Microbial processes and arsenic mobilization in mine tailings and shallow aquifers J. Routh Department of Geology and Geochemistry, Stockholm University, Stockholm, Sweden A. Saraswathy Department of Biology, West Virginia State College, West Virginia, USA ABSTRACT: Microbial processes play an important role in transforming and mobilizing As in the sub-surface. Enrichment cultures indicated several As tolerant species, which actively reduced As(V) to As(III). No change in As speciation occurred in the controls, thereby confirming As(V) reduction was biologically mediated, and active metabolism was a prerequisite for reduction. Different growth and As(V) reduction rates were noted under oxic to sub-oxic conditions, and a zero-order model best fits the As(V) reduction data. Arsenic concentrations in the microcosms seem to affect biomass yield and As(V) reduction rates in some of the strains. Arsenic reduction in these microorganisms probably occurs for respiratory or detoxification purposes. It is likely that microbial mobilization of As may have an impact on groundwater remediation treatment in these environments. 1 INTRODUCTION Arsenic (As) compounds are highly toxic (Nriagu 2002). People have known about its lethal proper- ties since antiquity, and have often used arsenic trioxide (As 2 O 3 ) for homicide purposes. Renewed interest in the metalloid has increased dramatically, but for different reasons. A recent paper by Bhattacharya et al. (2002) details the As crisis, which affects the lives of general population en masse in several countries. The problem is nowhere as serious as it is in Bangladesh and India, where more than 70 million people are at risk from drinking As-rich groundwater (Harvey et al. 2003). The first reports on clinical manifestation of As toxicity from this region came up around 1978, and thereafter, chronic cases of As poisoning (including fatalities) have been reported since 1982 (Saha 1984, Goriar 1984). Arsenic commonly occurs in the environment as inorganic trivalent As(III) and pentavalent As(V) species (Cullen & Reimer 1989). The trivalent arsenous acid is more dominant under reducing conditions, whereas its pentavalent counterpart, in the form of arsenic acid is common under oxi- dizing conditions. Mobility of As(III) is higher compared to As(V) in sedimentary and aqueous environments. The difference in mobility is attributed to the high affinity of As(V) for insoluble species such as hydrous ferric and manganese oxides (Cullen & Reimer 1989). Additionally, several methylated forms of organoarsenicals (e.g., methylarsonic, methylarsonus, and dimethylarsenic acid) are also found in water as breakdown or excretory byproducts (Cullen & Reimer 1989, Sohrin et al. 1997). Although As(III) is not thermodynamically stable under oxidizing conditions, there are several incidences where As(III) occurs as the dominant species in the water column (Aurillo et al. 1994, Sohrin et al. 1997). Prevalence of As(III) in these studies was correlated with phytoplankton abundance suggesting non-equilibrium conditions were microbially mediated. Arsenic enters the terrestrial and aquatic environments through both natural and anthropogenic activities. Natural processes can contribute to the widespread distribution of As through weathering 145 Natural Arsenic in Groundwater: Occurrence, Remediation and Management – Bundschuh, Bhattacharya and Chandrasekharam (eds) © 2005, Taylor & Francis Group, London, ISBN 04 1536 700 X Copyright © 2005 Taylor & Francis Group plc, London, UK of As bearing rocks and minerals, microbial activity, and volcanic eruptions. In contrast, anthro- pogenic point sources are localized and include inputs from mining of base metals, smelter slag, coal combustion, production of paints and dyes, tanning, wood preservation, and pesticides. The net output of As from anthropogenic processes is high compared to natural processes (Bhattacharya et al. 2002), but ironically, it is the natural processes that are involved in dispersion of As, which is of most concern to human beings. 1.1 Arsenic toxicity and microbial resistance Arsenic compounds readily accumulate in living tissues due to their affinity for proteins and lipids or cause breakdown of oxidative phosphorylation (Oremland & Stolz 2003). Many prokaryotes and eukaryotes have however, developed unique inter-cellular reaction mechanisms to rid them- selves of As, and excrete it as waste byproducts. Some even generate energy during this process. For example, higher eukaryotes reduce As(V) to As(III) followed by methylation resulting in mono and dimethylarsonoic acids (MMA, DMA). Fungi produce trimethylarsines and bacteria produce MMA and DMA (Diorio et al. 1995, Sohrin et al. 1997). The physical excretion of these byproducts often occur