FISHNanoSIMS技术在环境微生物生态学上的应用研究陈晨

36120151ENVIＲONMENTALSCIENCEVol．36No．1Jan．2015FISH-NanoSIMS122*211．7100552．100085．-fluorescenceinsituhybridization-NanosecondaryionmassspectroscopyFISH-NanoSIMS13C-C6H12O6、15N-NH4ClCNPseudomonassp．QJX-1．FISH-NanoSIMS12C－、13C－、12C14N－、12C15N－．13C、15NPseudomonassp．QJX-113C-C6H12O615N-NH4Cl．Pseudomonassp．QJX-1．FISHNanoSIMS．FISH-NanoSIMSX172A0250-3301201501-0244-08DOI10．13227/j．hjkx．2015．01．0322014-06-102014-07-1020140902913Z03ESPCＲ1991～[e-mail]*[e-mail]-NanoSIMSTechniqueinEnvironmentalMicrobialEcologyStudyCHENChen12BAIYao-hui2*LIANGJin-song2YUANLin-jiang11．SchoolofEnvironmentalandMunicipalEngineeringXi'anUniversityofArchitectureandTechnologyXi'an710055China2．StateKeyLaboratoryofEnvironmentalAquaticScienceＲesearchCenterforEco-EnvironmentalSciencesChineseAcademyofSciencesBeijing100085ChinaAbstractWiththedevelopmentofmicrobialecologytechniquesitispossibletoanalyzethedistributionandfunctionofmicroorganismssimultaneouslyincomplexecosystems．ToexploretheapplicationofFISH-NanoSIMSinenvironmentalmicrobialecologystudyourstudyusedthestableisotopelabeledcompounds13C-C6H12O6and15N-NH4ClasCandNsourcesforcultivatingthepureculturemanganeseoxidizingbacteriaPseudomonassp．QJX-1andenvironmentalsamplestheshallowsoilandanaerobicsludge．FISH-NanoSIMSwasusedtodetectthedistributionofmicroorganismsandrelativelyquantifysecondaryions12C－13C－12C14N－12C15N－incultivatedsamplesinordertoexploretheutilizationofCandNisotopessourcesbythepurecultureandmicroorganismsinenvironmentsamples．Theresultsshowedthatthecontentsof13Cand15Nintheareaofbacteriaweresignificantlygreaterthanthenaturalabundanceinallsamples．ItindicatedthatPseudomonassp．QJX-1andsomespecificbacteriainenvironmentalsamplescouldmetabolize13C-C6H12O6and15N-NH4Cl．FurthermorethisstudyrevealedthatforPseudomonassp．QJX-1themanganeseoxidationonlyoccurredwhenthecarbonandnitrogenwereconsumedtoalowlevel．Forenvironmentalsamplesthebacterialnitrificationanddenitrificationwerebothobservedintheshallowsoilandanaerobicsludge．InawordourstudydemonstratedthatthecombinationofFISHandNanoSIMScouldsimultaneouslyexaminemicrobialdistributionandmicrobialmetabolicactivityinenvironmentalsampleswhichwillhelpustoobtaintheeco-physiologyinformationofmicrobialcommunity．KeywordsFISH-NanoSIMSmicrobialecologyisotopelabelingcarbonandnitrogensourcesmanganeseoxidation12．PCＲDNAPCＲ1FISH-NanoSIMS3．PCＲ4．FISH-SIMSfluorescenceinsituhybridizationFISHsecondaryionmassspectrometrySIMS．FISHCLSMSIMS、5．2001OrphanFISHSIMSSIMS2．nanosecondaryionmassspectroscopyNanoSIMSSIMS．