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The interaction of sediment bacteria with arsenic compounds Jaafar, Jafariah 1992-12-31

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THE INTERACTION OF SEDIMENT BACTERIA WITH ARSENICCOMPOUNDSbyJAFARIAH JAAFARB.A. California State University, Chico,1981M.Sc. University of Kentucky,1983A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESCHEMISTRYWe accept this thesis as conformingto the required standardTHE TJNIVEI’ITY OF BRITISH COLUMBIAFebruary 19920Jafariah Jaafar, 1992In presenting this thesis in partial fulfilment of the requirementsfor anadvanced degree at The University of British Columbia,I agree that theLibrary shall make it freely available for referenceand study. I further agreethat permission for extensive copying of this thesisfor scholarly purposesmay be granted by the Head of myDepartment or by his or herrepresentatives. It is understood that copying orpublication of this thesis forfinancial gain shall not be allowed without my writtenpermission.CHEMISTRYThe University of British Columbia2075 Wesbrook PlaceVancouver, CanadaV6T 1W5Date: 26 FEBRUARY 1992ABSTRACTIn general, bacteria are capable of biotransforming inorganic arsenicinto methylarsenic acids and arsines. The microbial activity of lakesediments was examined with respect to the mobilization of mine tailingsthat have a high arsenic content. Aerobic and anaerobic mixed microbialpopulations were isolated from Kam Lake, Yellowknife, N.W.T.An aerobic microbial population from 5 cm sediment depth, the layerimmediately above the contaminated mine tailings, was capable oftransforming arsenicals. Speciation of arsenicals in the culture medium,determined by using hydride generation - gas chromatography - atomicabsorption spectrometry (HG-GC-AAS) shows that this bacterial populationis able to methylate arsenicals and subsequently demethylate the product.However, only methylation was observed in media containingdimethylarsinic acid.Anaerobic microbial populations, from all depths, produce a yellowprecipitate upon incubation with arsenate for 10-14 days. The precipitatewas identified asA52S3 bymicroanalysis and scanning electron microscope +energy dispersive x-ray (SEM + EDX). The anaerobic microbial population,which should not contain sulfate-reducing organisms, appears to be arsenictolerant; there is no evidence of methylation of arsenic.11TABLE OF CONTENTSAbstractList of TablesVList of FiguresviList of AbbreviationsviiiAcknowledgementsixDedicationxChapter IIntroduction11.1 Arsenic11.2 Biotransformation of Arsenic21.2.1 Arsenite and Arsenate RedoxTransformation 21.2.2 Biomethylation ofArsenic41.3 Arsenic-Sulfur Compounds81.4 Methods for the Speciation ofArsenic Compounds 91.5 Microbial Sampling111.6 Scope of Work11Chapter IIExperimental142.1 Reagents and Chemicals142.1.1 Chemicals142.1.2 Chromatographic Supplies142.1.3 Prepared Solutions142.1.3.1 Arsenate, arsenite, monomethylarsonicacid anddimethylarsinic acid standard solutions142.1.3.2 Potassium borohydride solution152.2 Instrumentation152.2.1 Chambers for Incubationof Anaerobic Cultures 152.2.2 Gas Chromatograph172.2.3 Atomic AbsorptionSpectrophotometer (AAS) 172.2.4 Autoclave172.2.5 Microscope172.2.6 Scanning ElectronMicroscope + Energy Dispersive X-ray(SEM+EDX)182.3 Culture procedures182.3.1 Media Preparation182.3.2 Bacterial Isolation21‘U2.3.3 Bacterial Enrichment.232.3.4 Bacterial Preservation232.4 Arsenic Metabolism242.4.1 Aerobic Experiment242.4.2 Anaerobic Experiment252.5 Analytical Methods. 252.5.1 Hydride Generation-GasChromatography-Atomic AbsorptionSpectrometry (HG-GC-AAS) Hydride Generation272.5.1.2 Gas Chromatography272.5.1.3 Atomic AbsorptionSpectrometry282.5.2 Wet Digestion of theFreeze-dried Yellow Precipitate282.5.3 SEM + EDX29Chapter IIIResults and Discussion303.1 Microbial Sampling303.1.1 Study Site303.1.2 Kam Lake CoreDescription313.2 Cultivation and Subculture313.3 HG-GC-AAS343.3.1 CalibrationAnd Limit of Detection373.4 Aerobic Arsenic Metabolism403.4.1 Effect of Arsenate403.4.2 Effect of DMAA453.4.3 Summaryof Aerobic Metabolism483.5 Anaerobic Arsenic Metabolism493.5.1 Medium B503.5.2 Characterization ofAnaerobic Metabolic Product503.5.2.1 Mass Spectrometry503.5.2.2 Elemental Analysis513.5.2.3 Wet Digestionof Freeze-dried Yellow PrecipitateandFlame-AAS Analysis523.5.2.4 SEM + EDX523.5.3 Arsenic Speciationof Supernatant533.5.4 Summary of AnaerobicMetabolism54Chapter 1VBibliography56ivLIST OF TABLESTABLE PAGE1.1 pKa values of arsenic species andreduction products 102.1 Tryptic Soy Broth (Medium A) 192.2 “Arsenate-reducer” (Medium B) 203.1 Oxidation-reduction potential(ORP)measurement for core #6 takenat Kam Lake on 3 August 1991 323.2 Oxidation-reduction potential (ORP), pH and temperaturemeasurements for core #3 taken at Kam Lake on 3 August 1991. .333.3 Arsenic concentration in Medium B 503.4 Elemental analysis of yellow precipitate 523.5 SEM + EDX analysis of the yellow precipitateobtained from Kam Lake microorganism 533.6 Flame-AAS and HG-GC-AAS of supernatant mediumafter anaerobic growth of Medium B 54VLIST OF FIGURESFIGUREPAGE1.1 Structure of selected arseniccompounds 31.2 Challenger’s mechanism for the biologicalmethylationof arsenic61.3 S-adenosylmethionine (SAM)62.1 Map of Yellowknife Lakes System,NorthwestTerritories222.2 Schematic of HG-GC-AAS System263.1 Chromatogram for 4 mL of a standardsolution of10 ppb As(V), MMAA and 20 ppb of DMAA363.2 Comparison of effects of 4M HC1and 4M CH3COOH onDMA signal 373.3 Calibration curve forthe determination of totalinorganic arsenic383.4 Calibration curve for the determinationof arsenite 383.5 Calibration curvefor the determination of MMAA 393.6 Calibration curvefor the determination of DMAA 393.7 Inorganic As (D),MMAA (•) and DMAA (•) productionby 5 cm aerobic heterotrophs from Kam Lakesediments423.8 Inorganic As (11), MMAA (+)and DMAA () productionby 9 cm aerobic heterotrophs from Kam Lakesediments423.9 Inorganic As(Ii), MMAA (4) and DMAA (•) productionby 13 cm aerobic heterotrophsfrom Kam Lakesediments433.10 Production of AsH3 inBlank (D)and Control (4) 443.11 Day-to-day variation oftotal arsenic in the 5 cm aerobicheterotrophs incubation443.12 Productionof AsH3(U), CH3AsH2(+) and (CH3)2AsH (I) bythe5 cm aerobicheterotrophs in MediumD443.13 Production ofAsH3 (U) from Blank and (CH3)2AsH(+)from Control media463.14 Trimethylarsineproduction from 5 cmmicrobialpopulation in MediumD483.15 Mass spectrumof yellow precipitate.Spectrum obtainedat330°C51viiLIST OF ABBREVIATIONSHPLC High performanceliquid chromatographyAAS Atomic absorptionspectrometryGC GaschromatographyICP Inductively coupledplasmaMMAA MonomethylarsonicacidDMAA DimethylarsinicacidTMAO TrimethylarsineoxideMMA MonomethylarsineDMA DimethylarsinePRAS Pre-reducedanaerobically sterileTSB Tryptic SoyBrothTSA Tryptic SoyAgarSEM Scanning electronmicroscopeEDX Energydispersive x-rayORP Oxidationreduction potentialpE - log of electronactivityr1uACKNOWLEDGEMENTI am grateful to my supervisors,Professors W.R. Cullenand K.J.Reimer, for their guidancethroughout the course of thiswork.I wish to give special thanksto Mr. Gary Hewitt forhelping me in thebacterial isolation and introducingme to the microbial world,and to Ms.Deepthi Hettipathirana for helpingme with the thesis stylesheet.Their timeand patience spent duringthis work are very much appreciated.I thank Ms. Eileen Cochienfor helping me with the bacterialpreservation during Summer1991, and Mr. Hao Li fortrouble shooting theHG-GC-AAS system. Ialso thank Ms. MaryMager for teaching