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PAGEBacterialSorptionofHeavyMetalsFourbacteria,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.MATERIALSANDMETHODSBacteriaandgrowthconditions.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)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,andmeanswereseparatedbytheleast-significant-differencemethod.RESULTSBacterialsorptionofCd2+andCu2+fromsolutionwasdescribedwellbythelinearizedFreundlichadsorptionisothermequation,log10S=log1OK+nlog1OC,whereSistheamountofmetaladsorbedinmicromolespergram,Cistheequilibriumsolutionconcentrationinmicromolesperliter,andKandnareFreundlichconstants.TheFreundlichconstantsforCd2+andCu2+sorptionbythefourbacteriaaregiveninTable1.TheconstantKrepresentsthepredictedquantityofmetalremovedinmicromolesofmetalpergramofdrycellsatanequilibriumconcentrationof1uM,andnistheslopeoftheisotherm.ExaminationofKvaluesforCdsorptionshowedthatthegram-negativebacteriumE.coliwasmostefficientatCdsorptionandP.aeruginosaalsotendedtosorbmoreCd2+thandidthegram-positivebacteria.B.subtilisremovedthemostCu2+atanequilibriumconcentrationof1uM.However,onlya1.9-folddifferenceinCusorptionwasobservedbetweenthemostandleastefficientbacteria,B.subtilisandB.cereus,respectively.RepresentativeplotsofadsorptionisothermsforCd2+andCu2+areshowninFig.1.Analysisofcovarianceindicatedthattheslopesoftheisothermsweredifferentforthefourbacteria;athighequilibriumconcentrations,P.aeruginosaandB.cereusweremostandleastefficient,respectively,atremovingCd2+andCu2+fromsolution.FreundlichisothermswerenotusefulfordescribingtheremovalofAg+andLa3+fromsolutionbecausethereweretoofewdatumpointsoverawiderangeofconcentrations.EquilibriumconcentrationsofLa3+werebelowdetectionlimitswhentheinitialconcentrationwas10uM.SeveraloftheobservationsforAg+equilibriumconcentrationswerealsobelowdetectionlimitsatthe10uMconcentration.Whentheequilibriumconcentrationwasbelowdetectionlimits,thetotalmetalboundonadry-weightbasiswascalculatedwiththeassumptionthatessentiallyalloftheAg+orLa3+insolutionwasbound.A10mMtreatmentwasincludedforthesemetalstoextendtheconcentrationrangeexamined.AgoodrelationshipwasfoundforremovalofAg+fromsolutionasafunctionoftheinitialAg+concentrationfrom10to1,000p.M(Fig.2).TherewerenosignificantdifferencesinAg+removalamongbacteria.SaturationofthecellswithAg+apparentlyoccurredinthe10mMAg+treatment,asthetotalAg+boundincreasedonlyabout2-foldovera10-foldincreaseinAg+concentration.SignificantdifferencesamongthebacteriaforLa3+removalwerefoundinthe1mMtreatmentwhen33,70,114,and144p.molg-1wereremovedbyB.cereus,E.coli,B.subtilis,P.aeruginosa,respectively(Fig.3).TherewerenosignificantdifferencesamongbacteriaforLa3+removalfromthe10or100uMsolutions.ThebacterialcellswereevidentlysaturatedwithLaatthe1mMconcentration,asverylittleadditionalLa3+wasboundbythesecellsfromthe10mMLa3+treatment(Fig.3).Silverwasremovedfromsolutionmuchmoreefficientlythanweretheothermetalsatthe1and0.1mMconcentrations(Table2).Anaverageof99%ofthetotalAg+wasremovedfromsolutioninthe0.1mMtreatment.Cadmiumwasboundbythecellstoamuchlesserextent,withonly12and23%ofthetotalCd2+removedfromthe1and0.1mMtreatments,respectively.Eveninthe0.001mMtreatment,anaverageof46%oftheaddedCd2+remainedinsolution(datanotshown).Electronmicrographsofmetal-treatedcellsshowedthatAgwasassociatedwiththecellprimarilyasdiscreteparticlesatornearthecellwallsofthebacteria,whethertheyweregrampositiveorgramnegative(Fig.4).Energy-dispersiveX-rayanalysisconfirmedthattheparticlesweresilver.Attemptswerealsomadetoidentifytheformofsilverintheparticlesbyusingselect-areaelectrondiffraction;however,theparticlesweretoosmalltoproduceusefultransforms.Lanthanumwasalsoeasilyobservedonthecellsinthinsections.TheboundLawasobservedasneedlelikeprecipitatesdepositeduniformlyaroundthecellwallperiphery(Fig.5).Energy-dispersiveX-rayanalysisconfirmedthattheboundmetalwasLa,andselect-areaelectrondiffractionanalysisindicatedthattheprecipitatewascrystalline.TherewasnoevidenceofuniformdispersalofeitherAgorLainthecytoplasm,indicativeofenergizeduptake.However,discreteAgparticleswereoccasionallyfoundinthecytoplasm(ca.1%ofthecells),generallynearthecellplasmamembrane,anditispossiblethatthesecellsrepresentnonviablebacteriawithinthepopulation.NeitherCu2+norCd2+providedenoughelectronscatteringtopositivelyidentifysitesofdeposition.Bothmetalswereevidentlydiffuselyscatteredthroughoutthecellwallsofthebacteria.Energy-dispersiveX-rayanalysisindicatedthatCuwaspresentmostlyinthecellwalls,althoughsmallamountswerealsodetectedinthecytoplasm.Cadmiumwasboundinsuchlowquantitiesthatnonewasdetectedinthinsections.Potassiumwasaconstantelementdetectedinallcells,andoneoftheKlinesintheenergy-dispersivespectrumoverlapsthatofCd(K=3.31keV;Cd=3.13keV)(32);thiscouldhaveobscuredaCdpeak.However,CdwasdetectedinwholemountsofcellsasabroadeningoftheKpeak(datanotshown).Theaffinityofthebacterialcellsforthesecationsatthe1mMconcentrationdecreasedintheorderAg+>La3+>Cu2+>Cd2+.Whencellswereequilibratedwiththemetalsindividually,sorptionsofCu2+andLa3+wereessentiallyequal.However,whencellswereequilibratedwithamixtureofallfourmetals,La3+sorptionalwaysexceededCu2+sorption(Table3).Bindingofeachofthemetalswasreducedwhentheothercationswerepresent,indicatingsomecompetitionformetal-bindingsitesonthecellsurfaces.Onaverage,thesorbedmetalwas61%Ag,22%La,13%Cu,and4%Cd.DISCUSSIONTheobjectiveofthisresearchwastoevaluatethemetalsorptioncapacitiesofselectedgram-positiveandgramnegativebacteriaandtodeterminewhetherFreundlichadsorptionisothermsmightbeusefulforevaluatingbacterialmetalbinding.Althoughadsorptionisothermshavebeentraditionallyusedtodescribestoichiometricsolute-solidinteractions,suchasadsorption,chemisorption,andionexchange,theyhavealsobeenusedtodescribetheremovalofvariouscationsfromarangeofsolutionconcentrationsbymicroorganismsandbacterialexopolymers.However,whenusingintactbacterialcells,onemustconsiderthatotherprocessesbesidessurfaceadsorptioncouldoccur.Thesealternateprocessesincludeactiveuptakeofthemetalsintothecytoplasmthroughnonspecificcationtransportsystemsandprecipitationofmetalatthecellsurface.Forexample,cadmiumhasbeenshowntobetransportedintocellsofB.subtilisviaanenergy-dependentmanganesetransportsystem.Thetermsorptionisusedheretoindicatethatmetalwasremovedbyoneormoreoftheseprocesses.WebelievethatFreundlichisothermsdescribedsorptionofcadmiumandcopperaccurately.Differencesinthesorptioncapacitiesforcadmiumandcopperwerepredictedamongthebacteria;however,thesedifferenceswererelativelysmall,particularlyatlowconcentrations.Thegramnegativebacteria,particularlyE.coli,removedmorecadmiumfromsolutionatlowconcentrations,aspredictedbytheFreundlichKconstants.Nogeneralizationsregardingdifferencesbetweengram-negativeandgram-positivebacteriaforcoppersorptioncanbemadeonthebasisofourwork.Atanequilibriumconcentrationof1uM,onlya1.9-folddifferenceinpredictedcoppersorptionwasobservedbetweenthemostandleastefficientbacteria,B.subtilisandB.cereus.Athigherconcentrations,themostefficientbacteriumforcoppersorptionwasP.aeruginosa.Theminutedifferencesbetweengram-positiveandgram-negativebacteriaareincontrasttoobservationsofmetalbindingbyisolatedcellwallsandenvelopesofthesebacterialgroups.BeveridgeandFyfe(3)reportedthatcellwallsofB.subtilisandB.licheniformisbound28to33timesmoreCu2+thandidE.colienvelopes.Atthesametime,theirB.subtiliswallpreparationscomplexedmuchmorecopperonamole-to-dry-weightbasisthandidthoseusedinthewhole-cellexperimentsreportedhere(2,990umolg-1;3).Therearegoodexplanationsforthis.Intactcellsareobviouslymuchmorechemicallycomplexthanpurifiedwallsorenvelopes.Mostofthedrymassofabacteriumresidesinthecytoplasmasadiversespectrumofproteins,nucleicacids,lipids,carbohydrates,andinorganiccomponents.Toxicmetalrarelybreachesanenergizedplasmamembraneunlessitpassesthroughspecializedporterstobedetoxifiedwithinthecellorrapidlypumpedoutagain(26).Theformermustfirstbeinducedandthenchromosomallyexpressed,whichisunlikelytohaveoccurredduringourexperiments.Accordingly,duringourexperiments,retentionofanenergizedmembranewouldensurenegligibleconcentrationsofcytoplasmicheavymetals,afactconfirmedbyenergy-dispersiveX-rayanalysis.SincemostCuwasassociatedwiththebacterialsurfaceandmostbacterialmasswasassociatedwiththecytoplasm,themetal-to-massratioofintactcellsincomparisonwiththatofpurifiedcellwallpreparationswouldtendtobelow.ThisargumentassumesthatCubindingisessentiallycontrolledbyinteractionwithnegativelychargedcarboxylandphosphorylgroupsonthecellsurfacewithlittleornoprecipitation.ThisassumptionissupportedbythegoodfitoftheCu(andCd)sorptiondatatotheFreundlichisotherms.Inadditiontothemetal-to-massconsiderations,itispossiblethatsomesurface-boundmetalwassloughedofftogetherwithsolublewallpolymeraswallturnoverandautolysisprogressed.Althoughmetal-bindingexperimentswereconductedatlowtemperaturesandtoxic-metalconcentrations,somemetabolicprocesses,suchaswallturn-over,wouldproceeduntilallautolyticenzymeshadbeendenatured.Thesloughed-offmetal-wallpolymerwouldbesolubleandfreeofthebacteria;thiscomplexedmetalwouldnotbedetectedasbacterium-boundmetalinourexperiments.MonitoringoftotalCdinsolutionbyinductivelycoupledargonplasmaspectroscopyandfreeCd2+byaCd-specificionelectrodehasindicatedlowerconcentrationsoffreeCd2+thantotalCd(M.D.Mullen,unpublisheddata).ThediscrepancybetweentotalCdandthefreeCd2+concentrationisindicativeofsolubleorganicCdcomplexesinsolution.
细菌吸附重金属对蜡状芽孢杆菌,枯草杆菌,大肠杆菌,绿脓杆菌四种细菌,通过批量平衡方法从溶液中去除Ag+,Cd2+,Cu2+,和La3+的能力进行研究。镉和铜吸附浓度范围在0.001到1毫摩尔每升(mmol/L),通过Freundlich等温线被描述。在Cd2+的和Cu2+的浓度均为1毫摩尔每升时,在金属去除上绿脓杆菌和蜡状芽孢杆菌分别是最有效和最无效的细菌。FreundlichK常数表明,在Cd2+的去除上大肠杆菌最有效的和Cu2+的去除上枯草芽孢杆菌最有效的。用细菌从溶液去除Ag+是非常有效的;从1毫摩尔每升的溶液中平均89%的总Ag+被去除,而只有12%的总Cd2+,29%的总Cu2+,和27%的总La3+,分别从1毫摩尔每升的溶液中被吸附。电子显微镜观察表明,La3+沉积在细胞表面上如针状,析出结晶。在细胞表面上,偶尔在细胞质中银沉淀如离散的胶状聚集体。无论Cd2+还是Cu2+都不能提供足够的电子散射,以确定吸附的位置。细菌清除这些金属的亲和力序列下降顺序为Ag>La>Cu>Cd。结果表明,细菌细胞能够结合大量不同的金属。吸附方程可用于描述细菌金属如镉、铜等金属的相互作用,但是,当金属沉淀出现时,这种方法可能并不充分。在土壤环境中的有毒金属离子的结局在很大程度上取决于这些金属离子与无机和有机表面的相互作用。金属阳离子与这些表面相互作用的程度,决定于在溶液中的金属离子的浓度,因此,可能运动进入地下水或通过植物吸收。已做的大量工作,目的是通过土壤和土壤成分来评估各种重金属的吸附或络合,如粘土和有机质组分。一个潜在重要的有机表面已经得到人们的关注即土壤微生物种群。土壤微生物通常与粘土和土壤微环境中的有机组分联系在一起,并预计将分享金属动态,通常是归因于这些组分。细菌具有很高的表面面积与体积比,正如一个严格的物理细胞界面,应该有一个从溶液中吸附金属的高容量。有证据表明,细菌细胞比干重的基础上的粘土矿物在金属去除效果上更有效。Kurek和同事观察到由一个副球菌sp.的死细胞吸附的Cd2+,当固体溶液的比例是细菌和粘土相同时,粘质沙雷氏菌大于蒙脱石。活细胞积累了相同数量的Cd2+,象粘土。几次调查表明,由藻类、细菌和真菌络合的金属离子的数量较大。通过孤立的革兰氏阳性和革兰氏阴性细菌细胞壁结合的金属也被评估。观察到革兰阳性细菌枯草芽孢杆菌、b.地衣的细胞壁结合几种金属的数量比革兰氏阴性细菌大肠杆菌的细胞被膜结合的多。我们对土壤环境中微生物作用的各种重金属的行为感兴趣。这项工作的目标是,确定两种革兰氏阳性和两种革兰氏阴性菌的完整细胞的金属结合能力,以确定是否是一个均衡模型,符合Freundlich吸附等温线,就充分说明细菌吸附金属。研究蜡状芽孢杆菌、枯草芽孢杆菌和绿脓杆菌作为从土壤中经常分离的常见品种的代表。大肠杆菌也被用来作为第二种革兰氏阴性菌,因为它是一个有良好的特点的微生物,其细胞被膜已经被证明比枯草芽孢杆菌细胞壁绑定的金属少。在本次调查中使用的四种金属离子为Ag+、Cd2+、Cu2+和La3+。镉和铜都是环境确定的毒离子。个别的单价和三价重金属代表的银和镧,也有毒但不经常在环境中发现的。物料和方法细菌的生长条件。在这些实验中使用的细菌是蜡状芽孢杆菌ATCC11778、铜绿假单胞菌ATCC14886,获得都来自美国典型培养物保藏;枯草芽孢杆菌168和大肠杆菌K-12strainAB264,都从圭尔夫大学获得。细菌是在5倍脑心浸膏培养基(洗液微生物系统)修改的HEPES2.4克(N-2-hydroxyethylpiperazine-N-2-乙磺酸)2.0g/L的MES(2-Nmorpholinoethanesulfonic酸)1升缓冲介质酸度中常规培养成的。用0.5mol/LKOH中等酸度调整pH值为6.8。