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Arsenic release from gold mine rocks mediated by the activity of indigenous bacteria
Hydrometallurgy 104 (2010) 437–442
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Hydrometallurgy j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / h yd r o m e t
Arsenic release from gold mine rocks mediated by the activity of indigenous bacteria L. Drewniak 1, R. Matlakowska 1, B. Rewerski 1, A. Sklodowska ⁎ Laboratory of Environmental Pollution Analysis, Faculty of Biology, University of Warsaw, Poland
a r t i c l e
i n f o
Available online 17 June 2010 Keywords: Arsenic mobilization Siderophores Arsenite oxidizers Arsenate dissimilatory reducers
a b s t r a c t The ancient gold mine at Zloty Stok is a source of serious arsenic contamination of underground and ground water, and soil. The objectives of this study were to investigate the impact of arsenic-metabolizing bacteria on arsenic mobilization from natural minerals and to estimate their potential role in the arsenic biogeochemical cycle in Zloty Stok gold mine. Using simple cultivation methods it was found that bacteria isolated from this gold mine could directly and indirectly influence arsenic mobilization from primary and secondary minerals. The dissimilatory arsenate reducers Shewanella sp. O23S and Aeromonas sp.O23A were particularly active in direct mobilization of the arsenic. Siderophores produced by Pseudomonas spp. isolated from a mine rock biofilm were shown to play a significant role in indirect arsenic mobilization. A potential mechanism of arsenic release from arsenate minerals, mediated by arsenite oxidizers, dissimilatory arsenate reducers and arsenic-resistant bacteria is proposed. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Microorganisms that metabolize arsenic belong to different groups of prokaryotes from across the bacterial domain (Oremland and Stoltz, 2005). Their physiological properties connected with arsenic are also diverse. Some microorganisms can oxidize arsenite with arsenite oxidase, either as a detoxification process alone (Gihring and Banfield, 2001), or using arsenite as an electron donor (Rhine et al., 2006; Santini et al., 2000). Others can use arsenate as the terminal acceptor in dissimilatory arsenate respiration (Malasarn et al., 2008; Stoltz et al., 2006). Microbial reduction of arsenate to arsenite may occur not only as an energy-generating process, but also as a detoxification mechanism. Many microorganisms have evolved strategies for arsenic detoxification, which require the ars genes. These genes encode proteins that catalyze the reduction of less toxic arsenate to more toxic arsenite via cytoplasmic arsenate reductase, followed by As (III) removal from the cell via an efflux pump (Cervantes et al., 1994; Silver and Phung, 2005). Based on current knowledge about arsenic microbes, using three different types of selective medium, a total of twenty two bacterial strains were isolated from rock biofilms on the walls of the Gertruda Adit (Fig. 1A,B) and seven from arsenic-rich bottom sediments (Fig. 1C) sampled from the Zloty Stok (SW Poland) gold mine (Drewniak et al., 2009). Phylogenetic analysis divided these 29 strains into 17 genera belonging to 5 classes: α-Proteobacteria, γ-Proteobac⁎ Corresponding author. E-mail addresses: [email protected] (L. Drewniak), [email protected] (R. Matlakowska), [email protected] (B. Rewerski), [email protected] (A. Sklodowska). 1 Tel./fax: + 48 22 541006. 0304-386X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2010.02.025
teria, Flavobacteria, Bacilli and Actinobacteria. All of the isolated bacteria were resistant to both inorganic arsenic species [As (III) and As (V)]. Some also showed extreme tolerance to arsenite (up to 20 mM) and arsenate (up to 500 mM). Arsenate reductase activity and the arsenate reductase gene (arsC) were detected in all of the tested isolates (Drewniak et al., 2008a). Anaerobic respiratory arsenic transformation was carried out only by two of the bottom sediment strains: Shewanella sp. O23S and Aeromonas sp. O23A. Both of these isolates were able to grow in the absence of oxygen by employing energy-generating As (V) respiration coupled with lactate oxidation (Drewniak et al., 2009). A dissimilatory arsenate reductase gene (arrA) was identified in both of these arsenate-respiring strains. A respiratory process based on the oxidation of arsenite (electron donor) to arsenate was performed by only one bottom sediment isolate: Sinorhizobium sp. M14 (Drewniak et al., 2008b). A second arsenite oxidizer strain (Pseudomonas sp. S1) was also able to oxidize As (III), but it could not use carbon dioxide (CO2) or bicarbonate (HCO− 3 ) as the carbon source. However, an arsenite oxidase gene (aoxB) was identified in both strains (Drewniak et al., 2009). The objectives of the present study were (i) to investigate the impact of isolated arsenic-metabolizing microbes on arsenic mobilization from natural minerals, and (ii) to define their potential role in the arsenic biogeochemical cycle in Zloty Stok gold mine. 2. Area description, methods, and material studied 2.1. Area description The ancient Zloty Stok gold mine in south-west Poland with some 300 km of underground passages on 21 levels was closed in 1962. Most of the underground galleries and shafts are now flooded with
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Fig. 1. Biofilms occurring in the deepest section of Gertruda Adit in Zloty Stok gold mine: A, B — rock biofilms on the walls of the adit; C — microbial mats in the bottom sediments.