as encrustations on the cell wall (Saraswathy et al. 2004) or transferred into the water column at the sediment-water interface (Ahmann et al. 1997, Martin & Pedersen, 2002, Routh et al. 2004). Additionally, As can be converted to benign products as arsenobetaine and As containing sugars found in marine algae, animals, and higher plants (Cullen & Reimer 1989). Reduction of As(V) to As(III) in anoxic environments primarily occurs via dissimilatory As reduction (Ahmann et al. 1997, Newman et al. 1997a,b), whereby microorganisms utilize As(V) as the terminal electron acceptor. The reaction is energetically favorable and coupled to oxidation of organic matter. Two important prerequisites for such microorganisms are the presence of strict anoxic conditions and high As levels (Newman et al. 1997b). Dissimilatory As reduction occurs in bacteria scattered throughout the bacterial domain representing ␥-, ␦-, -Proteobacteria, low-GC gram-positive bacteria, thermophilic Eubacteria, and Crenoarchea (Oremland & Stolz 2003). However, microorganisms may also possess reduction mechanisms that are not coupled to respi- ratory processes, but instead, impart resistance to As toxicity (e.g., Jones et al. 2000, Macur et al. 2001). Enzymes involved in the detoxification pathway are transcribed by the ars operon resulting in inter-cellular reduction of As(V), and the subsequent efflux of As(III) via a trans-membrane pump (Cervantes et al. 1994, Rosen 2002). 1.2 Arsenic mobilization Different biogeochemical mechanisms have been proposed to explain As mobilization in sedimen- tary environments. These processes involve: (1) oxidation of pyrite, (2) reduction of Fe-oxyhydroxides coupled to oxidation of organic matter and release of As(V), (3) exchange of As(V) with phosphate based fertilizers (e.g., Roy Chowdury et al. 1999, Nickson et al. 1998, Harvey et al. 2003). More recently, researchers have increasingly focused on the role of microorganisms in mobilizing As in sedimentary environments (e.g., Ahmann et al. 1997, Cummings et al. 1999, Jones et al. 2000, Macur et al. 2001, Islam et al. 2004). Transformation of As by microorganisms has important environ- mental implications because As(V) and As(III) have different sorption and toxicological properties. Primarily such studies have mostly focused on sites contaminated by mining, pesticides or other related anthropogenic activities, and they all demonstrate enhanced microbial As mobilization on short time scales. Here, we present data from our ongoing investigations on mine tailings in northern Sweden and shallow aquifers in West Bengal, India. The environments are completely different in terms of physiographic settings, sub-surface geology, and environmental conditions, but both places indicate high As concentrations in ground and surface water. Although different processes in the sub-surface may substantially influence the biogeochemical cycling of As, we focused on microbial processes and their affect As cycling. The different microbial processes important in As cycling underscore the need for our continued inquiry regarding As transformations, in hopes, that we can develop 146 Copyright © 2005 Taylor & Francis Group plc, London, UK greater predictability of its behavior. To the best of our knowledge, microbial processes affecting As mobilization has not been investigated by other researchers at these sites, and may thus, pro- vide new insights. Moreover, microbial processes involved in As(V) reduction and mobilization are many times faster than chemical transformation (e.g., Sohrin et al. 1997, Jones et al. 2000). If microbial processes are indeed active, this may have important environmental implications on As remediation for groundwater treatment and management at these sites. 2MATERIALS AND METHODS 2.1 Sampling sites Adak mine tailings: The abandoned mine tailings at Adak in Västerbotten district of northern Sweden extends over 1500 m ϫ 2000 m ϫ 5 m. The site is affected by acid mine drainage and has low pH (ϳ3–4). The tailings have high concentrations of As, Cu, and Zn, and they have been mixed with glacial till to reduce surficial weathering (Jacks et al. 2003). The tailings are underlain by peat bogs, and extend close to the shores of Lake Ruttjejaure. Shallow streams running adjacent to the tailings deposits, drain into Lake Ruttjejaure carrying washouts of mine tailings. Sampling was done using a gravity corer to extract undisturbed sediment cores from the lake. Details on sam- pling and different geochemical and microbiological assays are further discussed in Bhattacharya (2004). Ambikanagar groundwater aquifer: Ambikanagar is located in the Deganga Block of North 24 Parganas in West Bengal, India. The water table occurs at a depth of 5-m, and rainfall in summer is the principal