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

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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 19920 Jafariah Jaafar, 1992In presenting this thesis in partial fulfilment of the requirements for anadvanced degree at The University of British Columbia, I agree that theLibrary shall make it freely available for reference and study. I further agreethat permission for extensive copying of this thesis for scholarly purposesmay be granted by the Head of my Department or by his or herrepresentatives. It is understood that copying or publication of this thesis forfinancial gain shall not be allowed without my written permission.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 as A52S3 by microanalysis 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 Tables VList of Figures viList of Abbreviations viiiAcknowledgements ixDedication xChapter IIntroduction 11.1 Arsenic 11.2 Biotransformation of Arsenic 21.2.1 Arsenite and Arsenate Redox Transformation 21.2.2 Biomethylation ofArsenic 41.3 Arsenic-Sulfur Compounds 81.4 Methods for the Speciation of Arsenic Compounds 91.5 Microbial Sampling 111.6 Scope of Work 11Chapter IIExperimental 142.1 Reagents and Chemicals 142.1.1 Chemicals 142.1.2 Chromatographic Supplies 142.1.3 Prepared Solutions Arsenate, arsenite, monomethylarsonic acid anddimethylarsinic acid standard solutions Potassium borohydride solution 152.2 Instrumentation 152.2.1 Chambers for Incubation of Anaerobic Cultures 152.2.2 Gas Chromatograph 172.2.3 Atomic Absorption Spectrophotometer (AAS) 172.2.4 Autoclave 172.2.5 Microscope 172.2.6 Scanning Electron Microscope + Energy Dispersive X-ray(SEM+EDX) 182.3 Culture procedures 182.3.1 Media Preparation 182.3.2 Bacterial Isolation 21‘U2.3.3 Bacterial Enrichment .232.3.4 Bacterial Preservation 232.4 Arsenic Metabolism 242.4.1 Aerobic Experiment 242.4.2 Anaerobic Experiment 252.5 Analytical Methods . 252.5.1 Hydride Generation-Gas Chromatography-Atomic AbsorptionSpectrometry (HG-GC-AAS) Hydride Generation Gas Chromatography Atomic Absorption Spectrometry 282.5.2 Wet Digestion of the Freeze-dried Yellow Precipitate 282.5.3 SEM + EDX 29Chapter IIIResults and Discussion 303.1 Microbial Sampling 303.1.1 Study Site 303.1.2 Kam Lake Core Description 313.2 Cultivation and Subculture 313.3 HG-GC-AAS 343.3.1 Calibration And Limit of Detection 373.4 Aerobic Arsenic Metabolism 403.4.1 Effect of Arsenate 403.4.2 Effect of DMAA 453.4.3 Summary of Aerobic Metabolism 483.5 Anaerobic Arsenic Metabolism 493.5.1 Medium B 503.5.2 Characterization of Anaerobic Metabolic Product 503.5.2.1 Mass Spectrometry 503.5.2.2 Elemental Analysis 513.5.2.3 Wet Digestion of Freeze-dried Yellow Precipitate andFlame-AAS Analysis 523.5.2.4 SEM + EDX 523.5.3 Arsenic Speciation of Supernatant 533.5.4 Summary of Anaerobic Metabolism 54Chapter 1VBibliography 56ivLIST 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 FIGURESFIGURE PAGE1.1 Structure of selected arsenic compounds 31.2 Challenger’s mechanism for the biological methylationof arsenic 61.3 S-adenosylmethionine (SAM) 62.1 Map of Yellowknife Lakes System, NorthwestTerritories 222.2 Schematic of HG-GC-AAS System 263.1 Chromatogram for 4 mL of a standard solution of10 ppb As(V), MMAA and 20 ppb of DMAA 363.2 Comparison of effects of 4M HC1 and 4M CH3OOH onDMA signal 373.3 Calibration curve for the determination of totalinorganic arsenic 383.4 Calibration curve for the determination of arsenite 383.5 Calibration curve for the determination of MMAA 393.6 Calibration curve for the determination of DMAA 393.7 Inorganic As (D), MMAA (•) and DMAA (•) productionby 5 cm aerobic heterotrophs from Kam Lakesediments 423.8 Inorganic As (11), MMAA (+) and DMAA () productionby 9 cm aerobic heterotrophs from Kam Lakesediments 423.9 Inorganic As (Ii), MMAA (4) and DMAA (•) productionby 13 cm aerobic heterotrophs from Kam Lakesediments 433.10 Production of AsH3 in Blank (D)and Control (4) 443.11 Day-to-day variation of total arsenic in the 5 cm aerobicheterotrophs incubation 443.12 Production of AsH3 (U), CH3As2(+) and (CH3)2As (I) by the5 cm aerobic heterotrophs in Medium D 443.13 Production of AsH3 (U) from Blank and (CH3)2As (+)from Control media 463.14 Trimethylarsine production from 5 cm microbialpopulation in Medium D 483.15 Mass spectrum of yellow precipitate. Spectrum obtained at330°C 51viiLIST OF ABBREVIATIONSHPLC High performance liquid chromatographyAAS Atomic absorption spectrometryGC Gas chromatographyICP Inductively coupled plasmaMMAA Monomethylarsonic acidDMAA Dimethylarsinic acidTMAO Trimethylarsine oxideMMA MonomethylarsineDMA DimethylarsinePRAS Pre-reduced anaerobically sterileTSB Tryptic Soy BrothTSA Tryptic Soy AgarSEM Scanning electron microscopeEDX Energy dispersive x-rayORP Oxidation reduction potentialpE - log of electron activityr1uACKNOWLEDGEMENTI am grateful to my supervisors, Professors W.R. Cullen and K.J.Reimer, for their guidance throughout the course of this work.I wish to give special thanks to Mr. Gary Hewitt for helping me in thebacterial isolation and introducing me to the microbial world, and to Ms.Deepthi Hettipathirana for helping me with the thesis stylesheet. Their timeand patience spent during this work are very much appreciated.I thank Ms. Eileen Cochien for helping me with the bacterialpreservation during Summer 1991, and Mr. Hao Li for trouble shooting theHG-GC-AAS system. I also thank Ms. Mary Mager for teaching me theSEM+EDX. Thanks also