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The interaction of sediment bacteria with arsenic compounds 1992

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THE INTERACTION OF SEDIMENT BACTERIA WITH ARSENIC COMPOUNDS by JAFARIAH JAAFAR B.A. California State University, Chico,1981 M.Sc. University of Kentucky,1983 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES CHEMISTRY We accept this thesis as conforming to the required standard THE TJNIVEI’ITY OF BRITISH COLUMBIA February 1992 0 Jafariah Jaafar, 1992 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. CHEMISTRY The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: 26 FEBRUARY 1992 ABSTRACT In general, bacteria are capable of biotransforming inorganic arsenic into methylarsenic acids and arsines. The microbial activity of lake sediments was examined with respect to the mobilization of mine tailings that have a high arsenic content. Aerobic and anaerobic mixed microbial populations were isolated from Kam Lake, Yellowknife, N.W.T. An aerobic microbial population from 5 cm sediment depth, the layer immediately above the contaminated mine tailings, was capable of transforming arsenicals. Speciation of arsenicals in the culture medium, determined by using hydride generation - gas chromatography - atomic absorption spectrometry (HG-GC-AAS) shows that this bacterial population is able to methylate arsenicals and subsequently demethylate the product. However, only methylation was observed in media containing dimethylarsinic acid. Anaerobic microbial populations, from all depths, produce a yellow precipitate upon incubation with arsenate for 10-14 days. The precipitate was 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 arsenic tolerant; there is no evidence of methylation of arsenic. 11 TABLE OF CONTENTS Abstract List of Tables V List of Figures vi List of Abbreviations viii Acknowledgements ix Dedication x Chapter I Introduction 1 1.1 Arsenic 1 1.2 Biotransformation of Arsenic 2 1.2.1 Arsenite and Arsenate Redox Transformation 2 1.2.2 Biomethylation ofArsenic 4 1.3 Arsenic-Sulfur Compounds 8 1.4 Methods for the Speciation of Arsenic Compounds 9 1.5 Microbial Sampling 11 1.6 Scope of Work 11 Chapter II Experimental 14 2.1 Reagents and Chemicals 14 2.1.1 Chemicals 14 2.1.2 Chromatographic Supplies 14 2.1.3 Prepared Solutions 14 Arsenate, arsenite, monomethylarsonic acid and dimethylarsinic acid standard solutions 14 Potassium borohydride solution 15 2.2 Instrumentation 15 2.2.1 Chambers for Incubation of Anaerobic Cultures 15 2.2.2 Gas Chromatograph 17 2.2.3 Atomic Absorption Spectrophotometer (AAS) 17 2.2.4 Autoclave 17 2.2.5 Microscope 17 2.2.6 Scanning Electron Microscope + Energy Dispersive X-ray (SEM+EDX) 18 2.3 Culture procedures 18 2.3.1 Media Preparation 18 2.3.2 Bacterial Isolation 21 ‘U 2.3.3 Bacterial Enrichment .23 2.3.4 Bacterial Preservation 23 2.4 Arsenic Metabolism 24 2.4.1 Aerobic Experiment 24 2.4.2 Anaerobic Experiment 25 2.5 Analytical Methods . 25 2.5.1 Hydride Generation-Gas Chromatography-Atomic Absorption Spectrometry (HG-GC-AAS) 25 Hydride Generation 27 Gas Chromatography 27 Atomic Absorption Spectrometry 28 2.5.2 Wet Digestion of the Freeze-dried Yellow Precipitate 28 2.5.3 SEM + EDX 29 Chapter III Results and Discussion 30 3.1 Microbial Sampling 30 3.1.1 Study Site 30 3.1.2 Kam Lake Core Description 31 3.2 Cultivation and Subculture 31 3.3 HG-GC-AAS 34 3.3.1 Calibration And Limit of Detection 37 3.4 Aerobic Arsenic Metabolism 40 3.4.1 Effect of Arsenate 40 3.4.2 Effect of DMAA 45 3.4.3 Summary of Aerobic Metabolism 48 3.5 Anaerobic Arsenic Metabolism 49 3.5.1 Medium B 50 3.5.2 Characterization of Anaerobic Metabolic Product 50 Mass Spectrometry 50 Elemental Analysis 51 Wet Digestion of Freeze-dried Yellow Precipitate and Flame-AAS Analysis 52 SEM + EDX 52 3.5.3 Arsenic Speciation of Supernatant 53 3.5.4 Summary of Anaerobic Metabolism 54 Chapter 1V Bibliography 56 iv LIST OF TABLES TABLE PAGE 1.1 pKa values of arsenic species and reduction products 10 2.1 Tryptic Soy Broth (Medium A) 19 2.2 “Arsenate-reducer” (Medium B) 20 3.1 Oxidation-reduction potential (ORP)measurement for core #6 taken at Kam Lake on 3 August 1991 32 3.2 Oxidation-reduction potential (ORP), pH and temperature measurements for core #3 taken at Kam Lake on 3 August 1991. .33 3.3 Arsenic concentration in Medium B 50 3.4 Elemental analysis of yellow precipitate 52 3.5 SEM + EDX analysis of the yellow precipitate obtained from Kam Lake microorganism 53 3.6 Flame-AAS and HG-GC-AAS of supernatant medium after anaerobic growth of Medium B 54 V LIST OF FIGURES FIGURE PAGE 1.1 Structure of selected arsenic compounds 3 1.2 Challenger’s mechanism for the biological methylation of arsenic 6 1.3 S-adenosylmethionine (SAM) 6 2.1 Map of Yellowknife Lakes System, Northwest Territories 22 2.2 Schematic of HG-GC-AAS System 26 3.1 Chromatogram for 4 mL of a standard solution of 10 ppb As(V), MMAA and 20 ppb of DMAA 36 3.2 Comparison of effects of 4M HC1 and 4M CH3OOH on DMA signal 37 3.3 Calibration curve for the determination of total inorganic arsenic 38 3.4 Calibration curve for the determination of arsenite 38 3.5 Calibration curve for the determination of MMAA 39 3.6 Calibration curve for the determination of DMAA 39 3.7 Inorganic As (D), MMAA (•) and DMAA (•) production by 5 cm aerobic heterotrophs from Kam Lake sediments 42 3.8 Inorganic As (11), MMAA (+) and DMAA () production by 9 cm aerobic heterotrophs from Kam Lake sediments 42 3.9 Inorganic As (Ii), MMAA (4) and DMAA (•) production by 13 cm aerobic heterotrophs from Kam Lake sediments 43 3.10 Production of AsH3 in Blank (D)and Control (4) 44 3.11 Day-to-day variation of total arsenic in the 5 cm aerobic heterotrophs incubation 44 3.12 Production of AsH3 (U), CH3As2(+) and (CH3)2As (I) by the 5 cm aerobic heterotrophs in Medium D 44 3.13 Production of AsH3 (U) from Blank and (CH3)2As (+) from Control media 46 3.14 Trimethylarsine production from 5 cm microbial population in Medium D 48 3.15 Mass spectrum of yellow precipitate. Spectrum obtained at 330°C 51 vii LIST OF ABBREVIATIONS HPLC High performance liquid chromatography AAS Atomic absorption spectrometry GC Gas chromatography ICP Inductively coupled plasma MMAA Monomethylarsonic acid DMAA Dimethylarsinic acid TMAO Trimethylarsine oxide MMA Monomethylarsine DMA Dimethylarsine PRAS Pre-reduced anaerobically sterile TSB Tryptic Soy Broth TSA Tryptic Soy Agar SEM Scanning electron microscope EDX Energy dispersive x-ray ORP Oxidation reduction potential pE - log of electron activity r1u ACKNOWLEDGEMENT I 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 the bacterial isolation and introducing me to the microbial world, and to Ms. Deepthi Hettipathirana for helping me with the thesis stylesheet. Their time and patience spent during this work are very much appreciated. I thank Ms. Eileen Cochien for helping me with the bacterial preservation during Summer 1991, and Mr. Hao Li for trouble shooting the HG-GC-AAS system. I also thank Ms. Mary Mager for teaching me the SEM+EDX. Thanks also to all my lab colleagues for their friendly and helpful endeavors. Finally, I thank the Public Services Department, Malaysia for the financial support and Universiti Teknologi Malaysia for granting me the study leave. ix DEDICATION Affandi, Anna and Azman x CHAPTER I INTRODUCTION 1.1 ARSENIC Arsenic ranks as