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n-butyl phthalate by Xiangjiang River sediment and microflora analysis

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n-butyl phthalate by Xiangjiang River sediment and microflora analysisby,n,River,and,butyl

    n-butyl phthalate by Xiangjiang River

    sediment and microflora analysis J.Cent.SouthUniv.Techno1.(2009)16:09480953

    DoI:10.1007/sl1771009-0158-4

    Aerobicbiodegradationof

    XiangjiangRiversediment

    di--n--butylphthalateby

    andmicrofloraanalysis

    ZHOUHong_bo(周洪波),LINFeng(k~k).,HUPei.1ei(胡培磊),JINGDecal(~TJ-),

    RENHong?qiang(任洪强),ZHAOJing(~_),QIUGuan.zhou(邱冠周)

    Springer

    (1.SchoolofMineralsProcessingandBioengineering,CentralSouthUniversity,Changsha410083,China;

    2.StateKeyLaboratoryofPollutionControlandResourcesReuse,NanjingUniversity,Nanjing210008,China;

    3.SchoolofLifeScienceandTechnology,HuazhongUniversityofScienceandTechnology ,Wuhan430074,China)

    Abstract:Di

    _but),1phthalate(DBP),oneofphthalateacidesters(PAEs),wasinvestigatedtodetermineitsbiodegradationrate

    usingXiangjiangRiversedimentandfindpotentialDBPdegradersintheenrichmentcultureofthesediment.Thesedimentsample

    was1ncubatedwithaninitialconcentrationofDBPof100mg/Lfor5d.ThebiodegradationrateofDBPwasdetectedusingHPLC

    andthedegradedproductswereanalyzedbyGC/MS.Subsequently,themicrobialdiversityoftheenrichmentculturewasanalyzed

    bypolymerasechainreaction-restrictionfragmentlengthpolymorphism(PCR-RFLP).The

resultsrevealthatalmost100%ofDBPis

    degradedaftermerely3d,generatingtwomaindegradedproducts:mono-butylphthalate(M

    BP)and9octadecenoicacid.Atiera

    six

    monthenrichmentperiodunderthepressureofDBP,thedominantfamilyinthefinaIenrichme

    ntcultureisclusteredwiththe

    Comamonassp.,theremainingareaffiliatedwithSphingomonassp.,Hydrogenophagasp.,R

    hizobiumsp.,andAcidovoraxsp.The

    resultsshowthepotentialofthesebacteriatobeusedinthebioremediationofDBPintheenviro

    nment.

    Keywords:bioremediation;dinbutylphthalate;sediment;polymerasechainreactionrestrictionfragmentlen~hpolymorphism

    microbialdiversity

    lIntrOducti0n

    Phthalateacidesters(PAEs)haveenjoyedawide

    applicationintheindustrialproductionaswellas

    people'sdaily1ifesincethemiddleofthe20thcentury. Theybelongtoaclassofrefractoryorganiccompounds extensivelyusedinplasticindustry,occupying

    80%-85%oftheglobalplasticizermarket[1].AsPAEs canenterenvironmentdirectly,theyarewidely

    distributedindifferentenvironmentsatconcentrations rangingfrom0.3to98u2/Linsurfacewater,0.2to

    8.4mg/kginsediment,and28to154mg/l(ginsewage

    sludge[2].what'sworse.someofthemaretoxicto

    mammalsandaquaticorganisms[3],interferingwith theirreproductivesyste:msandendocriliefunction[4]. Furthermore,certainPAEsaresuspectedofexerting adverseeffectsonhumanhealth51.

    Di..butylphthalaterDBP)thatbelongstothe

    familyofPAEsisusedinthepreparationofinsect repellentsandpapercoatings,andasanagentfortextile 1ubrication.withdevelopmentalandreproductive toxicityonanimals[61.Asoneofthemostfrequently identifiedPAEsindiverseenvironmentalsamples,DBP islistedasatop.priorityenvironmentalpollutantby severalregulatorybodies.suchasU.S.Environment ProtectionAgency,EuropeanUnionandChinaNational EnvironmentalMonitoringCenter[7].

