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第33卷第4期2007年12月延边大学学报(自然科学版)JournalofYanbianUniversity(NaturalScience)Vol.33No.4Dec.2007收稿日期:2007208222基金项目:教育部留学回国人员科研启动基金资助项目(20052383号)作者简介:尹成日(1963),男(朝鲜族),博士,教授,研究方向为环境污染物的生物处理.文章编号:100424353(2007)0420259205TreatmentofWastewaterContainingChloroguaiacolsUsingTwo2stageAnaerobicFixedBedReactorYinCheng2ri1,GAOZai2yuan2(1.KeyLaboratoryofOrganismFunctionalFactorsoftheChangbaiMountain,MinistryofEducation,YanbianUniversity;2.LibraryofYanbianUniversity:YanjiJilin133002,China)Abstract:Anaerobictreatmentofwastewatercontainingchloroguaiacolswasstudiedwithfocusontheremovalofchlorinatedguaiacols.Experimentswerecarriedoutusingalaboratory2scaleanaerobicfixedbedreactoroperatedatdifferenthydraulicretentiontimes(HRTs).TheprocessefficientlyremovedfourtypesofchlorinatedguaiacolswhentheHRTwas24h.Inthiscondition,allchlorinatedguaiacolswerealmostcompletelyremoved.Themicrobialcommunitystructureinthereactorwasnotchangedsignificantlyduringoperatingperiod.Keywords:anaerobictreatment;chloroguaiacols;fixedbedreactorCLCnumber:X703.1Documentcode:A1IntroductionTraditionally,theconcernoverdischargesfromthepulpandpaperindustryhasbeenfocusedontheriskoffiberdepositionsandoxygendeficiency.Inrecentyears,however,interesthasshiftedtowardstheenvironmentalimpactofchlorinatedorganiccompoundsintheeffluents,resultingfrompulpbleachingwithchlorine2containingchemicals.Manychlorinatedorganiccompoundsoflowmolecu2larmassareknowntobetoxic,bioaccumulable,mutagenicandpersistentanditisthereforeimportanttoavoidreleasingsuchcompoundsintotheenvironment.Conventionalaerobicbiologicaltreatmentemployingaeratedlagoonsandactivatedsludgeprocessesiscommonlyusedatpulpandpapermillstoremovebiochemicaloxygendemand(BOD)fromtheefflu2ents.Thesetreatmentmethodsremovealsoasignificantpartofabsorbableorganichalogens(AOX);theAOXremovalrangingfrom20%to60%hasbeenreported1.However,aerobictreatmentgeneral2lyhasapooreffectonpolychlorinatedcompounds.Underaerobicconditions,chlorinatedguaiacolsandphenolscanbeO2methylatedtothecorrespondinganisolesandveratroles,whicharemoreresistanttobiodegradationandhavehigherpotentialforbioaccumulationinfish2.Andmanyvolatilechloro2organiccompounds,suchaschloroform,arelikelytoberemovedfromtheeffluentbystrippingratherthanbybiodegradation,resultinginanuncontrolledemissionofthesecompoundsintotheenvironment.Studiesonanaerobictreatmentofbleachingplanteffluenthaveshownthistreatmenttohaveseveraladvantagesascomparedtoaerobictreatment:polychlorinatedchlorophenolicsareremovedsignificantly,ahigherAOXremovalisachievedandthephytotoxicchlorateeliminated.Furthermore,anaerobictreatmentshouldbeasignificantlymorecost2efficientwaythanaerobictreatmenttoremoveBOD,providingthatanefficientconversionoforganicmatterintobiogascanbemaintainedintheprocess.Thusfar,mostinvestigationshavebeenfocusedontheremovalofunspecifiedAOX.However,thedegradationofspecificcompounds,e.g.chlorinatedguaiacolsintheanaerobictreatmentprocesshasnot1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.