the twentieth most abundant element in the earth’s crust.1 It is widely dispersed at low levels, ranging from 0.1 to several hundred ppm (jiglg), depending on several factors such as geographical location and anthropogenic input.2 The average arsenic concentration in the earth’s crust is about 3 ppm,1 but much higher concentrations, ranging from 0.4 to 6000 ppm, are found in sulfur containing ores that contain arsenopyrite (FeAsS), realgar (As4S)and orpiment (As2S3). Arsenic has been found to be a common contaminant of both freshwater and seawater in many parts of the world. Arsenic compounds have different properties and uses.4-5 Arsenic trioxide is well known as a poison. Both inorganic and organic arsenic compounds were widely used in medicine.6 Arsenic, like mercury, has been used extensively in agriculture as herbicides and pesticides, and in industrial applications as wood preservatives. High arsenic concentrations in soils have been related to the mining industries, in particular, gold mining. It is recognized that the toxic effects of arsenic depend on the concentrations of individual arsenic species, so the development of analytical techniques that enable the speciation of arsenicals has become an important objective for individuals and organizations interested in the biogeocheniistry of the element. 1 1.2 BIOTRANSFORMATION OF ARSENIC Three types of arsenic biotransformation have been found to occur in the environment: (1) oxidation and reduction between inorganic arsenite, As(III) and arsenate, As(V); (2) biomethylation of arsenic compounds to methylarsenic acids and the more volatile and toxic methylarsines; (3) biosynthesis of more complex organoarsenic compounds, such as arsenobetaine, arsenocholine and arsenosugars. This third class of transformation is carried out by organisms higher up along the food chain, and not, apparently, by microorganisms. However, Hanaoke et al.7, reported the degradation of arsenobetaine to trimethylarsine oxide and dimethylarsinic acid, probably by the intestinal microorganisms in the chiton and only in an aerobic medium. A list of arsenic compounds is shown in Figure 1.1. 1.2.1 Arsenite and Arsenate Redox Transformation In the aquatic environment, arsenic exists in two different oxidation states. In oxygenated water, arsenate is the dominant species, existing as HA5O42-. Arsenite becomes the main species in anoxic basins and inlets, for example, Saanich Inlet, B.C.8 In air saturated waters the redox condition is set by the 02/1120 couple, with a pE of about 12.6.9 Thermodynamic calculations for an oxygenated seawater at pH 8.1 predict the As(V)/As(III) ratio to be about 1026.10 However, significant amounts of arsenite are found in oxygenated surface waters, and conversely, arsenate is found in anoxic waters. 2 HO—As—OH HO—As—OH OH OH Arsenate Arsenite O 0 II II HO — As— CR3 CH3 As— CH3 I I OH OH Monomethylarsonic acid Dimethylarsinic acid CH3 o CH3 CH3— Ask— CR2— C - O CH3— As— CR2— CH,- OH CH3 CR3 Arsenobetaine Arsenocholine 1aYOH, R0SO3H IbY=OH. R=OH IcY=OH, R=SO,H Me2 CM2 o .0—CH2—CH(Y)—CHR Id Y NH2. R = SO3H H’\ /H H Ic YOH, R0P—0CH2 OHOH OH CHOH CH,OH Arsenosugars (1) Figure 1.1 Structure of Selected Arsenic Compounds 3 Studies11’2 found the As(V)/As(III) ratio to be in the range of 0.1 to 100, suggesting that the redox pair is not in thermodynamic equilibrium. In oxygenated seawater, bacteria and phytoplankton have been shown to be responsible for the presence of arsenite, by reduction of arsenate.12-4 Johnsonl2 showed that a mixed population of marine bacteria carries out this reduction in seawater medium. Arsenite is slowly oxidized to arsenate in seawater, even under sterile conditions.15 Some bacteria are also responsible for this oxidation, and Turner et al.l6 in 1954 demonstrated that bacteria from arsenical cattle- dipping fluids could oxidize arsenite to arsenate. The processes that control the oxidation of arsenite to arsenate in natural waters still need further study. 1.2.2 Biomethylation of Arsenic The biomethylation of arsenic compounds has been known for many years; however, interest in this phenomenon has increased, in association with the discovery that microorganisms in natural lake sediments are able to methylate mercury to form a highly toxic methylmercury species.17 The discovery that mercury is readily cycled from the sediments and concentrated by organisms along the food chain18, has led to speculation that similar cycles may occur with arsenic. Certain fungi, yeasts and bacteria are known to methylate arsenic to volatile arsines. Poisoning incidents resulting from fumes emitted from wall paper coverings were first investigated by Gosio in 1892.19 He identified one 4 mould species, Penicillium brevicaule (current name: Scopulariopsis brevicaulis) 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 the presence of arsenic compounds.19 Challenger and coworkers further suggested that methylarsonic acid and climethylarsinic acid were intermediates in the production of trimethylarsine. He favored the hypothesis that the methylation of arsenic involved the transfer of a methyl group from some already methylated compound such as betaine, choline or methionine. Challenger proposed a mechanism for the conversion of arsenate to trimethylarsine, shown in Figure 1.2. The arsenic (III) intermediates in braces are unknown. His proposed mechanism involved alternating oxidation and reduction steps. du Vigneaud and coworkers’ study on transmethylation2O led Challenger to further study trimethylarsine production by S.brevicaulis in the presence of labelled precursors.21 Only14CH3-labelled methionine was found to transfer its label to arsenite to a significant extent. The result indicated that an “active methionine”, S-adenosylmethionine (SAM), Figure 1.3, is the methyl donor. Work by Cullen and co-workers.225supported the idea that SAM is the source of CH3+. They demonstrated that the CD3 group in L methionine-methyl-d3, CD3SCH2CH2CH(NH2)COOH, is transferred intact by cultures of S. brevicaulis. 5 2e [) H3AsO4 As(OH)3 CH3AsO(OH)2 arsenite monomethylarsonje acid CH3AsO(OH)2 {CH3As(O )) ‘(CH3)2AsO(OH) dimethylarsinic acid (CH3)2AsO(OH) 2e ((CH3)As(O )) [cH3](CH3)AsO trimethylarsine oxide (CH3)As trimethylarsine Figure 1.2 Challenger’s mechanism for the biological methylation of arsenic — a—c2—c2— HO I Figure 1.3 S-adenosylmethionine (SAM) arsenate OH 6 Cox and Alexander26isolated microorganisms from soil and sewage in cultures containing various arsenic compounds. The organisms were able to produce trimethylarsine from arsenite, arsenate, methylarsonic acid and dlimethylarsinic acid when grown at pH 5. They suggested that the acidic condition in sewage might promote trimethylarsine production. Shariatpanahi and coworkers27 studied bacteria that were isolated from the environment and grown in the presence of arsenate ( lOOug/mL). Under aerobic conditions, they found the transformation of arsenate to dimethyl and trimethyl species. In experiments with lake and river sediments using pure cultures of the bacteria Aeromonas and Flavobacterium sp. and Escherichia coli, Wong et al.28 observed the production of monomethylarsonic acid, dimethylarsinic acid and trimethylarsine oxide. They also observed the production of dimethyl- and trimethylarsines. The sediments were incubated with or without high levels of arsenic (at least three orders of magnitude higher than a typical non-polluted environment). These results were questioned by Andreae,5 who suggested that this biomethylation is a response to extreme arsenic stress, and not a general process carried out by aerobic bacteria in the environment. Additional work is required to understand the biomethylation process. The mechanism proposed by Challenger seems likely to occur in the aquatic environment with the reduction of arsenate and methylation to monomethylarsonic acid and dimethylarsinic acid. Trimethylarsine is not always the end product. 