芳香族降解.docx

芳香族降解.docx

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芳香族降解

Preferentialdegradationofaromatichydrocarbonsinkerosenebyamicrobialconsortium

HernandoBacosa*,KoichiSuto,ChihiroInoue

GraduateSchoolofEnvironmentalStudies,TohokuUniversity,Aoba6-6-20,Aramaki,Aoba-ku,Sendai,Miyagi980-8579,Japan

Abstract:

Numerousstudiesonthebiodegradationofpetroleumproductsusingtotalpetroleumhydrocarbons（TPH）havebeencarriedout;however,thebiodegradationofequivalentcarbonnumber（EC）basedhydrocarbonfractionsinmineraloilhasattractedlittleattention.ThisstudyinvestigatedtheabilityofamicrobialconsortiumtodegradetheECfractionsinkerosene,whichwasusedasarepresentativemineraloil.Basedonthecloningandsequencingofthe16SrRNAgene,themicrobialcommunitywaspredominantlyidentifiedasBetaproteobacteriaofthegeneraAchromobacter,Alcaligenes,andCupriavidus.Degradationexperimentsinsealed120-mlvialscontaining1%（wv_1）kerosenerevealedthataromaticfractionsweredegradedfasterthanaliphaticfractions.AromaticfractionsEC>7e8andEC>8e10werecompletelydegradedafterthreedayswhilealiphaticfractionsEC>6e8andEC>8e10wereonlypartiallydegraded.ThearomaticEC>10e12fractionwasthethirdmostdegraded,andthealiphaticEC>10e12andEC>12e16fractionsweretheleastdegradedfractions.Thefirst-orderrateconstantsforthearomaticfractionsrangedfrom0.12d_1to0.51d_1andfrom0.06d_1to0.32d_1forthealiphaticfractions.Themicrobialconsortiumpreferentiallyutilizedaromaticfractions,whicharemoretoxicthanaliphaticfractions.Thisfindingisusefulwhenconsideringrisk-basedbioremediation:

AmicrobialcommunitycouldpotentiallydegradethemoreToxicaromatichydrocarboncomponentsinroleum-contaminatedenvironments.

1.Introduction

Thecontaminationofsoil,groundwater,andmarineecosystemswithpetroleumproductsasaresultofhumanandindustrialActivitiesisawidespreadenvironmentalproblem.Petrochemicalmixtures,suchasmineraloil,maycontainhundredsofaliphaticandaromatichydrocarboncompoundswithawiderangeofpropertiesandtoxicities（Blombergetal.,1997;PotterandSimmons,1998）.Aromatichydrocarbonsaremoretoxicandmorepersistentintheenvironmentthanarealiphaticcompounds.Thetremendouscomplexityofpetroleummixturesmakesitdifficult,ifnotimpossible,toidentifytheindividualcomponentsusingcurrentlyavailableanalyticalmethods.Thus,thecomplexityoftheoilproductsandthelackofatoxicityreferencemakecleanupapproachesandbioremediationstrategiesincontaminatedsitesanoneroustask.

ThefractionationapproachoftheTotalPetroleumHydrocarbonCriteriaWorkingGroup（TPHCWG）providesapracticalsolutiontotheselimitations.TheTPHCWGhasgroupedpetroleumhydrocarbonsintoaromaticandaliphaticgroups,whicharefurthercategorizedinto13fractionsbasedontheirequivalentcarbonnumber（EC）（Edwardsetal.,1997;Gustafsonetal.,1997）.TheECisrelatedtotheboilingpointofthehydrocarboncompoundsnormalizedtotheboilingpointofthen-alkanesortheirretentiontimeinaboilingpointgaschromatography（GC）column.CompoundsthatbelonginthesameECrangearepresumablysimilarintheirenvironmentalbehaviors,degradability,andtoxicity.TheTPHCWGhasassignedtoxicity-basedlimitvaluestoeachoftheECfractions,whichcanbeusedtoestimatethehumannon-carcinogenichealthrisks.TheTPHCWGevaluatedriskbasedontwocriteriadthereferencedosage（RfD）andthereferenceconcentration（RfC）dbothofwhichtermswereoriginallycoinedbytheUnitedStatesEnvironmentalProtectionAgency（USEPA）.TheRfDis“anestimate（withuncertaintyspanningperhapsanorderofmagnitude）ofdailyexposurelevelforthehumanpopulation,includingsensitivesubgroups,thatislikelytobewithoutappreciableriskofdeleteriouseffectsduringalifetime”（USEPA,1989）.TheRfCis“anestimate（withuncertaintyspanningperhapsanorderofmagnitude）ofacontinuousinhalationexposuretothehumanpopulation,includingsensitivesubgroups,thatislikelytobewithoutanappreciableriskofdeleteriouseffectsduringalifetime”（USEPA,1994）.EachfractionhasacorrespondingRfDandRfCthatareusedtoevaluatethenon-carcinogeniceffectsofexposuretothatfraction（Vorheesetal.,1999）.Furthermore,aromaticcompounds,includingbenzene,toluene,ethylbenzene,andxylene（collectivelyknownasBTEX）,aswellaspolycyclicaromatichydrocarbons（PAHs）,canposeamoreseriousrisktohumanhealthbecauseoftheircarcinogenicandmutagenicpotentials（Kästneretal.,1998;Lópezetal.,2008）.

