-1226BrandiGSistiMAmaglianiG．2000．Evaluationoftheenvironmentalimpactofmicrobialaerosolsgenerat