source of recharge for aquifers. Reconnaissance work by our group indicated that As concentrations in the shallow wells are often above the permissible drinking water limit (50 g/L; Routh et al. 2003). The underlying thick Quaternary alluvium consists of cycles of com- plete or partly truncated fining-upward sequences dominated by coarse to medium sand, fine sand, silt, and clay. Aquifer sediments in the deep wells are mostly coarse sands, whereas the shallow wells usually consist of fine-to-medium grained sands. Air jet drilling was used to install an 18-m deep well. The aquifer sediments were collected in Anero™ (Mitsubishi, Inc.) bags and shipped to Stockholm for microbial assays. The experiments were started within 4-days after sampling. 2.2 Microcosm experiments The microcosm experiments were a two-step process: (1) enrichment studies to isolate bacteria tol- erant to high As levels, and (2) determination of the As mobilizing capacity of isolated pure microbial cultures. The sediments were inoculated into sterile minimal medium (Turpeinen et al. 1999). The medium contained lactate as the carbon source, and it was spiked with As. The microcosms were sampled, and a specific volume of sample sacrificed periodically for different microbiological and geochemical assays. The sediment slurries extracted under aseptic conditions through the rubber septa were centrifuged at 907 G (3000 rpm). The aqueous and sediment phases obtained were sep- arately analyzed for As(III) and As(V) species, and compared to the heterotrophic plate counts of the corresponding day. The heterotrophic plate counts were performed by serially diluting the samples using phosphate buffered saline solution. The bacterial culture was spread on Tryptic Soy Agar plates spiked with As, and incubated for 72 hrs at 22°C. The bacterial colonies were selected and replated until pure cultures were obtained. The isolates were identified using the API and 16S rRNA techniques. The pure cultures were inoculated into sterile basal salts medium under oxic to sub-oxic (2–3 mg/L dissolved oxygen; obtained by bubbling Ar through the medium and storing samples in N 2 filled box) conditions containing 1mM, 2mM, and 5mM As(V) and lactate. During sampling Eh, pH, and oxygen were measured in the microcosms. Replicate samples were sacrificed period- ically. Microbial growth was determined by measuring change in optical density (600 nm) and dry weight over the duration of the microcosm experiment. As(III) and As(V) species in the medium 147 Copyright © 2005 Taylor & Francis Group plc, London, UK was measured by modifying existing spectrophotometric methods (Johnson & Pilson 1972, Cummings et al. 1999). Optical density was calibrated for each strain. Additional samples were set up as controls after treating the samples with HgCl 2 and formaldehyde. Specific details of these procedures are provided in Collins et al. (2004) and Routh et al. (2004). 3 RESULTS 3.1 Adak mine tailings Microbes enhanced dissolution of As in enrichment cultures by increasing As(III) concentrations in the aqueous phase and mobilizing ϳ27–51% of As present in contaminated sediments (Bhattacharya et al. 2003). Several bacteria were isolated from the enrichment studies, and identification of different microbial strains is ongoing. Here, we have focused on two microbial strains where we have generated complete data for: (1) arsenic transformation and mobilization, and (2) API and 16S rRNA identification. Arsenicicoccus bolidensis is hitherto a new species of actinomycete and it is a gram-positive, facultatively anaerobic, coccus-shaped microorganism (Fig. 1; Collins et al. 2004). The microcosm experiments indicated a fall in As(V) coupled to increase in As(III) and heterotrophic growth (indicated as increase in optical density and dry weight). A. bolidensis reduced 0.06–0.20mM/day As(V) under sub-oxic conditions (Saraswathy et al. 2004). Arsenic reduced by the bacteria occurs as encrustations on bacterial cells as shown by EDAX X-emission spectrum (Fig. 1). The As(V) reduction values are low compared to other As reducing microorganisms (Routh et al. 2004). As(V) reduction is however, related to growth in A. bolidensis implying that respiration and/or detoxification pathways may be involved in As(V) transformation. Notably, this is the first report of an actinomycete involved in As reduction in sedimentary environments. A novel species of facultatively anaerobic Chromobacterium occurring as rod-shaped microor- ganisms were isolated from the Adak sediments (Fig. 1). Bacterial growth in the culture decreased after 12 days corresponding to the conversion of 74% of 1mM As(V) to As(III). This bacterium was able to reduce 0.7–0.22 mM/day of As(V) (Fig. 2). Oxygen levels as high as 0.025 mM did not affect bacterial growth or As(V) reduction. In the presence of other electron acceptors in com- petition experiments involving lactate and a combination of As(V) with sulfate or nitrate, only As(V) concentrations varied. 