Arsenic-Microbe-Mineral Interactions in Mining-Affected Environments

The toxic element arsenic (As) occurs widely in solid and liquid mine wastes. Aqueous forms of arsenic are taken up in As-bearing sulfides, arsenides, sulfosalts, oxides, oxyhydroxides, Fe-oxides, -hydroxides, -oxyhydroxides and -sulfates, and Fe-, Ca-Feand other arsenates. Although a considerable body of research has demonstrated that microbes play a significant role in the precipitation and dissolution of these As-bearing minerals, and in the alteration of the redox state of As, in natural and simulated mining environments, the molecular-scale mechanisms of these interactions are still not well understood. Further research is required using traditional and novel mineralogical, spectroscopic and microbiological techniques to further advance this field, and to help design remediation schemes.


Introduction
Throughout the world, the weathering of solid mine wastes contaminates waters with potentially toxic components, such as arsenic (As), in concentrations that may pose serious hazards to human and ecosystem health.The stability and potential mobility of As in mine wastes is controlled by their uptake in minerals [1,2].Many of the reactions that result in the dissolution of primary As-bearing minerals, and formation of secondary products, are mediated by prokaryotes (i.e., Bacteria and/or OPEN ACCESS Archaea).This review describes important As-bearing minerals and As-oxidizing and -reducing prokaryotes in mining environments, discusses how these organisms interact with the minerals, and outlines avenues for further research in this area.

Arsenic-Bearing Minerals in Mining-Affected Environments
Arsenic occurs in more than 300 minerals.These include arsenates (ca.60% of the 300 As-bearing minerals), sulfides and sulfosalts (ca.20%), oxides (ca.10%) and arsenates, arsenides, native elements and metal alloys (ca.10%) [1,3].Examples of As-bearing minerals that occur in mining-affected environments are summarized in Table 1.Native As and As-bearing sulfides are regarded as primary minerals in mining-affected environments, since they are produced by the hydrothermic, mesothermic or diagenetic processes that generate the ores.In addition, their weathering and alteration by ore processing generates secondary oxides and arsenates (Table 1).The most common As-bearing sulfides in mining-affected environments are pyrite and arsenopyrite (Figure 1a) [4][5][6].Realgar and orpiment are less common, while enargite and tennantite occur in Cu-rich deposits [7,8], cobaltite occurs in Co-rich deposits [9] and gersdorffite occurs in Ni-rich deposits [10].The dominant forms of As in these primary phases are As(−I), As(III) and As(V) (Table 1).Secondary As-bearing minerals are abundant in mine wastes.These are produced by the natural weathering of primary sulfides, or by metal extraction processes (e.g., roasting, [12,13]).Secondary As-bearing minerals containing structural As include Fe arsenates such as scorodite [2] and kankite [14], Fe sulfoarsenates such as tooeleite [14,15], Ca-Fe arsenates such as yukonite (Figure 1b) and amorphous forms [14,16,17], and Ca-Mg arsenates such as pharmacolite [18].Arsenic can also be incorporated in trace amounts in a variety of minerals such as Fe, Mn or Al oxides (Figure 1c), hydroxides and sulfates, other hydrous and anhydrous sulfates, phosphates and hydrous silicates [4,5,13,14,[19][20][21][22][23].In these cases, the As is taken up by sorption onto the mineral surface [24], or incorporated into the mineral structure by co-precipitation.The most common of these occurs in mine wastes include Fe oxides and hydroxides (Table 1) [2,5,14,25], and minerals of the jarosite family [4,5,19,25,26].The dominant form of As in these secondary phases is As(V), but As(III) has been reported in roaster oxide phases [12] (Table 1), in secondary oxide and oxyhydroxide phases (claudetite, manganarsite, Table 1) in an underground gold mine [11] and in products of experiments involving bacteria and acid mine drainage water bacteria-acid mine drainage water experiments [27].