在3升的锥形瓶中两个2毫升的指数后期阶段发酵样本被用来接种800毫升肉汤。细胞后期指数阶段是生长在室温下150转的轨道摇床上(约23℃)。以这种方式种植的蜡状芽孢杆菌细胞仍保持在营养状况。收获的细胞,通过离心和用0.5mol/L的硝酸调整pH为4.0的冷的10mmol/L的Ca(NO3)2洗涤2次。洗涤细胞被悬浮在浓度为12mg/mL(干重)的Ca(NO3)2溶液中,并储存在2-5°C下4h后再使用。Ca(NO3)2溶液也被用来补足所有的金属溶液,并作为离子强度缓冲液。金属吸附的研究。在这项研究中使用的四种金属盐类是AgNO3,Cd(NO3)2·4H2O,Cu(NO3)2·2.5H2O和La(NO3)2·6H2O。金属溶液是用0.5mol/L的硝酸调整pH值至4.0,以避免从CuCO3析出Cu。在pH4.0的溶液中完成这些金属的鉴定是通过GEOCHEM计算机程序。在pH值4.0的一个CA(NO3)2基质中,对这些金属离子进行了预测,发现主要是自由离子的形式(在所有情况下>97%)。在这些研究中使用的塑料制品是被浸在3mol/L的硝酸中,并用双去离子水冲洗数次,在使用前要避免金属污染。一个批次的平衡方法被用于确定由细菌吸附的金属。2毫升的洗涤细胞被放置在含有6毫升的冷10mmol/L的Ca(NO3)2的10毫升聚丙烯离心管,并添加1毫升的金属储备溶液。试管被限制放置在一个反相振动筛上,并在5°C下平衡2h。两小时后,通过离心从溶液中分离出细胞,并收集上清液,用于金属分析。均衡金属浓度是通过电感耦合氩等离子体光谱在Thermo贾雷尔灰等离子300光谱仪被测定。在干重的基础上,金属量的去除是通过细胞被确定的。为了确定吸附等温线,最终Cd2+的金属浓度分别为1,0.1,0.01,0.001mmol/L。初步实验结果表明,从0.001mmol/L处理中吸附的Cu2+会导致平衡浓度低于检测限。随后,Cu2+的使用最稀的浓度为0.005mmol/L。从0.01和0.001mmol/L的溶液中吸附的Ag+和La3+也通常导致浓度低于检测限。银、镧评价的浓度为10、1、0.1、0.01mmol/L。作为一个不平衡的四四格成立的吸附实验,随着时间的推移,重复三次。在所有四种金属的浓度中每个区组内重复包含了四个不同的细菌金属组合。在适当的时候,通过DAO等概述的方法和方差分析的统计程序程序构建吸附等温线。电子显微镜。通过电子显微镜可观察到细菌细胞中金属的位置。如上所述,使用1mmol/L的金属溶液均衡细胞,固定30分钟,在室温下5%戊二醛(EM级;Polysciences公司)含有金属的浓度约等于平衡浓度。接着用戊二醛的金属溶液洗涤细胞,穿过2%琼脂(琼脂实验室),脱水通过乙醇—环氧丙烷氧化物,并嵌入在SPURR中(polysciences股份有限公司)。在赖克特Ultracut电子超薄切片机上对嵌入式细胞进行轻薄切片,并在高聚物涂层200目铜或铝格栅上收集切片。部分金属处理的细胞不着色;作为一个对比药剂通过吸附金属来提供电子散射。有些抑制的细胞被淡染,用2%醋酸铀淡染2分钟,以提供更好的可视化效果。电子显微镜在100千伏的配有EDAXX射线能量分散光谱仪和TracorNorthern多通道分析仪的飞利浦EM-400下进行的。能量色散X射线分析被用来确认细胞中金属成分。亲和系列的制定。为了进一步阐明细菌细胞对这些金属的亲和力,在初始浓度为1mmol/L单一金属或所有的四种金属浓度为1mmol/L溶液中放置2小时5摄氏度温度下,细胞达到均衡。如上所述,所有金属的溶液是在PH值为4的10mmol/LCa(NO3)2下被制成的。离心获得细胞后,通过电感耦合氩等离子体光谱测定上清液中的金属。实验重复三次。运用SAS统计程序的方差程序,和最少显著差法分离手段进行数据分析。研究结果通过Freundlich吸附等温方程线性化,更好地描述细菌从溶液中吸附Cu2+和Cd2+的情况,log10S=log10K+nlog10C表1由细菌吸附Cd2+和Cu2+的Freundlich等温线logK是截距,n是回归线的斜率。常数K代表在1umol/L的平衡浓度下微摩尔每克吸附的金属量(logC=0)。这里S是微摩尔每克吸附的金属量,C为微摩尔每升平衡溶液的浓度,K和n是Freundlich方程常数。表1中给出的四种细菌吸附Cd2+和Cu2+的Freundlich方程的常数。常数K代表预计在1umol/L的平衡浓度下金属微摩尔每克干细胞中移除的金属量,n为等温线的斜率。镉吸附的K值的检查结果显示,革兰氏阴性菌大肠杆菌在Cd吸附上是最有效的,绿脓杆菌也倾向于比革兰氏阳性菌吸收更多的Cd2+。1umol/L平衡浓度下枯草杆菌去除Cu2+最多。然而,在铜的吸附中只有1.9倍的差异,观察之间的最高和最低的有效的细菌,分别是枯草芽孢杆菌和蜡状芽孢杆菌。Cd2+和Cu2+的吸附等温线代表图,如图1所示。协方差分析表明,等温线的斜率分别是不同的四种细菌;在高平衡浓度下,从溶液中去除Cd2+和Cu2+最高和最低有效分别是绿脓杆菌和蜡状芽孢杆菌。图1通过蜡状芽孢杆菌和绿脓杆菌吸附镉(a)和铜(b)的Freundlich等温线。虚线代表等温线约95%置信区间。