NanoSIMS50L、6、、7～9．NanoSIMS/SEM/TEM、FISH10～1213～15．FISH-NanoSIMS．11.11.1.1QJX-1Pseudomonassp．QJX-1．PYG16peptone-yeastextract-glucose、、0.25g·L－160mg·L－1CaCl2·2H2O0.5g·L－1MgSO4·7H2O10mmol·L－1HEPESN-2-Hydroxyethylpiperazine-N'-2-ethanesulfonicacid4-0.22μmpH7.0～7.5．3mL－80℃27mLPYG100mL30℃170r·min－124h3mL．10mL90mLPYG250mLMn2+5mg·L－113C-C6H12O6、15N-NH4ClC、N100∶530℃170r·min－12d、10d、20d．3Mn2+．1.1.2．．4g4g100mL13C-C6H12O6、15N-NH4ClC、N200∶530℃10d．1.22.0mL8000r·min－12min1mL50mmol·L－1phosphatebuffersolutionPBS38000r·min－12min0.2mLPBS0．6mL4%paraformaldehydePFA4℃3h8000g2minPBS3200μLPBS200μL－20℃．1.310mm10%2h18～25℃12h2μL50%、80%、100%/PBS3min．1FA%54236TBD1419、EUB338．．2mL．9μL1μL50ng·μL－146℃30min9μL46℃12r·min－13.5h．3.5h．45°、4℃12hDAPI10mg·L－1Sigma10minDAPI12h．1Table1Probesanddyesusedintheexperiment5'-3'EUB338GCTGCCTCCCGTAGGAGT338-355Cy517TBD1419ACTTCTGCCAGATTCCAC1419-1436Cy318DAPI181.4FISHCLSMLSM78010×7×720×．1.5NanoSIMSNanoSIMS50LCameca．N－CN－12C－、13C－、12C14N－、12C15N－．．1nA100nm1.0pACs+．150nm256×25620ms10μm×10μm～25μm×25μm．．CLSMNanoSIMS．13C－12C15N－．ImageJ．13C、15N13C－12C－12C15N－12C14N－13C/12C、12C15N/12C14N31913C/12C=0.011015N/14N=0.00370．22.12.1.1NanoSIMSFISH-NanoSIMS1.51A、BNanoSIMS50L1A1～A4、B1～B4．FISH13C、15N13C-C6H12O615N-NH4Cl．Amann2013C-．FISH-NanoSIMS10dMn2+Mn2+313C/12C、12C15N/12C14N2．410%．FISH-NanoSIMS．6421FISH-NanoSIMSA、B10dPseudomonassp．QJX-1AMn2+BMn2+a、bFISHDAPIEUB338．A1～A4、B1～B4A、BNanoSIMS1Pseudomonassp．QJX-1FISH-NanoSIMSFig．1FISH-NanoSIMSanalysisofPseudomonassp．QJX-12Pseudomonassp．QJX-1NanoSIMSTable2NanoSIMSstatisticsanalysisforPseudomonassp．QJX-1Mn2+Mn2+/%±/%/%±/%18809.0025099.0013C/12C22073.0020189.33±1689.21±8.3724345.0026038.67±2311.49±8.8819686.0028672.0010039.009337.0012C15N/12C14N10282.009822.00±598.76±6.0912407.0010346.67±1784.42±1.729145.009296.002.1.2Pseudomonassp．QJX-1Pseudomonassp．QJX-1．32．20d20d．Mn2+Pseudomonassp．QJX-1．Pseudomonassp．QJX-1、、b、KS21．2.22.2.1NanoSIMSFISH-NanoSIMS1.53C、DNanoSIMS50L7423632Pseudomonassp．QJX-1Fig．2ContentsofcarbonandnitrogenisotopesduringcultivationofPseudomonassp．QJX-1C、D10d、c、dFISHDAPIEUB338TBD1419C1～C4、D1～D4C、DNanoSIMS3FISH-NanoSIMSFig．3FISH-NanoSIMSanalysisofcomplexenvironmentalsamples8421FISH-NanoSIMS3C1～C4、D1～D4．C、DFISH13C、15N13C-C6H12O6、15N-NH4Cl．Li2213C-CH2OH．FISH-NanoSIMS10d313C/12C、12C15N/12C14N3．410%～20%FISH-NanoSIMS．3NanoSIMSTable3NanoSIMSstatisticsanalysisforenvironmentalsamples/%±/%/%±/%6646.005987.0013C/12C8599.007729.67±993.99±12.875659.005305.00±915.48±17.267944.004263.0013220.005221.0012C15N