me theSEM+EDX. Thanks alsoto all my lab colleagues for theirfriendly andhelpful endeavors.Finally, I thank the Public ServicesDepartment, Malaysia for thefinancial support and UniversitiTeknologi Malaysia for granting methestudy leave.ixDEDICATIONAffandi, Anna and AzmanxCHAPTER IINTRODUCTION1.1 ARSENICArsenic ranks as the twentiethmost abundant element in theearth’scrust.1 It is widely dispersedat low levels, rangingfrom 0.1 to severalhundred ppm (jiglg), dependingon several factors such asgeographicallocation and anthropogenicinput.2 The average arsenicconcentration in theearth’s crust is about 3 ppm,1but much higher concentrations,ranging from0.4 to 6000 ppm,are found in sulfur containingores that containarsenopyrite (FeAsS), realgar(As4S)and orpiment (As2S3).Arsenic hasbeen found to be a commoncontaminant of both freshwaterand seawater inmany parts of the world.Arsenic compounds have differentproperties and uses.4-5 Arsenictrioxide is well known as a poison.Both inorganic and organicarseniccompounds were widely usedin medicine.6 Arsenic, like mercury,has beenused extensively in agricultureas herbicides and pesticides,and in industrialapplications as wood preservatives.High arsenic concentrations in soilshavebeen related to the mining industries,in particular, gold mining.It is recognized thatthe toxic effects of arsenicdepend on theconcentrations of individualarsenic species, so the developmentof analyticaltechniques that enable thespeciation of arsenicals has becomean importantobjective for individuals andorganizations interestedin the biogeocheniistryof the element.11.2 BIOTRANSFORMATION OF ARSENICThree types of arsenic biotransformation have been found to occur inthe environment: (1) oxidation and reduction between inorganic arsenite,As(III) and arsenate, As(V);(2) biomethylation of arsenic compounds tomethylarsenic acids and the more volatile and toxic methylarsines;(3)biosynthesis of more complex organoarsenic compounds, suchasarsenobetaine, arsenocholine and arsenosugars. This third class oftransformation is carried out by organisms higher up along the food chain,and not, apparently, by microorganisms. However, Hanaoke et al.7,reportedthe degradation of arsenobetaine to trimethylarsine oxide anddimethylarsinic acid, probably by the intestinal microorganisms in the chitonand only in an aerobic medium.A list of arsenic compounds is shown in Figure Arsenite and Arsenate Redox TransformationIn the aquatic environment, arsenic exists in two different oxidationstates. In oxygenated water, arsenate is the dominant species, existing asHA5O42-. Arsenite becomesthe main species in anoxic basins and inlets, forexample, Saanich Inlet, B.C.8In air saturated waters the redox condition is set by the02/1120couple, with a pE of about 12.6.9 Thermodynamic calculationsfor anoxygenated seawater at pH 8.1 predict the As(V)/As(III) ratio to beabout1026.10However, significant amounts of arsenite are found in oxygenatedsurface waters, and conversely, arsenate is found in anoxic waters.2HO—As—OHHO—As—OHOHOHArsenate ArseniteO0IIIIHO — As—CR3CH3 As— CH3IIOHOHMonomethylarsonic acid Dimethylarsinic acidCH3oCH3CH3— Ask— CR2— C - OCH3— As— CR2— CH,- OHCH3 CR3Arsenobetaine Arsenocholine1aYOH, R0SO3HIbY=OH. R=OHIcY=OH, R=SO,HMe2 CM2o .0—CH2—CH(Y)—CHRId Y NH2. R = SO3HH’\/HHIc YOH, R0P—0CH2OHOHOH CHOHCH,OHArsenosugars (1)Figure 1.1 Structure of Selected ArsenicCompounds3Studies11’12 found the As(V)/As(III) ratio to bein the range of 0.1 to100, suggesting that the redox pair is notin thermodynamic equilibrium. Inoxygenated seawater, bacteria and phytoplanktonhave been shown to beresponsible for the presence of arsenite, byreduction of arsenate.12-14Johnsonl2 showed that a mixed population of marine bacteria carriesout thisreduction in seawater medium.Arsenite is slowly oxidized to arsenatein seawater, even under sterileconditions.15 Some bacteria are also responsiblefor this oxidation, andTurner et al.l6 in 1954 demonstratedthat bacteria from arsenical cattle-dipping fluids could oxidize arsenite toarsenate. The processes that controlthe oxidation of arsenite to arsenatein natural waters still need furtherstudy.1.2.2 Biomethylation of ArsenicThe biomethylation of arsenic compoundshas been known for manyyears; however, interest in this phenomenonhas increased, in associationwith the discovery that microorganismsin natural lake sediments are able tomethylate mercury to form a highly toxicmethylmercury species.17 Thediscovery that mercury is readily cycledfrom the sediments and concentratedby organisms along the food chain18,has led to speculation that similarcycles may occur with arsenic.Certain fungi, yeasts and bacteriaare known to methylate arsenic tovolatile arsines. Poisoningincidents resulting from fumes emittedfrom wallpaper coverings were firstinvestigated by Gosio in1892.19 He identified one4mould species, Penicillium brevicaule (current name: Scopulariopsisbrevicaulis) as the producer of the gas which has a characteristic garlic odor,and incorrectly identified the gas as diethylarsine,(C2H5)2A5H.Challenger in 1932 correctly identified the gas as trimethylarsine,which was emitted by the mould S.brevicaulis growing on bread in thepresence of arsenic compounds.19 Challenger and coworkers furthersuggested that methylarsonic acid and climethylarsinic acid wereintermediates in the production of trimethylarsine. He favored thehypothesis that the methylation of arsenic involved the transfer of a methylgroup from some already methylated compound such as betaine, choline ormethionine. Challenger proposed a mechanism for the conversion of arsenateto trimethylarsine, shown in Figure 1.2. The arsenic (III) intermediates inbraces are unknown. His proposed mechanism involved alternatingoxidation and reduction steps.du Vigneaud and coworkers’ study on transmethylation2O ledChallenger to further study trimethylarsine production by S.brevicaulis inthe presence of labelled precursors.21 Only14CH3-labelled methionine wasfound to transfer its label to arsenite to a significant extent. The resultindicated that an “active methionine”, S-adenosylmethionine(SAM), Figure1.3, is the methyl donor.Work by Cullen and co-workers.22-25supported the idea that SAM isthe source of CH3+. They demonstrated that the CD3 group in Lmethionine-methyl-d3, CD3SCH2CH2CH(NH2)COOH, is transferred intactby cultures of S. brevicaulis.52e[)H3AsO4As(OH)3CH3AsO(OH)2arsenite monomethylarsonjeacidCH3AsO(OH)2{CH3As(OH))‘(CH3)2AsO(OH)dimethylarsinic acid(CH3)2AsO(OH)2e((CH3)2As(OH))[cH3](CH3)AsOtrimethylarsine oxide(CH3)AstrimethylarsineFigure 1.2 Challenger’s mechanismfor thebiological methylationof arsenic— a—c2—c2—HO IFigure 1.3 S-adenosylmethionine(SAM)arsenateOH6Cox and Alexander26isolated microorganismsfrom soil and sewageincultures containingvarious arsenic compounds.The organisms wereable toproduce trimethylarsinefrom arsenite, arsenate,methylarsonic acidanddlimethylarsinic acidwhen grown at pH 5.They suggested thatthe acidiccondition in sewage might promotetrimethylarsine production.Shariatpanahiand coworkers27 studiedbacteria that wereisolatedfrom the environmentand grown in the presenceof arsenate ( lOOug/mL).Under aerobic conditions,they found the transformationof arsenate todimethyl and trimethylspecies.In experiments withlake and river sediments usingpure cultures ofthe bacteria Aeromonasand Flavobacterium sp.and Escherichia coli,Wonget al.28 observedthe production of monomethylarsonicacid, dimethylarsinicacid and trimethylarsineoxide. They also observedthe production ofdimethyl- and