1、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)0420259205TreatmentofWastewaterContainingChloroguaiacolsUsingTwo2stageAnaerobicFixedBedReactorYinC
2、heng2ri1/GAOZai2yuan2(1/KeyLaboratoryofOrganismFunctionalFactorsoftheChangbaiMountain/MinistryofEducation/YanbianUniversity；2/LibraryofYanbianUniversity/YanjiJilin133002/China)Abstract/Anaerobictreatmentofwastewatercontainingchloroguaiacolswasstudiedwithfocusontheremovalofchlorinatedguaiacols/Experi
3、mentswerecarriedoutusingalaboratory2scaleanaerobicfixedbedreactoroperatedatdifferenthydraulicretentiontimes(HRTs)/TheprocessefficientlyremovedfourtypesofchlorinatedguaiacolswhentheHRTwas24h/Inthiscondition/allchlorinatedguaiacolswerealmostcompletelyremoved/Themicrobialcommunitystructureinthereactorw
4、asnotchangedsignificantlyduringoperatingperiod/Keywords/anaerobictreatment；chloroguaiacols；fixedbedreactorCLCnumber/X703/1Documentcode/A1IntroductionTraditionally/theconcernoverdischargesfromthepulpandpaperindustryhasbeenfocusedontheriskoffiberdepositionsandoxygendeficiency/Inrecentyears/however/int
5、eresthasshiftedtowardstheenvironmentalimpactofchlorinatedorganiccompoundsintheeffluents/resultingfrompulpbleachingwithchlorine2containingchemicals/Manychlorinatedorganiccompoundsoflowmolecu2larmassareknowntobetoxic/bioaccumulable/mutagenicandpersistentanditisthereforeimportanttoavoidreleasingsuchcom
6、poundsintotheenvironment/Conventionalaerobicbiologicaltreatmentemployingaeratedlagoonsandactivatedsludgeprocessesiscommonlyusedatpulpandpapermillstoremovebiochemicaloxygendemand(BOD)fromtheefflu2ents/Thesetreatmentmethodsremovealsoasignificantpartofabsorbableorganichalogens(AOX)；theAOXremovalranging
7、from20%to60%hasbeenreported1/However/aerobictreatmentgeneral2lyhasapooreffectonpolychlorinatedcompounds/Underaerobicconditions/chlorinatedguaiacolsandphenolscanbeO2methylatedtothecorrespondinganisolesandveratroles/whicharemoreresistanttobiodegradationandhavehigherpotentialforbioaccumulationinfish2/A
8、ndmanyvolatilechloro2organiccompounds/suchaschloroform/arelikelytoberemovedfromtheeffluentbystrippingratherthanbybiodegradation/resultinginanuncontrolledemissionofthesecompoundsintotheenvironment/Studiesonanaerobictreatmentofbleachingplanteffluenthaveshownthistreatmenttohaveseveraladvantagesascompar
9、edtoaerobictreatment/polychlorinatedchlorophenolicsareremovedsignificantly/ahigherAOXremovalisachievedandthephytotoxicchlorateeliminated/Furthermore/anaerobictreatmentshouldbeasignificantlymorecost2efficientwaythanaerobictreatmenttoremoveBOD/providingthatanefficientconversionoforganicmatterintobioga
10、scanbemaintainedintheprocess/Thusfar/mostinvestigationshavebeenfocusedontheremovalofunspecifiedAOX/However/thedegradationofspecificcompounds/e/g/chlorinatedguaiacolsintheanaerobictreatmentprocesshasnot1994-2009ChinaAcademicJournalElectronicPublishingHouse/Allrightsreserved/http//延边大学学报(自然科学版)第33卷bee
11、nsystematicallystudied/Theaimofthepresentstudywastoevaluatethepossibilityofachievingchloroguaiacols(tetrachloroguaiacol/TeCG/4/5/62trichloroguaiacol/4/5/62TriCG/4/52dichloroguaiacol/4/52DCG/and42chloroguaiacol/42CG)removalinthetwo2stageanaerobicfixedbedreactor/2Materialsandmethods2/1Syntheticwastewa
12、tercontainingchloroguaiacolsSyntheticwastewatercontainedmixturesof42CG/4/52DCG/4/5/62TriCG/andTeCG/Thewastewaterwassuppliedwith0/2gyeastextract/0/1gNH4Cl/27mgKH2PO4/25mgMgSO47H2O/11/3mgCaCl2/2/8mgFeSO4H2O/5/5mgMnCl2H2O/0/68mgZnCl2/1/2mgCoCl26H2O/1/2mgNiSO44H2O/0/025mgNa2MoO42H2Oand0/026mgNa2SeO35H2O
13、l-1/Theconcentrationofallfourchloroguaiacolsinthesyntheticwastewaterwas0/05mM/Glucoseaselectrondonorforanaerobicdechlorinationwasused/Thecompoundwasaddedtothesyntheticwastewaterinanamountcorrespondingto1000mgCODl-1/2/2AnaerobicdigestersludgeTheanaerobicdigestersludgehadbeenobtainedfrom3500m3anaerobi