3.2 Ambikanagar shallow aquifer Enrichment cultures indicated several As tolerant species, which actively reduced As(V). Specific details regarding the geochemical trends indicated by these bacteria are discussed in Routh et al. (2004). Continued increase in As concentrations in the enrichment cultures affected bacterial growth resulting in a decrease in plate counts and As(V) reduction. During the experiment, oxy- gen and Eh levels decreased, whereas pH increased. Amongst the eleven microbial strains isolated from the TSA plates, five of them were morphologically most distinct. These strains were selected for 16S rRNA characterization and As mobilization experiments. The bacteria were identified as: Acinetobacter johnsonii, Citrobacter freundii, Comamonas testosteroni, Enterobacter cloacae, and Sphingobium yanoikuyae. These bacteria range from aerobic to facultative anaerobic species. Similarity of the 16S rRNA sequence with GenBank varies between 98 and 100%. Different growth and As(V) reduction rates were noted on inoculating the basal salts medium. Maximum growth and As(V) reduction was noted in the bacteria A. johnsonii (Fig. 2), which cor- responded with initial As(V) concentration and biomass yield. Compared to other As(V) reducing microorganisms, bacteria isolated in this study indicated lower reduction rates (0.11–0.25 mM/day). Notably, C. testosteroni and S. yanoikuyae did not indicate a direct correlation between As(V) concentration versus growth rate and biomass yield. It is likely that As(V) reduction in these bacteria was related to detoxification (e.g. Macur et al. 2001). 148 Copyright © 2005 Taylor & Francis Group plc, London, UK 4 DISCUSSION 4.1 As(V) reduction and growth Our investigations on As cycling in the mine tailings and shallow aquifers show several interest- ing results. First, in situ microorganisms play an important role in transforming and mobilizing As at both locations. The microbial strains indicated general resistance to As toxicity under oxic to sub-oxic conditions, and reduced As(V) to As(III). Increase in optical density and dry weight was directly correlated to growth in the microcosms inoculated with individual microbial strains. Notably, As speciation remained unchanged and no microbial growth occurred in the controls (data not shown). This clearly confirms that As(V) reduction was ‘biologically mediated’ and microorganisms play a role in As cycling. Because the enrichment cultures did not isolate iron and sulfate reducing bacteria (which are also capable of mobilizing As e.g., Cummings et al. 2000, Jones et al. 2000, Kuhn & Sigg 1993) their role in As cycling at these sites can only be specula- tive. Although we have clearly established As(V) reduction and mobilization in the sediment microcosms in absence of Fe and sulfur reducing bacteria, it is unknown if this process is affective in natural environments. While some of these microorganisms isolated in this study are new (e.g., 149 Figure 1. ESEM imaging in wet mode illustrating the Arsenicicoccus bolidensis and Chromobacterium cluster. Representative EDAX X-emission spectrum quantification collected from electron dense particles on bacterial surface as encrustations (inset). The S, O, and P speaks are due to background from the support- ing grid. 0 1 2 3 4 5 6 0369121518 0.0 0.2 0.4 0.6 0.8 Chromobacterium Conc. (mM) Days As(V) As(III) O.D 1.0 O.D. (600 nm) 024681012 0 1 2 3 4 5 6 Conc. (mM) Days As(V) As(III) O.D 0.0 0.2 0.4 0.6 0.8 1.0 O.D. (600 nm) Acinetobacterjohnsonii Figure 2. Growth of Chromobacterium and Acinetobacter johnsonii in minimal medium with 5 mM As(V). Note that change in As speciation corresponds with enhanced growth represented as optical density measure- ments in the microcosm cultures (experiments were conducted in duplicate). Copyright © 2005 Taylor & Francis Group plc, London, UK A. bolidensis and Chromobacterium), others have been associated with As cycling in sedimentary environments (e.g. S. yanoikuyae; Macur et al. 2001). Inasmuch as it is important to expect other biogeochemical conditions to play a role in affect- ing As(V) reduction, in situ microbial processes are probably more affective on short-term basis. Researchers have indicated that abiotic reduction of As(V) may occur due to sulfides (Kuhn & Sigg 1993), but no odor for H 2 S was detected during sampling or change in color noted in the sed- iments to suggest presence of iron sulfides. Moreover, both laboratory and field measurements indicate that abiotic reduction of As(V) is kinetically a slow process (Kuhn & Sigg 