7 The 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 be energetically favorable for the organisms to transmethylate arsenic (and other metals) rather than to synthesize methane. This methylation might take place in the upper layer of the sediments. This process might be responsible for mobilizing methylarsines from sediments to solutions and to the food chain. 1.3 ARSENIC-SULFUR COMPOUNDS In anoxic water systems, bacterial action would create a reducing environment and the pE will be set by the reduction of S042 to HS.9 Under this reducing condition (pE value approximately -4) arsenite is the dominant species. Stable arsenic sulfides, realgar (As4S)and orpiment (As2S3), will precipitate in the presence of sulfur under acidic anoxic conditions.3° Microorganisms play an important role in geochemical processes, and in the cycling of sulfur. Ehrlich31’2investigated the degradation of sulfide minerals, orpiment and arsenopyrite, by microorganisms. The rate of arsenic released by bacterial oxidation was twice as great as by spontaneous oxidation. The bacterial action released arsenate and arsenite. The biodegradation process results in the solubilization of metal compounds and can be applied practically for metal recovery.33 Another role of microorganisms is in the partial generation of sulfide minerals. However, limited study has been done in this area. 8 1.4 METHODS FOR THE SPECL4TION OF ARSENIC COMPOUNDS Total arsenic has been monitored in environmental samples by using many analytical techniques: atomic absorption spectrometry,3438 atomic emission spectrometry,39voltammetry,40 and neutron activation analysis.41 Currently, there is an increased interest in determining the concentration of individual arsenic species, largely because of their different toxicities. The toxicity decreases in the order: arsines > arsenite > arsenate> alkylarsenic acids > arsonium compounds > metallic arsenic.1 A variety of techniques for the speciation of arsenicals has been reported. Generally, hyphenated techniques such as HPLC-AAS,42 HPLC ICP-MS,43 GC-AAS,4446 and GC-MS47 combined with selective hydride generation and cold-trapping,48’9 are used. However, the problems associated with these techniques are long analysis time and their non-routine nature. A number of arsenic compounds are volatile , such as arsine, or can be converted into volatile arsines by reduction with sodium borohydride. The reduction of arsenic compounds with sodium borohydride is pH-dependent,49 and is related to the pKa of the individual arsenic acids (Table 1.1). Braman et al.38 investigated the effect of pH on the fraction of undissociated arsenic acids present at equilibrium. A number of workers44’6 have used the piCa differences of arsenate and arsenite to speciate them. However, Hinners50 found that MMAA and DMAA interfered with the determination of As(III) and As(V). 9 Anderson et al.49 investigated several factors that influence the arsenic species determination. They used a continuous hydride generation with sodium borohydride and AAS for detection. Several combinations have shown promising selectivity towards the reduction of As(IH), As(V), M.MAA and 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 from solution by sweeping them with an inert gas, collected on a liquid nitrogen cold trap, and separated by using gas chromatography. The separated arsines are then detected by a quartz-cuvette atomic absorption spectrophotometer. Table 1.1: pKa values of arsenic species and reduction products Species piCa’s Reduction Reaction b.p. pH Product (°C) Arsenate 2.2 1 AsH3 -55 Arsenite 9.2 1-6 AsH3 -55 MMAA 4.1 1 CHAs2 2 DMAA 6.2 1-2 (CH3)As 35.6 TMAO - 1-4 (CHAs 70 10 1.5 MICROBIAL SAMPLING Microorganisms, either mixed communities or single species, for laboratory study are isolated by growing them under well-defined conditions as cultures. In nature, bacteria invariably occur as mixed communities containing more than one kind of microorganism. In the present work, enriched cultures of a mixed microbial population were isolated using a selective liquid media containing arsenate. The enrichment technique tends to select for the microorganism with the highest growth rate, by changing conditions in favor of the desired organism. This is because of a direct competition for nutrients among the mixed developing population. Two techniques that are frequently used for the cultivation of strict anaerobes are the roll tube technique, developed by Hungate,56 and the anaerobic glove box technique. In the roll tube method, agar medium is distributed as a thin layer over the internal surface of test tubes charged with an anaerobic atmosphere. Specimens are streaked on the surface of the agar 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 the glove box is more practical since all manipulations are performed in the reducing atmosphere of the box. 1.6 SCOPE OF WORK Arsenic has frequently been concentrated in certain areas as a result of anthropogenic activities. For example, gold mining operations for years dumped arsenic as a waste by-product in their tailings. Mine sites, such as 11 those along the B.C. coast or those adjacent to lakes, frequently dumped tailings directly into these waters, and over time sediments rich in arsenic built up around the effluent outflow. Recent improvements in gold separation methods have reduced arsenic concentrations in effluents and cleaner tailings now lie on top of arsenic-laden layers. Bacteria appear to be capable of mobilizing deposited arsenic into water soluble arsenic acids and arsines. MMAA, DMAA, TMAO and the highly toxic and volatile arsine and methylarsines are known products of bacterial metabolism. 19, 26-28 To initiate an investigation of microbial mobilization of historically deposited arsenic, we decided to see if mixed microbial cultures obtained from sediments in a lake associated with the NercoCon Mine in Yellowknife, N.W.T. could metabolize arsenic. Kam Lake was chosen because it has a well defined sub-stratum with high arsenic concentration:51 the layer resulted from a large spill of process effluent that would normally have been treated prior to disposal but was instead diverted to Kam Lake. In a typical sediment, there is a predictable progression of nutritional microbial types that result from the lower oxygen concentration and increasing reducing condition as depth increases. Aerobes may predominate at the surface, whereas a variety of anaerobic types will predominate beneath the surface. In the present study, two microbial populations were initially sought that would represent aerobic and anaerobic microbial activity. Each mixed population was assayed for its ability to metabolize arsenate and DM.AA. Hydride generation - gas chromatography - atomic absorption 12 spectrometry (HG-GC-AAS) was the analytical method used for arsenic speciation and characterization. 