Bioremediationisanattractiveandenvironmentalfriendlyapproachforthecleanupofcontaminatedsites.Itexploitsthepotentialofnaturallyoccurringmicrobialpopulationsor,insomecases,introducesmicroorganismswithaknownabilitytodegradethecontaminants.Theapplicationofamicrobialconsortiumwithawidearrayofenzymaticabilitiesismoreeffectiveinthedegradationofpetroleumusingasingleisolate（RichardandVogel,1999;Rahmanetal.,2002）.Bioremediationofpetroleumhydrocarboncontaminatedsiteshasbeenthefocusofmanystudiesandthetechniquehasbeenrecentlyapplied;theseinvestigationsusedthetotalpetroleumhydrocarbons（TPH）concentrationtodeterminethetotalmassofthecontaminantintheenvironmentalandasameasureofbiodegradation（Al-Awadhietal.,1996;Mailaetal.,2005;Iturbeetal.,2007;Machackovaetal.,2008）.Similarly,numerouslaboratorystudiesdealingwithpetroleumhydrocarbonbiodegradationhavealsousedtheTPHconcentrationtomeasurethedegradationofthecontaminant（Hozumietal.,2000;Okohetal.,2001;Jamrahetal.,2007;AdamsandGuzmán-Osorio,2008）.However,thismethoddoesnotconsiderthecomplexityofthepollutantandhasneglectedtoconsiderthathydrocarboncomponentsareassociatedwitharisktohumanhealthandtheenvironment.Conventionalcleanuporbioremediationapproachthatdonotconsidertherisktohumansortheenvironmentarecostlyanddonotensuresuccessorthecompleterecoveryofthecontaminatedsite（KhanandHusain,2001）.AstheTPHvaluedoesnotprovideinformationonthecompositionofthespilledoil,evaluatingtheefficiencyofbiodegradationbasedonECfractionsisausefulapproachinrisk-basedandcost-effectivebioremediationefforts.

However,studiesonthedegradabilityofeachcompoundandECfractioninmineraloilsarelimited.Moreover,notmuchisknownaboutthedegradationofECfractionsinmineraloilinrelationtomicrobialpopulationsandspecificenvironments.ThisstudyinvestigatedtheabilityofamicrobialconsortiumtodegradethearomaticandaliphaticECfractionsofkerosene,whichwasusedasarepresentativemineraloil,inliquidcultures.

2.Materialsandmethods

2.1.Chemicalsandmedia

Naphthalene,acenaphthylene,anhydroussodiumsulfate,dichloromethane,andn-hexanewereobtainedfromWakoPureChemicalsIndustries（Osaka,Japan）.ASTMD2887aliphaticmixstandardandsilicagelgrade923with100e200meshwereprocuredfromSigma-Aldrich（Mo.,USA）.TolueneandethylbenzenewerepurchasedfromGLSciences（Tokyo,Japan）.Allchemicalswereofthehighestpurityavailable,generallygreaterthan96%aslistedbymanufacturers.Kerosenewasobtainedfromalocalgasstation.

BushnellHaasmedium（BHM）wasusedastheculturemediuminthisstudy.BHMiscomposedofthefollowing:

1gl_1NH4NO3,1gl_1K2HPO4,1gl_1KH2PO4,200mgl_1MgSO4$7H2O,50mgl_1FeCl3,and20mgl_1CaCl2.ThepHwasadjustedto7.0with1.0MNaOH.Themediawassterilizedbyautoclavingat121_Cfor15min.Aftercooling,1mloffilter-sterilized（0.2-mmfilter）traceelementsolutionwasadded.Thetraceelementsolutioncontained3mgl_1MnSO4$7H2O,3mgl_1ZnSO4$7H2O,1mgl_1CoSO4$7H2O,and1mgl_1（NH4）6Mo7O24$4H2O.

2.2.Microbialconsortium

Themicrobialconsortiumwasobtainedthroughenrichmenttechniquesfrompetroleum-contaminatedsoilatYabaseOilFieldinAkitaPrefecture,Japan.Soilsamples（10geach）wereaddedto120-mlserumbottlescontaining20mlofBHMand1%wv_1kerosene.Aftertwoweeksat30_Cinanorbitalshaker,1-mlaliquotsweretransferredto19mloffreshBSMcontaining1%wv_1keroseneasthesolecarbonandenergysource.Toattainastablekerosenedegradingconsortium,theculturewasenrichedthroughsuccessivetransferseverytwoweeksoversixmonths.

2.3.DNAextraction

Bacterialcellswereharvestedfromthekerosene-adaptedmicrobialconsortiumbycentrifugationat10,000rpmfor10mininsterileplastictubes.Thesupernatantwasdiscardedandcellpelletsweresuspendedin40mlofsterilizedwater.Freeze/thawcyclesfor15minat_80_Cand15minatroomtemperaturewereperformedtwice.TenmicrolitersofProteinaseK（1mgml_1）and50mlofTTNEbuffer（40mMTriseHCl,1%Tween20,0.2%NonidetP-40,0.2mMEDTA）wereaddedforatotalreactionvolumeof100ml.TheproteinaseKwasactivatedbyincubatingthesolutionat60_Cfor20min.TEsaturatedphenol（100ml）wasaddedbeforethesamplewasvortexedfor30s.Thesamplewasthencentrifugedat10,000rpmfor10min.Thelayercontainingthenucleicacidswasremovedandpurifiedbyethanolprecipitation.DNAwasdissolvedin1/10dilutedTEbufferandstoredat_20_Cuntiluse.

2.4.PCRamplification

Nearlyfull-length16SrRNAgenefragmentswereamplifiedbyPCRusingtheuniversalprimersEu10F（5’-AGAGTTTGATCCTGGCTCAG-3’）correspondingtoEscherichiacolipositions8e27asaforwardprimer,andEu1500R（50-GGTTACCTTGTTACGACTT-30）correspondingtoE.colipositions1510e1492asareverseprimer.PCRreactionswereperformedinatotalvolumeof50mlcontaining25mlofPCRMasterMix（Promega,USA）,20pmolo