trimethylarsines.The sediments wereincubated with orwithout high levelsof arsenic (at least threeorders of magnitudehigher thana typical non-polluted environment).These results werequestioned byAndreae,5who suggestedthat this biomethylationis a response to extremearsenic stress, and not ageneral process carriedout by aerobic bacteriain theenvironment.Additional work is requiredto understand the biomethylationprocess.The mechanism proposed byChallenger seemslikely to occur in the aquaticenvironment withthe reductionof arsenate and methylationtomonomethylarsonic acidand dimethylarsinicacid. Trimethylarsineis notalways the end product.7The biological function of methylated arseniccompounds is unknown,however, it is possible that the methylationserves as a detoxification process.Ferguson and Gavis29 suggested thatin an anaerobic environment, it maybeenergetically favorable forthe organisms to transmethylatearsenic (andother metals) rather than tosynthesize methane. This methylationmighttake place in the upper layerof the sediments. This processmight beresponsible for mobilizing methylarsinesfrom sediments to solutions andtothe food chain.1.3 ARSENIC-SULFUR COMPOUNDSIn anoxic water systems, bacterial actionwould create a reducingenvironment and the pE will be set bythe reduction of S042 to HS.9Underthis reducing condition (pE valueapproximately -4) arsenite is the dominantspecies. Stable arsenic sulfides,realgar (As4S)and orpiment (As2S3),willprecipitate in the presence of sulfurunder acidic anoxic conditions.3°Microorganisms play an importantrole in geochemical processes, andin the cycling of sulfur.Ehrlich31’32investigated the degradation ofsulfideminerals, orpiment and arsenopyrite, by microorganisms.The rate of arsenicreleased by bacterial oxidation wastwice as great as by spontaneousoxidation. The bacterial actionreleased arsenate and arsenite. Thebiodegradation process results inthe solubilization of metal compounds andcan be applied practicallyfor metal recovery.33 Anotherrole ofmicroorganisms is inthe partial generation of sulfide minerals.However,limited study has been donein this area.81.4 METHODS FORTHE SPECL4TION OFARSENIC COMPOUNDSTotal arsenic hasbeen monitored in environmentalsamples by usingmany analytical techniques:atomic absorptionspectrometry,3438atomicemission spectrometry,39voltammetry,40andneutron activationanalysis.41Currently, there isan increased interestin determining the concentrationofindividual arsenicspecies, largely becauseof their differenttoxicities. Thetoxicity decreases inthe order: arsines> arsenite > arsenate>alkylarsenicacids > arsonium compounds> metallic arsenic.1A variety of techniquesfor the speciationof arsenicals has beenreported. Generally,hyphenated techniquessuch as HPLC-AAS,42HPLCICP-MS,43 GC-AAS,4446and GC-MS47 combinedwith selective hydridegeneration and cold-trapping,48’49are used. However,the problemsassociated with thesetechniques arelong analysis time and theirnon-routinenature.A number of arseniccompounds are volatile, such as arsine, or canbeconverted into volatilearsines by reductionwith sodium borohydride.Thereduction of arsenic compoundswith sodium borohydrideis pH-dependent,49and is related to thepKa of the individualarsenic acids (Table1.1). Bramanet al.38 investigatedthe effect of pHon the fraction of undissociatedarsenicacids present at equilibrium.A number of workers44’46have used the piCadifferences of arsenateand arsenite to speciatethem. However, Hinners50found that MMAA and DMAAinterfered with thedetermination of As(III)and As(V).9Anderson et al.49 investigated several factors that influencethearsenic species determination. They used a continuoushydride generationwith sodium borohydride and AAS for detection. Severalcombinations haveshown promising selectivity towards the reductionof As(IH), As(V), M.MAAand DMAA: (a) 5M HC1 for the determination of totalinorganic arsenic; (b)citric acid-sodium citrate buffer at pH 6for the determination of As(III); (c)0.1GM citric acid for the determination of DMAA.The volatile property of the arsines enabledthem to be removed fromsolution by sweeping them with an inertgas, collected on a liquid nitrogencold trap, and separated by using gas chromatography.The separatedarsines are then detected by a quartz-cuvetteatomic absorptionspectrophotometer.Table 1.1: pKa values of arsenic speciesandreduction productsSpecies piCa’s Reduction Reaction b.p.pH Product (°C)Arsenate 2.2 1 AsH3-55Arsenite 9.2 1-6 AsH3-55MMAA 4.1 1 CHAsH22DMAA 6.2 1-2 (CH3)AsH 35.6TMAO - 1-4 (CHAs 70101.5 MICROBIAL SAMPLINGMicroorganisms, eithermixed communitiesor single species,forlaboratory study are isolated bygrowing them under well-definedconditionsas cultures. Innature, bacteria invariablyoccur as mixed communitiescontaining more thanone kind of microorganism.In the present work,enriched culturesof a mixed microbial populationwere isolated using aselective liquid media containingarsenate. The enrichmenttechnique tendsto select for the microorganismwith the highest growth rate, bychangingconditions in favor ofthe desired organism.This is because of a directcompetition for nutrientsamong the mixed developingpopulation.Two techniques thatare frequently used for thecultivation of strictanaerobes are theroll tube technique, developedby Hungate,56 andtheanaerobic glove box technique.In the roll tube method, agarmedium isdistributed as a thinlayer over the internalsurface of test tubes chargedwith an anaerobic atmosphere.Specimens are streaked onthe surface of theagar layer on thewall of the tube. This techniqueprovides a low oxidation-reduction potential of -150mV and an oxygen-free atmosphere.Use of theglove box is more practicalsince all manipulationsare performed in thereducing atmosphere of thebox.1.6 SCOPE OF WORKArsenic has frequentlybeen concentratedin certain areas as a result ofanthropogenic activities.For example, gold miningoperations for yearsdumped arsenic as awaste by-productin their tailings. Mine sites,such as11those along the B.C.coast or those adjacent tolakes, frequently dumpedtailings directly intothese waters, andover time sediments richin arsenicbuilt up around theeffluent outflow.Recent improvementsin goldseparation methods havereduced arsenicconcentrations in effluentsandcleaner tailings nowlie on top of arsenic-ladenlayers.Bacteria appear to becapable of mobilizingdeposited arsenic intowater soluble arsenicacids and arsines. MMAA,DMAA, TMAO and thehighly toxic and volatilearsine and methylarsinesare known productsofbacterial metabolism.19, 26-28To initiate an investigationof microbial mobilizationof historicallydeposited arsenic, we decidedto see if mixed microbialcultures obtained fromsediments in a lake associatedwith the NercoConMine in Yellowknife,N.W.T. could metabolizearsenic. Kam Lake waschosen because it has a welldefined sub-stratumwith high arsenic concentration:51the layer resultedfrom a large spill ofprocess effluent thatwould normally havebeen treatedprior to disposal but wasinstead diverted to KamLake.In a typical sediment,there is a predictable progressionof nutritionalmicrobial typesthat result from thelower oxygen concentrationandincreasing reducing conditionas depth increases. Aerobesmay predominateat the surface, whereasa variety of anaerobictypes will predominatebeneaththe surface. In thepresent study, two microbialpopulations were initiallysought that would representaerobic and anaerobic microbialactivity. Eachmixed population wasassayed for its abilityto metabolize arsenateandDM.AA. Hydridegeneration - gas