延边大学学报(自然科学版)第33卷beensystematicallystudied.Theaimofthepresentstudywastoevaluatethepossibilityofachievingchloroguaiacols(tetrachloroguaiacol:TeCG,4,5,62trichloroguaiacol:4,5,62TriCG,4,52dichloroguaiacol:4,52DCG,and42chloroguaiacol:42CG)removalinthetwo2stageanaerobicfixedbedreactor.2Materialsandmethods2.1SyntheticwastewatercontainingchloroguaiacolsSyntheticwastewatercontainedmixturesof42CG,4,52DCG,4,5,62TriCG,andTeCG.Thewastewaterwassuppliedwith0.2gyeastextract,0.1gNH4Cl,27mgKH2PO4,25mgMgSO47H2O,11.3mgCaCl2,2.8mgFeSO4H2O,5.5mgMnCl2H2O,0.68mgZnCl2,1.2mgCoCl26H2O,1.2mgNiSO44H2O,0.025mgNa2MoO42H2Oand0.026mgNa2SeO35H2Ol-1.Theconcentrationofallfourchloroguaiacolsinthesyntheticwastewaterwas0.05mM.Glucoseaselectrondonorforanaerobicdechlorinationwasused.Thecompoundwasaddedtothesyntheticwastewaterinanamountcorrespondingto1000mgCODl-1.2.2AnaerobicdigestersludgeTheanaerobicdigestersludgehadbeenobtainedfrom3500m3anaerobicdigesterreactor(inopera2tionfor10yearsatthehydraulicretentiontimeof25days)ofTaejonsewagetreatmentplantinKorea.Thesludgewasstoredat4ingas2tightplasticcontainerforamaximumperiodoffivemonths.Intheassay,17.4gvolatilesuspendedsolidl-1(ratioofVSStoSSwas0.561)oftheanaerobicsludgewasused.2.3ReactordesignandoperationTwoidenticalreactors(R1andR2),designedasanaerobicfixedbedreactorandconstructedofpolyvinylchloride(PVC),wereusedinthestudy.Eachreactorwas7.0cmindiameter,andheightfromtheinlettotheoutletwas48.0cm.Thisprovidedaneffectivebedvolumeof1.4L.Atthetimeofexperimentation,thesludgedepthwasapproximately34.0cm.Thereactorswereoperatedathydraulicretentiontimes(HRT),basedonthereactorliquidvolume,of12,and24hours,placedinatemperaturecontrolledroomat30.ThepHofthebleachingplanteffluentwasmeasuredandadjustedto7.5with2MH2SO4.Thereactorswerefilledwithceramicspheres(diameter5mm)assupportmediumforanaerobicdigestersludge.Wastewaterwasfedtothesystembyaperistalticpump.Initially,thereactorswereinoculatedwithanaerobicdigestersludge,andthenstartedupandoperatedforaweekonthesyntheticwastewateratalowfeedrate(HRT=48h)toallowthesludgetoacclimatizetothechlorinatedguaiacols.2.4NucleicacidextractionandPCRamplificationof16SrDNADNAwasextractedwithhotsodiumdodecyl2sulfate2phenolandwaspurifiedbyusingphenol2chloroform2isoamylalcohol(25241,V/V/V),followedbyethanolprecipitation;about100mg(freshweight)ofsludgeandtheprotocolofOelmulleretal.3wereused.Fragmentsofthe16SribosomalDNA(rDNA)suitablefordenaturinggradientgelelectrophoresis(DGGE)analysiswereobtainedbyusingtwodifferentprimercombinationsfordifferentphylogeneticlineages(Table1);onecombinationwasusedformembersofthedomainBacteria4,theothercombinationwasusedformembersofthedomainArchaea5.ThelengthsofthePCRproductswere585and624bp,respectively.Recently,researchershaveshownthatthebacterialprimercombinationconsistingofprimers341Fand907Rusedhereprovidesthemostreliableresults6.ThePCRconditionsusedforthebacterialprimersetshavebeendescribedpreviouslybyMuyzeretal.4Toamplifythearchaealgenes,weusedatouchdownprotocolfor20cycleswithtemperaturesrangingfrom71to61;theannealingtemperaturewasreduced1everytwocycles.Thisprocedurewasfollowedby15additionalcyclesatanannealingtemperatureof61.Exceptforthe0621994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.第4期尹成日,等:两段式固定床厌氧反应器处理含愈创木酚废水的研究initialdenaturationstep(94,5min),denaturationandannealingphasestepswere1minlong,whilemostofthepolymerizationphasestepswere3minlong(theonlyexceptionwasthefinalcycle,whichwas10minlong).Table1Sequences,targetsites,andspecificitiesoftheprimersusedinthisstudyPrimeraTargetsitebSequence(5to3)Specificity341F2GCc341357CCTACGGGAGGCAGCAGMostBacteria907R907926CCGTCAATTCCTTTGAGTTTMostknownorganismsARC344F2GC344363ACGGGGYGCAGCAGGCGCGAMostArchaeaARC915R915934GTGCTCCCCCGCCAATTCCTMostArchaeaaF(forward)andR(reverse)indicatetheorientationoftheprimersinrelationtotherRNAsequences.bEscherichiacolinumberingofBrosiusetal.7.cGCisa402nucleotideGC2richsequenceattachedtothe59endoftheprimerasdescribedbyMuyzeretal.4.TheGCsequenceis52CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG23.2.5DGGEanalysisofPCRproductsDGGEwasperformedfor4.0hataconstantvoltageof200V.About600ngofPCRproductwasdepositedineachwell.Afterelectrophoresis,thegelswerestainedwithGelStarNucleicAcidStain(Takara,Japan)andphotographedwithUVtransillumination(wavelength,314nm)withaPolaroidcamera.2.6AnalysisofchloroguaiacolsandVSSThehexaneextractsofacetylatedsamplesweretheninjectedintothegaschromatography,forchlorinatedguaiacolsanalysis.AllgaschromatographyexperimentswereperformedwithaHewlettPackardmodel5890seriesinstrumentequippedwithaflameionizationdetector.Afusedsilicacapillarycolumn(30m0.53mm2.65m)wasused.Theinitialoventemperaturewasheldat100for1minandthenincreasedatarateof6/mintoafinaltemperatureof260.Injectionport,anddetectorwereheldat275.Heliumwasusedasacarriergasataflowrateof30mL/min.VSSweredeterminedaccordingtothestandardmethods8.2.7ChemicalsChlorinatedguaiacolsthatareusuallypresentinratherhighconcentrationsinbleachingplanteffluentwereanalyzedandusedasindicatorsoftreatmenteffect.Thechlorinatedguaiacols(purity:tetrachlo2roguaiacol99+,4,5,62trichloroguaiacol99+,4,52dichloroguaiacol99+,42chloroguaiacol90%95%)werepurchasedfromHelixBiotech.Corp.(Columbia,NewWestminster,BC,Canada).Otherchemicalswerereagentgradeandwerepurchasedfromtheusualcommercialsources.Nitrogengasof99.999%(V/V)puritywasusedwithoutanytreatment.3ResultsanddiscussionHydraulicretentiontime(HRT)isonethemostimportantfactorsaffectingthebioreactorefficiency.ToinvestigatethebiodegradationefficiencyofchlorinatedguaiacolsinresponsetothevarianceofHRT,syntheticwastewatercontainingchloroguaiacolswascontinuouslyfedintothetwo2stageanaerobicfixedbedreactor,varyingtheHRTfrom24to12hours.DuringtheoperationalperiodthepHremainedcon2stant,between7.0and8.0.AsshowninFig.1andFig.2,the4,5,62TriCGand4,52DCGwereremovedmorethan90%,butTeCGand42CGshowedunstableremovalinR1.TheremovalratesofTeCGand42CGwere75%85%.However,allchlorinatedguaiacolswerealmostcompletelyremovedinR2.After2weeks,HRTwasdecreasedfrom24(totalloadingrateoffourtypesofchlorinatedguaiacolswas42.1g/(m3d)to12hours.Duringthisperiod,although4,5,62TriCGand4,52DCGwerealsoremovedcompletely,completeremovalofTeCGcouldnotbeattained.However,afterincreasingHRTto24hoursagain,removal1621994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.延边大学学报(自然科学版)第33卷efficienciesofchlorinatedguaiacolswerequicklyrecovered.Theremovalefficienciesofchlorinatedguaiacolsinthetwo2stageanaerobicfixedbedsystemwereinthefollowingorder:4,52DCG4,5,62TriCG42CGTeCG.Thisisinaccordancewiththeresultobtainedfrombatchtest.:Influentconcentration:concentrationofR1effluent:concentrationofR2effluentFig.1RemovalratesofTeCG(A)and4,5,62TriCG(B)Fig.2Removalratesof4,52DCG(A)and42CG(B)intwo2stageanaerobicfixedbedreactorintwo2stageanaerobicfixedbedreactorFromtheseexperimentsitispossibletoconcludethattheanaerobicfixedbedreactorisanefficienttreatmentmethodforwastewatercontainingchloroguaiacols.However,finaleffluentshouldundergoanadditionaltreatmente.g.anaerobicprocesstocompletelyeliminatetheresidualfractionofchlorinatedguaiacols2derivedmetabolites.HaggblomandSalkinoja2Salonen9treatedkraft2bleachingwastewaterinananaerobicfl

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