14、cdigesterreactor(inopera2tionfor10yearsatthehydraulicretentiontimeof25days)ofTaejonsewagetreatmentplantinKorea/Thesludgewasstoredat4ingas2tightplasticcontainerforamaximumperiodoffivemonths/Intheassay/17/4gvolatilesuspendedsolidl-1(ratioofVSStoSSwas0/561)oftheanaerobicsludgewasused/2/3Reactordesignan
15、doperationTwoidenticalreactors(R1andR2)/designedasanaerobicfixedbedreactorandconstructedofpolyvinylchloride(PVC)/wereusedinthestudy/Eachreactorwas7/0cmindiameter/andheightfromtheinlettotheoutletwas48/0cm/Thisprovidedaneffectivebedvolumeof1/4L/Atthetimeofexperimentation/thesludgedepthwasapproximately
16、34/0cm/Thereactorswereoperatedathydraulicretentiontimes(HRT)/basedonthereactorliquidvolume/of12/and24hours/placedinatemperaturecontrolledroomat30/ThepHofthebleachingplanteffluentwasmeasuredandadjustedto7/5with2MH2SO4/Thereactorswerefilledwithceramicspheres(diameter5mm)assupportmediumforanaerobicdige
17、stersludge/Wastewaterwasfedtothesystembyaperistalticpump/Initially/thereactorswereinoculatedwithanaerobicdigestersludge/andthenstartedupandoperatedforaweekonthesyntheticwastewateratalowfeedrate(HRT=48h)toallowthesludgetoacclimatizetothechlorinatedguaiacols/2/4NucleicacidextractionandPCRamplification
18、of16SrDNADNAwasextractedwithhotsodiumdodecyl2sulfate2phenolandwaspurifiedbyusingphenol2chloroform2isoamylalcohol(25241/V/V/V)/followedbyethanolprecipitation；about100mg(freshweight)ofsludgeandtheprotocolofOelmulleretal/3wereused/Fragmentsofthe16SribosomalDNA(rDNA)suitablefordenaturinggradientgelelect
19、rophoresis(DGGE)analysiswereobtainedbyusingtwodifferentprimercombinationsfordifferentphylogeneticlineages(Table1)；onecombinationwasusedformembersofthedomainBacteria4/theothercombinationwasusedformembersofthedomainArchaea5/ThelengthsofthePCRproductswere585and624bp/respectively/Recently/researchershav
20、eshownthatthebacterialprimercombinationconsistingofprimers341Fand907Rusedhereprovidesthemostreliableresults6/ThePCRconditionsusedforthebacterialprimersetshavebeendescribedpreviouslybyMuyzeretal/4Toamplifythearchaealgenes/weusedatouchdownprotocolfor20cycleswithtemperaturesrangingfrom71to61；theanneali
21、ngtemperaturewasreduced1everytwocycles/Thisprocedurewasfollowedby15additionalcyclesatanannealingtemperatureof61/Exceptforthe0621994-2009ChinaAcademicJournalElectronicPublishingHouse/Allrightsreserved/http//第4期尹成日/等/两段式固定***厌氧反应器处理含愈创木酚废水的研究initialdenaturationstep(94/5min)/denaturationandannealingphase
22、stepswere1minlong/whilemostofthepolymerizationphasestepswere3minlong(theonlyexceptionwasthefinalcycle/whichwas10minlong)/Table1Sequences/targetsites/andspecificitiesoftheprimersusedinthisstudyPrimeraTargetsitebSequence(5to3)Specificity341F2GCc341357CCTACGGGAGGCAGCAGMostBacteria907R907926CCGTCAATTCCT
23、TTGAGTTTMostknownorganismsARC344F2GC344363ACGGGGYGCAGCAGGCGCGAMostArchaeaARC915R915934GTGCTCCCCCGCCAATTCCTMostArchaeaaF(forward)andR(reverse)indicatetheorientationoftheprimersinrelationtotherRNAsequences/bEscherichiacolinumberingofBrosiusetal/7/cGCisa402nucleotideGC2richsequenceattachedtothe59endoft
24、heprimerasdescribedbyMuyzeretal/4/TheGCsequenceis52CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG23/2/5DGGEanalysisofPCRproductsDGGEwasperformedfor4/0hataconstantvoltageof200V/About600ngofPCRproductwasdepositedineachwell/Afterelectrophoresis/thegelswerestainedwithGelStarNucleicAcidStain(Takara/Japan)andph
25、otographedwithUVtransillumination(wavelength/314nm)withaPolaroidcamera/2/6AnalysisofchloroguaiacolsandVSSThehexaneextractsofacetylatedsamplesweretheninjectedintothegaschromatography/forchlorinatedguaiacolsanalysis/AllgaschromatographyexperimentswereperformedwithaHewlettPackardmodel5890seriesinstrume