chromatography- atomic absorption12spectrometry (HG-GC-AAS) was the analyticalmethod used for arsenicspeciation and characterization.13CHAPTER IIEXPERIMENTAL2.1 REAGENTS AND CHEMICALS2.1.1 ChemicalsUnless otherwise stated, all chemicals were obtained from commercialsources and were of analytical grade. Tryptic Soy Agar (TSA) and TrypticSoy Broth (TSB) were obtained from Difco Laboratories.As2S3and A54S4were prepared by using literature methods.52,532.1.2 Chromatographic SuppliesTeflon tubing (1 mm i.d. by 600 mm length) was purchasedcommercially. Porapak PS (80/100 mesh) was obtained from WatersChromatography Division/Milhipore Corporation. Silyl 8, a GC columnconditioner, was obtained from Pierce Chemical Co.2.1.3 Prepared Solutions2.1.3.1 Arsenate, arsenite, monomethylarsonic acid and dimethylarsinic acidstandard solutions.Stock solutions of 1000 pg/mL As were prepared by weighing theappropriate amount of Na2HA5O4.7H20,NaAsO2, CH3AsO(ONa)2.6H20and (CH3)2AsO(OH) into separate 100.0 mL volumetric flasks, and diluting14with deionized water (Aquanetics Aqua Media System). The arsenate,arsenite and MMAA stock solutions were prepared as needed. The DMAAstock solution was prepared every two months.Intermediate stock solutions of 10 .tg/mL were prepared by serialdilution. The DMAA solution was prepared daily.A working solution of 10 ng/mL each of As(V), As(III) and MMAA, and20 ng/mL of DMAA was prepared daily by serial dilution. Potassium borohydride solution.A 4% solution was prepared by weighing 2.OOg of KBH4 in apolyethylene bottle. One pellet of sodium hydroxide was added. The solidswere dissolved in deionized water and the solution was diluted to 50.0 mL.2.2 INSTRUMENTATION2.2.1 Chambers for Incubation of Anaerobic Cultures.Incubation of anaerobes requires pre-reduced media and elimination ofoxygen from the growth environment. Oxygen can be scavenged within asealed jar by catalytic hydrogenation over a palladium catalyst.BBL anaerobic chambers (Becton Dickinson Microbiology) andchambers built by the Mechanical Engineering Service, Department ofChemistry, U.B.C. based on the design of Baich et al.54 were used inconjunction with BBL GasPak H2 plus CO2 generator envelopes for allanaerobic incubations. GasPak envelopes contain a tablet of sodium15borohydride and a tablet of sodiumbicarbonate plus citric acid. TogenerateH2and C02, a top corner is cut to allowaddition of 10 mL of water. Thewater is wicked through a channel- the time required is sufficient to allowchambers to be sealed before gas generation begins -to the two tablets, andupon contact the gases are evolved. The hydrogenreacts with oxygen over Pdcoated alumina pellets (118”; AldrichChemical Co.) held in a wirebasketsecured to the underside of the chamberheadplate to produce water. Theappearance of condensate on the chamberwalls is an indication of reactionprogress. Anaerobic indicator strips (GasPak)soaked with methylene blueindicator change from blue to colorless to confirmthe removal of oxygen.Carbon dioxide does not participatein the development of anaerobiosis but isa required nutrient for some anaerobes(e.g. methanogens).All anaerobic sample and culture manipulations wereperformed underconditions designed to minimize oxygenexposure. Initial sediment coresamples were acquired in sealed gravity coresampler tubes55 which werelater opened in a glove bag (Instruments for Researchand Industry) under anatmosphere of oxygen-free nitrogen.Subsequent culture manipulations wereperformed in a modified glove box(KSE, Adrian, Michigan) charged with agas mixture of C02 (5%), H2 (10%)andN2 (85%)and supplied with Pdcoated alumina pellets. Anaerobicindicator strips were used to confirmanaerobic conditions, and catalyst was replacedroutinely.162.2.2 Gas ChromatographTwo types of gas chromatograph were used: a HewlettPackard 5830Amodel with a 18850A GC terminal, and aHewlett Packard Series 5890.Ahome-packed column of Porapak PS was prepared.2.2.3 Atomic Absorption Spectrophotometer (AAS)The AAS was used as the detector for the GC effluent.A Jarrell AshModel 810 AAS (Fisher Scientific Co.)was used for arsenic speciation andwas equipped with Waters QA-1 Data System.Total arsenic was determined on Varian modelAA 1275 series. TheAA hollow cathode lamp was operating at 8mA and the monochromator wasset at 193.7 nm, one of the resonancelines of arsenic. The spectrophotometerwas equipped with a deuterium backgroundcorrector and a Hewlett Packard82905A printer.2.2.4 AutoclaveAmsco, Eagle Series-3041 Gravity autoclavewas used to sterilize allculture vessels and heat-stable media.2.2.5 MicroscopeThe microbial growth was observed byusing an Olympus BH-2microscope.172.2.6 ScpnningElectron Microscope+ Energy DispersiveX-ray (SEM+ EDX)A Hitachi S-570 ScanningElectron Microscope(SEM) was used. Itwas equippedwith a Kevex Super8000Energy-DispersiveX-ray Analyser(EDX). This instrumentis located in the Metalsand Materials EngineeringDepartment, UBC.2.3 CULTURE PROCEDURES2.3.1 Media PreparationAll aerobic media were preparedon the open bench,and sterile mediawere handled in aClass II (laminar flow)biohazard cabinet(MechanicalEngineering Services,Department ofChemistry, UBC). Pre-reducedanaerobically sterile(PRAS)5657 media wereprepared by flushingfilter-sterilized (glass woolpacked tube) nitrogen gasover the headspaceof theboiled media in an air-tightPyrex bottle.All transfers were donein an anaerobic glove boxcontaining palladiumcatalysts and underan atmosphere of 5% C02,10% H2 and 85% N2 (fromacommercial cylinder).A methylene blue indicatorstrip was used to checkforanaerobic condition.All glassware was flushedwith nitrogen gas beforebeing sterilized. All anaerobicincubations weredone in the anaerobicchamber.Arsenate augmentedTryptic Soy Broth at quarterstrength (MediumA) was preparedby adding 10.0mL of 100.0 j.ig/mL As(V)solution to 7.50 g of18the commercially availablepowder (Table 2.1) anddissolving this in 1000 rnLof distilled water.Ten mL aliquots of this brothwere pipetted into screwcaptest tubes (16 mmdiameter by 120 mm) andautoclaved at 121°Cfor 15 mm.The final pH of this mediumwas 7.0.Table 2.1 Tryptic SoyBroth (Medium A)Bacto Tryptone (PancreaticDigest of Casein)15 gBacto Sytone (PancreaticDigest of Soybean Meal)5 gSodium chloride5 g“Arsenate-reducer” medium (MediumB) was prepared fromtheingredients shownin Table 2.2. All these (exceptsodium bicarbonate)weredissolved in water anddiluted to 960 mL. Themedium was preparedPRAS,and autoclaved byusing a gravity cycle at 121°Cfor 15 mm., with no dryingtime. The sodium bicarbonatewas filter-sterilized byusing a 0.45 pmMillipore membranefilter (HV type). The autoclavedsolution was cooled,placed in the anaerobicglove box, and 40mL of the sterile sodiumbicarbonate was addedaseptically. The final pHof the medium was adjustedto 7.4 with 4M HC1 or NaOH.This medium was pipetted(10.0 mL portions)into screw cap test tubesand allowed to standovernight in the glove box.The tubes were then placedin the anaerobic chamberwhich was chargedwith CO2 and H2 from aGasPak envelope, and removedfrom the glove box.19Table 2.2 “Arsenate-reducer” (MediumB)Ingredients gILNaHCO3 2.5CaC12.2H0 0.1KC1 0.1NH4C1 1.5NaH2PO.H0 0.6NaCH3COO 2.7Difco yeast extract 0.05NaC1 0.1MgC12.6H0 0.1MgSO4.7H 0.1MnC12.4H0 0.005NaMoO4.2H 0.001FeSO.7H20 0.001NaHAsO4.7H 3.12202.3.2 BacterialIsolationThe microbiologicalsampling was done inthe field by Mr.Gary Hewittof Department of Chemistry,UBC and Dr. DougBright of RRMC.Sediment core samplestaken from Station 1,Kam Lake, Yellowknife,Northwest Territories(Figure 