13 CHAPTER II EXPERIMENTAL 2.1 REAGENTS AND CHEMICALS 2.1.1 Chemicals Unless otherwise stated, all chemicals were obtained from commercial sources and were of analytical grade. Tryptic Soy Agar (TSA) and Tryptic Soy Broth (TSB) were obtained from Difco Laboratories. As2S3 and A54S4 were prepared by using literature methods.52,3 2.1.2 Chromatographic Supplies Teflon tubing (1 mm i.d. by 600 mm length) was purchased commercially. Porapak PS (80/100 mesh) was obtained from Waters Chromatography Division/Milhipore Corporation. Silyl 8, a GC column conditioner, was obtained from Pierce Chemical Co. 2.1.3 Prepared Solutions Arsenate, arsenite, monomethylarsonic acid and dimethylarsinic acid standard solutions. Stock solutions of 1000 pg/mL As were prepared by weighing the appropriate amount of Na2HA5O4.7H20, NaAsO2, CH3AsO(ONa)2.6H20 and (CH3)2AsO(OH) into separate 100.0 mL volumetric flasks, and diluting 14 with deionized water (Aquanetics Aqua Media System). The arsenate, arsenite and MMAA stock solutions were prepared as needed. The DMAA stock solution was prepared every two months. Intermediate stock solutions of 10 .tg/mL were prepared by serial dilution. The DMAA solution was prepared daily. A working solution of 10 ng/mL each of As(V), As(III) and MMAA, and 20 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 a polyethylene bottle. One pellet of sodium hydroxide was added. The solids were dissolved in deionized water and the solution was diluted to 50.0 mL. 2.2 INSTRUMENTATION 2.2.1 Chambers for Incubation of Anaerobic Cultures. Incubation of anaerobes requires pre-reduced media and elimination of oxygen from the growth environment. Oxygen can be scavenged within a sealed jar by catalytic hydrogenation over a palladium catalyst. BBL anaerobic chambers (Becton Dickinson Microbiology) and chambers built by the Mechanical Engineering Service, Department of Chemistry, U.B.C. based on the design of Baich et al.54 were used in conjunction with BBL GasPak H2 plus CO2 generator envelopes for all anaerobic incubations. GasPak envelopes contain a tablet of sodium 15 borohydride and a tablet of sodium bicarbonate plus citric acid. To generate H2 and C02, a top corner is cut to allow addition of 10 mL of water. The water is wicked through a channel - the time required is sufficient to allow chambers to be sealed before gas generation begins - to the two tablets, and upon contact the gases are evolved. The hydrogen reacts with oxygen over Pd coated alumina pellets (118”; Aldrich Chemical Co.) held in a wire basket secured to the underside of the chamber headplate to produce water. The appearance of condensate on the chamber walls is an indication of reaction progress. Anaerobic indicator strips (GasPak) soaked with methylene blue indicator change from blue to colorless to confirm the removal of oxygen. Carbon dioxide does not participate in the development of anaerobiosis but is a required nutrient for some anaerobes (e.g. methanogens). All anaerobic sample and culture manipulations were performed under conditions designed to minimize oxygen exposure. Initial sediment core samples were acquired in sealed gravity core sampler tubes55 which were later opened in a glove bag (Instruments for Research and Industry) under an atmosphere of oxygen-free nitrogen. Subsequent culture manipulations were performed in a modified glove box (KSE, Adrian, Michigan) charged with a gas mixture of C02 (5%), H2 (10%) and N2 (85%) and supplied with Pd coated alumina pellets. Anaerobic indicator strips were used to confirm anaerobic conditions, and catalyst was replaced routinely. 16 2.2.2 Gas Chromatograph Two types of gas chromatograph were used: a Hewlett Packard 5830A model with a 18850A GC terminal, and a Hewlett Packard Series 5890. A home-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 Ash Model 810 AAS (Fisher Scientific Co.) was used for arsenic speciation and was equipped with Waters QA-1 Data System. Total arsenic was determined on Varian model AA 1275 series. The AA hollow cathode lamp was operating at 8 mA and the monochromator was set at 193.7 nm, one of the resonance lines of arsenic. The spectrophotometer was equipped with a deuterium background corrector and a Hewlett Packard 82905A printer. 2.2.4 Autoclave Amsco, Eagle Series-3041 Gravity autoclave was used to sterilize all culture vessels and heat-stable media. 2.2.5 Microscope The microbial growth was observed by using an Olympus BH-2 microscope. 17 2.2.6 Scpnning Electron Microscope + Energy Dispersive X-ray (SEM + EDX) A Hitachi S-570 Scanning Electron Microscope (SEM) was used. It was equipped with a Kevex Super8000 Energy-Dispersive X-ray Analyser (EDX). This instrument is located in the Metals and Materials Engineering Department, UBC. 2.3 CULTURE PROCEDURES 2.3.1 Media Preparation All aerobic media were prepared on the open bench, and sterile media were handled in a Class II (laminar flow) biohazard cabinet (Mechanical Engineering Services, Department of Chemistry, UBC). Pre-reduced anaerobically sterile (PRAS)5657 media were prepared by flushing filter- sterilized (glass wool packed tube) nitrogen gas over the headspace of the boiled media in an air-tight Pyrex bottle. All transfers were done in an anaerobic glove box containing palladium catalysts and under an atmosphere of 5% C02, 10% H2 and 85% N2 (from a commercial cylinder). A methylene blue indicator strip was used to check for anaerobic condition. All glassware was flushed with nitrogen gas before being sterilized. All anaerobic incubations were done in the anaerobic chamber. Arsenate augmented Tryptic Soy Broth at quarter strength (Medium A) was prepared by adding 10.0 mL of 100.0 j.ig/mL As(V) solution to 7.50 g of 18 the commercially available powder (Table 2.1) and dissolving this in 1000 rnL of distilled water. Ten mL aliquots of this broth were pipetted into screw cap test 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 g Bacto Sytone (Pancreatic Digest of Soybean Meal) 5 g Sodium chloride 5 g “Arsenate-reducer” medium (Medium B) was prepared from the ingredients shown in Table 2.2. All these (except sodium bicarbonate) were dissolved 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 drying time. The sodium bicarbonate was filter-sterilized by using a 0.45 pm Millipore membrane filter (HV type). The autoclaved solution was cooled, placed in the anaerobic glove box, and 40 mL of the sterile sodium bicarbonate was added aseptically. The final pH of the medium was adjusted to 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 charged with CO2 and H2 from a GasPak envelope, and removed from the glove box. 19 Table 2.2 “Arsenate-reducer” (Medium B) Ingredients gIL NaHCO3 2.5 CaC12.2H0 0.1 KC1 0.1 NH4C1 1.5 NaH2PO.0 0.6 NaCH3COO 2.7 Difco yeast extract 0.05 NaC1 0.1 MgC12.6H0 0.1 MgSO4.7H 0.1 MnC12.4H0 0.005 NaMoO.2H 0.001 FeSO4.7H0 0.001 Na2HAsO.7H 3.12 20 2.3.2 Bacterial Isolation The microbiological sampling was done in the field by Mr. Gary Hewitt of 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 gravity core sampler from an aluminum power boat. Plastic core tubes for assay of pH, temperature and oxidation-reduction potential (ORP) and for microbial sampling had 1 cm holes drilled each 2 cm of their length. The holes were covered with duct