water and are partially or totally inaccessible. One of the main and the most well preserved galleries is transportation Gertruda Adit. The deepest section of Gertruda Adit has collapsed at its end, is separated by a low sluice gate and partially filled with water. At this location there is a stable low air temperature (10.4–11.1 °C), a reduced concentration of oxygen (17.2%), almost total humidity (∼100%), and the water is slightly alkaline (pH 7.4–8.1). High arsenic concentrations in the rocks, rock biofilms (19–59 mg/kg) and water (1.58–3.51 mg/kg), and the presence of AsH3 (1.52–3.23 mg/m3) in the air have been detected (Drewniak et al., 2008a). Moreover, a deficit of iron ions (ferric and ferrous) in the mine water, was noted. 2.2. Bacterial strains One chemolithoautotrophic strain — Sinorhizobium sp. M14 (EF442029), two dissimilatory arsenate reducers — Aeromonas sp. O23A (GU191138) and Shewanella sp. O23S (GU191139) (isolated from bottom sediments) and two arsenic-resistant strains — Pseudomonas sp. OS8 (EF491958) and Pseudomonas sp. OS20 (EF491969) (isolated from rock biofilm), were chosen as representative microorganisms characteristic of the mine microflora. This selection was based on the results of studies on the biodiversity and physiology of Zloty Stok gold mine bacteria (Drewniak et al., 2009, 2008a).
anaerobic conditions. The media for test cultures and controls were prepared in triplicate. The cells for inoculation were grown to exponential phase in Luria Bertrani medium (LB) then harvested by centrifugation (10,000 ×g, 4 °C, 10 min) and washed three times in MSM to remove residual LB. The prepared media were then inoculated to a density of about 106 cells/ml. The cultures of Pseudomonas spp. OS8 and OS20, Aeromonas sp. O23A and Shewanella sp. O23S were incubated at 22 °C for 21 days with shaking (110 rpm) and sampled at the start and the end of the experiment. The Sinorhizobium sp. M14 culture was incubated at 22 °C for 7 days. Non-inoculated controls were incubated and sampled as described for the test cultures. 2.5. Test for dissolution of mine rock and arsenopyrite by siderophores produced by arsenic-resistant bacteria To determine the mine rock and arsenopyrite dissolution abilities of bacterial siderophores, Pseudomonas strains OS8 and OS20 isolated
2.3. Media and growth conditions Bacterial strains were cultivated in minimal salts medium (MSM) (Santini et al., 2000) or in GASN medium (Bultreys and Gheysen, 2000) at 22 °C. For heterotrophic growth, Luria Bertrani (LB) medium (Sambrook and Russell, 2001) or MSM supplemented with yeast extract (0.04% w/v) was used. For aerobic growth, 100 ml cultures were incubated in 300 ml Erlenmeyer flasks. For anaerobic growth, MSM medium was supplemented with 5 mM sodium lactate and 100 ml serum bottles with argon injected into the headspace were used. These bottles were closed with rubber stoppers secured by aluminum crimp seals. 2.4. Arsenic mobilization experiments The ability of arsenic-metabolizing isolates to release arsenic from arsenopyrite (FeAsS) and rocks (mixture of arsenopyrite — FeAsS, loellingite — FeAs2, traces of scorodite — FeAsO4·2H2O and origin ore) from Zloty Stok mine was examined. The mineral or mine rock specimens were crushed and washed with 6N HCl and sterilized, then 5 g was added to the respective growth media (dependent on which arsenic-metabolizing bacteria was tested). To examine the arsenic mobilization abilities of Sinorhizobium sp. M14 (arsenite oxidizer) and Pseudomonas spp. OS8 and OS20, arsenopyrite or mine rock were added to MSM as the electron donor. To examine the mobilization abilities of dissimilatory arsenate reducers Aeromonas sp. O23A and Shewanella sp. O23S, arsenopyrite or mine rock were added to MSM in
Fig. 2. Mobilization of arsenic from natural rocks by bacteria isolated from Zloty Stok gold mine. A — Arsenic released into the culture supernatant by bacteria growing on MSM (containing mine rock) under aerobic conditions, after 7 days of incubation. B — Arsenic released into the culture supernatant by bacteria growing on MSM (containing mine rock and 5 mM lactate) in anaerobic conditions, after 21 days of incubation.
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from the mine rock biofilm (Drewniak et al., 2008a) were used. These strains were grown in GASN medium for 24 h at room temperature. The cultures were then centrifuged (10,000 ×g, 10 min.) and the supernatants used as siderophore preparations. The siderophores in the supernatants were quantified by absorbance at 630 nm following a 1 h incubation with CAS assay solution (Schwyn and Neilands, 1987). The siderophore concentrations were estimated from a standard curve prepared using deferoxamine mesylate (DFOB) and expressed as mM DFOB (Fekete et al., 1989). In dissolution reactions, 10 ml of 0.2-mM bacterial siderophore, a 0.2-mM solution of DFOB (positive control) or sterile GASN medium (negative control) were added to 5 g of mine rock or natural
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arsenopyrite, and the concentrations of Fe and As in solution were measured after 96-h incubation. 2.6. Chemical analysis Arsenite transformation in culture supernatants was determined following filtration through sterile 0.22-μm pore size Durapore filters (Millipore). Arsenic species were separated by high-performance liquid chromatography on a reversed-phase polymeric resin (Waters IC-Pak™ Anion HC column, 150 by 4.6 mm, pore size 10 μm) and quantified by Graphite Furnace Atomic Absorption Spectrometry (GFAAS) (AA Solaar M6 Spectrometer, TJA Solutions, UK). Total
Fig. 3. Arsenopyrite (FeAsS) oxidation mediated by Sinorhizobium sp. M14. A — Growth of M14 in MSM containing arsenopyrite. B — Arsenic release following culture with arsenopyrite at 22 °C for 7 days. Sterile MSM containing arsenopyrite was used as a control. C — Scorodite (FeAsO4∙ 2H2O) precipitation on the arsenopyrite surface.
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arsenic and iron levels were quantified by GFAAS using ASA standards (Merck, Darmstadt, Germany) prepared in 3% HNO3. 3. Results and discussion 3.1. Arsenic mobilization from gold mine rocks Arsenic occurs in the Zloty Stok gold mine mainly in minerals such as loellingite (FeAs2), arsenopyrite (FeAsS) and scorodite (FeAsO4·2H2O). It is also present in mine water and bottom sediments as arsenite and arsenate, and in the Gertruda Adit air as arsenic hydride (Drewniak et al., 2008a). Because microbial activity is one of the main catalysts of the release of arsenic from natural minerals into the environment (Oremland and Stoltz, 2005, 2003; Zobrist et al., 2000), we decided to examine how arsenic-metabolizing microbes isolated from Zloty Stok mine participate in the mobilization of arsenic from rocks (containing arsenic minerals) to water and sediments. Aerobic and anaerobic arsenic mobilization experiments were conducted by inoculating MSM containing sterile mine rock samples with different arsenic-metabolizing microbes isolated from the Zloty Stok mine. These cultures were incubated for 7 (aerobic experiment) or 21 (anaerobic experiment) days. After 7 days of incubation in aerobic conditions, arsenic release from mine rock was observed in both the control (sterile MSM medium) and the test cultures inoculated with “arsenic bacteria” (Fig. 2A). The mean concentration of released arsenic in the controls was 88.8 mg/l, which was 33.5% lower than that in the Sinorhizobium sp. culture (133.5 mg/l) and 2fold lower than that in Pseudomonas spp. cultures (195.0 and 211.3 mg/l). This result indicates that the metabolic activity of Sinorhizobium sp. M14 and both Pseudomonas strains (OS8 and OS20) can increase arsenic release from natural rocks, and may enhance abiotic mobilization processes. As in aerobic conditions, arsenic release was observed under anaerobic conditions in both the sterile control and bacterial test cultures (Fig. 2B). Anaerobic arsenic mobilization promoted by the two dissimilatory arsenate reducers (Shewanella sp. O23S and Aeromonas sp. O23A) was much more efficient than in the cultures of other bacteria grown under anaerobic or aerobic conditions.
processes (Drewniak et al., 2009, 2008a), they were able to enhance the mobilization of arsenic from natural minerals. The isolates hypertolerant to As (V) produce iron-binding compounds (siderophores) that help them to uptake iron from insoluble minerals such as scorodite (Drewniak et al., 2008a). Siderophores bind Fe3+ in complexes that can be imported into the bacterial cell by active transport mechanisms. During this process, it is thought that As (V) is released from the solid to the aqueous phase and the toxic effects of this mobilized arsenic are neutralized by the activity of the bacterial arsenate reductase. As3+ is transported out of the cell by the ArsAB arsenic chemiosmotic efflux system. To confirm the hypothesis that siderophores produced by the tested bacterial strains are responsible for the release of arsenic from mine rocks into the environment, arsenic mobilization experiments were performed. These examined the release of arsenic and iron from gold mine rock or arsenopyrite, mediated by siderophores produced by the isolated strains. Two Pseudomonas strains, OS8 and OS20, isolated from mine rock biofilm were chosen for these experiments. These strains are hypertolerant to arsenic [12.5 mM As (III) and 350 mM As (V)], exhibit high arsenate reductase activity (Drewniak et al., 2008a) and under iron-limiting conditions produce chemically different siderophores: hydroxamate-type (OS8) and catechol-type (OS20) (unpublished data). Filtered supernatants from Pseudomonas spp. OS8 and OS20 cultures containing siderophores at a concentration adjusted to approximately 0.2 mM were added to samples of mine rock or arsenopyrite. Sterile GASN medium with or without added deferoxamine (DFOB), a common siderophore ligand (0.2 mM), was used as the positive and negative control, respectively. After 96 h incubation,
3.2. Arsenic minerals oxidation The analysis of test cultures containing mine rock or arsenopyrite at neutral pH inoculated with Sinorhizobium sp. M14 (bottom sediment strain) demonstrated that this bacterium can directly mobilize arsenic from minerals. Strain M14 was able to grow chemolithoautotrophically using arsenopyrite as a source of energy (Fig. 3A). After 7 days of incubation at 22 °C under aerobic conditions, 0.73 μg/l total arsenic was present in the sterile control, whereas in the bacterial test culture a concentration of 3.94 μg/l was detected (Fig. 3B). However, speciation analysis identified As (III) in the culture supernatant, demonstrating that oxidation of the released arsenic was incomplete. Furthermore, a dark orange scorodite-like precipitate formed on the surface of the arsenopyrite (Fig. 3C). We hypothesize that under circumneutral pH, the arsenic [As (V)], oxidized by the activity of strain M14, was co-precipitated with Fe (III) (released from arsenopyrite) and residual arsenate was reduced to arsenite by means of the M14 Ars system (responsible for arsenate resistance). The two Pseudomonas strains were unable to grow under these conditions and the release of arsenic was not detected (data not shown). 3.3. Dissolution of mine rock and arsenopyrite by siderophores produced by arsenic-resistant strains Despite the fact that none of the arsenic-resistant bacteria isolated from mine rock biofilms use arsenic compounds in respiratory
Fig. 4. Release of As and Fe from: A — natural mine rock B — arsenopyrite, after treatment with GASN medium (negative control), deferoxamine (DFOB, positive control) and siderophores produced by Pseudomonas sp. OS8 and OS20, after 96 h incubation time at 22 °C.