    Unfortunately,DBPisrelativelystableinthe environment.Thehydrolysishalf-lifeofDBPwas estimatedtobeabout20years.Besides.itsphotolysis half-lifeisalsoquietlong.whichisfromseveralmonths toafewyears[8].Apartfromphysicalandchemical ways,biologicalpathwayisindisputablyaneffectiveand efficientroutetoeliminatethiswidespreadpollutant.LI etal[91studiedthekineticsofDBPbiodegradationin soil.WhentheinitialconcentrationofDBPisbelow 50mg&g.thehalflifeofDBPisonly5.20d.YUAN etal[101reportedthatthebiodegradationofPAEsis moreeffectiveunderaerobicthananaerobicconditions. XiangjiangRiveristhelongestriverinHunan Province,China.Itisalsoseriouslypollutedby pollutantsincludingPAEssincethebiggestcitiesin Hunanarealllocatedalongthisriver.Inthiswork,the DBPbiodegradationabilityofXiangjiangRiver sedimentwastestedunderaerobicconditionandthe functionalmicroorganismsintheenrichmentculture werethenexplored.

    2Experimental

2.1Chemicais

    DBPwith99.5%puritywaspurchasedfromHunan HuihongChemicalsCorporationChina.Chemicals Founda~onitem:Project(50621063)supportedbytheNationalNamreScienceFoundationo

    fChina;Project(NCET-060691)supportedbytheProgramfor NewCenturyExcellentTalentsinUniversity Receiveddate:20090112;Accepteddate:20090520

    Correspondingauthor:ZHOUHong-bo,Professor;Tel:+86(7388877216;E

    mail:zhouhb@mail.CSU.edu.cn

    J.Cent.SouthUniv.Techno1.(2009)16:09480953949

    usedfordilutingandextractingDBPwere analyticalreagentgradeandwereredistilled.Methanol usedwasHPLCgrade(SKChemicals,Korea).Other chemicalsandsolventswereanalyticalreagentgrade.

    2.2Sedimentsampleandeultureconditions SedimentsamplewastakenfromXiangjiangRiver inChangshaCity,HunanProvince,China.Enrichment culturewasobtainedfromthesedimentsampleusing DBPasthesolecarbonsource.Theculturemedium containedthefollowingsalts(g/L):(NH4)2504(3.00), KCl(0.10),KH2PO4(3.oo),MgSO4'7H20(0.50), CaC12.2H2O(0.25),Na25203(10.oo).Theenrichment wasperformedforl80d.

    2.3BiOde2radatiOntrails

    12ofsedimentwasaddedtoa50mLflaskwith2O mLmedium.TheinitialconcentrationofDBPinthe systemwas100mg/L.Theflaskwasincubatedat30?

    f0r5dinarotaryshakeroperatedat150r/min. Samplingwasperformedevery24h.

    2.4HPLCandGC/MSanalysis

    DBPconcentrationsinthesampleswereanalyzed usingareverse.phaseHPLCsystem.Inordertomaintain ahomogenoussystemforaccuratequantificationofthe residualDBP,1.0mLtween80stocksolution(10g/L1 wasaddedtoeachflaskasasolubilizingagent.The flaskswerethenautoclavedatl15?for30minto

    ensureastrictlyaseptichomogenousenvironmentbe;fore theywereputinarotaryshakeratl50r/minfor24h. Atierequilibration.sampleswerecentrifugedatl2000 r/minfor30min.andthen1.0mLofeachsamplewas removedtoacleantubetowhich1.0mLof

    dichloromethanewasadded.Theaqueousandorganic phaseswereformedinthetubeaftervortexingfor1min andcentrifugationat12000r/minfor3min.After extractingfromtheaqueousphasetwotimes,the dichloromethanewasevaporatedtodrynessandthe residuewasredissolvedin1.0mLofmethano1. Approximately0.5mLoftheDBP.containingmethanol passedthrougha0.22Bmmembranefilterbeforea portionf20pL)wasinjectedintoHPLC.