to all my lab colleagues for their friendly andhelpful endeavors.Finally, I thank the Public Services Department, Malaysia for thefinancial support and Universiti Teknologi Malaysia for granting me thestudy leave.ixDEDICATIONAffandi, Anna and AzmanxCHAPTER IINTRODUCTION1.1 ARSENICArsenic ranks as the twentieth most abundant element in the earth’scrust.1 It is widely dispersed at low levels, ranging from 0.1 to severalhundred ppm (jiglg), depending on several factors such as geographicallocation and anthropogenic input.2 The average arsenic concentration in theearth’s crust is about 3 ppm,1 but much higher concentrations, ranging from0.4 to 6000 ppm, are found in sulfur containing ores that containarsenopyrite (FeAsS), realgar (As4S)and orpiment (As2S3). Arsenic hasbeen found to be a common contaminant of both freshwater and seawater inmany parts of the world.Arsenic compounds have different properties and uses.4-5 Arsenictrioxide is well known as a poison. Both inorganic and organic arseniccompounds were widely used in medicine.6 Arsenic, like mercury, has beenused extensively in agriculture as herbicides and pesticides, and in industrialapplications as wood preservatives. High arsenic concentrations in soils havebeen related to the mining industries, in particular, gold mining.It is recognized that the toxic effects of arsenic depend on theconcentrations of individual arsenic species, so the development of analyticaltechniques that enable the speciation of arsenicals has become an importantobjective for individuals and organizations interested in 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, such asarsenobetaine, 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 becomes the main species in anoxic basins and inlets, forexample, Saanich Inlet, B.C.8In air saturated waters the redox condition is set by the 02/1120couple, with a pE of about 12.6.9 Thermodynamic calculations for anoxygenated seawater at pH 8.1 predict the As(V)/As(III) ratio to be about1026.10 However, significant amounts of arsenite are found in oxygenatedsurface waters, and conversely, arsenate is found in anoxic waters.2HO—As—OH HO—As—OHOH OHArsenate ArseniteO 0II IIHO— As— CR3 CH3 As— CH3I IOH OHMonomethylarsonic acid Dimethylarsinic acidCH3 o CH3CH3— Ask— CR2— C - O CH3— As— CR2— CH,- OHCH3 CR3Arsenobetaine Arsenocholine1aYOH, R0SO3HIbY=OH. R=OHIcY=OH, R=SO,HMe2 CM2 o .0—CH2—CH(Y)—CHR Id Y NH2. R = SO3HH’\ /H HIc YOH, R0P—0CH2OHOHOH CHOHCH,OHArsenosugars (1)Figure 1.1 Structure of Selected ArsenicCompounds3Studies11’2 found the As(V)/As(III) ratio to be in the range of 0.1 to100, suggesting that the redox pair is not in thermodynamic equilibrium. Inoxygenated seawater, bacteria and phytoplankton have been shown to beresponsible for the presence of arsenite, by reduction of arsenate.12-4Johnsonl2 showed that a mixed population of marine bacteria carries out thisreduction in seawater medium.Arsenite is slowly oxidized to arsenate in seawater, even under sterileconditions.15 Some bacteria are also responsible for this oxidation, andTurner et al.l6 in 1954 demonstrated that bacteria from arsenical cattle-dipping fluids could oxidize arsenite to arsenate. The processes that controlthe oxidation of arsenite to arsenate in natural waters still need furtherstudy.1.2.2 Biomethylation of ArsenicThe biomethylation of arsenic compounds has been known for manyyears; however, interest in this phenomenon has increased, in associationwith the discovery that microorganisms in natural lake sediments are able tomethylate mercury to form a highly toxic methylmercury species.17 Thediscovery that mercury is readily cycled from the sediments and concentratedby organisms along the food chain18, has led to speculation that similarcycles may occur with arsenic.Certain fungi, yeasts and bacteria are known to methylate arsenic tovolatile arsines. Poisoning incidents resulting from fumes emitted from wallpaper coverings were first investigated by Gosio in 1892.19 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.225supported 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 [)H3AsO4 As(OH)3 CH3AsO(OH)2arsenite monomethylarsonje acidCH3AsO(OH)2 {CH3As(O )) ‘(CH3)2AsO(OH)dimethylarsinic acid(CH3)2AsO(OH) 2e ((CH3)As(O )) [cH3](CH3)AsOtrimethylarsine oxide(CH3)AstrimethylarsineFigure 1.2 Challenger’s mechanism for thebiological methylation of arsenic— a—c2—c2—HO IFigure 1.3 S-adenosylmethionine (SAM)arsenateOH6Cox and Alexander26isolated microorganisms from soil and sewage incultures containing various arsenic compounds. The organisms were able toproduce trimethylarsine from arsenite, arsenate, methylarsonic acid anddlimethylarsinic acid when grown at pH 5. They suggested that the acidiccondition in sewage might promote trimethylarsine production.Shariatpanahi and coworkers27 studied bacteria that were isolatedfrom the environment and grown in the presence of arsenate ( lOOug/mL).Under aerobic conditions, they found the transformation of arsenate todimethyl and trimethyl species.In experiments with lake and river sediments using pure cultures ofthe bacteria Aeromonas and Flavobacterium sp. and Escherichia coli, Wonget al.28 observed the production of monomethylarsonic acid, dimethylarsinicacid and trimethylarsine oxide. They also observed the production ofdimethyl- and trimethylarsines. The sediments were incubated with orwithout high levels of arsenic (at least three orders of magnitude higher thana typical non-polluted environment). These