Prokaryotes in Arsenic-Bearing, Mining-Affected Environments
The two soluble inorganic forms of arsenic, arsenate (As(V)) and arsenite (As(III)), occur widely in mining-affected environments.Even though these forms of As are highly toxic, some prokaryotes can gain energy from their reduction and oxidation, respectively [28].Prokaryotes that use As(V) as the terminal electron acceptor in anaerobic respiration are known as arsenate respirers, and they can couple As(V) reduction to the oxidation of both inorganic (e.g., sulfide) and organic (e.g., acetate) electron donors [29].Other organisms can use As(III) as the electron donor coupled to the reduction of oxygen, nitrate or chlorate [30].
Most prokaryotes that oxidize As(III) or respire As(V) are able to survive and grow in As-contaminated mining environments and many have acquired additional As-resistance mechanisms.The most well studied resistance mechanism is the Ars (arsenic resistance system) [31].This comprises, the arsenate reductase (ArsC) and the efflux pump (ArsB) which requires energy (as opposed to metabolism mentioned above) in the form of adenosine triphosphate (ATP) and contains an ATPase (ArsA) that hydrolyses the ATP to adenosine diphosphate (ADP).If As(V) enters the cells (most probably through the phosphate transport system) it is reduced to As(III) inside the cell (i.e., in the cytoplasm) and the As(III) is pumped out of the cell through ArsB coupled to ArsA-mediated ATP hydrolysis (Figure 2).If, on the other hand, As(III) enters the cell it can be pumped straight out via ArsB.Some organisms also detoxify inorganic As by methylation.This produces monomethyl arsonate (MMA(V)), methylarsonite (MMA(III)), dimethylarsinate (DMA(V)), dimethylarsenite (DMA(III)) and trimethylarseine oxide (TMAO), as well as volatile arsines (As-III) [31].Microbial oxidation of arsenite to arsenate is much more rapid than chemical oxidation of arsenite to arsenate [32].Therefore, rapid arsenite to arsenate oxidation in the environment can be attributed to arsenite-oxidizing bacteria.
Table 2 shows a selection of aerobic arsenite oxidizers belonging to two different sub-phyla of the Bacteria (Alpha and Betaproteobacteria) isolated from mining environments.These organisms are all mesophiles (grow at moderate temperatures) except for GM1 which is psychrotolerant (grows ≤ 20 °C) and include chemolithoautotrophs (obtain carbon from the fixation of carbon dioxide and energy from the oxidation of inorganic compounds) and heterotrophs (cannot fix carbon dioxide and instead use organic carbon for making cell material).Several anaerobic arsenate-respiring bacteria have also been isolated from arsenic-contaminated mining environments.For example, Chrysiogenes arsenatis str.BAL-1 T was isolated from anoxic mud from a tailings pond in the Ballarat goldfields, Victoria, Australia [41].C. arsenatis couples the reduction of As(V) to a variety of organic electron donors.Two As(V)-respiring bacteria were isolated from arsenic-contaminated anoxic muds from a tailings pond in Bendigo, Victoria, Australia.One of these organisms, Desulomicrobium sp.Ben-RB could concomitantly reduce As(V) and sulfate, coupling the reduction of both to the oxidation of lactate forming As 2 S 3 [42].The second organism, Bacillus macyae str.JMM-4 uses As(V) as the terminal electron acceptor and acetate as the electron donor [43,44].All three As(V) respirers were isolated in the presence of 10 mM arsenate and can probably tolerate much higher concentrations.
The organisms mentioned above gain energy from either the oxidation of arsenite coupled to the reduction of oxygen, nitrate or chlorate or the reduction of arsenate coupled to the oxidation of inorganic or organic electron donors.This means that they produce ATP which is then used for cell division.Two different enzymes (Aio and Arx) are responsible for the oxidation of arsenite to arsenate, depending on whether the organisms use oxygen or nitrate as electron acceptors [30,45,46].Aio is the most well-characterized and it is located in the periplasm.The arsenite which enters the periplasm is oxidized to arsenate by the Aio and then the electrons are transferred to other proteins involved in the electron transport chain which results in the production of ATP and the reduction of oxygen to water (Figure 3).On the other hand, the respiratory arsenate reductase (Arr), which is located in the periplasm or is attached to the cytoplasmic membrane is the terminal reductase in an electron transport chain and receives the electrons for arsenate reduction from various electron donors which are oxidized in the cytoplasm [29] (Figure 3).
Several organisms found in mining-impacted environments which are involved in the metabolism of other constituents of the minerals (e.g., oxidation of Fe, S, etc.) are often resistant to As either via the Ars system or another unknown mechanism, but do not metabolize it.These include iron-oxidizing (e.g., Acidithiobacillus ferrooxidans), iron-reducing (e.g., Shewanella putrefaciens), and sulfur-oxidizing organisms (e.g., Acidithiobacillus caldus) [47][48][49].Some species of Shewanella have, however, been shown to respire As(V) [50].