图2从溶液中去除银作为初始银浓度的函数。在浓度为10到1000umol/L的范围内,从所有基准点最小二乘法回归分析所得的线是logy=-0.446+0.980logx,r^2=0.986。去除La3+的细菌之间有着显著差异,发现在1mmol/L的处理时,蜡状芽孢杆菌、大肠杆菌、枯草芽孢杆菌、绿脓杆菌,分别去除33、70、114和144umol/g(图3)。从10或100umol/L的溶液中去除La3+的细菌之间没有显著差异。在1mmol/L浓度中细菌细胞与La明显饱和,正如从10mmol/L的La3+处理中很少额外的La3+被这些细胞绑定(图3)。图3由细菌从一个浓度范围内吸附的镧。线代表标准误差的平均值。在1和0.1mmol/L的浓度(表2)的溶液中去除白银比其他金属更有效。在0.1mmol/L处理中平均99%总的Ag+从溶液中被去除。镉由细胞的绑定程度要小得多,从1mmol/L和0.1mmol/L的处理中去除总Cd2+,分别只有12%和23%。即使在0.001mmol/L的处理中,溶液仍保持着平均46%的添加Cd2+(数据未显示)。表2从1和0.1mmol/L的Ag+、Cd2+、Cu2+和La3+的溶液中去除金属绑定的数量和总金属百分比吸附是指在P=0.05时通过最显着的差异的方法在遵循的列内,相同的字母都没有显著不同。括号内数字为总金属去除的百分比。这是所有测试细菌的平均数据。图4透射电子显微镜枯草杆菌细胞与1mmol/LAg+平衡。箭头表示与细胞相关的银聚集体。相似细胞的能量色散X射线分析证实,沉淀物组成是银条,100纳米。金属处理的细胞的电子显微镜照片显示,Ag与细胞相关联的主要是离散粒子,达到或接近细菌的细胞壁,不管他们是革兰氏阳性或革兰氏阴性(图4)。能量色散X-射线分析证实了其颗粒是银。也作了尝试,在颗粒中使用选择区电子衍射以确定银的形式;然而,颗粒太小,无法产生有用的转化。在细胞薄片上也很容易观察到镧。绑定的镧被观察到作为针状沉淀均匀的沉积在细胞壁外围(图5)。能量色散X-射线衍射分析证实,绑定的金属是镧,选择区电子衍射分析表明,沉淀是结晶。没有证据说明银或镧均匀分散在细胞质中,象征着被加强的吸收。然而,在细胞质中偶尔发现了离散的Ag颗粒(约1%的细胞),一般靠近细胞质膜,并有可能这些细胞代表无生存能力的细菌数量。图5透射电子显微镜铜绿假单胞菌细胞与1mmol/L的La3+平衡。相似细胞的能量色散X射线分析证实,沉淀物组成是La。无论是Cu2+或Cd2+都不能提供足够的电子散射肯定的确定沉积的部位。这两种金属显然是弥漫分布在整个细菌的细胞壁上。能量色散X射线分析表明,在细胞壁上目前主要是是铜,虽然在细胞质中也发现了少量的铜。检测薄片发现镉在这样低的数量下没有被绑定。钾是在所有细胞中检测到的常量元素,Cd在能量分散谱上和一个K线重叠(K=3.31千电子伏;CD=3.13千电子伏)(32);这可能会使一个Cd的顶峰被掩盖。然而,在整个支架的细胞中镉被检测到一个扩大的高峰(数据未显示)。这些离子的细菌细胞的亲和力在1mmol/L浓度中下降的顺序是Ag+>La3+>Cu2+>Cd2+。当细胞与单一金属平衡时,吸收的Cu2+和La3+本质上是相等的。然而,当细胞与所有四种金属的混合物平衡时,La3+吸附作用总是超过Cu2+的吸附作用(见表3)。当其他阳离子存在时,每种金属的结合减少,说明一些金属结合位点的竞争是在细胞表面上。平均而言,吸附金属为61%Ag,22%La,13%的Cu,和4%的Cd。表3从溶液中吸附金属,包含一种单一的金属或所有四种金属分离方式可以用下列公式计算:最显着的差异(0.05)=11umol/g。这些数据是所有四种细菌的平均值。每个金属的初始浓度为1mmol/L。讨论这项研究的目的是评估选定的革兰氏阳性和革兰氏阴性菌的金属吸附能力,并确定Freundlich吸附等温线是否可能被用于评估细菌金属的结合。虽然吸附等温线传统上被用来形容化学计量的固体溶质相互作用,如吸附、化学吸附、离子交换,但是在一定溶液浓度范围内它们也被用来形容通过微生物和细菌胞外聚合物去除各种离子的作用(17,14,23,24)。然而,当使用完整的细菌细胞时,除了可能发生表面的吸附外必须考虑其他进程。这些交替的过程,包括通过非特异性阳离子运输系统主动地吸收进入细胞质中的金属,和沉淀在细胞表面的金属。例如,镉已被证明是通过一个能源依赖锰的运输系统被运送到枯草杆菌的细胞之中(18)。这里使用的吸附表明,金属是在一个或多个这些进程中被去除。我们相信,Freundlich等温线描述镉和铜的吸附是准确。对细菌之间进行了预测,镉和铜的吸附能力存在着差异,但是,这些差异相对较小,尤其是在低浓度时。革兰氏阴性菌,特别是大肠杆菌,在低浓度的溶液中去除更多的镉,这符合FreundlichK常数的预测。在我们工作的基础上,对于铜的吸附,没有概括革兰氏阴性菌和革兰氏阳性菌之间的差异。平衡浓度为1mmol/L时,预计铜的吸附中只有1.9倍的差异,观察的最高和最低的有效的细菌,分别是枯草芽孢杆菌和
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