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Fig. 5. Possible mechanisms of arsenic mobilization from arsenate minerals in Zloty Stok gold mine mediated by indigenous bacteria. Solid lines — processes verified experimentally in this study; broken lines — hypothetical processes. Arsenite oxidizers present in oxic zone of bacterial mats (eg. Sinorhizobium sp. M14) transform arsenic minerals (arsenopyrite, loellingite) occur in the rocks to scorodite or goethite in oxidation processes. Then the dissimilatory arsenate reducers in anoxic zone (eg. Shewanella sp. O23S, Aeromonas sp. O23A) release As (III) which can be further reduced to arsine. Bacteria occurring in the rock biofilm utilize simple organic compounds from the air and produce siderophores for iron uptake (from scorodite, arsenopyrite etc.) causing high concentration of arsenic in the surrounding environment. As (III) is released in detoxification processes.
a change in the color was observed in the reactions with mine rock or arsenopyrite treated with the culture supernatants and in the DFOB positive controls. No alteration in color was apparent in the negative controls, suggesting that the changes were the result of the reaction of siderophores with iron from the mine rock or arsenopyrite. GFAAS analysis confirmed this hypothesis because dissolved Fe (iron) was detected in the reactions with the Pseudomonas spp. OS8 and OS20 culture supernatants and the positive controls. The iron concentration was much higher in these reactions than in the negative controls without siderophores (Fig. 4A,B). Analysis of the arsenic concentration in the solution also confirmed the release of arsenic from arsenopyrite only in all culture supernatants (siderophores solution) (Fig. 4A,B), and in the positive controls. In the case of mine rocks siderophore mediated arsenic release was observed only in Pseudomonas sp. OS20 culture filtrate opposite to iron release. This finding may be explained by lower abundance of arsenic species in rock material (mixture of arsenic and iron minerals and origin ore) than in almost pure crystals of arsenopyrite. Moreover arsenic in natural rocks occurs not only in minerals containing iron, while siderophores are specific mainly for iron. 4. Conclusions The results of this study demonstrate that indigenous bacteria can directly and indirectly promote the mobilization of arsenic in Zloty Stok gold mine. An experiment examining arsenic mobilization from natural mine rock and arsenopyrite conducted at neutral pH showed that the arsenite oxidizer Sinorhizobium sp. M14 can directly mobilize arsenic from such minerals. The dissimilatory arsenate reducers Shewanella sp. O23S and Aeromonas sp. O23A were particularly active in the direct mobilization of arsenic. The significant role of siderophores produced by Pseudomonas spp. isolated from mine rock
biofilm in indirect arsenic mobilization was confirmed. These findings suggest possible mechanisms of arsenic mobilization from arsenate minerals in Zloty Stok gold mine, mediated by indigenous arsenite oxidizers, dissimilatory arsenate reducers and arsenic-resistant bacteria (Fig. 5). Acknowledgements This research was supported by ordered research project No. PBZKBN-111/T09/2004 from the Polish Ministry of Science and Higher Education over the period 2005–2008. Lukasz Drewniak is scholarship holder in the Start Programme of Foundation of Polish Science. We gratefully acknowledge the help of Dr. John Gittins for his critical reading of the manuscript and English correction. References Bultreys, A., Gheysen, I., 2000. Production and comparison of peptide siderophores from strains of distantly related pathovars of Pseudomonas syringae and Pseudomonas viridiflava LMG 2352. Appl. Environ. Microbiol. 66, 325–331. Cervantes, C., Ji, G., Ramirez, J.L., Silver, S., 1994. Resistance to arsenic compounds in microorganisms. FEMS Microbiol. Rev. 15, 355–367. Drewniak, L., Styczek, A., Majder-Lopatka, M., Sklodowska, A., 2008a. Bacteria, hypertolerant to arsenic in the rocks of an ancient gold mine and their potential role in dissemination of arsenic pollution. Environ. Pollut. 156, 1069–1074. Drewniak, L., Matlakowska, R., Sklodowska, A., 2008b. Arsenite and arsenate metabolism of Sinorhizobium sp. M14 living in the extreme environment of the Zloty Stok gold mine. Geomicrobiol. J. 25, 363–370. Drewniak, L., Matlakowska, R., Sklodowska, A., 2009. Microbial impact on arsenic mobilization in Zloty Stok gold mine. Adv. Mater. Res. 71–73, 121–124. Fekete, F.A., Chandhoke, V., Jellison, J., 1989. Iron-binding compounds produced by wood-decaying basidiomycetes. Appl. Environ. Microbiol. 55, 2720–2722. Gihring, T.M., Banfield, J.F., 2001. Arsenite oxidation and arsenate respiration by a new Thermus isolate. FEMS Microbiol. Lett. 204, 335–340. Malasarn, D., Keeffe, J.R., Newman, D.K., 2008. Characterization of the arsenate respiratory reductase from Shewanella sp. strain ANA-3. J. Bacteriol. 190, 135–142. Oremland, R.S., Stoltz, J.F., 2003. The ecology of arsenic. Science 300, 939–944.
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Oremland, R.S., Stoltz, J.F., 2005. Arsenic, microbes and contaminated aquifers. Trends Microbiol. 13, 45–49. Rhine, E.D., Phelps, C.D., Young, L.Y., 2006. Anaerobic arsenite oxidation by novel denitrifying isolates. Environ. Microbiol. 8, 899–908. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Santini, J.M., Sly, L.I., Schnagl, R.D., Macy, J.M., 2000. A new chemolithoautotrophic arsenite-oxidizing bacterium isolated from a gold mine: phylogenetic, physiological, and preliminary biochemical studies. Appl. Environ. Microbiol. 66, 92–97.