    HPLCanalysiswascarriedoutusingEliteseries HPLC(Elite,China)consistingofanonlineDG230-2 degasser,aP230/P230ppump.andaUV230UVVis

    detectorsetat228nm.AHypersilBDSC18column

    (200mm×4.6mm,5p_m)wasusedfortheseparation. Themobilephaseconsistedofamethanolwatersolution

    (90:l0,volumeratio),andtheflowratewas0.5mL/min. GC,MSanalysiswasperformedonShimadzu

    GCMSQP2010analyzer.AnOV-5capillarycolumn

    (30.0m×0.32mm(i.d.),0-32I-tmfilmthickness)was used.Theinjectiontemperaturewas310?andthe

    samplewasinjectedinthesplitmodefor1min.Thesplit ratiowas10:1.Theoventemperaturewasprogrammed from100to280?.ForMSdetection.standardE1

    conditions(70eV)wereusedwithasourcetemperature of200?.Heliumwasusedascarriergaswim

    0.91mL/minofflowrate.Thecollectedscansof metaboliteswereidentifiedbycomparingwiththe publishedmassspectrumatNISTfNationalInstituteof StandardsandTechnology)Database.

    Eachsamplingwasrunintriplicate.Besides, additionalsedimentsampleswithoutaddingDBPwere usedtoeliminatetheeffectofthecompoundsinthe sedimenttotheHPLCandGC/MSanalysis.

    2.5DNAextractionand16SrDNAgeneamplification BacterialDNAwasextractedfromtheenrichment sampleusingE.Z.N.A.BacteriaDNAKitfOMEGA, USA).16SrDNAgenewasthenamplifiedbyPCRfrom theextractedDNA.PCRmixtureconsistedof5uLOf PCRbuffer(Mgplus),1ofdNTP(10mmol/L), 1uLofeachprimer:forwardprimer(27fi

    5.AGAGTTTGATCCTGGCTCAG3,5?lnol/L)and

    reverseprimerf1492r:5.GGTTACCTTGTTACGA

    CTT.3,5pmol/L),0-25uLofTaqpolymeraseand2pL ofDNAextractionproducts.Doubledistilledwaterwas

    addeduntilitsfinalvolumereached5OLlL.ThePCR conditionswereaninitialdenaturationstepat95?for

    6minfollowedbv36cyclesat94?for1min,56?

forlminand72?for2min.andafinalstepat72?

    f0rl0min.PCRproductswerepurifiedbvE.Z.N.A.

    GelExtractionKit(0MEGA.USA1.

    2.6Cloningandrestrictionfragmentlengthpoly- morphism(RFLP)analysis

    ThepurifiedPCRproductswereclonedinto

    PGEM.Tvectorsthatwereusedtotl'ansformDH5a Escherichiacolicompetentcells.Atierblue.white screening176whitecolonieswererandomlyselected andre.amplifiedbyPCR.72ofthepositivereamplified productsweredigestedbytherestrictionendonuclease eIIIandMspIewEnglandBiolabs,at37?

    overnight.ThesystemforthereactionofMspIand HaeIIIdigestionconsistedof1uLbur.O.25uLof eachenzyme,and5BLofpurifiedclonePCRproducts. Double.distilledwaterwasaddeduntilitsfinalvolume reachedl0LtL.Thedigestionproductswereanalyzedon a3%agarosegelprestainedwithethidiumbromideby elecophoresis(3h,80V).Restrictionprofilescanbe dividedinto7difierentgroups.Representativesofeach groupwereselectedforsequencing.

    2.7Sequencingandphylogeneticanalysis

    Sevenrepresentativeclonesweresequenced (completedbyBeijingSunbiotechCo,Ltd)andthese sequenceswerecomparedwiththeexistingGenBank 16SrDNAgenesequences.Theclosest16SrDNAgene 950J.Cent.SouthUniv.

    Techno1.(2009)16:09480953

    sequenceswerealignedwithCLUSTALX1.83. Phylogenetictreewasconstructedbytheneighbor

jpiningmethod,usingMEGA3.1[11].

