Immobilization of Arsenite and Ferric Iron by Acidithiobacillus ferrooxidans and Its Relevance to Acid Mine Drainage

Weathering of the As-rich pyrite-rich tailings of the abandoned mining site of Carnoulès (southeastern France) results in the formation of acid waters heavily loaded with arsenic. Dissolved arsenic present in the seepage waters precipitates within a few meters from the bottom of the tailing dam in the presence of microorganisms. An Acidithiobacillus ferrooxidans strain, referred to as CC1, was isolated from the effluents. This strain was able to remove arsenic from a defined synthetic medium only when grown on ferrous iron. This A. ferrooxidans strain did not oxidize arsenite to arsenate directly or indirectly. Strain CC1 precipitated arsenic unexpectedly as arsenite but not arsenate, with ferric iron produced by its energy metabolism. Furthermore, arsenite was almost not found adsorbed on jarosite but associated with a poorly ordered schwertmannite. Arsenate is known to efficiently precipitate with ferric iron and sulfate in the form of more or less ordered schwertmannite, depending on the sulfur-to-arsenic ratio. Our data demonstrate that the coprecipitation of arsenite with schwertmannite also appears as a potential mechanism of arsenite removal in heavily contaminated acid waters. The removal of arsenite by coprecipitation with ferric iron appears to be a common property of the A. ferrooxidans species, as such a feature was observed with one private and three collection strains, one of which was the type strain.     

Toxicity and metabolism of subcytotoxic inorganic arsenic in human renal proximal tubule epithelial cells (HK-2)

Arsenic is an environmental toxicant and a human carcinogen. The kidney, a known target organ of arsenic toxicity, is critical for both in vivo arsenic biotransformation and elimination. This study investigates the potential of an immortalized human proximal tubular epithelial cell line, HK-2, to serve as a representative model for low level exposures of the human kidney to arsenic. Subcytotoxic concentrations of arsenite (< or = 10 micromol/L) and arsenate (< 100 micromol/L) were determined by leakage of LDH from cells exposed for 24 h. Threshold concentrations of arsenite (between 1 and 10 micromol/L) and arsenate (between 10 and 25 micromol/L) were found to affect MTT processing by mitochondria. Biotransformation of subcytotoxic arsenite or arsenate was determined using HPLC-ICP-MS to detect metabolites in cell culture media and cell lysates. Following 24 h, analysis of media revealed that arsenite was minimally oxidized to arsenate and arsenate was reduced to arsenite. Only arsenite was detected in cell lysates. Pentavalent methylated arsenicals were not detected in media or lysates following exposure to either inorganic arsenical. The activities of key arsenic biotransformation enzymes--MMAV reductase and AsIII methyltransferase--were evaluated to determine whether HK-2 cells could reduce and methylate arsenicals. When compared to the activities of these enzymes in other animal tissues, the specific activities of HK-2 cells were indicative of a robust capacity to metabolize arsenic. It appears this human renal cell line is capable of biotransforming inorganic arsenic compounds, primarily reducing arsenate to arsenite. In addition, even at low concentrations, the mitochondria are a primary target for toxicity.     

Insights into the structure, solvation, and mechanism of ArsC arsenate reductase, a novel arsenic detoxification enzyme.

BACKGROUND: In Escherichia coli bearing the plasmid R773, resistance to arsenite, arsenate, antimonite, and tellurite is conferred by the arsRDABC plasmid operon that codes for an ATP-dependent anion pump. The product of the arsC gene, arsenate reductase (ArsC), is required to efficiently catalyze the reduction of arsenate to arsenite prior to extrusion. RESULTS: Here, we report the first X-ray crystal structures of ArsC at 1.65 A and of ArsC complexed with arsenate and arsenite at 1.26 A resolution. The overall fold is unique. The native structure shows sulfate and sulfite ions binding in the active site as analogs of arsenate and arsenite. The covalent adduct of arsenate with Cys-12 in the active site of ArsC, which was analyzed in a difference map, shows tetrahedral geometry with a sulfur-arsenic distance of 2.18 A. However, the corresponding adduct with arsenite binds as a hitherto unseen thiarsahydroxy adduct. Finally, the number of bound waters (385) in this highly ordered crystal structure approaches twice the number expected at this resolution for a structure of 138 ordered residues. CONCLUSIONS: Structural information from the adduct of ArsC with its substrate (arsenate) and with its product (arsenite) together with functional information from mutational and biochemical studies on ArsC suggest a plausible mechanism for the reaction. The exceptionally well-defined water structure indicates that this crystal system has precise long-range order within the crystal and that the upper limit for the number of bound waters in crystal structures is underestimated by the structures in the Protein Data Bank.     