Schwyn, B., Neilands, J.B., 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160, 47–56. Silver, S., Phung, L.T., 2005. A bacterial view of the periodic table: genes and proteins for toxic inorganic ions. J. Ind. Microbiol. Biotechnol. 32, 587–605. Stoltz, J.F., Basu, P., Santini, J.M., Oremland, R.S., 2006. Arsenic and selenium in microbial metabolism. Annu. Rev. Microbiol. 60, 107–130. Zobrist, J., Dowdle, P.R., Davis, J.A., Oremland, R.S., 2000. Mobilization of arsenite by dissimilatory reduction of adsorbed arsenate. Environ. Sci. Technol. 34, 4747–4753.
Contents lists available at ScienceDirect
Hydrometallurgy j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / h yd r o m e t
Arsenic release from gold mine rocks mediated by the activity of indigenous bacteria L. Drewniak 1, R. Matlakowska 1, B. Rewerski 1, A. Sklodowska ⁎ Laboratory of Environmental Pollution Analysis, Faculty of Biology, University of Warsaw, Poland
a r t i c l e
i n f o
Available online 17 June 2010 Keywords: Arsenic mobilization Siderophores Arsenite oxidizers Arsenate dissimilatory reducers
a b s t r a c t The ancient gold mine at Zloty Stok is a source of serious arsenic contamination of underground and ground water, and soil. The objectives of this study were to investigate the impact of arsenic-metabolizing bacteria on arsenic mobilization from natural minerals and to estimate their potential role in the arsenic biogeochemical cycle in Zloty Stok gold mine. Using simple cultivation methods it was found that bacteria isolated from this gold mine could directly and indirectly influence arsenic mobilization from primary and secondary minerals. The dissimilatory arsenate reducers Shewanella sp. O23S and Aeromonas sp.O23A were particularly active in direct mobilization of the arsenic. Siderophores produced by Pseudomonas spp. isolated from a mine rock biofilm were shown to play a significant role in indirect arsenic mobilization. A potential mechanism of arsenic release from arsenate minerals, mediated by arsenite oxidizers, dissimilatory arsenate reducers and arsenic-resistant bacteria is proposed. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Microorganisms that metabolize arsenic belong to different groups of prokaryotes from across the bacterial domain (Oremland and Stoltz, 2005). Their physiological properties connected with arsenic are also diverse. Some microorganisms can oxidize arsenite with arsenite oxidase, either as a detoxification process alone (Gihring and Banfield, 2001), or using arsenite as an electron donor (Rhine et al., 2006; Santini et al., 2000). Others can use arsenate as the terminal acceptor in dissimilatory arsenate respiration (Malasarn et al., 2008; Stoltz et al., 2006). Microbial reduction of arsenate to arsenite may occur not only as an energy-generating process, but also as a detoxification mechanism. Many microorganisms have evolved strategies for arsenic detoxification, which require the ars genes. These genes encode proteins that catalyze the reduction of less toxic arsenate to more toxic arsenite via cytoplasmic arsenate reductase, followed by As (III) removal from the cell via an efflux pump (Cervantes et al., 1994; Silver and Phung, 2005). Based on current knowledge about arsenic microbes, using three different types of selective medium, a total of twenty two bacterial strains were isolated from rock biofilms on the walls of the Gertruda Adit (Fig. 1A,B) and seven from arsenic-rich bottom sediments (Fig. 1C) sampled from the Zloty Stok (SW Poland) gold mine (Drewniak et al., 2009). Phylogenetic analysis divided these 29 strains into 17 genera belonging to 5 classes: α-Proteobacteria, γ-Proteobac⁎ Corresponding author. E-mail addresses: [email protected] (L. Drewniak), [email protected] (R. Matlakowska), [email protected] (B. Rewerski), [email protected] (A. Sklodowska). 1 Tel./fax: + 48 22 541006. 0304-386X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2010.02.025
teria, Flavobacteria, Bacilli and Actinobacteria. All of the isolated bacteria were resistant to both inorganic arsenic species [As (III) and As (V)]. Some also showed extreme tolerance to arsenite (up to 20 mM) and arsenate (up to 500 mM). Arsenate reductase activity and the arsenate reductase gene (arsC) were detected in all of the tested isolates (Drewniak et al., 2008a). Anaerobic respiratory arsenic transformation was carried out only by two of the bottom sediment strains: Shewanella sp. O23S and Aeromonas sp. O23A. Both of these isolates were able to grow in the absence of oxygen by employing energy-generating As (V) respiration coupled with lactate oxidation (Drewniak et al., 2009). A dissimilatory arsenate reductase gene (arrA) was identified in both of these arsenate-respiring strains. A respiratory process based on the oxidation of arsenite (electron donor) to arsenate was performed by only one bottom sediment isolate: Sinorhizobium sp. M14 (Drewniak et al., 2008b). A second arsenite oxidizer strain (Pseudomonas sp. S1) was also able to oxidize As (III), but it could not use carbon dioxide (CO2) or bicarbonate (HCO− 3 ) as the carbon source. However, an arsenite oxidase gene (aoxB) was identified in both strains (Drewniak et al., 2009). The objectives of the present study were (i) to investigate the impact of isolated arsenic-metabolizing microbes on arsenic mobilization from natural minerals, and (ii) to define their potential role in the arsenic biogeochemical cycle in Zloty Stok gold mine. 2. Area description, methods, and material studied 2.1. Area description The ancient Zloty Stok gold mine in south-west Poland with some 300 km of underground passages on 21 levels was closed in 1962. Most of the underground galleries and shafts are now flooded with
438
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Fig. 1. Biofilms occurring in the deepest section of Gertruda Adit in Zloty Stok gold mine: A, B — rock biofilms on the walls of the adit; C — microbial mats in the bottom sediments.