results were questioned byAndreae,5 who suggested that this biomethylation is a response to extremearsenic stress, and not a general process carried out by aerobic bacteria in theenvironment.Additional work is required to understand the biomethylation process.The mechanism proposed by Challenger seems likely to occur in the aquaticenvironment with the reduction of arsenate and methylation tomonomethylarsonic acid and dimethylarsinic acid. Trimethylarsine is notalways the end product.7The biological function of methylated arsenic compounds is unknown,however, it is possible that the methylation serves as a detoxification process.Ferguson and Gavis29 suggested that in an anaerobic environment, it may beenergetically favorable for the organisms to transmethylate arsenic (andother metals) rather than to synthesize methane. This methylation mighttake place in the upper layer of the sediments. This process might beresponsible for mobilizing methylarsines from sediments to solutions and tothe food chain.1.3 ARSENIC-SULFUR COMPOUNDSIn anoxic water systems, bacterial action would create a reducingenvironment and the pE will be set by the reduction of S042 to HS.9 Underthis reducing condition (pE value approximately -4) arsenite is the dominantspecies. Stable arsenic sulfides, realgar (As4S)and orpiment (As2S3), willprecipitate in the presence of sulfur under acidic anoxic conditions.3°Microorganisms play an important role in geochemical processes, andin the cycling of sulfur. Ehrlich31’2investigated the degradation of sulfideminerals, orpiment and arsenopyrite, by microorganisms. The rate of arsenicreleased by bacterial oxidation was twice as great as by spontaneousoxidation. The bacterial action released arsenate and arsenite. Thebiodegradation process results in the solubilization of metal compounds andcan be applied practically for metal recovery.33 Another role ofmicroorganisms is in the partial generation of sulfide minerals. However,limited study has been done in this area.81.4 METHODS FOR THE SPECL4TION OF ARSENIC COMPOUNDSTotal arsenic has been monitored in environmental samples by usingmany analytical techniques: atomic absorption spectrometry,3438 atomicemission spectrometry,39voltammetry,40 and neutron activation analysis.41Currently, there is an increased interest in determining the concentration ofindividual arsenic species, largely because of their different toxicities. Thetoxicity decreases in the order: arsines > arsenite > arsenate> alkylarsenicacids > arsonium compounds > metallic arsenic.1A variety of techniques for the speciation of arsenicals has beenreported. Generally, hyphenated techniques such as HPLC-AAS,42 HPLCICP-MS,43 GC-AAS,4446 and GC-MS47 combined with selective hydridegeneration and cold-trapping,48’9 are used. However, the problemsassociated with these techniques are long analysis time and their non-routinenature.A number of arsenic compounds are volatile , such as arsine, or can beconverted into volatile arsines by reduction with sodium borohydride. Thereduction of arsenic compounds with sodium borohydride is pH-dependent,49and is related to the pKa of the individual arsenic acids (Table 1.1). Bramanet al.38 investigated the effect of pH on the fraction of undissociated arsenicacids present at equilibrium. A number of workers44’6 have used the piCadifferences of arsenate and arsenite to speciate them. However, Hinners50found that MMAA and DMAA interfered with the determination of As(III)and As(V).9Anderson et al.49 investigated several factors that influence thearsenic species determination. They used a continuous hydride generationwith sodium borohydride and AAS for detection. Several combinations haveshown promising selectivity towards the reduction of As(IH), As(V), M.MAAand DMAA: (a) 5M HC1 for the determination of total inorganic arsenic; (b)citric acid-sodium citrate buffer at pH 6 for the determination of As(III); (c)0.1GM citric acid for the determination of DMAA.The volatile property of the arsines enabled them to be removed fromsolution by sweeping them with an inert gas, collected on a liquid nitrogencold trap, and separated by using gas chromatography. The separatedarsines are then detected by a quartz-cuvette atomic absorptionspectrophotometer.Table 1.1: pKa values of arsenic species andreduction 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 CHAs2 2DMAA 6.2 1-2 (CH3)As 35.6TMAO - 1-4 (CHAs 70101.5 MICROBIAL SAMPLINGMicroorganisms, either mixed communities or single species, forlaboratory study are isolated by growing them under well-defined conditionsas cultures. In nature, bacteria invariably occur as mixed communitiescontaining more than one kind of microorganism. In the present work,enriched cultures of a mixed microbial population were isolated using aselective liquid media containing arsenate. The enrichment technique tendsto select for the microorganism with the highest growth rate, by changingconditions in favor of the desired organism. This is because of a directcompetition for nutrients among the mixed developing population.Two techniques that are frequently used for the cultivation of strictanaerobes are the roll tube technique, developed by Hungate,56 and theanaerobic glove box technique. In the roll tube method, agar medium isdistributed as a thin layer over the internal surface of test tubes chargedwith an anaerobic atmosphere. Specimens are streaked on the surface of theagar layer on the wall of the tube. This technique provides a low oxidation-reduction potential of -150 mV and an oxygen-free atmosphere. Use of theglove box is more practical since all manipulations are performed in thereducing atmosphere of the box.1.6 SCOPE OF WORKArsenic has frequently been concentrated in certain areas as a result ofanthropogenic activities. For example, gold mining operations for yearsdumped arsenic as a waste by-product in their tailings. Mine sites, such as11those along the B.C. coast or those adjacent to lakes, frequently dumpedtailings directly into these waters, and over time sediments rich in arsenicbuilt up around the effluent outflow. Recent improvements in goldseparation methods have reduced arsenic concentrations in effluents andcleaner tailings now lie on top of arsenic-laden layers.Bacteria appear to be capable of mobilizing deposited arsenic intowater soluble arsenic acids and arsines. MMAA, DMAA, TMAO and thehighly toxic and volatile arsine and methylarsines are known products ofbacterial metabolism. 19, 26-28To initiate an investigation of microbial mobilization of historicallydeposited arsenic, we decided to see if mixed microbial cultures obtained fromsediments in a lake associated with the NercoCon Mine in Yellowknife,N.W.T. could metabolize arsenic. Kam Lake was chosen because it has a welldefined sub-stratum with high arsenic concentration:51 the layer resultedfrom a large spill of process effluent that would normally have been treatedprior to disposal but was instead diverted to Kam Lake.In a typical sediment, there is a predictable progression of nutritionalmicrobial types that result from the lower oxygen concentration andincreasing reducing condition as depth increases. Aerobes may predominateat the surface, whereas a variety of anaerobic types will predominate beneaththe surface. In the present study, two microbial populations were initiallysought that would represent aerobic and anaerobic microbial activity. Eachmixed population was assayed for its ability to metabolize arsenate andDM.AA. Hydride generation - gas chromatography - atomic absorption12spectrometry (HG-GC-AAS) was the analytical method 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. As2S3 and A54S4were prepared by using literature methods.52,32.1.2 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 sodium bicarbonate plus citric acid. To generateH2 and C02, a top corner is cut to allow addition 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 hydrogen reacts with oxygen over Pdcoated alumina pellets (118”; Aldrich Chemical Co.) held in a wire basketsecured to the underside of the chamber headplate to produce water. Theappearance of condensate on the chamber walls is an indication of reactionprogress. Anaerobic indicator strips (GasPak) soaked with methylene blueindicator change from blue to colorless to confirm the removal of oxygen.Carbon dioxide does not participate in the development of anaerobiosis but isa required nutrient for some anaerobes (e.g. methanogens).All anaerobic sample and culture manipulations were performed underconditions designed to minimize oxygen exposure. Initial sediment coresamples were acquired in sealed gravity core sampler tubes55 which werelater opened in a glove bag (Instruments for Research and 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%) and N2 (85%) and supplied with Pdcoated alumina pellets. Anaerobic indicator strips were used to confirmanaerobic conditions, and catalyst was replaced routinely.162.2.2 Gas ChromatographTwo types of gas chromatograph were used: a Hewlett Packard 5830Amodel with a 18850A GC terminal, and a Hewlett 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 model AA 1275 series. TheAA hollow cathode lamp was operating at 8 mA and the monochromator wasset at 193.7 nm, one of the resonance lines of arsenic. The spectrophotometerwas equipped with a deuterium background corrector and a Hewlett Packard82905A printer.2.2.4 AutoclaveAmsco, Eagle Series-3041 Gravity autoclave was used to sterilize allculture vessels and heat-stable media.2.2.5 MicroscopeThe microbial growth was observed by using an Olympus BH-2microscope.172.2.6 Scpnning Electron Microscope + Energy Dispersive X-ray (SEM+ EDX)A Hitachi S-570 Scanning Electron Microscope (SEM) was used. Itwas equipped with a Kevex Super8000 Energy-Dispersive X-ray Analyser(EDX). This instrument is located in the Metals and Materials EngineeringDepartment, UBC.2.3 CULTURE PROCEDURES2.3.1 Media PreparationAll aerobic media were prepared on the open bench, and sterile mediawere handled in a Class II (laminar flow) biohazard cabinet (MechanicalEngineering Services, Department of Chemistry, UBC). Pre-reducedanaerobically sterile (PRAS)5657 media were prepared by flushing filter-sterilized (glass wool packed tube) nitrogen gas over the headspace of theboiled media in an air-tight Pyrex bottle.All transfers were done in an anaerobic glove box containing palladiumcatalysts and under an atmosphere of 5% C02, 10% H2 and 85% N2 (from acommercial cylinder). A methylene blue indicator strip was used to check foranaerobic condition. All glassware was flushed with nitrogen gas beforebeing sterilized. All anaerobic incubations were done in the anaerobicchamber.Arsenate augmented Tryptic Soy Broth at quarter strength (MediumA) was prepared by adding 10.0 mL of 100.0 j.ig/mL As(V) solution to 7.50 g of18the commercially available powder (Table 2.1) and dissolving this in 1000 rnLof distilled water. Ten mL aliquots of this broth were pipetted into screw