26、ntequippedwithaflameionizationdetector/Afusedsilicacapillarycolumn(30m0/53mm2/65m)wasused/Theinitialoventemperaturewasheldat100for1minandthenincreasedatarateof6/mintoafinaltemperatureof260/Injectionport/anddetectorwereheldat275/Heliumwasusedasacarriergasataflowrateof30mL/min/VSSweredeterminedaccordi
27、ngtothestandardmethods8/2/7ChemicalsChlorinatedguaiacolsthatareusuallypresentinratherhighconcentrationsinbleachingplanteffluentwereanalyzedandusedasindicatorsoftreatmenteffect/Thechlorinatedguaiacols(purity/tetrachlo2roguaiacol99+/4/5/62trichloroguaiacol99+/4/52dichloroguaiacol99+/42chloroguaiacol90
28、%95%)werepurchasedfromHelixBiotech/Corp/(Columbia/NewWestminster/BC/Canada)/Otherchemicalswerereagentgradeandwerepurchasedfromtheusualcommercialsources/Nitrogengasof99/999%(V/V)puritywasusedwithoutanytreatment/3ResultsanddiscussionHydraulicretentiontime(HRT)isonethemostimportantfactorsaffectingthebi
29、oreactorefficiency/ToinvestigatethebiodegradationefficiencyofchlorinatedguaiacolsinresponsetothevarianceofHRT/syntheticwastewatercontainingchloroguaiacolswascontinuouslyfedintothetwo2stageanaerobicfixedbedreactor/varyingtheHRTfrom24to12hours/DuringtheoperationalperiodthepHremainedcon2stant/between7/
30、0and8/0/AsshowninFig/1andFig/2/the4/5/62TriCGand4/52DCGwereremovedmorethan90%/butTeCGand42CGshowedunstableremovalinR1/TheremovalratesofTeCGand42CGwere75%85%/However/allchlorinatedguaiacolswerealmostcompletelyremovedinR2/After2weeks/HRTwasdecreasedfrom24(totalloadingrateoffourtypesofchlorinatedguaiac
31、olswas42/1g/(m3d)to12hours/Duringthisperiod/although4/5/62TriCGand4/52DCGwerealsoremovedcompletely/completeremovalofTeCGcouldnotbeattained/However/afterincreasingHRTto24hoursagain/removal1621994-2009ChinaAcademicJournalElectronicPublishingHouse/Allrightsreserved/http//延边大学学报(自然科学版)第33卷efficienciesof
32、chlorinatedguaiacolswerequicklyrecovered/Theremovalefficienciesofchlorinatedguaiacolsinthetwo2stageanaerobicfixedbedsystemwereinthefollowingorder/4/52DCG4/5/62TriCG42CGTeCG/Thisisinaccordancewiththeresultobtainedfrombatchtest//Influentconcentration/concentrationofR1effluent/concentrationofR2effluent
33、Fig/1RemovalratesofTeCG(A)and4/5/62TriCG(B)Fig/2Removalratesof4/52DCG(A)and42CG(B)intwo2stageanaerobicfixedbedreactorintwo2stageanaerobicfixedbedreactorFromtheseexperimentsitispossibletoconcludethattheanaerobicfixedbedreactorisanefficienttreatmentmethodforwastewatercontainingchloroguaiacols/However/
34、finaleffluentshouldundergoanadditionaltreatmente/g/anaerobicprocesstocompletelyeliminatetheresidualfractionofchlorinatedguaiacols2derivedmetabolites/HaggblomandSalkinoja2Salonen9treatedkraft2bleachingwastewaterinananaerobicfluidizedbedandobtainedbetween16%61%removalofchlorinatedguaiacols(TeCG/3/4/52
35、TriCG/4/5/62TriCG/and4/52DCG)/Parkeretal/10alsostudiedtheremovalofchlorinatedphenolicsfromkraftmillbleach()freshsludge/()R1sludge/()R2sludgeFig/3DGGEpatternsof16SrDNAfrag2mentsfromfreshsludge/R1andR2sludgeinthetwo2stageanaerobicfixedbedreactor(R1andR2)treatingchloroguaiacols2containingwastewaterplan
36、teffluentsinanadaptedanaerobicsludgeblanketreactor/Inthepresenceoftheco2substratesmethanolandethanol/toxicityremovalwasabout93%andabout65%withouttheco2substrates/Inthesamestudy/42chloroguaiacol/4/52dichloroguaiacol/and4/5/62trichloroguaiacolwerealmostcompletelyremovedinthepresenceofco2substrates(95%
37、)/Microbialcommunitystructureinsludgesampledfromtwo2stageanaerobicfixedbedreactortreatingchloroguaiacols2containingwastewaterwasassessedbyPCR2DGGEanalysisof16SrDNAfragments/PCR2DGGEanalysisshoweddidnotsignificantdifferencesinArchaeamicrobialcommunitystructurebetweenthesludgestudied(Fig/3)/Anadditionalband3fromR2sludgecomparedtofreshsludgewasfoundinEubacteria2621994-2009ChinaAcademicJournalElectronicPublishingHouse/Allrightsreserved/http//
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