water and are partially or totally inaccessible. One of the main and the most well preserved galleries is transportation Gertruda Adit. The deepest section of Gertruda Adit has collapsed at its end, is separated by a low sluice gate and partially filled with water. At this location there is a stable low air temperature (10.4–11.1 °C), a reduced concentration of oxygen (17.2%), almost total humidity (∼100%), and the water is slightly alkaline (pH 7.4–8.1). High arsenic concentrations in the rocks, rock biofilms (19–59 mg/kg) and water (1.58–3.51 mg/kg), and the presence of AsH3 (1.52–3.23 mg/m3) in the air have been detected (Drewniak et al., 2008a). Moreover, a deficit of iron ions (ferric and ferrous) in the mine water, was noted. 2.2. Bacterial strains One chemolithoautotrophic strain — Sinorhizobium sp. M14 (EF442029), two dissimilatory arsenate reducers — Aeromonas sp. O23A (GU191138) and Shewanella sp. O23S (GU191139) (isolated from bottom sediments) and two arsenic-resistant strains — Pseudomonas sp. OS8 (EF491958) and Pseudomonas sp. OS20 (EF491969) (isolated from rock biofilm), were chosen as representative microorganisms characteristic of the mine microflora. This selection was based on the results of studies on the biodiversity and physiology of Zloty Stok gold mine bacteria (Drewniak et al., 2009, 2008a).
anaerobic conditions. The media for test cultures and controls were prepared in triplicate. The cells for inoculation were grown to exponential phase in Luria Bertrani medium (LB) then harvested by centrifugation (10,000 ×g, 4 °C, 10 min) and washed three times in MSM to remove residual LB. The prepared media were then inoculated to a density of about 106 cells/ml. The cultures of Pseudomonas spp. OS8 and OS20, Aeromonas sp. O23A and Shewanella sp. O23S were incubated at 22 °C for 21 days with shaking (110 rpm) and sampled at the start and the end of the experiment. The Sinorhizobium sp. M14 culture was incubated at 22 °C for 7 days. Non-inoculated controls were incubated and sampled as described for the test cultures. 2.5. Test for dissolution of mine rock and arsenopyrite by siderophores produced by arsenic-resistant bacteria To determine the mine rock and arsenopyrite dissolution abilities of bacterial siderophores, Pseudomonas strains OS8 and OS20 isolated
2.3. Media and growth conditions Bacterial strains were cultivated in minimal salts medium (MSM) (Santini et al., 2000) or in GASN medium (Bultreys and Gheysen, 2000) at 22 °C. For heterotrophic growth, Luria Bertrani (LB) medium (Sambrook and Russell, 2001) or MSM supplemented with yeast extract (0.04% w/v) was used. For aerobic growth, 100 ml cultures were incubated in 300 ml Erlenmeyer flasks. For anaerobic growth, MSM medium was supplemented with 5 mM sodium lactate and 100 ml serum bottles with argon injected into the headspace were used. These bottles were closed with rubber stoppers secured by aluminum crimp seals. 2.4. Arsenic mobilization experiments The ability of arsenic-metabolizing isolates to release arsenic from arsenopyrite (FeAsS) and rocks (mixture of arsenopyrite — FeAsS, loellingite — FeAs2, traces of scorodite — FeAsO4·2H2O and origin ore) from Zloty Stok mine was examined. The mineral or mine rock specimens were crushed and washed with 6N HCl and sterilized, then 5 g was added to the respective growth media (dependent on which arsenic-metabolizing bacteria was tested). To examine the arsenic mobilization abilities of Sinorhizobium sp. M14 (arsenite oxidizer) and Pseudomonas spp. OS8 and OS20, arsenopyrite or mine rock were added to MSM as the electron donor. To examine the mobilization abilities of dissimilatory arsenate reducers Aeromonas sp. O23A and Shewanella sp. O23S, arsenopyrite or mine rock were added to MSM in
Fig. 2. Mobilization of arsenic from natural rocks by bacteria isolated from Zloty Stok gold mine. A — Arsenic released into the culture supernatant by bacteria growing on MSM (containing mine rock) under aerobic conditions, after 7 days of incubation. B — Arsenic released into the culture supernatant by bacteria growing on MSM (containing mine rock and 5 mM lactate) in anaerobic conditions, after 21 days of incubation.
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from the mine rock biofilm (Drewniak et al., 2008a) were used. These strains were grown in GASN medium for 24 h at room temperature. The cultures were then centrifuged (10,000 ×g, 10 min.) and the supernatants used as siderophore preparations. The siderophores in the supernatants were quantified by absorbance at 630 nm following a 1 h incubation with CAS assay solution (Schwyn and Neilands, 1987). The siderophore concentrations were estimated from a standard curve prepared using deferoxamine mesylate (DFOB) and expressed as mM DFOB (Fekete et al., 1989). In dissolution reactions, 10 ml of 0.2-mM bacterial siderophore, a 0.2-mM solution of DFOB (positive control) or sterile GASN medium (negative control) were added to 5 g of mine rock or natural
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arsenopyrite, and the concentrations of Fe and As in solution were measured after 96-h incubation. 2.6. Chemical analysis Arsenite transformation in culture supernatants was determined following filtration through sterile 0.22-μm pore size Durapore filters (Millipore). Arsenic species were separated by high-performance liquid chromatography on a reversed-phase polymeric resin (Waters IC-Pak™ Anion HC column, 150 by 4.6 mm, pore size 10 μm) and quantified by Graphite Furnace Atomic Absorption Spectrometry (GFAAS) (AA Solaar M6 Spectrometer, TJA Solutions, UK). Total
Fig. 3. Arsenopyrite (FeAsS) oxidation mediated by Sinorhizobium sp. M14. A — Growth of M14 in MSM containing arsenopyrite. B — Arsenic release following culture with arsenopyrite at 22 °C for 7 days. Sterile MSM containing arsenopyrite was used as a control. C — Scorodite (FeAsO4∙ 2H2O) precipitation on the arsenopyrite surface.