Arsenic-Microbe-Mineral Interactions in Mining-Affected Environments
Microbes play a significant role in the precipitation (i.e., biomineralization) and dissolution of As-bearing minerals, and in the alteration of the redox state of As, in natural and simulated mining environments.For example, the oxidation of Fe(II), contained in sterilized acid mine drainage water containing hundreds of mg of Fe(II) and tens of mg of As [mainly As(III)], by various Acidithiobacillus ferrooxidans strains results in the trapping of As(III) by newly precipitated Fe(III) minerals [27].X-ray Diffraction (XRD) and Extended X-Ray Absorption Fine Structure (EXAFS) spectroscopic analysis showed that tooeleite and As(III)-sorbed schwertmannite are formed in the presence of the bacteria in the acid mine drainage (AMD) water.The nature, proportion and sequence of the biominerals formed were controlled by the type of bacterial strain used and the strain cell numbers.Arsenic-bearing schwertmannite was also found in a biomat formed 0.3 m downstream of discharging arsenic-bearing mine water of pH 4.7 (Figure 4a) [51].The schwertmannite was characterized by scanning particle induced X-ray emission analysis, XRD and X-ray Absorption Near Edge Structure (XANES), which showed that it contained As(V).Another unnamed Fe-S phase contained As(III).Phylogenetic identification of the organisms by 16S recombinant DNA (rDNA) analysis of the discharged mine water showed that Gallionella species were the dominant bacteria present, but it was not shown whether these organisms were directly responsible for the biomineral formation.Benzerara et al. [52], using transmission electron microscopy and scanning transmission X-ray microscopy, showed that As(III)-bearing tooeleite and an As(V)-Fe(III) amorphous phase formed extracellularly or within the periplasm of microbial cells in the Carnoulè s (France) AMD system (Figure 4b).Acidithiobacillus ferrooxidans strain CC1, isolated from acid mine drainage of the former Carnoulè s mining site, was shown to oxidize Fe(II), resulting in the formation of poorly ordered schwertmannite that co-precipitated arsenite from the AMD [53].An As-bearing Fe(III) phase was shown to form on the surfaces of cells of Acidithiobacillus ferrooxidans during the bioleaching of realgar [54], but the redox state of the As was not determined (Figure 4c).Various strains of Acidithiobacillus ferrooxidans also oxidized Fe(II) to Fe(III) in the presence of high concentrations of dissolved As(III), resulting in the formation of tooeleite [15].
Microbes solubilize minerals directly (through attachment) to secure essential nutrients, or indirectly (without attachment) by changing aqueous solution chemistry so that reactive solutes are produced.Attachment is particularly effective because soluble electron carriers can then diffuse through the periplasm (Figure 2).Mineral dissolution rates can then be increased or decreased due to the production of pH gradients at the mineral surface, or to the improvement of the kinetics of secondary mineral formation [55,56].Experimental studies have demonstrated that bacteria are closely involved in the oxidative and reductive dissolution of As-bearing mine waste minerals.For example, As-bearing sulfide minerals such as arsenopyrite, enargite, orpiment and realgar can be oxidized by aerobic bacteria such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans (e.g., [47,[57][58][59]).Such bacteria, which typically occur in acid mine drainage environments, are described in detailed reviews by Hallberg and Johnson [60], Baker and Banfield [61], Gonzá lez-Pastor and Mirete [62], Yelton et al. [63] and Drewniak and Sklodowska [64].Islam et al. [65] have also demonstrated that native arsenic can be dissolved at alkaline pH (~8) with the assistance of microbes.The involvement of bacteria in the oxidation of arsenopyrite has been comprehensively reviewed by Corkhill and Vaughan [66].The studies cited in their work demonstrated that oxidation of arsenopyrite by acidophilic Fe-and S-oxidizing bacteria is considerably more effective than abiotic oxidation.For example, the oxidative dissolution of arsenopyrite was shown to be much more rapid and extensive when it occurred in the presence of Leptospirillum ferrooxidans than abiotically [59].This bacterium produced dissolution pits in the mineral surface (Figure 5a), and As was oxidized more rapidly than S or Fe.The oxidation of arsenopyrite and As-bearing pyrite by Acidithiobacillus ferrooxidans can also result in pitting on the mineral surfaces, and of thin coatings of jarosite [67].The oxidation of orpiment, with the concomitant release of arsenite, can also occur abiotically, but is much more rapid in the presence of Ferrobacillus ferrooxidans TM [58].It should be noted, however, that in these cases the bacteria are oxidizing the Fe and/or S, making the As more accessible to abiotic oxidation.Several As-bearing minerals found in mine wastes have been shown to undergo microbially-assisted reductive dissolution.The dissimilatory arsenate reducers Shewanella sp.O23S and Aeromonas sp.O23A play a significant role in releasing As(V) from As-bearing minerals in the Zloty Stok gold mine [68].The experimental anaerobic dissolution of Pb-As-jarosite (PbFe 3 (SO 4 ,AsO 4 ) 2 (OH) 6 ) by Shewanella putrefaciens at circumneutral pH resulted in immediate Fe(III) reduction followed by As(V) reduction after 72 h [48].
It was suggested that the Fe(III) reduction was thermodynamically driven, but the aqueous As(V) reduction was due to the Ars bacterial detoxification response (see Figure 2).Secondary As(III)-and Pb-bearing Fe(II)-O precipitates formed as a result of this process.A microbial community containing autotrophic dissimilative reducers of As and SO 4 was shown to promote the dissolution of As-bearing Fe(III) hydroxides, pharmacosiderite, arseniosiderite, and the precipitation of realgar in a natural Au-As geochemical anomaly at Mokrsko, Czech Republic [69].Dissimilatory Fe(III)-reducing bacteria such as Shewanella alga and Desulfuromonoas palmitatis have also been shown to reduce scorodite, releasing dissolved arsenate [70,71] and in some cases, producing secondary symplesite [71].