captest tubes (16 mm diameter by 120 mm) and autoclaved at 121°C for 15 mm.The final pH of this medium was 7.0.Table 2.1 Tryptic Soy Broth (Medium A)Bacto Tryptone (Pancreatic Digest of Casein) 15 gBacto Sytone (Pancreatic Digest of Soybean Meal) 5 gSodium chloride 5 g“Arsenate-reducer” medium (Medium B) was prepared from theingredients shown in Table 2.2. All these (except sodium bicarbonate) weredissolved in water and diluted to 960 mL. The medium was prepared PRAS,and autoclaved by using a gravity cycle at 121°C for 15 mm., with no dryingtime. The sodium bicarbonate was filter-sterilized by using a 0.45 pmMillipore membrane filter (HV type). The autoclaved solution was cooled,placed in the anaerobic glove box, and 40 mL of the sterile sodiumbicarbonate was added aseptically. The final pH of the medium was adjustedto 7.4 with 4M HC1 or NaOH. This medium was pipetted (10.0 mL portions)into screw cap test tubes and allowed to stand overnight in the glove box.The tubes were then placed in the anaerobic chamber which was chargedwith CO2 and H2 from a GasPak envelope, and removed from the glove box.19Table 2.2 “Arsenate-reducer” (Medium B)Ingredients gILNaHCO3 2.5CaC12.2H0 0.1KC1 0.1NH4C1 1.5NaH2PO.0 0.6NaCH3COO 2.7Difco yeast extract 0.05NaC1 0.1MgC12.6H0 0.1MgSO4.7H 0.1MnC12.4H0 0.005NaMoO.2H 0.001FeSO4.7H0 0.001Na2HAsO.7H 3.12202.3.2 Bacterial IsolationThe microbiological sampling was done in the field by Mr. Gary Hewittof Department of Chemistry, UBC and Dr. Doug Bright of RRMC.Sediment core samples taken from Station 1, Kam Lake, Yellowknife,Northwest Territories (Figure 2.1) were obtained by hand lowering a gravitycore sampler from an aluminum power boat. Plastic core tubes for assay ofpH, temperature and oxidation-reduction potential (ORP) and for microbialsampling had 1 cm holes drilled each 2 cm of their length. The holes werecovered with duct tape for sample acquisition and for sealed transport back tothe laboratory. Standard core tubes were of 3.75 cm diameter and 60 to 80cm long. Cores for microbial sampling were transported from Kam Lake tothe Department of Indian and Northern Affairs’ water testing laboratory inYellowknife, and opened in a glove bag (Instruments for Research andIndustry) under an atmosphere of oxygen-free nitrogen. The holes wereuncovered and sediment samples from different depths were asepticallyremoved with a sterile spatula after the layer closest to the wall wasremoved. Roughly 1 mL of sediment was then inoculated into each testmedium.Cultures of aerobic heterotrophs, Medium A, were removed from theglove bag and incubated with loose caps. Cultures in “arsenate-reducer”medium (Medium B) were placed in an anaerobic chamber before removalfrom the glove bag. All cultures were transported to the Department ofChemistry, UBC for 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 were made by serial subculture. The mixed aerobicculture in Medium A was enriched once by transferring 1.0 mL of the culture,to a sterile, 10.0 mL of Medium A. Medium A was incubated for two days.One mL of the mixed anaerobic culture was transferred, in eachenrichment step, into a sterile 10.0 mL of Medium B. Medium B wasincubated in the anaerobic chamber for 10-14 days between each of the threeenrichment steps.2.3.4 Bacterial PreservationThe two-day old aerobic subculture was centrifuged for 30 mm at aspeed setting of 80 in a Dynac Centrifuge (Becton Dickinson Co.) and thesupernatant was discarded. One mL each of TSB (double strength) and 20%glycerol were added to the bacterial cell to achieve a final concentration of10% (v/v) glycerol and single strength TSB. The cells were resuspended and1.0 mL aliquots were dispensed into cryovials (1.2 mL capacity, NUNC), precooled on dry ice, and finally stored in a cryogenic refrigerator (TaylorWarton, model 34 HC) charged with liquid nitrogen.Enriched anaerobic cultures were centrifuged by the same procedureand returned to the glove box. The supernatant was discarded and 1.0 mLeach of Medium B and 20% glycerol (PRAS) was added to resuspend the cells.One mL of the cell suspension was dispensed into cryovials and sealed. Thetubes were removed from the glove box, pre-cooled on dry ice andimmediately preserved in the cryogenic refrigerator.232.4 ARSENIC METABOLISM2.4.1 Aerobic ExperimentThe medium used was TSB containing 50 ng/mL As(V) solution(Medium C). Twenty mL portions of the sterile medium was pipettedaseptically into a series of Evergreen tubes (50 mL capacity, polypropylenetube).The preserved aerobic heterotrophs were thawed and grown on TrypticSoy Agar (TSA) + 1.0 As(V) plates. The plates were incubated at roomtemperature for 2 days to allow colonies to develop. Approximately 7 mL ofMedium C was added to the plate and any growth was scraped to resuspendit. The cell suspension was pipetted to a test tube containing Medium C andthe turbidity was measured spectrophotometrically at 420 nm with aSpectronic 20. Appropriate volumes of the cell suspension and Medium Cwere mixed to obtain an absorbance reading of 1.0. Two mL each of thestandardized inoculum was added to 20.0 mL Medium D in 7 Evergreentubes. The tubes were incubated aerobically at room temperature. Allcultures were shaken daily to resuspend the growth. One tube wascentrifuged each day, and the ‘snift test’ was carried out to detect theproduction of arsines, and then the supernatant was transferred to a steriletube and frozen prior to analysis for arsenic compounds. The solid phase andbacterial