tape for sample acquisition and for sealed transport back to the laboratory. Standard core tubes were of 3.75 cm diameter and 60 to 80 cm long. Cores for microbial sampling were transported from Kam Lake to the Department of Indian and Northern Affairs’ water testing laboratory in Yellowknife, and opened in a glove bag (Instruments for Research and Industry) under an atmosphere of oxygen-free nitrogen. The holes were uncovered and sediment samples from different depths were aseptically removed with a sterile spatula after the layer closest to the wall was removed. Roughly 1 mL of sediment was then inoculated into each test medium. Cultures of aerobic heterotrophs, Medium A, were removed from the glove bag and incubated with loose caps. Cultures in “arsenate-reducer” medium (Medium B) were placed in an anaerobic chamber before removal from the glove bag. All cultures were transported to the Department of Chemistry, UBC for further work. 21 Subsequent culture manipulations were performed in a glove box as described in section 2.2.1. Room temperature (approximately 21°C) was used for all incubations. Figure 2.1 Map of Yeflowknife Lakes System, Northwest Territories. B indicates sampling site. 22 2.3.3 Bacterial Enrichment Enrichments were made by serial subculture. The mixed aerobic culture 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 each enrichment step, into a sterile 10.0 mL of Medium B. Medium B was incubated in the anaerobic chamber for 10-14 days between each of the three enrichment steps. 2.3.4 Bacterial Preservation The two-day old aerobic subculture was centrifuged for 30 mm at a speed setting of 80 in a Dynac Centrifuge (Becton Dickinson Co.) and the supernatant was discarded. One mL each of TSB (double strength) and 20% glycerol were added to the bacterial cell to achieve a final concentration of 10% (v/v) glycerol and single strength TSB. The cells were resuspended and 1.0 mL aliquots were dispensed into cryovials (1.2 mL capacity, NUNC), pre cooled on dry ice, and finally stored in a cryogenic refrigerator (Taylor Warton, model 34 HC) charged with liquid nitrogen. Enriched anaerobic cultures were centrifuged by the same procedure and returned to the glove box. The supernatant was discarded and 1.0 mL each 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. The tubes were removed from the glove box, pre-cooled on dry ice and immediately preserved in the cryogenic refrigerator. 23 2.4 ARSENIC METABOLISM 2.4.1 Aerobic Experiment The medium used was TSB containing 50 ng/mL As(V) solution (Medium C). Twenty mL portions of the sterile medium was pipetted aseptically into a series of Evergreen tubes (50 mL capacity, polypropylene tube). The preserved aerobic heterotrophs were thawed and grown on Tryptic Soy Agar (TSA) + 1.0 As(V) plates. The plates were incubated at room temperature for 2 days to allow colonies to develop. Approximately 7 mL of Medium C was added to the plate and any growth was scraped to resuspend it. The cell suspension was pipetted to a test tube containing Medium C and the turbidity was measured spectrophotometrically at 420 nm with a Spectronic 20. Appropriate volumes of the cell suspension and Medium C were mixed to obtain an absorbance reading of 1.0. Two mL each of the standardized inoculum was added to 20.0 mL Medium D in 7 Evergreen tubes. The tubes were incubated aerobically at room temperature. All cultures were shaken daily to resuspend the growth. One tube was centrifuged each day, and the ‘snift test’ was carried out to detect the production of arsines, and then the supernatant was transferred to a sterile tube and frozen prior to analysis for arsenic compounds. The solid phase and bacterial cells were discarded. The process was repeated for 7 days. Controls, containing Medium C, and blanks (TSB) were carried through the same incubation condition and frozen daily for arsenic analysis. 24 A 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 aerobic heterotrophs were added to Media D and E. The tubes were incubated as before. One each of eleven days supernatant samples were frozen for arsenic speciation. Controls (Media D and E) and TSB blanks were similarly prepared. 2.4.2 Anaerobic Experiment One mL of the anaerobic culture solution from the third enrichment step was added to 10.0 mL Medium B. The test tubes were incubated in the anaerobic chamber for 10-14 days. A heavy yellow precipitate formed and was separated from the solution by centrifugation for 30 mm at a speed setting of 80. The precipitate was washed three times with distilled water, freeze-dried and its composition was investigated by using mass spectrometry, elemental analysis, and SEM + EDX. The supernatant was analyzed for arsenic compounds by HG-GC-AAS. 2.5 ANALYTICAL METHODS 2.5.1 Hydride Generation-Gas Chromatography-Atomic Absorption Spectrometry (HG-GC-AAS) Arsenic speciation studies involved a selective hydride generation of volatile arsines, which were then concentrated by cryogenic trapping. The 25 arsines were separated by gas chromatography and detected by atomic absorption spectrophotometer. The HG-GC-AAS system is shown in Figure 2.2. Figure 2.2 Schematic of HG-GC-AAS System Trapping Ar.cines Valve Sample Liquid N2 Trap Valve :He ,“ Atomic Absorption •‘ Spectrometer dry-ice I alcohol Water Trap S S S S S Water BathDrain Reaction Vessel Re1easin’ Arsines Gas Chromatograph 26 Hydride Generation One to two mL of the sample solution was injected into the Teflon reaction vessel containing 50 mL deionized water and 4 mL, 4M HC1. The mixture was flushed with a helium flow for 5 mm. Four mL of 4% KBH4was slowly injected into the vessel and the generated arsines were swept by a helium flow through an acetone-dry ice trap to remove water and were collected for 5 mm in a trap made of Teflon tubing immersed in liquid nitrogen. The liquid nitrogen was removed and the tubing was warmed in a water bath and the arsines were flushed onto the GC column. Gas Chromatography A Porapak PS column was prepared as follows: A 600 mm length of Teflon 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 was hand-spun for 5 minutes to ensure tight packing. The open end of the column was then plugged with Teflon wool. To condition the column, one end of the packed column was connected to the injection line, while the exit was left unconnected in the GC oven. The column was conditioned by flushing the column with helium gas as the temperature was raised from 60°C to 150°C, at a rate of 300/min. The temperature was then held at 150°C for an hour and subsequently cooled to 60°C. One end of the column was injected with 50 mL of Silyl-8. The column was heated again at 150°C for 45 mm. The column silanization was repeated for the other end of the column. 27 Temperature progrimming was used to enhance the separation of the arsines, by the column and the program used was as follows: Temp 1 60°C Time 1 6 sec Rate 30°C/mm Temp 2 150°C Time 2 3 mm. Atomic Absorption Spectrometry The effluent from the GC was detected in a hydrogen-air flame in a quartz-cuvette of the AAS. Arsines produced from inorganic arsenic [As(V) + As(III)], MEMAA, DMAA and TMAO were detected. Standards arsenicals were injected to establish the retention times and the standard calibration curves. 