细菌吸附重金属外文翻译大学论文.docx

细菌吸附重金属外文翻译大学论文.docx

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细菌吸附重金属外文翻译大学论文

BacterialSorptionofHeavyMetals

Fourbacteria,Bacilluscereus,B.subtilis,Escherichiacoli,andPseudomonasaeruginosa,wereexaminedfortheabilitytoremoveAg+,Cd2+,Cu2+,andLa3+fromsolutionbybatchequilibrationmethods.CdandCusorptionovertheconcentrationrange0.001to1mMwasdescribedbyFreundlichisotherms.At1mMconcentrationsofbothCd2+andCu2+,P.aeruginosaandB.cereuswerethemostandleastefficientatmetalremoval,respectively.FreundlichKconstantsindicatedthatE.coliwasmostefficientatCd2+removalandB.subtilisremovedthemostCu2+.RemovalofAg+fromsolutionbybacteriawasveryefficient;anaverageof89%ofthetotalAg+wasremovedfromthe1mMsolution,whileonly12,29,and27%ofthetotalCd2+,Cu2+,andLa3+,respectively,weresorbedfrom1mMsolutions.ElectronmicroscopyindicatedthatLa3+accumulatedatthecellsurfaceasneedlelike,crystallineprecipitates.Silverprecipitatedasdiscretecolloidalaggregatesatthecellsurfaceandoccasionallyinthecytoplasm.NeitherCd2+norCu2+providedenoughelectronscatteringtoidentifythelocationofsorption.TheaffinityseriesforbacterialremovalofthesemetalsdecreasedintheorderAg>La>Cu>Cd.Theresultsindicatethatbacterialcellsarecapableofbindinglargequantitiesofdifferentmetals.Adsorptionequationsmaybeusefulfordescribingbacterium-metalinteractionswithmetalssuchasCdandCu;however,thisapproachmaynotbeadequatewhenprecipitationofmetalsoccurs.

Thefateoftoxicmetalliccationsinthesoilenvironmentdependslargelyontheinteractionsofthesemetalswithinorganicandorganicsurfaces.Theextenttowhichametalliccationinteractswiththesesurfacesdeterminestheconcentrationofmetalinsolutionand,consequently,thepotentialformovementintogroundwateroruptakebyplants.Aconsiderableamountofworkhasbeendonetoevaluatetheadsorptionorcomplexationofvariousheavymetalsbysoilsandsoilconstituents,suchasclaysandorganicmatterfractions.Onepotentiallyimportantorganicsurfacewhichhasreceivedlittleattentionisthatofthesoilmicrobialpopulation.Soilmicroorganismsaretypicallyassociatedwiththeclayandorganicfractionsofthesoilmicroenvironmentsandwouldbeexpectedtoparticipateinthemetaldynamicstypicallyascribedtothesefractions.Bacteriahaveahighsurfacearea-to-volumeratioand,asastrictlyphysicalcellularinterface,shouldhaveahighcapacityforsorbingmetalsfromsolution.Thereisevidencethatbacterialcellsaremoreefficientatmetalremovalthanclaymineralsonadry-weightbasis.Kurekandco-workersobservedthatsorptionofCd2+bydeadcellsofaParacoccussp.andSerratiamarcescenswasgreaterthanthatofmontmorillonitewhenthesolid-to-solutionratiowasthesameforbothbacteriaandclay.LivecellsaccumulatedaboutthesamequantityofCd2+asdidclay.

Severalinvestigationshaveshownthatrelativelylargequantitiesofmetalliccationsarecomplexedbyalgae,bacteria,andfungi.Metalbindingbyisolatedgram-positiveandgram-negativebacterialcellwallshasalsobeenevaluated.Cellwallsofthegram-positivebacteriaBacillussubtilisandB.licheniformiswereobservedtobindlargerquantitiesofseveralmetalsthancellenvelopesofthegram-negativebacteriumEscherichiacoli.

Weareinterestedintheroleofmicroorganismsinthebehaviorofvariousheavymetalsinthesoilenvironment.Theobjectivesofthisworkweretodeterminethemetal-bindingcapacitiesofwholecellsoftwogram-positiveandtwogram-negativebacteriaandtodeterminewhetheranequilibriummodel,theFreundlichadsorptionisotherm,wouldadequatelydescribebacterialmetalsorption.B.cereus,B.subtilis,andPseudomonasaeruginosawereexaminedasrepresentativesofcommonspeciesfrequentlyisolatedfromsoils.E.coliwasalsousedasasecondgram-negativebacteriumbecauseitisawell-characterizedmicroorganismanditscellenvelopehasbeenshowntobindlessmetalthandoB.subtiliscellwalls.ThefourmetallicionsusedinthisinvestigationwereAg+,Cd2+,Cu2+,andLa3+.Cadmiumandcopperarebothtoxiccationsofenvironmentalimportance.Silverandlanthanum,representativeofmonovalentandtrivalentheavymetals,respectively,arealsotoxicbutarelessfrequentlyfoundintheenvironment.