Abundant and diverse arsenic-metabolizing microorganisms in peatlands treating arsenic-contaminated mining wastewaters

Mining operations produce large quantities of wastewater. At a mine site in Northern Finland, two natural peatlands are used for the treatment of mining-influenced waters with high concentrations of sulphate and potentially toxic arsenic (As). In the present study, As removal and the involved microbial processes in those treatment peatlands (TPs) were assessed. Arsenic-metabolizing microorganisms were abundant in peat soil from both TPs (up to 10⁸ cells g_dw⁻¹), with arsenate respirers being about 100 times more abundant than arsenite oxidizers. In uninhibited microcosm incubations, supplemented arsenite was oxidized under oxic conditions and supplemented arsenate was reduced under anoxic conditions, while little to no oxidation/reduction was observed in NaN₃-inhibited microcosms, indicating high As-turnover potential of peat microbes. Formation of thioarsenates was observed in anoxic microcosms. Sequencing of the functional genemarkers aioA (arsenite oxidizers), arrA (arsenate respirers) and arsC (detoxifying arsenate reducers) demonstrated high diversity of the As-metabolizing microbial community. The microbial community composition differed between the two TPs, which may have affected As removal efficiencies. In the present situation, arsenate reduction is likely the dominant net process and contributes substantially to As removal. Changes in TP usage (e.g. mine closure) with lowered water tables and heightened oxygen availability in peat might lead to re-oxidation and re-mobilization of bound arsenite.     

Arsenic hazards: strategies for tolerance and remediation by plants

Arsenic toxicity has become a global concern owing to the ever-increasing contamination of water, soil and crops in many regions of the world. To limit the detrimental impact of arsenic compounds, efficient strategies such as phytoremediation are required. Suitable plants include arsenic hyperaccumulating ferns and aquatic plants that are capable of completing their life cycle in the presence of high levels of arsenic through the concerted action of arsenate reduction to arsenite, arsenite complexation, and vacuolar compartmentalization of complexed or inorganic arsenic. Tolerance can also be conferred by lowering arsenic uptake by suppression of phosphate transport activity, a major pathway for arsenate entry. In many unicellular organisms, arsenic tolerance is based on the active removal of cytosolic arsenite while limiting the uptake of arsenate. Recent molecular studies have revealed many of the gene products involved in these processes, providing the tools to improve crop species and to optimize phytoremediation; however, so far only single genes have been manipulated, which has limited progress. We will discuss recent advances and their potential applications, particularly in the context of multigenic engineering approaches.     

Microbial oxidation of arsenite and occurrence of arsenite‐oxidizing bacteria in acid mine water from a sulfur‐pyrite mine

The acid mine waters (pH 2.0–2.4) discharged from the Matsuo sul‐fur‐pyrite mine contained high concentrations of dissolved inorganic arsenic (2–13 ppm). Arsenic in the superficial acid mine waters was predominantly in the (V) state (arsenate); however, the element in the water from a deep mine drift was almost in the (III) state (arsenite). Microbial arsenite oxidation occurred in the acid mine waters and along the stream of the river, which was contaminated with a large volume of the mine drift water. Arsenite (500 ppm As)‐resistant bacteria (0–27 colonies/ml) were detected in the water samples and 208 slant cultures were obtained. Arsenite‐oxidizing activities of all the cultures were determined and six strains with strong arsenite‐oxidizing activity were isolated. These bacteria were acidophilic (optimum growth pH, 3—4), gram‐negative, aerobic, and rod‐shaped. They could not oxidize ferrous iron and elemental sulfur as a sole energy source and not derive the energy for chemoautotrophic growth from arsenite oxidation.     