    3Resultsanddiscussion

    3.1DBPbi0degradatiOn

    Inordertomakeapreliminaryevaluationofthe biodegradationabi1itVofthesediment.itsoriginal potentialtodegradeDBPwastestedunderaerobic condition.ThebiodegradationofDBPbvthesediment samplewasmonitoredbydetectingtheconcenationof

    missubstrateintheflasksystemthroughHPLC.The degradationpaRernofDBPisshowninFig.1.

    tha

    Fig,1DegradationpaRemofDBPinflasksystem Inthefirst24h,thedegradationrateisrelatively slow.Subsequentl~thedegradationratebecomesquite rapidandalmost100%ofDBPisdegradedin72h. Duringthisperiod.themicroflorainthesediment probablyhasacclimatizedtothenewsubstrate(DBP), resultingintherapiddegradationofDBELowsubstrate degradationrateduringtheinitialstagecanbeattributed totheacclimatizationphase,whichisaprerequisitefor bacteriatobeacclimatedtothenewenvironment. GC/MSwasusedtoidentifythemetabolitesand intermediatesOfDBPduringthebiodegradationprocess performedbythemicrolflorainthesedimentsample. Threemaincompoundsareidentifiedasmono-butVl phthalate(MBP),DBPand9.pctadecenoicacid, respectively(Fig.2,bycomparingeachmassspectrum withthepublishedmassspectrumintheNational InstituteofStandardsandTechnology(NIST)Database. Basedontheintermediatecompoundsdetectedby

    GC/MS.apreliminarypathwayformetabolism0fDBP bytheriversedimentisproposedandillustratedas follows:

    DBP

    OOC4Hq

    OOC4H9

    MBP

    9.octadecenoicacid

    4H9

    COOHCO2+H2O

    (1)

    OnebiochemicaldegradationpathwayofDBP proposedbyXUetal[12]isthatthehydrolysisofthe esterslinkageOfDBPwouldformMBPandsubsequent phthalicacid(PA)andprotocatechuicacid(PCA),and thenPAcanbefurthermetabolizedtoproducecarbon dioxideandwater.ThedetectionofMBPasintermediate OfDBPdegradationinthisworkisconsistentwith previousreports.However,themostfrequentlyreported productssuchasPAandPCAf131arenotdetected. CHANGetal[14]pointedoutthatthehighdegradation rateOfDBPmightbethereasonforthatPAwasnot detectedbyGC/MS.Besides,akindoffattyacidnamed 9.octadecenoicacidwasdetected.Actually,itwas reportedthatvariousfattyacidssuchastetradecanoic andhexadecanoicweredetectedduringmetabolization ofDBPbyfungi15].Nevertheless,itisstil1notclear whatrolethefattyacidsmightplayinthepathwayof DBPbiodegradation.Asstatedabove.theformer proposedbiochemicaldegradationpathwayforDBPby

    isolatedbacteriaissimplebutuncompleted.Accordingto thiswork.thedegradationpathwayofDBPbythe microflorainsedimentisprobablymorecomplicated. Thus.furtherinvestigationisnecessarytorevea1more informationaboutthepathwaybythemicrobial communityinthesediment.

    3.2Phylogeneticanalysis

    TherelativerapidDBPbiodegradationratesbythe sedimentrevealthattherearemicroorganismsthat probablypossessstrongcapabilitytodegradethe pollutant.Itis.however,noteasytofindthosereal functionalmicroorganismsdirectlyfromthesediment, consideringitscomplicatedmicrobialcommunity composition.TheenrichmentprocessCallserveasagood methodtoreducethiscomplicityandhelpustofoCUSon therightmicroorganisms.

    72positiveclonesfromtheenrichmentsamplewere analyzedbyPCR-RFLP,presentingsevenoperational taxonomicunitsr0TUs).Rare:factioncurve(dataarenot shown)evaluationoftheclonesrevealsthat72clones aresufficienttorepresentthemicrofloradiversityinthe finalenrichmentsystem.Representativeclonesofthe sevenrestrictionprofileswereselectedrespectivelyfor sequencing.Thesequenceinformationofthetested clonesislistedinTable1.Thelibrarywhichconsistsof fivegeneraisdominatedbyclonesrelatedto

    Comamonas(xJL67,XJL79,andXJL144).Fig.3

    showsthedetailedinformationoftheabundanceofeach genusintheenrichmentsystem.Basedonallthe availablesequences,phylogenetictreeofthemicroflora

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