MATERIALSANDMETHODS

Bacteriaandgrowthconditions.ThebacteriausedintheseexperimentswereB.cereusATCC11778;P.aeruginosaATCC14886,bothobtainedfromtheAmericanTypeCultureCollection;B.subtilis168;andE.coliK-12strainAB264,bothfromtheUniversityofGuelph.Thebacteriawereroutinelyculturedin0.5xbrainheartinfusionbroth（BBLMicrobiology.Systems）am.ended.with.2.4g.of.HEPES（N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid）liter-1and2.0gofMES（2-N-morpholinoethanesulfonicacid）liter-1tobufferthemediumacidity.ThemediumaciditywasadjustedtopH6.8with0.5MKOH.Two2-millilitersamplesoflate-exponential-phasestartercultureswereusedtoinoculate800mlofbrothin3-literErlenmeyerflasks.Cellsweregrowntothelate-exponentialphaseatroomtemperature（approximately23°C）onanorbitalshakerat150rpm.CellsofB.cereusgrowninthismannerremainedinthevegetativestate.Thecellswereharvestedbycentrifugationandwashedtwicewithcold10mMCa（NO3）2whichhadbeenadjustedtopH4.0with0.5MHNO3.ThewashedcellswereresuspendedintheCa（NO3）2solutionataconcentrationofapproximately12mg（dryweight）ml-1andstoredfor2to4hat5°Cbeforeuse.TheCa（NO3）2solutionwasalsousedtomakeupallofthemetalsolutionsandactedasanionicstrengthbuffer.

Metalsorptionstudies.ThefourmetalsaltsusedinthisstudywereAgNO3，Cd（NO3）2·4H2O，Cu（NO3）2·2.5H2OandLa（NO3）2·6H2O.ThemetalsolutionswereadjustedtopH4.0with0.5MHNO3toavoidprecipitationofCuasCuCO3.IdentificationofthesemetalsinsolutionatpH4.0wasdonewiththeGEOCHEMcomputerprogram.AtpH4.0inaCa（NO3）2matrix,thesemetalliccationswerepredictedtobefoundprimarilyinthefreeionicform（>97%inallcases）.

Alloftheplasticwareusedinthesestudieswasleachedin3MHNO3andrinsedseveraltimeswithdouble-deionizedwaterbeforeusetoavoidmetalcontamination.Abatchequilibrationmethodwasusedtodeterminesorptionofmetalsbybacteria.Twomillilitersofwashedcellswasplacedina10-mlpolypropylenecentrifugetubecontaining6mlofcold10mMCa（NO3）2;and1mlofmetalstocksolutionwasadded.Thetubeswerecapped,placedonaninvertingshaker,andequilibratedfor2hat5°C.After2h,thecellswereremovedfromsolutionbycentrifugationandthesupernatantwascollectedandusedformetalanalysis.EquilibriummetalconcentrationsweredeterminedbyinductivelycoupledargonplasmaspectroscopyonaThermoJarrell-AshPlasma300spectrometer.Theamountofmetalremovedbythecellswasdeterminedonadry-weightbasis.Todeterminesorptionisotherms,finalmetalconcentrationsforCd2+were1,0.1,0.01,and0.001mM.InitialexperimentsindicatedthatsorptionofCu2+fromthe0.001Mmtreatmentresultedinequilibriumconcentrationsbelowthedetectionlimit.Subsequently,themostdiluteconcentrationofCu2+usedwas0.005mM.SorptionofAg+andLa3+from0.01and0.001mMsolutionsalsotypicallyresultedinconcentrationsbelowthedetectionlimit.TheconcentrationsofAgandLaevaluatedwere10,1,0.1,and0.01mM.

Thesorptionexperimentwassetupasanunbalancedfour-by-fourlatticewiththreereplicationsovertime）.Eachblockwithinreplicatescontainedfourdifferentbacterium-metalcombinationsatallfourmetalconcentrations.Whenappropriate,thesorptionisothermswereconstructedbythemethodsoutlinedbyDaoetal.withtheGLMprocedureoftheSASstatisticalprogram.

Electronmicroscopy.Electronmicroscopywasdonetovisualizethelocationofmetalsonthebacterialcells.Cellswereequilibratedwith1mMmetalsolutionsasdescribedaboveandfixedfor30minatroomtemperaturein5%glutaraldehyde（EMgrade;PolysciencesInc.）containingthemetalofinterestataconcentrationapproximatelyequaltotheequilibrationconcentration.Thecellswerethenwashedfreeofglutaraldehydewiththemetalsolution,enrobedin2%Nobleagar（DifcoLaboratories）,dehydratedthroughanethanol-propyleneoxideseries,andembeddedinSPURR（PolysciencesInc.）.TheembeddedcellswerethinsectionedonaReichertUltracutEultramicrotome,andsectionswerecollectedonFormvarcarbon-coated200-meshcopperoraluminumgrids.Sectionsofmetal-treatedcellswerenotstained;theelectronscatteringprovidedbythesorbedmetalsactedasacontrastingagent.Somecontrolcellswerelightlystainedwith2%uranylacetatefor2mintoprovidebettervisualizationofthesecells.Electronmicros-copywasperformedat100kVonaPhilipsEM-400TequippedwithanEDAXenergy-dispersiveX-rayspectrometerinterfacedwithaTracorNorthernmultichannelanalyzer.Energy-dispersiveX-rayanalysiswasusedtoconfirmtheidentitiesofmetalsonthecells.

Affinityseriesdetermination.Tofurtherelucidatetheaffinityofthebacterialcellsforthesemetals,thecellswereequilibratedfor2hat5°Cinsolutionscontainingeitherthesinglemetalataninitialconcentrationof1mMorallfourmetalsat1mMeach.Asdescribedabove,allmetalsolutionsweremadeinpH4.010mMCa（NO3）2.Afterthecellswereharvestedbycentrifugation,metalsinthesupernatantweredeterminedbyinductivelycoupledargonplasmaspectroscopy.Theexperimentwasreplicatedthreetimes.DataweresubjectedtoanalysisofvarianceproceduresoftheSASstatisticalprogram,andmeanswereseparated