2.1) were obtained byhand lowering a gravitycore samplerfrom an aluminumpower boat. Plasticcore tubes for assayofpH, temperatureand oxidation-reductionpotential (ORP) andfor microbialsampling had 1cm holes drilled each2 cm of theirlength. The holes werecovered with ducttape for sampleacquisition and forsealed transport back tothe laboratory.Standard core tubeswere of 3.75 cm diameterand 60 to 80cm long. Coresfor microbial samplingwere transportedfrom Kam Lake tothe Department ofIndian and NorthernAffairs’ water testinglaboratory inYellowknife, and openedin a glove bag(Instruments for ResearchandIndustry) underan atmosphereof oxygen-free nitrogen.The holes wereuncovered andsediment samplesfrom different depthswere asepticallyremoved with asterile spatulaafter the layer closestto the wall wasremoved. Roughly1 mL of sedimentwas then inoculatedinto each testmedium.Cultures of aerobicheterotrophs, MediumA, were removed fromtheglove bag and incubatedwith loose caps. Culturesin “arsenate-reducer”medium (MediumB) were placed inan anaerobic chamberbefore removalfrom the glove bag.All cultures weretransported to the DepartmentofChemistry, UBCfor further work.21Subsequent culture manipulations were performed in a glove box asdescribed in section 2.2.1. Room temperature (approximately 21°C) was usedfor all incubations.Figure 2.1 Map of Yeflowknife Lakes System,Northwest Territories. B indicates samplingsite.222.3.3 Bacterial EnrichmentEnrichments weremade by serialsubculture. Themixed aerobicculture in MediumA was enrichedonce by transferring 1.0mL of the culture,to a sterile, 10.0mL of Medium A.Medium A was incubatedfor two days.One mL of the mixedanaerobic culturewas transferred,in eachenrichment step,into a sterile 10.0mL of Medium B.Medium B wasincubated in theanaerobic chamberfor 10-14 days betweeneach of the threeenrichment steps.2.3.4 BacterialPreservationThe two-day oldaerobic subculture wascentrifuged for 30mm at aspeed setting of 80in a Dynac Centrifuge (BectonDickinson Co.) andthesupernatant wasdiscarded. One mL eachof TSB (double strength)and 20%glycerol were addedto the bacterial cellto achieve a finalconcentration of10% (v/v) glyceroland single strength TSB.The cells were resuspendedand1.0 mL aliquotswere dispensed intocryovials (1.2 mL capacity, NUNC),precooled on dry ice,and finally storedin a cryogenic refrigerator(TaylorWarton, model 34 HC)charged with liquidnitrogen.Enriched anaerobic cultureswere centrifuged bythe same procedureand returned to theglove box. The supernatantwas discarded and 1.0mLeach of Medium Band 20% glycerol(PRAS) was added to resuspendthe cells.One mL of the cell suspensionwas dispensed intocryovials and sealed.Thetubes were removedfrom the glovebox, pre-cooledon dry ice andimmediately preservedin the cryogenic refrigerator.232.4 ARSENIC METABOLISM2.4.1 Aerobic ExperimentThe medium used wasTSB containing 50 ng/mLAs(V) solution(Medium C).Twenty mL portions ofthe sterile medium waspipettedaseptically into a series ofEvergreen tubes (50 mL capacity,polypropylenetube).The preserved aerobic heterotrophswere thawed and grown onTrypticSoy Agar (TSA) + As(V) plates. The plateswere incubated at roomtemperature for 2 daysto allow colonies todevelop. Approximately 7mL ofMedium C was added tothe plate and any growth wasscraped to resuspendit. The cell suspension waspipetted to a test tubecontaining Medium C andthe turbidity was measuredspectrophotometrically at420 nm with aSpectronic 20. Appropriatevolumes of the cell suspensionand Medium Cwere mixed to obtain anabsorbance reading of1.0. Two mL each of thestandardized inoculum wasadded to 20.0 mLMedium D in 7 Evergreentubes. The tubes wereincubated aerobically atroom temperature. Allcultures were shaken dailyto resuspend the growth.One tube wascentrifuged each day,and the ‘snift test’ wascarried out to detect theproduction of arsines, andthen the supernatant was transferredto a steriletube and frozen prior toanalysis for arsenic compounds.The solid phase andbacterial cells were discarded.The process was repeatedfor 7 days.Controls, containingMedium C, and blanks(TSB) were carriedthrough the same incubationcondition and frozen dailyfor arsenic analysis.24A second set of cultures wasprepared. In addition tothe 50 ppb As(v)in half strength TSB(Medium D), 50 ppb DMAAin half strength TSB(Medium E)were prepared. Standardizedinoculum of the aerobicheterotrophs were added toMedia D and E. The tubeswere incubated asbefore. One each of elevendays supernatant sampleswere frozen for arsenicspeciation. Controls (MediaD and E) andTSB blanks were similarlyprepared.2.4.2 Anaerobic ExperimentOne mL of the anaerobicculture solution fromthe third enrichmentstep was added to10.0 mL Medium B. The testtubes were incubatedin theanaerobic chamber for10-14 days. A heavyyellow precipitate formed andwas separated fromthe solution by centrifugationfor 30 mm at a speedsetting of 80. The precipitatewas washed threetimes with distilled water,freeze-dried and itscomposition was investigatedby using massspectrometry, elementalanalysis, and SEM +EDX. The supernatant wasanalyzed for arsenic compoundsby HG-GC-AAS.2.5 ANALYTICAL METHODS2.5.1 HydrideGeneration-Gas Chromatography-AtomicAbsorptionSpectrometry (HG-GC-AAS)Arsenic speciation studiesinvolved a selective hydridegeneration ofvolatile arsines, which werethen concentrated bycryogenic trapping. The25arsines were separated by gas chromatographyand detected by atomicabsorption spectrophotometer. The HG-GC-AAS systemis shown in Figure2.2.Figure 2.2 Schematic of HG-GC-AAS SystemTrapping Ar.cinesValveSampleLiquid N2TrapValve:He,“ Atomic Absorption•‘ Spectrometerdry-iceI alcoholWater TrapSSSSSWater BathDrainReaction Vessel Re1easin’ ArsinesGas Chromatograph262.5.1.1 Hydride GenerationOne to two mL of the sample solution was injected into the Teflonreaction vessel containing 50 mL deionized water and 4 mL, 4M HC1. Themixture was flushed with a helium flow for 5 mm. Four mL of 4% KBH4wasslowly injected into the vessel and the generated arsines were swept by ahelium flow through an acetone-dry ice trap to remove water and werecollected for 5 mm in a trap made of Teflon tubing immersed in liquidnitrogen. The liquid nitrogen was removed and the tubing was warmed in awater bath and the arsines were flushed onto the GC column. Gas ChromatographyA Porapak PS column was prepared as follows: A 600 mm length ofTeflon tubing was plugged at one end with a small piece of Teflon wool.Porapak PS (80/100 mesh) was packed into the tubing and the tube washand-spun for 5 minutes to ensure tight packing. The open end of thecolumn was then plugged with Teflon wool.To condition the column, one end of the packed column was connectedto the injection line, while the exit was left unconnected in the GC oven. Thecolumn was conditioned by flushing the column with helium gas as thetemperature was raised from 60°C to 150°C, at a rate of 300/min. Thetemperature was then held at 150°C for an hour and subsequently cooled to60°C. One end of the column was injected with 50mL of Silyl-8. The columnwas heated again at 150°C for 45mm. The column silanization was repeatedfor the other end of the column.27Temperatureprogrimming was used to enhance theseparation of thearsines, by the column andthe program used was as follows:Temp 1 60°CTime 1 6 secRate 30°C/mmTemp 2 150°CTime 2 3 mm. Atomic AbsorptionSpectrometryThe effluent from the GC wasdetected in a hydrogen-airflame in aquartz-cuvette of the AAS.Arsines produced frominorganic arsenic [As(V) +As(III)], MEMAA, DMAAand TMAO were