Optimization of an Iron Intercalated Montmorillonite Preparation for the Removal of Arsenic at Low Concentrations

A series of iron intercalated montmorillonites (Fe‐Monts) were prepared using (i) ion exchange of native sodium and calcium ions with iron ions, (ii) base hydrolysis of inserted iron ions in montmorillonite suspension, and (iii) insertion of pre‐hydrolyzed iron colloid in montmorillonite. The materials were characterized by X‐ray diffraction and gas adsorption‐desorption techniques. The basal d(001)‐spacing and BET specific surface area increased after the intercalation of iron species in montmorillonite. Local iron structure studied by X‐ray absorption fine structure (XAFS) spectroscopy showed an unsaturation of the Fe···Fe coordination number (N 2.5) of the intercalated iron species as compared to the bulk iron oxyhydroxides (N 6). The Fe‐Monts were employed for arsenic removal from aqueous solutions at low concentration (0.2–16 mg/L). Among the Fe‐Monts, the one prepared by the hydrolysis of inserted iron ions, was the best in performance. The saturation adsorption amount of the optimized iron‐montmorillonite was 4 and 28 times higher for the removal of arsenite and arsenate, respectively, as compared to bulk iron oxyhydroxide (goethite). Compared with bulk iron oxyhydroxide, the Fe‐Monts were superior for arsenate uptake and comparable for arsenite. In addition, arsenite adsorbed on the Fe‐Monts was found to be oxidized to arsenate based on XAFS spectroscopy.     

Brevibacterium sp. strain CS2: A potential candidate for arsenic bioremediation from industrial wastewater

A multiple metal-resistant Brevibacterium sp. strain CS2, isolated from an industrial wastewater, resisted arsenate and arsenate upto 280 and 40 mM. The order of resistance against multiple metals was Arsenate > Arsenite > Selenium = Cobalt > Lead = Nickel > Cadmium = Chromium = Mercury. The bacterium was characterized as per morphological and biochemical characteristics at optimum conditions (37 ℃ and 7 pH). The appearance of brownish color precipitation was due to the interaction of silver nitrate confirming its oxidizing ability against arsenic. The strain showed arsenic processing ability at different temperatures, pH, and initial arsenic concentration which was 37% after 72 h and 48% after 96 h of incubation at optimum conditions with arsenite 250 mM/L (initial arsenic concentration). The maximum arsenic removal ability of strain CS2 was determined for 8 days, which was 32 and 46% in wastewater and distilled water, respectively. The heat-inactivated cells of the isolated strain showed a bioremediation efficiency (E) of 96% after 10 h. Genes cluster (9.6 kb) related to arsenite oxidation was found in Brevibacterium sp. strain CS2 after the genome analysis of isolated bacteria through illumine and nanopore sequencing technology. The arsenite oxidizing gene smaller subunit (aioB) on chromosomal DNA locus (Prokka_01508) was identified which plays a role in arsenite oxidation for energy metabolism. The presence of arsenic oxidizing genes and an efficient arsenic oxidizing potential of Brevibacterium sp. strain CS2 make it a potential candidate for green chemistry to eradicate arsenic from arsenic-contaminated wastewater.     

Rapid biotransformation of arsenate into oxo-arsenosugars by a freshwater unicellular green alga, Chlamydomonas reinhardtii

We examined the short-term metabolic processes of arsenate for 24 h in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125. The arsenic species in the algal extracts were identified by high-performance liquid chromatography/inductively coupled plasma mass spectrometry after water extraction using a sonicator. Speciation analyses of arsenic showed that the levels of arsenite, arsenate, and methylarsonic acid in the cells rapidly increased for 30 min to 1 h, and those of dimethylarsinic acid and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment. These results indicate that this alga can rapidly biotransform arsenate into oxo-arsenosugar-glycerol for at least 10 min and then oxo-arsenosugar-phosphate through both reduction of incorporated arsenate to arsenite and methylation of arsenite and/or arsenate retained in the cells to dimethylarsinic acid via methylarsonic acid as an possible intermediate.     

