97Vol.9No.720157ChineseJournalofEnvironmentalEngineeringJul.2015*300457316SrRNARS1、RS2RS3、。337℃、3%、pH832℃、1%、pH842℃、1%、pH6。330d39.69%、31.13%53.29%58.08%RS120.74%RS38.08%GC-MSn-C12~n-C34、、b。16SrRNAPAHsX172A1673-9108201507-3538-07IsolationofpetroleumhydrocarbondegradingbacteriafromoilysludgeanditsdegradingcharacteristicsRenLijunLiuXianbinTianShengyanTianjinUniversityofScience&TechnologyTianjinKeyLaboratoryofMarineResourcesandChemistryTianjin300457ChinaAbstractThreebacteriastrainsRS1RS2andRS3wereisolatedfrompetrochemicaloilysludgeofBohaiSeaOilfieldTianjinChinaandidentifiedasCorynebacteriumsp.Brevibacteriumsp.andPseudomonassp.through16SrRNAsequencesimilaritysearch.Theoptimumdegradationconditionsofthreebacteriawerestudiedbysinglefactorexperimentandtheresultswere37℃salinity3%pH832℃salinity1%pH842℃salinity1%pH6respectively.ThedegradationresultsshowedthatthedegradationratesofTPHinoilysludgebyRS1RS2andRS3were39.69%31.13%and53.29%andtheeffectofmixedbacterialcultureswithadegradationrateof58.08%wassignificantlybetterthanthesinglebacteria.Thedegradingabilityofbacteriavarywithoilcomponent.RS1hadthebestdegradationeffectonaliphatichydrocarbonitsdegradationratewas20.74%andRS3hadthebestdegradationeffectonaromatichydrocarbonanditsdegradationratewas8.08%.GC-MSanaly-sisshowedthatn-C12~n-C34n-alkanesweresignificantlydegradedbymixedbacteriawhichhavestrongdegra-dationcapacitywithPAHsincludingnaphthaleneacenaphthenechryseneandbenzobfluoranthene.Keywordsoilysludgemicrobialdegradation16SrRNAn-alkanesPAHsKJXH2012-232014-05-252014-07-111989—。E-mailnana.ren@163.com*E-maillxb0688@tust.edu.cn1。、。、、NSO2。3。、、、4。。3。711.170.55%pH6.43.0%。1.21.2.13g/LNaCl5g/LMgSO4·7H2O0.25g/LNH42SO41g/LK2HPO410g/LKH2PO44g/LpH7.2~7.4。1.2.2LB。1.2.33g/LNaCl5g/LMgSO4·7H2O0.25g/LNH42SO41g/LK2HPO410g/LKH2PO44g/L10g/LpH7.2~7.4。1.3110g40mL80-1‰v/v1~2h。1mL9mL3。100μL37℃24h。250mL150mL337℃、100r/min24h。32。4℃。。SEM。1.416SrRNA。AXYGENDNA3DNADNA16SrRNAPCRPCR27F5'-AGAGT-TTGATCATGGCTCAG-3'1492R5'-CTACGGTTAC-CTTGTTACGAC-3'PCR50μLMix25μL2.0μL1.0μLddH2O21μL。PCR94℃4min94℃1min55℃1min72℃2min3072℃10min4℃。PCR1%。1.550mL150mL、pH7d3。5η=-×100%1、pH122、27、32、37、42、4750℃20.5%、1%、2%、3%、5%、7%8%3pH4、5、6、7、8、9、1011。1.61.6.1TPH337℃24hRS1RS2RS31∶1∶130mL100g330d3TPHTPH-。1.6.25g7d350mL20、2010mL30min。WhatmanGF/F10mL1mL。1cm30cm25cm1cm1mL30mL、50mL-1∶1、。935391。1mLGC-MS。PAHsAgilent7890A-5975CDB-5MS30m×0.25mm×0.25μm。1PAHs290℃70℃1min10℃/min260℃4min5℃/min300℃4min。280℃230℃EISIM。He1mL/min。2220℃250℃250℃。22.13RS1、RS2RS3。RS10.6~0.8μm×0.8~1.4μmRS21.0~1.2μm×2.0~3.2μmRS30.6~0.8μm×1.2~2.0μmRS1RS2RS3。