2.5.2 Wet Digestion of the Freeze-dried Yellow Precipitate The dried precipitate (5 mg) was added to concentrated nitric acid (3 mL), concentrated sulfuric acid (1 mL) and 30% hydrogen peroxide (3 niL) in a 100 mL round bottom flask. The flask was fitted with Teflon stopper and air condenser. The flask was heated at reflux for 2 hours by using a heating mantle. The flask was then cooled to room temperature and the digested sample 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 50 ppm As(V) standards. 28 2.5.3 SEM + EDX The solid sample was fixed with carbon Dag (colloidal carbon in mixed alcohols) onto a spectrographic-grade graphite stub and the surface was coated with carbon (200A) in a vacuum evaporator (Japan Electron Optic Laboratory). The stub was examined with a SEM using an accelerating potential of 30 KeV and a working distance of 35 mm. The x-rays generated by the electron beam were detected, identified and quantified by using the computer associated software package (Kevex Quantex IV). 29 CHAPTER III RESULTS AN]) DISCUSSION 3.1 MICROBIAL SAMPLING 3.1.1 Study Site An investigation was initiated by Reimer and coworkers on the Yellowknife Lakes system to investigate the speciation, redistribution and bioavailability of arsenic compounds from contaminated mine tailings.51 As part of this program, the microbial activity of lake sediments was examined with respect to the mobilization of mine tailings that contain a high arsenic content. Details of the geochemical cycling or arsenic and various metals in these lakes are presented in a report.51 Kam Lake was chosen as one study site. It has a maximum depth of 12 meters. According to Wagemann et al.59 the dissolved arsenic concentration ranges from approximately 2000 ppb to more than 5000 ppb in the water column of Kam Lake. Bright and coworkers51observed an arsenic concentration ranging from 2200 ppm in the upper layer of the sediment to about 50 ppm in deeper layers. The sediment of Kam Lake sampled by Bright and coworkers51in 1990 showed a profile that runs from an oxygenated surface layer to a sub-one zone and finally an anoxic layer. This typical profile is likely to promote a sequence of microbially mediated redox processes in which the aerobes dominate at the surface and anaerobes below the surface. 30 3.1.2 Kam Lake Core Description The sediment core (core #6) that was used for the microbiological sampling was obtained on 3 August 1991. Table 3.1 gives a detailed description of the core at various depths. The temperature and pH of the core were 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) and temperature of 18°C to 20°C (Table 3.2). 3.2 CULTiVATION AND SUBCULTURE Once collected the cores were kept cool (0°C) for shipment to the laboratory in Yellowknife. The core was then exposed and sampled in a glove bag under an atmosphere of oxygen-free nitrogen. Less than 8 hours elapsed between the time of sample acquisition and inoculation into test media in Yellowknife. All cultures were transported back to UBC, Vancouver for subsequent studies. The microbial growth in liquid media was monitored by visual observation of physical changes. The aerobes took about 48 hours to produce a turbid suspension. The anaerobes did not produce a turbid solution, however, the formation of a heavy yellow precipitate was used as the growth indicator. It took about 10-14 days for the anaerobes to produce the precipitate. The amount of precipitate formed decreased slightly with each subculture. 31 Table 3.1 Oxidation-reduction potential (ORP) measurement for core #6 taken at Kam Lake on 3 August 1991. Hole# Sediment Deptha (cm) ORP (mV) 1 1 - 2 3 -225 3_ 5 - 4 7 -425 5 9 - 6 11 -358 7 13 - 8 15 -328 9 17 - 10 19 - 11 21 -364 12 - - 13 25 -392 14 - - 15 29 - 16 31 -377 0-8 cm: oxidized green-brown sediment of fine silt/sand (hole #1) gives way 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 fibrous material (hole #7). 17 cm: transition band from slate grey tailings to more brown colored natural sediments beneath (hole #9). 18-32 cm: brown colored natural sediments: no dark patches, no H2S smell. -: no readings taken. a: Water depth 7 m. 32 Table 3.2 Oxidation-reduction potential (ORP), pH and temperature measurements for core #3 taken at Kam Lake Hole# Sediment ORP (mV) pH T°C Deptha (cm) 1 - - 7.38 20.1 2 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 19.0 8 15 -262 7.07 - 9 17 -281 - - 10 19 -270 - - 11 21 - - - 12 23 - 6.97 19.1 13 25 - - - 14 27 - 6.93 - 15 29 - - - 16 31 - 6.69 18.2 - : no readings taken. a Water depth 6.5 m Sediment 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 result that aerobic microbial growth occurred at all depths. The redox potentials of the 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. This is again surprising because Medium B has a redox potential of 130 mV. 33 Addition of sediment and the metabolism of bacteria in the anaerobic medium could be expected to lower the redox potential but only slightly, and not to the -300 mV necessary for strict anaerobes. Possibly the mixed anaerobic population comprises obligate anaerobes at the surface ranging to strict anaerobes at the bottom of the core. The anaerobes probably have oxygen-protective enzymes to enable them to survive in the oxygenated surface layer. 3.3 HG-GC-AAS The hydride generation technique was used for the determination of arsenite, arsenate, monomethylarsonic acid, dimethylarsinic acid and trimethylarsine oxide. The reducing system employed for arsine production is an alkaline potassium borohydride solution. It is customary to use an aqueous solution of KBH4 (0.5-8% wlv) in NaOH medium. The reaction in acidic 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 hydride generation. In the continuous flow technique,28,4619 a pump is used for the transport of sample solution and KBH4 solution to a reaction coil where the hydride formation occurs. A continuous signal is observed by the AAS. In the batch type apparatus,596Othe KBH4 solutior is mixed with the acidic sample in a reaction vessel. Reaction takes place immediately and the 34 arsines are swept by an inert carrier gas to the AAS. A discontinuous signal is observed by the AAS. The batch type system was used in the present study to determine arsenic speciation. The reaction mixture is buffered to pH 0.5 with 4M HC1 and the addition of KBH4 reduced inorganic arsenic [As(III) + As(V)] to AsH3, monomethylarsonic acid (MMAA) to monomethylarsine (MMA), dimethylarsinic acid (DMAA) to dimethylarsine (DMA), and trimethylarsine oxide (TMAO) to trimethylarsine (TMA). The reaction mixture is subsequently buffered to pH 6 with 1M citrate in 10% citric acid whereupon addition of KBH4 converts arsenite [As(III)] only to AsH3. The arsenate concentration can be determined from the difference between the arsine signals 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 a gas chromatographic column such as a silanized Porapak PS packed column44 interfaced to the trap. Temperature programming is used to enhance the separation. The effluent from the GC is swept into a quartz cuvette of an AAS where it is combusted in a hydrogen-air flame. Retention times are used for the identification of arsines and peak areas are used to calculate the concentration of As(III), As(V), MMAA, DMAA and TMAO in the sample solutions. A typical chromatogram of the generated arsines is shown in Figure 3.1. Arsine (AsH3) has a retention time of 0.96 mm, monomethylarsine (CH3ASH2) of 1.66 mm and dimethylarsine [(CH3)2AsHI of 2.41 mm. 35 12 10• C? rj C C? C 6 C? C? 4 2- 0 1 3 Figure 3.1 Chromatogram for 4 mL of a standard solution of 10 ppb As(V), MMAA and 20 ppb of DMAA. The dimethylarsine signal from the HC1 buffered reaction is the least reliable in terms of sensitivity and reproducibility, as has been previously reported.44 This has been attributed to incomplete reduction, condensation in the transport tubes or incomplete atomization.49 I • I 2 Time (mm) 36 Changing the acid system to 4M acetic acid (pH 2.5) improves the DMA signal (Figure 3.2) but the problem of long term reproducibility remains to be solved. 