cells were discarded. The process was repeated for 7 days.Controls, containing Medium C, and blanks (TSB) were carriedthrough the same incubation condition and frozen daily for arsenic analysis.24A second set of cultures was prepared. In addition to the 50 ppb As(v)in half strength TSB (Medium D), 50 ppb DMAA in half strength TSB(Medium E) were prepared. Standardized inoculum of the aerobicheterotrophs were added to Media D and E. The tubes were incubated asbefore. One each of eleven days supernatant samples were frozen for arsenicspeciation. Controls (Media D and E) and TSB blanks were similarlyprepared.2.4.2 Anaerobic ExperimentOne mL of the anaerobic culture solution from the third enrichmentstep was added to 10.0 mL Medium B. The test tubes were incubated in theanaerobic chamber for 10-14 days. A heavy yellow precipitate formed andwas separated from the solution by centrifugation for 30 mm at a speedsetting of 80. The precipitate was washed three times with distilled water,freeze-dried and its composition was investigated by using massspectrometry, elemental analysis, and SEM + EDX. The supernatant wasanalyzed for arsenic compounds by HG-GC-AAS.2.5 ANALYTICAL METHODS2.5.1 Hydride Generation-Gas Chromatography-Atomic AbsorptionSpectrometry (HG-GC-AAS)Arsenic speciation studies involved a selective hydride generation ofvolatile arsines, which were then concentrated by cryogenic trapping. The25arsines were separated by gas chromatography and detected by atomicabsorption spectrophotometer. The HG-GC-AAS system is 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 50 mL of Silyl-8. The columnwas heated again at 150°C for 45 mm. The column silanization was repeatedfor the other end of the column.27Temperature progrimming was used to enhance the separation of thearsines, by the column and the program used was as follows:Temp 1 60°CTime 1 6 secRate 30°C/mmTemp 2 150°CTime 2 3 mm. Atomic Absorption SpectrometryThe effluent from the GC was detected in a hydrogen-air flame in aquartz-cuvette of the AAS. Arsines produced from inorganic arsenic [As(V) +As(III)], MEMAA, DMAA and TMAO were detected. Standards arsenicalswere injected to establish the retention times and the standard calibrationcurves.2.5.2 Wet Digestion of the Freeze-dried Yellow PrecipitateThe dried precipitate (5 mg) was added to concentrated nitric acid (3mL), concentrated sulfuric acid (1 mL) and 30% hydrogen peroxide (3 niL) ina 100 mL round bottom flask. The flask was fitted with Teflon stopper andair condenser. The flask was heated at reflux for 2 hours by using a heatingmantle. The flask was then cooled to room temperature and the digestedsample was diluted to 100 mL with deionized water in a volumetric flask.Total arsenic was determined by flame-AAS, after calibration with 0.5 to 50ppm 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 pH with 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 hours elapsedbetween 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 turbid solution,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-reduction potential(ORP) measurement for core #6 taken at KamLake on 3 August 1991.Hole# Sediment Deptha (cm) ORP (mV)1 1 -2 3 -2253_ 5 -4 7 -4255 9 -6 11 -3587 13 -8 15 -3289 17 -10 19 -11 21 -36412 - -13 25 -39214 - -15 29 -16 31 -3770-8 cm: oxidized green-brown sediment of fine silt/sand (hole #1) givesway to an olive green layer just above patches of black (hole #3).8-16 cm: black patches in a matrix at the top of the grey tailings layer(hole #5): tailings zone with predominantly black interior of core with fibrousmaterial (hole #7).17 cm: transition band from slate grey tailings to more brown colorednatural sediments beneath (hole #9).18-32 cm: brown colored natural sediments: no dark patches, no H2Ssmell.-: no readings taken.a: Water depth 7 m.32Table 3.2 Oxidation-reduction potential(ORP), pH and temperature measurementsfor core #3 taken at Kam LakeHole# Sediment ORP (mV) pH T°CDeptha (cm)1 - - 7.38 20.12 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 7.11 19.08 15 -262 7.07 -9 17 -281 - -10 19 -270 - -11 21 - - -12 23 - 6.97 19.113 25 - - -14 27 - 6.93 -15 29 - - -16 31 - 6.69 18.2- : no readings taken.a Water depth 6.5 mSediment samples that were inoculated into Media A and B from 1, 5,9, 13, 19 and 29 cm depths, all showed growth. It is an interesting resultthat aerobic microbial growth occurred at all depths. The redox potentials ofthe sediment ranged from -225 to -425 mV and at this low redox potential,aerobes are not expected to thrive.The anaerobes were also successfully cultivated from all depths. Thisis again surprising because Medium B has a redox potential of 130 mV.33Addition of sediment and the metabolism of bacteria in the anaerobicmedium could be expected to lower the redox potential but only slightly, andnot to the -300 mV necessary for strict anaerobes. Possibly the mixedanaerobic population comprises obligate anaerobes at the surface ranging tostrict anaerobes at the bottom of the core. The anaerobes probably haveoxygen-protective enzymes to enable them to survive in the oxygenatedsurface layer.3.3 HG-GC-AASThe hydride generation technique was used for the determination ofarsenite, arsenate, monomethylarsonic acid, dimethylarsinic acid andtrimethylarsine oxide. The reducing system employed for arsine productionis an alkaline potassium borohydride solution. It is customary to 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,28,4619 a 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 