1993, Newman et al. 1997b), and does not match with the As transformation rates in these sediments (e.g. Routh et al. 2004). The effect of As(V) concentration on bacterial growth was evaluated. A zero-order model with respect to As(V) concentration best fits our experimental data. The kinetic model applied to eval- uate As(V) reduction is similar to U(VI) reduction involving sulfate reducing bacteria (Spear et al. 2000). The model is represented as: (1) where As ϭ is the model predicted As(V) concentration, As 0 ϭ initial concentration of As(V), k 0 ϭ maximum specific reaction rate coefficient expressed as As(V) concentration/mg (dry weight) of cells/ml/h, X ϭ bacterial cell concentration in mg (dry weight)/ml, and t ϭ time in hours. Figure 3 indicates the trend for modeled and fitted k 0 values for Chromobacterium in microcosms containing 1, 2, and 5 mM As(V). The zero-order model is a simplification of Michaelis-Menten and Monod type kinetics at high substrate concentrations denoted by: (2) where V max ϭ Michaelis-Menten maximum substrate utilization rate constant expressed as the As(V) concentration/mg (dry weight) of cells/ml/day, m ϭ Monod maximum specific growth rate constant/h, and Y ϭ cell yield expressed as mass of cells in mg per mg of substrate used. 150 0 1 2 3 4 5 6 0 3691215 Days Observed 1 mM Fitted 1 mM Observed 2 mM Fitted 2 mM Observed 5 mM Fitted 5 mM As(V) reduction (mM) Figure 3. Time course of As(V) reduction by Chromobacterium first with a zero-order model. The plot is based on calculation of As concentration and k 0 values (see equations 1 and 2 in text). Each set of data points represents at least two experiments conducted over a 15-days period. Copyright © 2005 Taylor & Francis Group plc, London, UK Microbial growth and reduction rates varied between individual species, but compared to other microorganisms the reduction rates were low (Routh et al. 2004). In this context, an important distinction from other laboratory simulated studies (e.g., Ahmann et al. 1997, Newman et al. 1997 a, b among others) is the fact that we used carbon levels (external inputs to the cultures) that was substantially low (0.01mM versus 1 to 10mM in other studies). While the low organic carbon con- centration is more reflective of the natural organic matter content in these environments, it may have resulted in low As(V) reduction rates. Organic matter breakdown products in the microcosms were not measured, but consistent increase in pH, As(III), and microbial numbers support reaction pathways involving breakdown of lactate (e.g. Zobrist et al. 2000) or natural sedimentary organic matter. Other researchers have also indicated fall in As(V) reduction with decrease in organic substrate in microcosms (e.g. Ahmann et al. 1997, Harvey et al. 2003, Islam et al. 2004). The results reiterate the importance of organic matter for the survival of these heterotrophic microbial communities in the sub-surface. One of the confounding issues in this study is underpinning whether these microorganisms reduce As(V) for detoxification or respiratory purposes. This partly arises due to the aerobic or strict anaerobic habitats preferred by the respective microbial colonies involved in As(V) reduc- tion (Newman et al. 1997b). The general conditions in this study were sub-oxic, and some of the microorganisms probably use As(V) as an alternate electron acceptor. Interestingly, recent studies by other researchers imply that presence of an enzymatic detoxification pathway does not preclude the As(V) respiration capability in bacteria. For example, both detoxifying and respiratory As (V) reductases occur in the microaerophile Bacillus selenitireducens (Switzer-Blum et al. 1998) and the As(V)-respiring anaerobe Shewanella ANA-3 strain (Saltikov & Newman 2003). Similar possibilities may exist in some of the bacteria discussed here (e.g., A. johnsonii, A. bolidensis), but genetic evidence (on same lines as in Saltikov & Newman 2003) is presently unavailable to support this idea. Nonetheless, the fact that some these microorganisms are capable of using O 2 or switch to other terminal electron acceptor during respiration implies that they are generally opportunistic by character. By means of complex inter-cellular processes these microorganism are able to survive under conditions that are generally less preferable to others in the sub-surface. The study implies the potential impact such microorganisms could have on As cycling at these sites. 