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arsenic and iron levels were quantified by GFAAS using ASA standards (Merck, Darmstadt, Germany) prepared in 3% HNO3. 3. Results and discussion 3.1. Arsenic mobilization from gold mine rocks Arsenic occurs in the Zloty Stok gold mine mainly in minerals such as loellingite (FeAs2), arsenopyrite (FeAsS) and scorodite (FeAsO4·2H2O). It is also present in mine water and bottom sediments as arsenite and arsenate, and in the Gertruda Adit air as arsenic hydride (Drewniak et al., 2008a). Because microbial activity is one of the main catalysts of the release of arsenic from natural minerals into the environment (Oremland and Stoltz, 2005, 2003; Zobrist et al., 2000), we decided to examine how arsenic-metabolizing microbes isolated from Zloty Stok mine participate in the mobilization of arsenic from rocks (containing arsenic minerals) to water and sediments. Aerobic and anaerobic arsenic mobilization experiments were conducted by inoculating MSM containing sterile mine rock samples with different arsenic-metabolizing microbes isolated from the Zloty Stok mine. These cultures were incubated for 7 (aerobic experiment) or 21 (anaerobic experiment) days. After 7 days of incubation in aerobic conditions, arsenic release from mine rock was observed in both the control (sterile MSM medium) and the test cultures inoculated with “arsenic bacteria” (Fig. 2A). The mean concentration of released arsenic in the controls was 88.8 mg/l, which was 33.5% lower than that in the Sinorhizobium sp. culture (133.5 mg/l) and 2fold lower than that in Pseudomonas spp. cultures (195.0 and 211.3 mg/l). This result indicates that the metabolic activity of Sinorhizobium sp. M14 and both Pseudomonas strains (OS8 and OS20) can increase arsenic release from natural rocks, and may enhance abiotic mobilization processes. As in aerobic conditions, arsenic release was observed under anaerobic conditions in both the sterile control and bacterial test cultures (Fig. 2B). Anaerobic arsenic mobilization promoted by the two dissimilatory arsenate reducers (Shewanella sp. O23S and Aeromonas sp. O23A) was much more efficient than in the cultures of other bacteria grown under anaerobic or aerobic conditions.
processes (Drewniak et al., 2009, 2008a), they were able to enhance the mobilization of arsenic from natural minerals. The isolates hypertolerant to As (V) produce iron-binding compounds (siderophores) that help them to uptake iron from insoluble minerals such as scorodite (Drewniak et al., 2008a). Siderophores bind Fe3+ in complexes that can be imported into the bacterial cell by active transport mechanisms. During this process, it is thought that As (V) is released from the solid to the aqueous phase and the toxic effects of this mobilized arsenic are neutralized by the activity of the bacterial arsenate reductase. As3+ is transported out of the cell by the ArsAB arsenic chemiosmotic efflux system. To confirm the hypothesis that siderophores produced by the tested bacterial strains are responsible for the release of arsenic from mine rocks into the environment, arsenic mobilization experiments were performed. These examined the release of arsenic and iron from gold mine rock or arsenopyrite, mediated by siderophores produced by the isolated strains. Two Pseudomonas strains, OS8 and OS20, isolated from mine rock biofilm were chosen for these experiments. These strains are hypertolerant to arsenic [12.5 mM As (III) and 350 mM As (V)], exhibit high arsenate reductase activity (Drewniak et al., 2008a) and under iron-limiting conditions produce chemically different siderophores: hydroxamate-type (OS8) and catechol-type (OS20) (unpublished data). Filtered supernatants from Pseudomonas spp. OS8 and OS20 cultures containing siderophores at a concentration adjusted to approximately 0.2 mM were added to samples of mine rock or arsenopyrite. Sterile GASN medium with or without added deferoxamine (DFOB), a common siderophore ligand (0.2 mM), was used as the positive and negative control, respectively. After 96 h incubation,
3.2. Arsenic minerals oxidation The analysis of test cultures containing mine rock or arsenopyrite at neutral pH inoculated with Sinorhizobium sp. M14 (bottom sediment strain) demonstrated that this bacterium can directly mobilize arsenic from minerals. Strain M14 was able to grow chemolithoautotrophically using arsenopyrite as a source of energy (Fig. 3A). After 7 days of incubation at 22 °C under aerobic conditions, 0.73 μg/l total arsenic was present in the sterile control, whereas in the bacterial test culture a concentration of 3.94 μg/l was detected (Fig. 3B). However, speciation analysis identified As (III) in the culture supernatant, demonstrating that oxidation of the released arsenic was incomplete. Furthermore, a dark orange scorodite-like precipitate formed on the surface of the arsenopyrite (Fig. 3C). We hypothesize that under circumneutral pH, the arsenic [As (V)], oxidized by the activity of strain M14, was co-precipitated with Fe (III) (released from arsenopyrite) and residual arsenate was reduced to arsenite by means of the M14 Ars system (responsible for arsenate resistance). The two Pseudomonas strains were unable to grow under these conditions and the release of arsenic was not detected (data not shown). 3.3. Dissolution of mine rock and arsenopyrite by siderophores produced by arsenic-resistant strains Despite the fact that none of the arsenic-resistant bacteria isolated from mine rock biofilms use arsenic compounds in respiratory
Fig. 4. Release of As and Fe from: A — natural mine rock B — arsenopyrite, after treatment with GASN medium (negative control), deferoxamine (DFOB, positive control) and siderophores produced by Pseudomonas sp. OS8 and OS20, after 96 h incubation time at 22 °C.