detected.Standards arsenicalswere injected to establishthe retention times andthe standard calibrationcurves.2.5.2 Wet Digestionof the Freeze-dried YellowPrecipitateThe dried precipitate (5mg) was added to concentratednitric acid (3mL), concentratedsulfuric acid (1 mL) and 30%hydrogen peroxide (3 niL) ina 100 mL round bottomflask. The flask was fitted withTeflon stopper andair condenser. The flask washeated at reflux for 2 hours byusing a heatingmantle. The flask wasthen cooled to room temperatureand the digestedsample was diluted to100 mL with deionizedwater in a volumetric flask.Total arsenic was determinedby flame-AAS, aftercalibration with 0.5 to50ppm As(V) standards.282.5.3 SEM + EDXThe solid sample was fixed with carbon Dag (colloidal carbon in mixedalcohols) onto a spectrographic-grade graphite stub and the surface wascoated with carbon (200A) in a vacuum evaporator (Japan Electron OpticLaboratory).The stub was examined with a SEM using an accelerating potential of30 KeV and a working distance of 35 mm. The x-rays generated by theelectron beam were detected, identified and quantified by using the computerassociated software package (Kevex Quantex IV).29CHAPTER IIIRESULTS AN]) DISCUSSION3.1 MICROBIAL SAMPLING3.1.1 Study SiteAn investigation was initiated by Reimer and coworkers on theYellowknife Lakes system to investigate the speciation, redistribution andbioavailability of arsenic compounds from contaminated mine tailings.51 Aspart of this program, the microbial activity of lake sediments was examinedwith respect to the mobilization of mine tailings that contain a high arseniccontent. Details of the geochemical cycling or arsenic and various metals inthese lakes are presented in a report.51 Kam Lake was chosen as one studysite. It has a maximum depth of 12 meters. According to Wagemann et al.59the dissolved arsenic concentration ranges from approximately 2000 ppb tomore than 5000 ppb in the water column of Kam Lake. Bright andcoworkers51observed an arsenic concentration ranging from 2200 ppm in theupper layer of the sediment to about 50 ppm in deeper layers.The sediment of Kam Lake sampled by Bright and coworkers51in 1990showed a profile that runs from an oxygenated surface layer to a sub-onezone and finally an anoxic layer. This typical profile is likely to promote asequence of microbially mediated redox processes in which the aerobesdominate at the surface and anaerobes below the surface.303.1.2 Kam Lake Core DescriptionThe sediment core (core #6) that was used for the microbiologicalsampling was obtained on 3 August 1991. Table 3.1 gives a detaileddescription of the core at various depths. The temperature and pH of the corewere not recorded in order to maintain the sterility of the sample. However,a similar core (core #3) showed a decreasing pHwith depth (pH 7.4 - 6.7) andtemperature of 18°C to 20°C (Table 3.2).3.2 CULTiVATION AND SUBCULTUREOnce collected the cores were kept cool (0°C) for shipment to thelaboratory in Yellowknife. The core was then exposed and sampled in a glovebag under an atmosphere of oxygen-free nitrogen. Less than 8 hourselapsedbetween the time of sample acquisition and inoculation into test media inYellowknife. All cultures were transported back to UBC, Vancouver forsubsequent studies.The microbial growth in liquid media was monitored by visualobservation of physical changes. The aerobes took about 48 hours to producea turbid suspension. The anaerobes did not produce a turbidsolution,however, the formation of a heavy yellow precipitate was used as the growthindicator. It took about 10-14 days for the anaerobes to produce theprecipitate. The amount of precipitate formed decreased slightly with eachsubculture.31Table 3.1 Oxidation-reductionpotential(ORP) measurement for core #6taken at KamLake on 3 August 1991.Hole# SedimentDeptha(cm) ORP (mV)11-2 3-2253_5-4 7-42559-611 -358713-815 -328917-1019-1121 -36412--1325 -39214--1529-1631 -3770-8 cm: oxidized green-brownsediment of fine silt/sand (hole#1) givesway to an olive greenlayer just above patches of black(hole #3).8-16 cm: black patchesin a matrix at the top of the grey tailingslayer(hole #5): tailingszone with predominantlyblack interior of core with fibrousmaterial (hole #7).17 cm: transition band fromslate grey tailings to more browncolorednatural sediments beneath(hole #9).18-32 cm: brown colorednatural sediments: no dark patches,no H2Ssmell.-: no readings taken.a:Water depth 7 m.32Table 3.2 Oxidation-reductionpotential(ORP), pH and temperature measurementsfor core #3 taken at Kam LakeHole# SedimentORP (mV) pHT°CDeptha(cm)1 -- 7.3820.12 3-220 7.15 -3 5-290 7.00 -4 7-415 - -5 9-400 6.95 -6 11-336 7.00-7 13-363 7.1119.08 15-262 7.07 -917 -281 --10 19-270 - -11 21- - -12 23- 6.9719.113 25- - -14 27- 6.93 -15 29- - -16 31- 6.6918.2- : no readings taken.aWater depth 6.5 mSediment samples that were inoculatedinto Media A and B from 1, 5,9, 13, 19 and 29 cm depths,all showed growth. It is an interestingresultthat aerobic microbial growthoccurred at all depths. Theredox potentials ofthe sediment ranged from -225 to -425mV and at this low redoxpotential,aerobes are not expected tothrive.The anaerobes were also successfullycultivated from all depths. Thisis again surprising becauseMedium B has a redox potentialof 130 mV.33Addition of sediment and the metabolism ofbacteria in the anaerobicmedium could be expected to lower the redox potential but only slightly,andnot to the -300 mV necessary for strict anaerobes. Possiblythe mixedanaerobic population comprises obligate anaerobesat the surface ranging tostrict anaerobes at the bottom of the core. The anaerobes probablyhaveoxygen-protective enzymes to enable them to survive in theoxygenatedsurface layer.3.3 HG-GC-AASThe hydride generation technique was used for the determination ofarsenite, arsenate, monomethylarsonic acid, dimethylarsinicacid andtrimethylarsine oxide. The reducing system employed for arsineproductionis an alkaline potassium borohydride solution. It is customaryto use anaqueous solution of KBH4 (0.5-8% wlv) in NaOH medium. The reaction inacidic medium is:As(OH)3 + BH4- + H+ ---> A5H3 + B(OH)3+ H2.The excess of the reagent is decomposed by the acidic solution:BH4-+ 3H20 + H± ---> B(OH)3 + 4H2.Two main types of apparatus have been used for the hydridegeneration. In the continuous flow technique,28,46149a pump is used for thetransport of sample solution and KBH4 solution to a reaction coil where thehydride formation occurs. A continuous signal is observed by the AAS.In the batch type apparatus,596Othe KBH4 solutior ismixed with theacidic sample in a reaction vessel. Reaction takes place immediately and the34arsines are swept by an inertcarrier gas to the AAS.A discontinuous signalis observed by the AAS.The batch type system was usedin the present study to determinearsenic speciation. The reactionmixture is buffered to pH 0.5 with4M HC1and the addition of KBH4 reducedinorganic arsenic [As(III) + As(V)]toAsH3, monomethylarsonic acid(MMAA) to monomethylarsine (MMA),dimethylarsinic acid (DMAA) todimethylarsine (DMA), and trimethylarsineoxide (TMAO) to trimethylarsine (TMA).The reaction mixture issubsequently buffered topH 6 with 1M citrate in 10% citric acidwhereuponaddition of KBH4 converts arsenite[As(III)] only to AsH3. The arsenateconcentration can be determinedfrom the difference between the arsinesignals in the two bufferedsystems.The generated arsines (Table 1.1) arecollected by cryogenic trapping.Efficient and reproducible separationof the arsines is achieved byusing agas chromatographic