Conclusions
This review outlines our present understanding of arsenic-microbe-mineral interactions in mining-affected environments.Although a considerable body of research exists on this topic, there are still several avenues for future research and discovery.The character of secondary minerals produced by microbial oxidation and particularly, reduction of As-bearing minerals in natural environments is still not well known.Although several As(III)-oxidizing and As(V)-reducing bacteria have been isolated and identified from mining-affected environments, these represent a very small proportion of those that likely exist.The specific role of bacteria in the dissolution of primary As-bearing minerals and formation of secondary As-bearing minerals is still not well understood.Additional work involving the use of novel surface mineral characterization techniques such as low-vacuum scanning electron microscopy, atomic force microscopy, and synchrotron-based techniques such as scanning X-ray microscopy, is required to advance knowledge in this field.

Figure 1 .
Figure 1.Examples of As-bearing minerals in mine wastes.(A) Tailings from the 1998 Aznalcóllar (Spain) tailings dam spill that contain arsenopyrite, described in [5].(B) Microbial biofilm sampled from Giant Mine, Yellowknife, NWT, Canada.The mineral yukonite, a Ca-Fe arsenate, is shown by the reddish-brown coloration.From [11].And (C) Electron microprobe X-ray map of secondary Fe-As-Sb-O phase in solids from Bolivian tailings weathering column experiments.Reprinted with permission from [2], Mineralogical Society of Great Britain & Ireland.

Figure 2 .
Figure 2. Conceptual diagram of the bacterial arsenic resistance mechanism.See text for explanation.

Figure 3 .
Figure 3. Conceptual diagram of aerobic arsenite oxidase.Arsenite enters the periplams and is oxidized to arsenate by Aio.See text for discussion.

Figure 4 .
Figure 4. Examples of mineralization of bacterial cells by As-bearing phases.(A) Transmission electron microscope (TEM) images of a stalk-like structure (A in a), a sheath-like structure (B in a), and a granular compound (C in b) with electron diffraction patterns of the sheath-like and granular ones in a biomat from an abandoned arsenic mine in the Gunma prefecture of Japan.The diffraction patterns and energy dispersive spectroscopy (EDS) analysis suggest that one of the minerals in the precipitates is schwertmannite, formed in the stalk-like and sheath-like compounds.Reprinted with permission from[51], Elsevier.(B) TEM image and analysis of ultramicrotomy thin sections in samples from Carnoulè s (France).The TEM image shows a section of a microbial cell with a layered, concentric inner cell wall and massive outer cell wall encrustation by Fe-and As-bearing minerals, together with energy dispersive X-ray spectroscopy (EDXS) analysis obtained from the outer cell wall.Reprinted with permission from Figure6in[52], Elsevier.And (C) Arsenic-rich Fe(III) precipitates on the outer cells of A. ferrooxidans BY-3 after the experimental bioleaching of realgar.From[54].

Figure 5 .
Figure 5. Microbial dissolution of As-bearing minerals found in mine wastes (A) Pits in arsenopyrite arising from the oxidative dissolution by Leptospirillum ferrooxidans.Reprinted with permission from Figure 7i in [59], Elsevier.And (B) Mechanism of simultaneous reduction of structural Fe(III) and aqueous As(V) during the dissolution of synthetic Pb-As jarosite by Shewanella putrefaciens.Solid Fe(II) phases are formed as a result of this process.Reprinted with permission from [48], Elsevier.

Table 1 .
Examples of As-bearing minerals in mining-affected environments.Where known, speciation is shown for redox-sensitive elements.