80 4M Acetic c60 •/ 40. 4MHCI 0 20 40 60 80 DMAA Concentration (ng) Figure 3.2 Comparison of effects of 4M HC1 and 4M CH3OOH on DMA signaL 3.3.1 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 shown inFigures3.3-3.6. 37 60 C? C? [Inorganic As] (ppb) 40 Figure 3.3 Calibration curve for the determination of total inorganic arsenic 50 Figure 3.4 Calibration curve for the determination of arsenite 50 40 30 20 0 10 20 30 0 10 20 30 40 As(III) Concentration (ppb) 38 80 C) C) cl Figure 3.5 Calibration curve for the determination of MMAA C) I C) rj) Figure 3.6 Calibration curve for the determination of DMAA 60 0 10 20 30 M11AA Concentration (ng) 40 80 60 40 20 0 0 20 40 60 80 DMAA Concentration (ng) 39 The calibration curves show a linear dependence of the AAS peak for the concentration range studied. The absolute limit of detection, defined as the analyte concentration producing a signal equal to the blank plus three standard deviation of the blank, is 0.12 ng (of As) for As(III), 0.25 ng for As(V) and the methylarsenic acids. 3.4 AEROBIC ARSENIC METABOLISM According to Bright et al.,51 the concentration of methylated arsenic species in porewater from a Kam Lake sediment core is highest (about 5.2 ppb As) at a depth of 8-13 cm. From Tables 3.1 and 3.2, this layer corresponds to the deposited tailings from the mine. 3.4.1 Effect of Arsenate The mixed bacterial populations from 5, 9 and 13 cm depth were chosen for the initial assay of arsenic metabolizing activity. The inoculum used was standardized by spectrophotometric measurement of the turbidity of a cell suspension. The mixed populations were grown in arsenic augmented broth (50 ppb As(V) solution). Samples taken daily from a series of Evergreen-tube cultures over a period of 1-7 days were analyzed for arsenic species. Figures 3.7 - 3.9 show the results of the initial experiment. The mixed population from the 5 cm depth (Figure 3.7) shows a pattern of methylation and demethylation. The concentration of added arsenate in the media decreases rapidly within 2 days, reaching zero by 5 days. The added arsenate 40 is transformed into MMAA and DMAA over the 7 days of incubation. The formation of DMAA increases from day 1 to day 4, giving way to a progressive decline on days 5-7. This pattern of variation indicates a shift from production to decomposition of DMAA, indicating that demethylating microorganisms are replacing the methylators in the mixed community. The 9 cm and 13 cm microbial cultures show very small amounts of methylated arsenic species (Figures 3.8 and 3.9). The amount of added arsenate decreases with time with no apparent transformation to MMAA or DMAA. Comparisons of the blank and control media (Figure 3.10) with the cultured media show that the mixed microorganism from the 5 cm depth have 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 recovered fully in the supernatant analyzed. The total arsenic concentration also varies from day to day (Figure 3.11). The observed decrease could be due to: (1) the added arsenic could be transformed and adsorbed on the bacterial cells and thus lost when the solid phase was discarded after centrifugation; (2) uptake of arsenic by the bacteria could cause the concentration to drop; (3) there are other species of arsenic in the supernatant that are undetected by the hydride generation technique; (4) formation of volatile arsines, that escape detection by the “snift test”. The “snift test” is based on the intense, garlic like odor of the arsines and arsine production can be evaluated by cautious sniffing of a sample of the headspace of the test tube.30,44 41 30 1 r;j2 20 10 0 2 4 6 8 Incubation Time (day) Figure 3.7 Inorganic As (CD, MMAA (+) and JJMAA (•) production by 5 cm aerobic heterotrophs from Kam Lake sediments 100• 80 60• 40 20 ‘I V 2 4 6 80 Incubation Time (day) Figure 3.8 Inorganic As (C]), MMAA (+) and DMAA (•) production by 9 cm aerobic heterotrophs from Kam Lake sediments 42 80 40 Cl) — — F — F - 0 2 4 6 8 Incubation Time (day) Figure 3.9 Inorganic As (D), MMAA (+) and DMAA () production by 13 cm aerobic heterotrophs from Kam Lake sediments 20 43 70 cI 60 C 40 30, c 20 10- Incubation Time (day) Figure 3.11 Day-to-day variation of total arsenic in the 5 cm aerobic heterotrophs incubation I • I • I 2 4 6 8 Incubation Time (day) Production of AsH3 in Blank (U) and Control (•) 0 0 Figure 3.10 70 60 50 40 30 20 10 0 0 2 4 6 8 44 Only 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 of the tailings layer and not in the tailings (9-16 cm) where the arsenic levels are anticipated to be highest. Probably some reaction (possibly microbial) takes place in the tailings layer that causes arsenic to be mobilized to the layers above, where the microbial population then transforms the inorganic arsenicals to methylarsenicals. The thickness of the tailings layer varies from one core sample to another and the arsenic methylators are probably present in the layer overlying the mine tailings. Following the results of the initial assay, the mixed population from the 5 cm depth was used for further investigation of the biotransformation of arsenic. The protein in the cultured medium caused severe foaming in the reaction vessel during the speciation by hydride generation and addition of 2- propanol, as an anti-foam agent, did not alleviate the problem. The culture medium was reduced to half strength TSB in subsequent experiments to alleviate this problem. The medium appears to be more favorable for the growth of arsenic methylators. 3.4.2 Effect of DMAA Because mixed microbial populations from 5 cm depth methylate the added arsenate to MMAA and DMAA, we would expect the microbes to demethylate any added DMAA to MMAA and inorganic arsenic. Instead, as shown in Figure 3.12, no MMAA is detected in the supernatant over a period of 1-11 days. The DMAA concentration fluctuates from day-to-day giving a 45 complex zigzag pattern with no hint of a trend. This pattern appears to indicate that DMAA is taken up by the mixed populations at a different rate each day, either transforming it and releasing it back to the solution or storing it inside the cells. %30 0 -. C.) C c-) 0 2 4 6 8 10 Incubation Time (day) Figure 3.12 Production of AsH3 (D), CH3As2(+)and (CH3)2As () by the 5 cm aer6bic heterotrophs in Medium D The DMAA concentration decreases with no evidence of transformation to MMAA or inorganic arsenic. The amount of inorganic arsenic in the supernatant is zero on day 2. However, the amount in the blank (Figure 3.13) is ten times higher, over the 11 days study period. 