is mixed with theacidic sample in a reaction vessel. Reaction takes place immediately and the34arsines are swept by an inert carrier gas to the AAS. A discontinuous signalis observed by the AAS.The batch type system was used in the present study to determinearsenic speciation. The reaction mixture is buffered to pH 0.5 with 4M HC1and the addition of KBH4 reduced inorganic arsenic [As(III) + As(V)] toAsH3, monomethylarsonic acid (MMAA) to monomethylarsine (MMA),dimethylarsinic acid (DMAA) to dimethylarsine (DMA), and trimethylarsineoxide (TMAO) to trimethylarsine (TMA). The reaction mixture issubsequently buffered to pH 6 with 1M citrate in 10% citric acid whereuponaddition of KBH4 converts arsenite [As(III)] only to AsH3. The arsenateconcentration can be determined from the difference between the arsinesignals in the two buffered systems.The generated arsines (Table 1.1) are collected by cryogenic trapping.Efficient and reproducible separation of the arsines is achieved by using agas chromatographic column such as a silanized Porapak PS packedcolumn44 interfaced to the trap. Temperature programming is used toenhance the separation. The effluent from the GC is swept into a quartzcuvette of an AAS where it is combusted in a hydrogen-air flame.Retention times are used for the identification of arsines and peakareas are used to calculate the concentration of As(III), As(V), MMAA, DMAAand TMAO in the sample solutions.A typical chromatogram of the generated arsines is shown in Figure3.1. Arsine (AsH3) has a retention time of 0.96 mm, monomethylarsine(CH3ASH2) of 1.66 mm and dimethylarsine [(CH3)2AsHI of 2.41 mm.351210•C?rjCC?C 6C?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 buffered reaction is the leastreliable in terms of sensitivity and reproducibility, as has been previouslyreported.44 This has been attributed to incomplete reduction, condensationin the transport tubes or incomplete atomization.49I • I2Time (mm)36Changing the acid system to 4M acetic acid (pH 2.5) improves theDMA signal (Figure 3.2) but the problem of long term reproducibility remainsto be solved.804M Aceticc60•/40.4MHCI0 20 40 60 80DMAA Concentration (ng)Figure 3.2 Comparison of effects of 4M HC1and 4M CH3OOH 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 30 40As(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 20 30M11AA Concentration (ng)408060402000 20 40 60 80DMAA Concentration (ng)39The calibration curves show a linear dependence of the AAS peak forthe concentration range studied. The absolute limit of detection, defined asthe 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. The inoculumused 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 from a 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 MMAA and DMAA over the 7 days of incubation. Theformation of DMAA increases from day 1 to day 4, giving way to a progressivedecline on days 5-7. This pattern of variation indicates a shift fromproduction to decomposition of DMAA, indicating that demethylatingmicroorganisms are replacing the methylators in the mixed community.The 9 cm and 13 cm microbial cultures show very small amounts ofmethylated arsenic species (Figures 3.8 and 3.9). The amount of addedarsenate decreases with time with no apparent transformation to MMAA orDMAA.Comparisons of the blank and control media (Figure 3.10) with thecultured media show that the mixed microorganism from the 5 cm depthhave a profound influence on the biomethylation of arsenic.From Figure 3.7, the amount of added arsenic (50 ppb As(V) solution,which is equivalent to approximately 95 units of peak area) is not recoveredfully in the supernatant analyzed. The total arsenic concentration also variesfrom day to day (Figure 3.11). The observed decrease could be due to: (1) theadded arsenic could be transformed and adsorbed on the bacterial cells andthus lost when the solid phase was discarded after centrifugation; (2) uptakeof arsenic by the bacteria could cause the concentration to drop; (3) there areother species of arsenic in the supernatant that are undetected by thehydride generation technique; (4) formation of volatile arsines, that escapedetection by the “snift test”. The “snift test” is based on the intense, garliclike odor of the arsines and arsine production can be evaluated by cautioussniffing of a sample of the headspace of the test tube.30,4441301r;j220100 2 4 6 8Incubation Time (day)Figure 3.7 Inorganic As (CD, MMAA (+) andJJMAA (•) production by 5 cm aerobicheterotrophs from Kam Lake sediments100•8060•4020‘I V2 4 6 80Incubation Time (day)Figure 3.8 Inorganic As (C]), MMAA (+) andDMAA (•) production by 9 cm aerobicheterotrophs from Kam Lake sediments428040Cl)—— F— F -0 2 4 6 8Incubation 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 AsH3 in Blank (U)and Control (•)00Figure 3.107060504030201000 2 4 6 844Only 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 mobilized to thelayers above, where the microbial population then transforms the inorganicarsenicals to methylarsenicals. The thickness of the tailings layer variesfrom one core sample to another and the arsenic methylators are probablypresent 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 in thereaction vessel during the speciation by hydride generation and addition of 2-propanol, as an anti-foam agent, did not alleviate the problem. The culturemedium 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 2 4 6 8 10Incubation Time (day)Figure 3.12 Production