Role of Aspergillus niger acrA in arsenic resistance and its use as the basis for an arsenic biosensor

Arsenic contamination of groundwater sources is a major issue worldwide, since exposure to high levels of arsenic has been linked to a variety of health problems. Effective methods of detection are thus greatly needed as preventive measures. In an effort to develop a fungal biosensor for arsenic, we first identified seven putative arsenic metabolism and transport genes in Aspergillus niger, a widely used industrial organism that is generally regarded as safe (GRAS). Among the genes tested for RNA expression in response to arsenate, acrA, encoding a putative plasma membrane arsenite efflux pump, displayed an over 200-fold increase in gene expression in response to arsenate. We characterized the function of this A. niger protein in arsenic efflux by gene knockout and confirmed that AcrA was located at the cell membrane using an enhanced green fluorescent protein (eGFP) fusion construct. Based on our observations, we developed a putative biosensor strain containing a construct of the native promoter of acrA fused with egfp. We analyzed the fluorescence of this biosensor strain in the presence of arsenic using confocal microscopy and spectrofluorimetry. The biosensor strain reliably detected both arsenite and arsenate in the range of 1.8 to 180 μg/liter, which encompasses the threshold concentrations for drinking water set by the World Health Organization (10 and 50 μg/liter).     

Rapid Biotransformation of Arsenate into Oxo-Arsenosugars by a Freshwater Unicellular Green Alga,Chlamydomonas reinhardtii

We examined the short-term metabolic processes of arsenate for 24 h in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125. The arsenic species in the algal extracts were identified by high-performance liquid chromatography/inductively coupled plasma mass spectrometry after water extraction using a sonicator. Speciation analyses of arsenic showed that the levels of arsenite, arsenate, and methylarsonic acid in the cells rapidly increased for 30 min to 1 h, and those of dimethylarsinic acid and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment. These results indicate that this alga can rapidly biotransform arsenate into oxo-arsenosugar-glycerol for at least 10 min and then oxo-arsenosugar-phosphate through both reduction of incorporated arsenate to arsenite and methylation of arsenite and/or arsenate retained in the cells to dimethylarsinic acid via methylarsonic acid as an possible intermediate.     

Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms

An enrichment culture from a boreal sulfide mine environment containing a low-grade polymetallic ore was tested in column bioreactors for simulation of low temperature heap leaching. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing revealed the enrichment culture contained an Acidithiobacillus ferrooxidans strain with high 16S rRNA gene similarity to the psychrotolerant strain SS3 and a mesophilic Leptospirillum ferrooxidans strain. As the mixed culture contained a strain that was within a clade with SS3, we used the SS3 pure culture to compare leaching rates with the At. ferrooxidans type strain in stirred tank reactors for mineral sulfide dissolution at various temperatures. The psychrotolerant strain SS3 catalyzed pyrite, pyrite/arsenopyrite, and chalcopyrite concentrate leaching. The rates were lower at 5 degrees C than at 30 degrees C, despite that all the available iron was in the oxidized form in the presence of At. ferrooxidans SS3. This suggests that although efficient At. ferrooxidans SS3 mediated biological oxidation of ferrous iron occurred, chemical oxidation of the sulfide minerals by ferric iron was rate limiting. In the column reactors, the leaching rates were much less affected by low temperatures than in the stirred tank reactors. A factor for the relatively high rates of mineral oxidation at 7 degrees C is that ferric iron remained in the soluble phase whereas, at 21 degrees C the ferric iron precipitated. Temperature gradient analysis of ferrous iron oxidation by this enrichment culture demonstrated two temperature optima for ferrous iron oxidation and that the mixed culture was capable of ferrous iron oxidation at 5 degrees C.     

Arsenic efflux governed by the arsenic resistance determinant of Staphylococcus aureus plasmid pI258.

The arsenic resistance operon of Staphylococcus aureus plasmid pI258 determined lowered net cellular uptake of 73As by an active efflux mechanism. Arsenite was exported from the cells; intracellular arsenate was first reduced to arsenite and then transported out of the cells. Resistant cells showed lower accumulation of 73As originating from both arsenate and arsenite. Active efflux from cells loaded with arsenite required the presence of the plasmid-determined arsB gene. Efflux of arsenic originating as arsenate required the presence of the arsC gene and occurred more rapidly with the addition of arsB. Inhibitor studies with S. aureus loaded with arsenite showed that arsenite efflux was energy dependent and appeared to be driven by the membrane potential. With cells loaded with 73AsO4(3-), a requirement for ATP for energy was observed, leading to the conclusion that ATP was required for arsenate reduction. When the staphylococcal arsenic resistance determinant was cloned into Escherichia coli, lowered accumulation of arsenate and arsenite and 73As efflux from cells loaded with arsenate were also found. Cloning of the E. coli plasmid R773 arsA gene (the determinant of the arsenite-dependent ATPase) in trans to the S. aureus gene arsB resulted in increased resistance to arsenite.     