40 20 10 12 46 60 c?30 C.? C 2O 10 . I • I • I 0 2 4 6 8 10 12 Incubation Time (day) Figure 3.13 Production of AsH3 (Ii) from Blank and (CH)2As (4) from Control media 47 Figure 3.14 Trimethylarsine production from 5 cm microbial population in Medium D Trimethylarsine oxide is produced on days 5-11 (Figure 3.14) from DMAA. (Due to the difficulty of quantification, the amount of trimethylarsine is reported in terms of AAS peak area). 10 8 C? 6 C? 4 Cf2 0 0 2 4 6 8 10 12 Incubation Time (day) 3.4.3 Summary of Aerobic Metabolism Microbial communities in sediments consist of co-existing species that interact with each other in a complex way. Microorganisms isolated from the layer overlying contaminated mine tailings are able to biomethylate arsenic. Addition of an external arsenic source (50 ppb As(V) solution) to this mixed community reveals a pattern of methylation and demethylation 48 suggesting a successive change between the methylating microbes and demethylating microbes, as shown in Figure 3.7. Addition of 50 ppb DMAA shows biomethylation to TMAO by the mixed microbes. No demethylation is observed. The reason for these observations is not understood at this time. Further work needs to be done to quantify the total arsenic in the solid and liquid phase. Probably there is an uptake by the microbes and storage inside the bacterial cells, and this could account for the above discrepancy. 3.5 ANAEROBIC ARSENIC METABOLISM The anaerobic “arsenate-reducer” produces a heavy yellow precipitate after 14 days of incubation. The yellow color of the precipitate indicates that it might be elemental sulfur, formed by the reduction of suifate. The low (0.04 mM) sulfate concentration in Medium B could just be sufficient to maintain the metabolic activity of the microorganisms. Moreover there is an almost inexhaustible supply of sulfate in the Kam Lake sediment which contain approximately 0.33 mM sulfate in its porewater.51 The sulfate reduction is usually carried out by dissimilatory sulfate-reducing bacteria, and the “arsenate-reducer” would have to compete successfully with the sulfate-reducing bacteria for the available substrates. A second likely product is an arsenic sulfide compound. Kam Lake contains high levels of arsenic, hence, it is possible that the mixed microbial population uses arsenate as a terminal electron acceptor. In the presence of 49 large amount of sulfur and in acidic medium, As2S3 and As4S precipitate out. 3.5.1 Medium B The arsenic concentration in Medium B was analyzed by AAS (Table 3.3). There appears to be a 10% loss of arsenic after sterilization. The medium is autoclaved in an air-tight Pyrex bottle, so the likelihood of loss by volatilization is minimized. To avoid the problem, whatever the cause, the arsenate solution should be filter-sterilized in future preparations of media to prevent its loss. Table 3.3 Arsenic concentration in Medium B Flame-AAS HG-GC-AAS [As] [AS]T [As(III)] [As(V)] gfL g/L Before autoclave 1.07 1.02 0.05 0.97 After autoclave 0.98 0.91 0.04 0.86 3.5.2 Characterization of the Anaerobic Metabolic Product Mass Spectrometry The mass spectrum of a freeze-dried precipitate was obtained at both low and high resolution EI-MS5O (Figure 3.14). The spectrum shows peaks 50 at mJz of 428 (As4S), 396 (As4S3), 300 (As4), and other peaks with combinations of As and S. The result shows that the molecules dissociates prior to ionization and thus the observed mass spectrum is that of the decomposition products. The results rule out the possibility that the precipitate is elemental sulfur or arsenic. Elemental Analysis Elemental analysis was done by Mr. Peter Borda, Chemistry Department, UBC. Table 3.4 shows the result of the analysis. The presence of arsenic interfered with the sulfur determination. I SE 90 89 70 60 59 40 39 20 19 0 75 499 197 90 80 79 69 59 49 39 29 IS S 199 125 299 Figure 3.15 Mass spectrum of yellow precipitate. Spectrum obtained at 330°C 225 259 51 Table 3.4 Elemental analysis of yellow precipitate C 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-AAS Analysis The amount of precipitate formed by the “arsenate-reducer” in the 10 niL test tube was small. The freeze-dried precipitate from the mixed populations of 5, 9 and 29 cm were combined for digestion. The precipitate is slightly soluble in nitric acid but a more elaborate digestion procedure that utilizes nitric acid, sulfuric acid and hydrogen peroxide was used. The precipitate dissolves completely and AAS analysis shows that the combined precipitate contain 59.2% As. (Calculated for As2S3:60% As). SEM÷EDX A flat surface of the precipitate on the carbon coated stub was scanned at 6.00K magnification. The scanning area of the sample must be smooth to enable the generated x-rays to reach the detector. Two or three different areas on the same stub were analyzed. Table 3.5 summarizes the results. 52 Table 3.5 SEM + EDX analysis of the yellow precipitate obtained from Kain Lake microorganism. Sample Weight Percent As S Ca 5 cm 57.0 38.2 4.83 ±2.6 ±2.0 ±0.7 9 cm 57.9 42.1 0.0 ±2.9 ±2.9 29 cm 56.1 40.1 3.70 ±0.9 ±0.5 ±0.4 Medium B + H2S 58.0 42.0 0.0 ±2.4 ±2.4 As2S3 57.6 42.4 ±4.3 ±4.3 As4S 70.3 29.3 Standards ofAs2S3and As4Swere similarly analyzed. The combined results indicate that the yellow precipitate is probably As2S3 with some “included” calcium and organic compounds. 3.5.3 Arsenic Speciation of Supernatant The supernatant media from cultures that produced the yellow precipitate was analyzed by HG-GC-AAS to determine arsenic speciation (Table 3.6). No methylarsenic acids were detected; approximately 76% of 53 arsenic exists as As(V) and 24% as As(III). Flame-AAS was also performed to compare total arsenic. Table 3.6 Flame-AAS and HG-GC-AAS of supernatant medium after anaerobic growth of Medium B Supernatant HG-GC-AAS (gIL) Flame-AAS (gIL) A5T As(III) As(V) AST 5cm - - - 0.52 9 cm 0.38 0.09 0.29 0.58 29 cm 0.22 0.09 0.13 - - not analyzed. A difference in total arsenic concentration of about 35% was observed between the two methods. The discrepencies could be due to the series of dilutions (i0 dilution) of the sample that were necessary for arsenic concentration to be within the working range of the HG-GC-AAS. Flame AAS is not as sensitive as the HG-GC-AAS and no dilution was necessary. 3.5.4 Summary of Anaerobic Metabolism The mixed anaerobic microbes from Kam Lake sediment produce a yellow precipitate which is identified as As2S3 from elemental and SEM + EDX analysis. The sulfide is likely to be an artifact, produced from either As(V) or As(III) when microbial growth produces a localized acidic 54 environment in the medium. In an independent experiment it was found that hydrogen sulfide does not precipitate any arsenic sulfide from the media unless the media is acidified. The source of sulfide is unknown. Presumably the microbes produce sulfide from the reduction of sulfate. The sulfate reduction is usually carried out by the sulfate-reducing bacteria, but these are unlikely to be present in this media which has a redox potential of 130 mV. The sulfate-reducers need media 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 not methylate the added arsenate although they may be reducing As(V) to As(III). Otherwise the microbes could be arsenic tolerant and be particularly insensitive to arsenic, with no uptake or metabolism of the arsenate added. 55 CHAPTER W BIBLIOGRAPHY 1. National Research Council. Arsenic; National Academy of Sciences; Washington, DC, 1977; p16. 2. Sanders, J.G. 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