of AsH3 (D),CH3As2(+)and (CH3)2As () 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 10 12Incubation Time (day)Figure 3.13 Production of AsH3 (Ii) fromBlank and (CH)2As (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 change between the methylating microbes anddemethylating microbes, as shown in Figure 3.7.Addition of 50 ppb DMAA shows biomethylation to TMAO by themixed microbes. No demethylation is observed. The reason for theseobservations is not understood at this time.Further work needs to be done to quantify the total arsenic in the solidand liquid phase. Probably there is an uptake by the microbes and storageinside the bacterial cells, and this could account for the above discrepancy.3.5 ANAEROBIC ARSENIC METABOLISMThe anaerobic “arsenate-reducer” produces a heavy yellow precipitateafter 14 days of incubation. The yellow color of the precipitate indicates thatit might be elemental sulfur, formed by the reduction of suifate. The low(0.04 mM) sulfate concentration in Medium B could just be sufficient tomaintain the metabolic activity of the microorganisms. Moreover there is analmost inexhaustible supply of sulfate in the Kam Lake sediment whichcontain approximately 0.33 mM sulfate in its porewater.51 The sulfatereduction is usually carried out by dissimilatory sulfate-reducing bacteria,and the “arsenate-reducer” would have to compete successfully with thesulfate-reducing bacteria for the available substrates.A second likely product is an arsenic sulfide compound. Kam Lakecontains high levels of arsenic, hence, it is possible that the mixed microbialpopulation uses arsenate as a terminal electron acceptor. In the presence of49large amount of sulfur and in acidic medium, As2S3 and As4S precipitateout.3.5.1 Medium BThe arsenic concentration in Medium B was analyzed by AAS (Table3.3). There appears to be a 10% loss of arsenic after sterilization. Themedium is autoclaved in an air-tight Pyrex bottle, so the likelihood of loss byvolatilization is minimized. To avoid the problem, whatever the cause, thearsenate solution should be filter-sterilized in future preparations 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 0.05 0.97After 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 was obtained at bothlow and high resolution EI-MS5O (Figure 3.14). The spectrum shows peaks50at mJz of 428 (As4S), 396 (As4S3), 300 (As4), and other peaks withcombinations of As and S. The result shows that the molecules dissociatesprior to ionization and thus the observed mass spectrum is that of thedecomposition products. The results rule out the possibility that 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 125 299Figure 3.15 Mass spectrum of yellowprecipitate. Spectrum obtained at 330°C225 25951Table 3.4 Elemental analysis of yellowprecipitateC H N S% found 4.88 1.60 1.60 29.3% expected - - - 40(for As2S3) Wet Digestion of Freeze-dried Yellow Precipitate and Flame-AASAnalysisThe amount of precipitate formed by the “arsenate-reducer” in the 10niL test tube was small. The freeze-dried precipitate from the mixedpopulations of 5, 9 and 29 cm were combined for digestion. The precipitate isslightly soluble in nitric acid but a more elaborate digestion procedure thatutilizes nitric acid, sulfuric acid and hydrogen peroxide was used. Theprecipitate dissolves completely and AAS analysis shows that the combinedprecipitate contain 59.2% As. (Calculated for As2S3:60% As). SEM÷EDXA flat surface of the precipitate on the carbon coated stub was scannedat 6.00K magnification. The scanning area of the sample must be smooth toenable the generated x-rays to reach the detector. Two or three differentareas on the same stub were analyzed. Table 3.5 summarizes 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% as As(III). Flame-AAS was also performed tocompare total arsenic.Table 3.6 Flame-AAS and HG-GC-AAS ofsupernatant medium after anaerobic growthof Medium BSupernatant HG-GC-AAS (gIL) Flame-AAS (gIL)A5T As(III) As(V) AST5cm - - - 0.529 cm 0.38 0.09 0.29 0.5829 cm 0.22 0.09 0.13 -- not analyzed.A difference in total arsenic concentration of about 35% was observedbetween the two methods. The discrepencies could be due to the series ofdilutions (i0 dilution) of the sample that were necessary for arsenicconcentration to be within the working range of the HG-GC-AAS. FlameAAS is not as sensitive as the HG-GC-AAS and no dilution was necessary.3.5.4 Summary of Anaerobic MetabolismThe mixed anaerobic microbes from Kam Lake sediment produce ayellow precipitate which is identified as As2S3 from elemental and SEM +EDX analysis. The sulfide is likely to be an artifact, produced from eitherAs(V) or As(III) when microbial growth produces a localized acidic54environment in the medium. In an independent experiment it was foundthat hydrogen sulfide does not precipitate any arsenic sulfide from the mediaunless the media is acidified.The source of sulfide is unknown. Presumably the microbes producesulfide from the reduction of sulfate. The sulfate reduction is usually carriedout by the sulfate-reducing bacteria, but these are unlikely to be present inthis media which has a redox potential of 130 mV. The sulfate-reducers needmedia with a low redox potential (about -300 mV).Speciation of arsenic in the supernatant shows 76% As(V) and 24%As(III), with no methylarsenic acids. Thus the mixed anaerobes do notmethylate the added arsenate although they may be reducing As(V) toAs(III). 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