column such as asilanized Porapak PS packedcolumn44 interfaced to the trap.Temperature programming is used toenhance the separation. The effluentfrom the GC is swept into aquartzcuvette of an AAS where it is combustedin a hydrogen-air flame.Retention times are usedfor the identification of arsines andpeakareas are used to calculate theconcentration of As(III), As(V), MMAA,DMAAand TMAO in the sample solutions.A typical chromatogram ofthe generated arsines is shown in Figure3.1. Arsine (AsH3) has a retentiontime of 0.96 mm, monomethylarsine(CH3ASH2)of 1.66 mm and dimethylarsine [(CH3)2AsHIof 2.41 mm.351210•C?rjCC?C6C?C?42-0 1 3Figure 3.1 Chromatogram for 4 mL of astandard solution of 10 ppb As(V), MMAA and20 ppb of DMAA.The dimethylarsine signal from the HC1 bufferedreaction is the leastreliable in terms of sensitivity and reproducibility, ashas been previouslyreported.44 This has been attributed to incompletereduction, condensationin the transport tubes or incomplete atomization.49I • I2Time (mm)36Changing the acid system to 4M acetic acid (pH 2.5) improvestheDMA signal (Figure 3.2) but the problem of long term reproducibilityremainsto be solved.804M Aceticc60•/40.4MHCI0 20 40 60 80DMAA Concentration (ng)Figure 3.2 Comparison of effects of 4M HC1and 4M CH3COOH on DMA signaL3.3.1 Calibration And Limit of Detection.Typical calibration plots of AAS peak area versus concentration(ppb)for inorganic arsenic [As(III) + As(V)], As(III), MMAA and DMAA are showninFigures3.3-3.6.3760C?C?[Inorganic As] (ppb)40Figure 3.3 Calibration curve for thedetermination of total inorganic arsenic50Figure 3.4 Calibration curve for thedetermination of arsenite504030200 10 20 300 10 20 3040As(III) Concentration (ppb)3880C)C)clFigure 3.5 Calibration curve for thedetermination of MMAAC)IC)rj)Figure 3.6 Calibration curve for thedetermination of DMAA600 10 2030M11AAConcentration(ng)408060402000 20 40 6080DMAA Concentration (ng)39The calibration curves show a linear dependence of the AASpeak forthe concentration range studied. The absolute limit of detection, definedasthe analyte concentration producing a signal equal to the blank plus threestandard deviation of the blank, is 0.12 ng(of As) for As(III), 0.25 ng forAs(V) and the methylarsenic acids.3.4 AEROBIC ARSENIC METABOLISMAccording to Bright et al.,51 the concentration of methylated arsenicspecies in porewater from a Kam Lake sediment core is highest(about 5.2ppb As) at a depth of 8-13 cm. From Tables 3.1 and 3.2, this layercorresponds to the deposited tailings from the mine.3.4.1 Effect of ArsenateThe mixed bacterial populations from 5, 9 and 13 cm depth werechosen for the initial assay of arsenic metabolizing activity. Theinoculumused was standardized by spectrophotometric measurement of the turbidityof a cell suspension. The mixed populations were grown in arsenicaugmented broth (50 ppb As(V) solution). Samples taken daily froma seriesof Evergreen-tube cultures over a period of 1-7 days were analyzed for arsenicspecies.Figures 3.7 - 3.9 show the results of the initial experiment. The mixedpopulation from the 5 cm depth (Figure 3.7) shows a pattern of methylationand demethylation. The concentration of added arsenate in the mediadecreases rapidly within 2 days, reaching zero by 5 days. The added arsenate40is transformed into MMAAand DMAA over the 7 daysof incubation.Theformation of DMAA increasesfrom day 1 to day 4, givingway to a progressivedecline on days 5-7. Thispattern of variation indicatesa shift fromproduction to decompositionof DMAA, indicatingthat demethylatingmicroorganisms are replacingthe methylators in the mixedcommunity.The 9 cm and 13cm microbial cultures showvery small amounts ofmethylated arsenic species(Figures 3.8 and 3.9). The amountof addedarsenate decreases withtime with no apparent transformationto MMAA orDMAA.Comparisons of the blankand control media (Figure 3.10)with thecultured media show that themixed microorganism from the 5cm depthhave a profound influenceon the biomethylation of arsenic.From Figure 3.7, the amountof added arsenic (50 ppb As(V)solution,which is equivalent to approximately95 units of peak area)is not recoveredfully in the supernatant analyzed.The total arsenic concentrationalso variesfrom day to day (Figure 3.11).The observed decrease could be dueto: (1) theadded arsenic could be transformedand adsorbed on the bacterialcells andthus lost when the solidphase was discarded after centrifugation;(2) uptakeof arsenic by the bacteriacould cause the concentrationto drop; (3) there areother species of arsenicin the supernatant thatare undetected by thehydride generation technique;(4) formation of volatilearsines, that escapedetection by the “snift test”.The “snift test” is based onthe intense, garliclike odor of the arsinesand arsine production canbe evaluated by cautioussniffing of a sampleof the headspace ofthe test tube.30,4441301r;j220100 2 4 6 8Incubation Time (day)Figure 3.7 Inorganic As (CD,MMAA (+) andJJMAA (•) production by 5 cmaerobicheterotrophs from Kam Lakesediments100•8060•4020‘IV2 4 6 80Incubation Time (day)Figure 3.8 Inorganic As(C]), MMAA (+) andDMAA (•) production by 9cm aerobicheterotrophs from Kam Lakesediments428040Cl)—— F — F -0 2 4 68Incubation Time (day)Figure 3.9 Inorganic As (D), MMAA (+) andDMAA()production by 13 cm aerobicheterotrophs from Kam Lake sediments204370cI60C4030,c2010-Incubation Time (day)Figure 3.11 Day-to-day variation of totalarsenic in the 5 cm aerobic heterotrophsincubationI • I • I2 4 6 8Incubation Time (day)Production of AsH3in Blank (U)and Control (•)00Figure 3.107060504030201000 24 6844Only the 5 cm microbial population shows strong methylation ability.From the original core profile (Table 3.1), the 5 cm depth is at the border ofthe tailings layer and not in the tailings (9-16 cm) where the arsenic levelsare anticipated to be highest. Probably some reaction (possibly microbial)takes place in the tailings layer that causes arsenic to be mobilizedto thelayers above, where the microbial population then transforms the inorganicarsenicals to methylarsenicals. The thickness of the tailingslayer variesfrom one core sample to another and the arsenic methylators areprobablypresent in the layer overlying the mine tailings.Following the results of the initial assay, the mixed population fromthe 5 cm depth was used for further investigation of the biotransformation ofarsenic. The protein in the cultured medium caused severe foaming inthereaction vessel during the speciation by hydride generation and additionof 2-propanol, as an anti-foam agent, did not alleviate the problem. Theculturemedium was reduced to half strength TSB in subsequent experiments toalleviate this problem. The medium appears to be more favorable for thegrowth of arsenic methylators.3.4.2 Effect of DMAABecause mixed microbial populations from 5 cm depth methylate theadded arsenate to MMAA and DMAA, we would expect the microbes todemethylate any added DMAA to MMAA and inorganic arsenic. Instead, asshown in Figure 3.12, no MMAA is detected in the supernatant over a periodof 1-11 days. The DMAA concentration fluctuates from day-to-day giving a45complex zigzag pattern with no hint of a trend. This pattern appears toindicate that DMAA is taken up by the mixed populations at a different rateeach day, either transforming it and releasing it back to the solution orstoring it inside the cells.