Bioleaching of realgar by Acidithiobacillus ferrooxidans using ferrous iron and elemental sulfur as the sole and mixed energy sources

The characteristics of the bioleaching of realgar by Acidithiobacillus ferrooxidans BY-3 (A. ferrooxidans) were investigated in this work. We examined the effects of using ferrous iron and elemental sulfur as the sole and mixed energy sources on the bioleaching of realgar. Under all experimental conditions, A. ferrooxidans BY-3 significantly enhanced the dissolution of realgar. Moreover, arsenic was more efficiently leached using A. ferrooxidans BY-3 in the presence of ferrous iron than in other culture conditions. A high concentration of arsenic was observed in the absence of alternative energy sources. This concentration was higher than that in cultures with sulfur only and lower than that in cultures with ferrous iron and sulfur. Linear or nonlinear models best fit the experimental data; the nonlinear model exhibited the dual effects of dissolution and removal on the bioleaching of realgar, whereas the linear model only applied to situations of slow bioleaching rather than removal.     

Relative toxicity of arsenite and arsenate on germination and early seedling growth of rice (Oryza sativa L.)

Elevated soil arsenic levels resulting from long-term use of arsenic contaminated ground for irrigation in Bangladesh may inhibit seed germination and seedling establishment of rice, the country's main food crop. A germination study on rice seeds and a short-term toxicity experiment with different concentrations of arsenite and arsenate on rice seedlings were conducted. Percent germination over control decreased significantly with increasing concentrations of arsenite and arsenate. Arsenite was found to be more toxic than arsenate for rice seed germination. There were varietal differences among the test varieties in response to arsenite and arsenate exposure. The performance of the dry season variety Purbachi was the best among the varieties. Germination of Purbachi was not inhibited at all up to 4 mg l^–1 arsenite and 8 mg l^–1 arsenate treatment. Root tolerance index (RTI) and relative shoot height (RSH) for rice seedlings decreased with increasing concentrations of arsenite and arsenate. Reduction of RTI caused by arsenate was higher than that of arsenite. In general, dry season varieties have more tolerance to arsenite or arsenate than the wet season varieties.     

Rhizosphere colonization and arsenic translocation in sunflower (Helianthus annuus L.) by arsenate reducing Alcaligenes sp. strain Dhal-L

In the present study, six arsenic-resistant strains previously isolated were tested for their plant growth promoting characteristics and heavy metal resistance, in order to choose one model strain as an inoculum for sunflower plants in pot experiments. The aim was to investigate the effect of arsenic-resistant strain on sunflower growth and on arsenic uptake from arsenic contaminated soil. Based on plant growth promoting characteristics and heavy metal resistance, Alcaligenes sp. strain Dhal-L was chosen as an inoculum. Beside the ability to reduce arsenate to arsenite via an Ars operon, the strain exhibited 1-amino-cyclopropane-1-carboxylic acid deaminase activity and it was also able to produce siderophore and indole acetic acid. Pot experiments were conducted with an agricultural soil contaminated with arsenic (214 mg kg^?1). A real time PCR method was set up based on the quantification of ACR3(2) type of arsenite efflux pump carried by Alcaligenes sp. strain Dhal-L, in order to monitor presence and colonisation of the strain in the bulk and rhizospheric soil. As a result of strain inoculation, arsenic uptake by plants was increased by 53 %, whereas ACR3(2) gene copy number in rhizospheric soil was 100 times higher in inoculated than in control pots, indicating the colonisation of strain. The results indicated that the presence of arsenate reducing strains in the rhizosphere of sunflower influences arsenic mobilization and promotes arsenic uptake by plant.