4.2 Environmental implications Of late, the thrust by different regulatory agencies is on developing As remediation methods that are supposed to be cost-affective and reaches out to a larger population. The most commonly used in situ techniques involve maintaining aerobic conditions through aeration or using chemical oxidants to convert As(III) to the less mobile and toxic As(V) species. In this context, microbial transformation and mobilization of As in the sub-surface may have important impli- cations on groundwater treatment. First, microbial As(V) reduction if it is common, then the pre- diction of As valence, and thus behavior, based solely on redox status may be problematic. For example, even under oxidizing conditions As(III) has been found as the predominant species in the Adak tailings deposit (Bhattacharya et al. 2003) similar to other studies (Aurillo et al. 1994, Sohrin et al. 1997). Second, efforts made to chemically oxidize As(III) to As(V) during groundwater treatment may be unproductive since As(V) is actively reduced to As(III) by in situ bacteria. This is largely bad news for researchers focusing on developing As remediation techniques. Most methods up to date have focused on manipulating the redox states and converting As(III) to As(V) (for review see Murcott 2001, Ahmed 2001). Many of these methods hardly take into account the role of in situ microbial activity. Hence, it is not surprising many groundwater treat- ment methods fail to work effectively under field conditions. Clearly, there is an urgent need to assess the occurrence and efficiency of these microbial processes in field-based pilot studies as a prerequisite to provide critical information before implementing specific remediation methods for removing As. 151 Copyright © 2005 Taylor & Francis Group plc, London, UK 5 CONCLUSIONS Microbial processes play a crucial role in As mobilization. Mobilization of As from sediments into the aqueous phase is mediated by eukaryotes, fungi, and bacteria. This involves reduction of As(V) into the more mobile and toxic As(III) species. Microbial reduction of As(V) mainly arises for detoxification or respiratory purposes. They are different biochemical pathways occurring under mostly oxic or strict anoxic conditions, respectively. Here, we have indicated the role of microorganisms in mobilizing As at sites contaminated by mine tailings (in northern Sweden) and shallow aquifers in West Bengal (India). We isolated several microorganisms (including two new species) that are involved in As reduction under oxic to sub- oxic conditions. These microorganisms are generally resistant to As toxicity. As (V) reduction in the microcosms, correspond with increase in dry weight and optical density measurements. In some of these microorganisms, the correspondence between As concentration, bacterial growth, and biomass yield is high. This leads us to believe that some of these microorganisms are prob- ably using As(V) for respiration in addition to detoxification purposes. Further genetic work is required to understand such complex biochemical pathways. Nonetheless, the study proves that the microorganisms whether they are present in mine tailings or groundwater aquifers; they are generally opportunistic by character. The microorganisms have developed unique survival skills, and in the process, created a microbial niche for themselves. Enhanced mobilization of As from sediments has important implications on groundwater treat- ment. Active microbial processes may result in disequilibrium conditions, whereby even under oxidizing conditions As(III) species may predominate. Additionally, groundwater treatment based on aeration and oxidation processes (if implemented at these sites) needs to be assessed critically. Given the fact that in both places, we have a thriving microbial community in the sub-surface, which is able to reduce As(V) under oxic to sub-oxic conditions, it raises questions about groundwater treatment methods suitable for these sites. ACKNOWLEDGEMENT We thank the Geological Survey of Sweden (SGU) and Swedish International Development Agency (Sida-SAREC) for providing the research funds to conduct our studies in Sweden and India. Gunnar Jacks, Sisir Nag, S.P. Sinha Ray, and Prosun Bhattacharya helped us with fieldwork. We thank Roger Herbert and Jim Saunders for their suggestions. 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Technol. 34: 4747–4753. 153 Copyright © 2005 Taylor & Francis Group plc, London, UK . reduction and growth Our investigations on As cycling in the mine tailings and shallow aquifers show several interest- ing results. First, in situ microorganisms play an important role in transforming. medium sand, fine sand, silt, and clay. Aquifer sediments in the deep wells are mostly coarse sands, whereas the shallow wells usually consist of fine-to-medium grained sands. Air jet drilling was. sub-oxic (2–3 mg/L dissolved oxygen; obtained by bubbling Ar through the medium and storing samples in N 2 filled box) conditions containing 1mM, 2mM, and 5mM As(V) and lactate. During sampling Eh,