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34720137ENVIＲONMENTALSCIENCEVol．34No．7Jul．2013A2O1222212*2*1．1000832．100085．A2O17．16SrDNArep-PCＲ．．16SrDNArep-PCＲX172A0250-3301201307-2912-062013-02-212013-05-15KZCX2-YW-JC407-3KZZD-EW-09-31986～E-mailshagao152@163.com*E-mailxiaweipeng@163.comzhbai@rcees．ac．cnDiversityofCulturableFilamentousBacteriaintheActivatedSludgefromA2OWastewaterTreatmentProcessGAOSha12JINDe-cai2ZHAOZhi-rui2QIＲong2PENGXia-wei12BAIZhi-hui21．CollegeofBiologicalSciencesandTechnologyBeijingForestryUniversityBeijing100083China2．ＲesearchCenterforEco-EnvironmentalSciencesChineseAcademyofSciencesBeijing100085ChinaAbstractTheanoxic-anaerobic-oxicA2Oprocessiswidelyusedinwastewatertreatmentplanthoweversludgebulkingandfoamingarethemostfrequentoperationalproblemsinthisprocess．ActivatedsludgebulkingiscausedbytheovergrowthofsometypesoffilamentousbacteriaespeciallyMicrothrixparvicella．Inthestudy17strainsoffilamentousbacteriawereisolatedfromthebulkingsludgeofA2OprocessusingGause'smedium．The16SrＲNAgenesofthe17isolatesweresequencedtoanalyzetheirdiversity．Theresultsshowedallofthe17isolateswereStreptomyces．Furtheranalysisofthesestrainsbytherepetitivesequencebasedonpolymerasechainreactionrep-PCＲtechnologyshowedthattherewasahighdiversityintheseisolatedStreptomyces．ThephysiologicalpropertiesofthemweredifferentfromMicrothrixparvicella．Thesettleabilityofactivatedsludgewasimprovedwhensomeoftheisolateswereinoculated．KeywordsbulkingsludgefilamentousbacteriaStreptomyces16SrDNArep-PCＲ12．．34．．5～8．910．．NocardiaNocardiaamaraeNocardiapinensiss、FlexibacterType0092、CytophagaType0411、Thiothrix、Beggiatoa、Type0914、Haliscomenobacterhydrossis、Microthrixparvicella、Sphaerotilusnatans．Microthrixparvicella、pH、DO11～13．．7A2O．14．．11.1A2O－20℃．1.21520g1g0.5g0.5g0.5g0.01g20g1000mL50mgpH7.2～7.4．．0.2g0.1g0.05g0.05g2g100mLpH7.2～7.4．100g1g0.5g0.5g0.5g0.01g20g1000mLpH7.2～7.4．1.35g45mL20min．10－4、10－5、10－6、10－7100μL28℃4～7d5．5～10d5～7d1000．1.4DNA16SrDNAPCＲDNA．16SrDNA27f1492r．16SrDNA-PCＲ50μL5μL10×Buffer4μLdNTP2.5nmol·L－11μL1.2μLDNA1μg0.5μLTaqDNA．PCＲ95℃5min94℃1min55℃1min72℃2min3072℃7min．PCＲ－20℃．1.516SrDNA16SrDNAPCＲ．NCBIGenBankBlastMEGA4N-J16～18．1.6Ｒep-PCＲ19PCＲBOXAIＲ5'-CTACGGCAAGGCGACGCTGACG-3'．25μL152.5μL10×Buffer2μLdNTP2.5nmol·L－12μL1.5μLDNA25μL．PCＲ95℃5min95℃45s44℃1min72℃3min3572℃10min．1112161.5%TBE6μL3μLloadingBuffer80V150min100V120min．1.727℃160r·min－12～3d．100mL150mL2%、5%、10%25℃60r·min－13hSVI10%．22.1171G+．17．191000DAPI．2.216SrDNA1716SrDNAGenBank1716SrDNA97%2．