%300-.C.)Cc-)0 24 68 10IncubationTime (day)Figure 3.12 Production of AsH3 (D),CH3AsH2(+)and (CH3)2AsH () by the 5 cmaer6bic heterotrophs in Medium DThe DMAA concentration decreases with no evidence of transformationto MMAA or inorganic arsenic. The amount of inorganic arsenic in thesupernatant is zero on day 2. However, the amount in the blank (Figure3.13) is ten times higher, over the 11 days study period.402010124660c?30C.?C2O10 . I • I • I0 2 4 6 8 1012Incubation Time (day)Figure 3.13 Production of AsH3(Ii) fromBlank and (CH3)2AsH (4) from Control media47Figure 3.14 Trimethylarsine production from5 cm microbial population in Medium DTrimethylarsine oxide is produced on days 5-11 (Figure 3.14) fromDMAA. (Due to the difficulty of quantification, the amount oftrimethylarsine is reported in terms of AAS peak area).108C?6C?4Cf200 2 4 6 8 10 12Incubation Time (day)3.4.3 Summary of Aerobic MetabolismMicrobial communities in sediments consist of co-existing species thatinteract with each other in a complex way. Microorganisms isolated from thelayer overlying contaminated mine tailings are able to biomethylate arsenic.Addition of an external arsenic source (50 ppb As(V) solution) to thismixed community reveals a pattern of methylation and demethylation48suggesting a successive changebetween the methylatingmicrobes anddemethylating microbes, as shownin Figure 3.7.Addition of 50 ppbDMAA shows biomethylation toTMAO by themixed microbes. No demethylationis observed. The reasonfor theseobservations is notunderstood at this time.Further work needs to be doneto quantify the total arsenicin the solidand liquid phase. Probablythere is an uptake bythe microbes and storageinside the bacterial cells, andthis could account for the abovediscrepancy.3.5 ANAEROBIC ARSENICMETABOLISMThe anaerobic “arsenate-reducer”produces a heavy yellow precipitateafter 14 days of incubation.The yellow color of theprecipitate indicates thatit might be elementalsulfur, formed by the reductionof suifate. The low(0.04 mM) sulfate concentrationin Medium B could just besufficient tomaintain the metabolicactivity of the microorganisms.Moreover there is analmost inexhaustiblesupply of sulfate in theKam Lake sediment whichcontain approximately 0.33mM sulfate in its porewater.51The sulfatereduction is usually carriedout by dissimilatory sulfate-reducingbacteria,and the “arsenate-reducer”would have to compete successfullywith thesulfate-reducing bacteriafor the available substrates.A second likely productis an arsenic sulfidecompound. Kam Lakecontains high levels ofarsenic, hence, it is possiblethat the mixed microbialpopulation uses arsenateas a terminalelectron acceptor. In thepresence of49large amount of sulfur and in acidic medium, As2S3 and As4Sprecipitateout.3.5.1 Medium BThe arsenic concentration in Medium B was analyzed byAAS (Table3.3). There appears to be a10% loss of arsenic after sterilization. Themedium is autoclaved in an air-tight Pyrex bottle, so the likelihoodof loss byvolatilization is minimized. To avoid the problem, whatever the cause,thearsenate solution should be filter-sterilized in futurepreparations of media toprevent its loss.Table 3.3 Arsenic concentration in Medium BFlame-AAS HG-GC-AAS[As][AS]T[As(III)] [As(V)]gfL g/LBefore autoclave 1.07 1.02 0.050.97After autoclave 0.98 0.91 0.04 0.863.5.2 Characterization of the Anaerobic Metabolic Product3.5.2.1 Mass SpectrometryThe mass spectrum of a freeze-dried precipitate wasobtained at bothlow and high resolution EI-MS5O (Figure 3.14). The spectrumshows peaks50at mJz of 428 (As4S), 396(As4S3), 300 (As4), and other peaks withcombinations of As and S. The result shows that the moleculesdissociatesprior to ionization and thus the observed mass spectrumis that of thedecomposition products. The results rule out the possibilitythat theprecipitate is elemental sulfur or arsenic. Elemental AnalysisElemental analysis was done by Mr. Peter Borda, ChemistryDepartment, UBC. Table 3.4 shows the result of the analysis. The presenceof arsenic interfered with the sulfur determination.I SE908970605940392019075 4991979080796959493929ISS199 125299Figure 3.15 Mass spectrum of yellowprecipitate. Spectrum obtained at 330°C225 25951Table 3.4 Elementalanalysis of yellowprecipitateC H NS% found4.88 1.60 1.6029.3% expected- - - 40(for As2S3) Wet Digestionof Freeze-dried YellowPrecipitate and Flame-AASAnalysisThe amount of precipitateformed by the “arsenate-reducer”in the 10niL test tube was small.The freeze-dried precipitatefrom the mixedpopulations of 5, 9 and29 cm were combinedfor digestion. The precipitateisslightly soluble innitric acid but a more elaboratedigestion procedure thatutilizes nitric acid,sulfuric acid andhydrogen peroxide was used.Theprecipitate dissolves completelyand AAS analysisshows that the combinedprecipitate contain59.2% As. (Calculatedfor As2S3:60% As). SEM÷EDXA flat surface of theprecipitate on the carboncoated stub was scannedat 6.00K magnification.The scanning area ofthe sample must be smooth toenable the generatedx-rays to reach the detector.Two or three differentareas on the samestub were analyzed. Table 3.5summarizes the results.52Table 3.5 SEM + EDX analysis of the yellowprecipitate obtained from Kain Lakemicroorganism.Sample Weight PercentAs S Ca5 cm 57.0 38.2 4.83±2.6 ±2.0 ±0.79 cm 57.9 42.1 0.0±2.9 ±2.929 cm 56.1 40.1 3.70±0.9 ±0.5 ±0.4Medium B + H2S 58.0 42.0 0.0±2.4 ±2.4As2S3 57.6 42.4±4.3 ±4.3As4S 70.3 29.3Standards ofAs2S3and As4Swere similarly analyzed. The combinedresults indicate that the yellow precipitate is probably As2S3 with some“included” calcium and organic compounds.3.5.3 Arsenic Speciation of SupernatantThe supernatant media from cultures that produced the yellowprecipitate was analyzed by HG-GC-AAS to determine arsenic speciation(Table 3.6). No methylarsenic acids were detected; approximately 76% of53arsenic exists as As(V) and 24% asAs(III). Flame-AAS was also performedtocompare total arsenic.Table 3.6 Flame-AAS andHG-GC-AAS ofsupernatant medium after anaerobicgrowthof Medium BSupernatant HG-GC-AAS(gIL) Flame-AAS (gIL)A5TAs(III) As(V)AST5cm - -- 0.529 cm 0.380.09 0.29 0.5829 cm 0.220.09 0.13 -- not analyzed.A difference in total arsenicconcentration of about 35% wasobservedbetween the two methods.The discrepencies could be due tothe series ofdilutions (i0 dilution) of thesample that were necessary for arsenicconcentration to be withinthe working range of the HG-GC-AAS.FlameAAS is not as sensitive asthe HG-GC-AAS and no dilution wasnecessary.3.5.4 Summary of AnaerobicMetabolismThe mixed anaerobic microbesfrom Kam Lake sediment produce ayellow precipitate which isidentified as As2S3 from elemental andSEM +EDX analysis. The sulfide islikely to be an artifact, produced fromeitherAs(V) or As(III) whenmicrobial growth produces a localizedacidic54environment in themedium. In an independentexperiment it was foundthat hydrogen sulfide doesnot precipitate any arsenic sulfidefrom the mediaunless the media is acidified.The source of sulfide isunknown. Presumably themicrobes producesulfide from the reductionof sulfate. The sulfatereduction is usually carriedout by the sulfate-reducingbacteria, but these areunlikely to be presentinthis media which has aredox potential of 130 mV.The sulfate-reducers needmedia with a low redox potential (about-300 mV).Speciation of arsenic in thesupernatant shows 76% As(V)and 24%As(III), with no methylarsenicacids. Thus the mixedanaerobes do notmethylate the added arsenatealthough they may bereducing As(V) toAs(III). Otherwise the microbescould be arsenic tolerantand be particularlyinsensitive to arsenic,with no uptake or metabolismof the arsenate added.55CHAPTER WBIBLIOGRAPHY1. 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