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Fig. 5. Possible mechanisms of arsenic mobilization from arsenate minerals in Zloty Stok gold mine mediated by indigenous bacteria. Solid lines — processes verified experimentally in this study; broken lines — hypothetical processes. Arsenite oxidizers present in oxic zone of bacterial mats (eg. Sinorhizobium sp. M14) transform arsenic minerals (arsenopyrite, loellingite) occur in the rocks to scorodite or goethite in oxidation processes. Then the dissimilatory arsenate reducers in anoxic zone (eg. Shewanella sp. O23S, Aeromonas sp. O23A) release As (III) which can be further reduced to arsine. Bacteria occurring in the rock biofilm utilize simple organic compounds from the air and produce siderophores for iron uptake (from scorodite, arsenopyrite etc.) causing high concentration of arsenic in the surrounding environment. As (III) is released in detoxification processes.
a change in the color was observed in the reactions with mine rock or arsenopyrite treated with the culture supernatants and in the DFOB positive controls. No alteration in color was apparent in the negative controls, suggesting that the changes were the result of the reaction of siderophores with iron from the mine rock or arsenopyrite. GFAAS analysis confirmed this hypothesis because dissolved Fe (iron) was detected in the reactions with the Pseudomonas spp. OS8 and OS20 culture supernatants and the positive controls. The iron concentration was much higher in these reactions than in the negative controls without siderophores (Fig. 4A,B). Analysis of the arsenic concentration in the solution also confirmed the release of arsenic from arsenopyrite only in all culture supernatants (siderophores solution) (Fig. 4A,B), and in the positive controls. In the case of mine rocks siderophore mediated arsenic release was observed only in Pseudomonas sp. OS20 culture filtrate opposite to iron release. This finding may be explained by lower abundance of arsenic species in rock material (mixture of arsenic and iron minerals and origin ore) than in almost pure crystals of arsenopyrite. Moreover arsenic in natural rocks occurs not only in minerals containing iron, while siderophores are specific mainly for iron. 4. Conclusions The results of this study demonstrate that indigenous bacteria can directly and indirectly promote the mobilization of arsenic in Zloty Stok gold mine. An experiment examining arsenic mobilization from natural mine rock and arsenopyrite conducted at neutral pH showed that the arsenite oxidizer Sinorhizobium sp. M14 can directly mobilize arsenic from such minerals. The dissimilatory arsenate reducers Shewanella sp. O23S and Aeromonas sp. O23A were particularly active in the direct mobilization of arsenic. The significant role of siderophores produced by Pseudomonas spp. isolated from mine rock
biofilm in indirect arsenic mobilization was confirmed. These findings suggest possible mechanisms of arsenic mobilization from arsenate minerals in Zloty Stok gold mine, mediated by indigenous arsenite oxidizers, dissimilatory arsenate reducers and arsenic-resistant bacteria (Fig. 5). Acknowledgements This research was supported by ordered research project No. PBZKBN-111/T09/2004 from the Polish Ministry of Science and Higher Education over the period 2005–2008. Lukasz Drewniak is scholarship holder in the Start Programme of Foundation of Polish Science. We gratefully acknowledge the help of Dr. John Gittins for his critical reading of the manuscript and English correction. References Bultreys, A., Gheysen, I., 2000. Production and comparison of peptide siderophores from strains of distantly related pathovars of Pseudomonas syringae and Pseudomonas viridiflava LMG 2352. Appl. Environ. Microbiol. 66, 325–331. Cervantes, C., Ji, G., Ramirez, J.L., Silver, S., 1994. Resistance to arsenic compounds in microorganisms. FEMS Microbiol. Rev. 15, 355–367. Drewniak, L., Styczek, A., Majder-Lopatka, M., Sklodowska, A., 2008a. Bacteria, hypertolerant to arsenic in the rocks of an ancient gold mine and their potential role in dissemination of arsenic pollution. Environ. Pollut. 156, 1069–1074. Drewniak, L., Matlakowska, R., Sklodowska, A., 2008b. Arsenite and arsenate metabolism of Sinorhizobium sp. M14 living in the extreme environment of the Zloty Stok gold mine. Geomicrobiol. J. 25, 363–370. Drewniak, L., Matlakowska, R., Sklodowska, A., 2009. Microbial impact on arsenic mobilization in Zloty Stok gold mine. Adv. Mater. Res. 71–73, 121–124. Fekete, F.A., Chandhoke, V., Jellison, J., 1989. Iron-binding compounds produced by wood-decaying basidiomycetes. Appl. Environ. Microbiol. 55, 2720–2722. Gihring, T.M., Banfield, J.F., 2001. Arsenite oxidation and arsenate respiration by a new Thermus isolate. FEMS Microbiol. Lett. 204, 335–340. Malasarn, D., Keeffe, J.R., Newman, D.K., 2008. Characterization of the arsenate respiratory reductase from Shewanella sp. strain ANA-3. J. Bacteriol. 190, 135–142. Oremland, R.S., Stoltz, J.F., 2003. The ecology of arsenic. Science 300, 939–944.
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