2.216SrRNA316SrRNA1422、14331415bp。GenBank1。1316SrRNABLASTTable1ResultsofBLASTof3strains16SrRNAgenesequenceGenBank%RS1KF894694Corynebacteriumglutamicum99RS2KF894695Brevibacteriumhalotolerans100RS3KF894696Pseudomonasstutzeri996-9。Pseudo-monascholoeaphtis10、Pseudo-monasputida1112、Pseudomonasaeruginosa13、Pseudomonasstutz-eri14。。15。。2.32.3.11RS110%27~42℃37℃RS232~37℃32℃RS2RS342℃32~42℃。1Fig.1Effectoftemperatureondegradationrate。3RS3RS1RS23。2.3.22RS10.5%~5%25%RS2RS30.5%~3%。5%RS125%。32045372Fig.2Effectofsalinityondegradationrate1617。2.3.3pHpH3RS3pH6~10RS1RS2pH6~8。3pHFig.3EffectofpHondegradationratepH。pHRS3pH1035%RS3。15℃50℃38℃PAHs18-21。342℃3%。2230℃、3%、pH8。RS342℃、1%、pH6。2.42.4.1TPHTPHRS1、RS2RS3TPH39.69%、31.13%53.29%58.08%。3TPH20d。4。4TPHFig.4Changesofdegradationrateovertime2324。。。。20d。2.4.253RS120.74%3RS38.08%。24.65%10.77%。145395Fig.5Degradationrateofdifferentoilcomponentsbystrains2526。2。27。2830d23%。RS33RS17d20.74%。2.4.3PAHsGC-MSGC-MS16PAHs。PAHs2。2Table2DegradationrateofPAHsbymixedstrainsPAHsng/gng/g%6.0773.37944.3935.4425.5927.79203.2183.39.79325.5524.075.7937.3255.07130.78b0.88830.538039.43a2.7022.5027.402、、b44.39%、27.79%、30.78%39.43PAHs。PAHs。PAHs。PAHsPAHs。RS329。PAHs30。、bb31。2.4.3GC-MS7dGC-MS6n-C12~n-C34。6m/z851~23n-C12~n-C34Fig.6M/z85chromatogramoftreatmentgroupandcontrolgroupn-C12~n-C34n-C12~n-C15、n-C27~n-C343233。GrossiV.346≤n-C25n-C30。24537。31RS1、RS2RS316SrRNACorynebacteriumsp.、Brevibac-teriumsp.Pseudomonassp.。2337℃、3%、pH832℃、1%、pH842℃、1%、pH6。RS1RS32。330dTPH58.08%。RS120.74%RS38.08%。4GC-MSn-C12~n-C34、、bPAHs。1CerqueiraV.S.HollenbachE.B.MaboniF.etal.Bio-degradationpotentialofoilysludgebypureandmixedbac-terialcultures.BioresourceTechnology20111022311003-110102SoodN.LalB.IsolationofanovelyeaststrainCandidadigboiensisTERIASN6capableofdegradingpetroleumhy-drocarbonsinacidicconditions.JournalofEnvironmentalManagement20099051728-17363BiswalB.K.TiwariS.N.MukherjiS.Biodegradationofoilinoilysludgesfromsteelmills.BioresourceTechnology200910041700-17034VasudevanN.RajaramP.Bioremediationofoilsludge-contaminatedsoil.EnvironmentInternational2001265-6409-4115.、.201154925-929WangHaifengLiYangZhaoLipingetal.Screenandi-dentificationofheavyoildegradingbacteriaandcommuni-cationconstruction.ChineseJournalofEnvironmentalEngi-neering201154925-929inChinese6..200526661-64WangZhiqiangWuQiangYeSiyuanetal.Studyonthegroundwaterpetroleumcontaminantbiodegradationbyhighefficientmicroorganism.EnvironmentalScience200526661-64inChinese7..200525123314-3322RenSuizhouGuoJ