3192341Table1CulturefeaturesoftheisolatedfilamentousstrainsonGause'smediumGS01GS02GS03GS04GS05GS06GS07GS08GS09GS10GS11GS12GS13GS14GS15GS16GS17172．ⅠGS12、GS16、GS02、GS03、GS01、GS14、GS11、GS17、GS07ⅡGS15、GS13、GS05、GS09、GS08、GS04、GS10、GS06．ⅠGS12、GS16、GS02、GS03、GS01、GS14Streptomycesmicroflavus126196JN180208、Streptomycescavourensis173883EU570437、Streptomycesmicroflavus2429JN180194、StreptomycesmicroflavusPM258JQ42216399%．GS03StreptomycesmicroflavusPM258100%GS11GS17StreptomycesrubiginosohelvolusNXPT24JN99992099.5%118Fig．1MicroscopicphotographoftheculturedisolatesandMicrothrixparvicella216SrDNATable216SrDNAsequenceanalysisoftheculturablefilamentousbacteria16SrDNAGenBankGenBank/%GS01JX430438StreptomycesflavofuscusJQ92441099.9GS02JX430439Streptomycesmicroflavus2429JN18019499.8GS03JX430440StreptomycesmicroflavusJQ422163100GS04JX430441StreptomycesphaeochromogenesEU59447199.5GS05JX430442Streptomycessp．XAS589GQ39524399.6GS06JX430443Streptomycessp．OA20JN94213299.9GS07JX430444Streptomycessp．CPE393JN96903499.6GS08JX430445Streptomycessp．CPE338JN96902599.5GS09JX430446StreptomycesrocheiFJ79257799.5GS10JX430447Streptomycessp．195018GU26387599.9GS11JX448396StreptomycesrubiginosohelvolusNXPT24JN99992099.5GS12JX679243StreptomycesmicroflavusJN18020897.8GS13JX465725Streptomycessp．XAS589GQ39524399.7GS14JX465726Streptomycessp．QZGY-A23JQ81208097.7GS15JX679244Streptomycessp．MJM3859EU60334799.7GS16JX679245StreptomyceslavendulaeAB18407999.9GS17JX465724StreptomycesrubiginosohelvolusNXPT24JN99992099.841927A2O216SrDNAN-JFig．2Neighborjoiningphylogenetictreeananlysisof16SrDNAofStreptomyces99.8%．GS07ⅠStreptomycessp．CPE393JN96903499.6%．Ⅱ82．GS157．GS15Streptomycessp．MJM3859EU60334799.7%799%．16SrDNA16SrDNAGenBank．2.3Ｒep-PCＲBOXAIＲ17rep-PCＲ3．4．ⅠGS10、GS06、GS08ⅡGS17、GS15ⅢGS07、GS11、GS03、GS12ⅣGS09、GS04、GS05、GS01、GS02、GS16．DNA、rep-PCＲ．rep-PCＲ．2.41720～23．．SVISVI60～100mL·g－1SVI150mL·g－1SVI300mL·g－124．2%、5%、10%17．GS05、GS14、GS175%4．45%GS05、GS14、GS17SVI3h．SVI．．．5192343BOX-PCＲFig．3FingerprintsofBOXAIＲ-PCＲelectrophoresis45%SVIFig．4EffectoftheisolatedStreptomycesstrainsonSVIofactivatedsludge2526．、pH、、、．．31．21716SrDNA．rep-PCＲ．3．致谢:．1．J．20123393197-3201．2．J．2007283546-550．3GuoFZhangT．ProfilingbulkingandfoamingbacteriainactivatedsludgebyhighthroughputsequencingJ．WaterＲesearch20124682772-2782．61927A2O4MartinsAMPPagillaKHeijnenJJetal．Filamentousbulkingsludge－acriticalreviewJ．WaterＲesearch2004384793-817．5CaravelliAGiannuzzLZaritzkyN．EffectofchlorineonfilamentousmicroorganismspresentinactivatedsludgeasevaluatedbyrespirometryandINT-dehydrogenaseactivityJ．WaterＲesearch20043892395-2405．6．J．201228315-19．7．J．2010361