Arsenic-Rich Acid Mine Water with Extreme ... - ACS Publications

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Arsenic-Rich Acid Mine Water with Extreme Arsenic Concentration: Mineralogy, Geochemistry, Microbiology, and Environmental Implications § ̌ Juraj Majzlan,*,† Jakub Plásǐ l,‡ Radek Skoda, Johannes Gescher,∥ Felix Kögler,⊥ Anna Rusznyak,⊥ ⊥ # Kirsten Küsel, Thomas R. Neu, Stefan Mangold,∇ and Jörg Rothe∇ †

Institute of Geosciences, Friedrich-Schiller-Universität, Burgweg 11, D-07749 Jena, Germany Institute of Physics ASCR, v.v.i., Na Slovance 2, CZ-182 21 Praha 8, Czech Republic § Department of Geological Sciences, Faculty of Science, Masaryk University, Kotlárš ká 2, CZ-611 37, Brno, Czech Republic ∥ Department of Applied Biology, Karlsruhe Institute of Technology, D-76131 Karlsruhe, Germany ⊥ Institute of Ecology, Friedrich-Schiller-Universität, Dornburger Strasse 159, D-07743 Jena, Germany # Helmholtz Centre for Environmental ResearchUFZ, D-39114 Magdeburg, Germany ∇ ANKA, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany ‡

S Supporting Information *

ABSTRACT: Extremely arsenic-rich acid mine waters have developed by weathering of native arsenic in a sulfide-poor environment on the 10th level of the Svornost mine in Jáchymov (Czech Republic). Arsenic rapidly oxidizes to arsenolite (As2O3), and there are droplets of liquid on the arsenolite crust with high As concentration (80,000−130,000 mg·L−1), pH close to 0, and density of 1.65 g·cm−1. According to the X-ray absorption spectroscopy on the frozen droplets, most of the arsenic is As(III) and iron is fully oxidized to Fe(III). The EXAFS spectra on the As K edge can be interpreted in terms of arsenic polymerization in the aqueous solution. The secondary mineral that precipitates in the droplets is kaatialaite [Fe3+(H2AsO4)3·5H2O]. Other unusual minerals associated with the arsenic lens are běhounekite [U4+(SO4)2·4H2O], štěpite [U4+(AsO3OH)2·4H2O], vysokýite [U4+[AsO2(OH)2]4·4H2O], and an unnamed phase (H3O)+2(UO2)2(AsO4)2·nH2O. The extremely low cell densities and low microbial biomass have led to insufficient amounts of DNA for downstream polymerase chain reaction amplification and clone library construction. We were able to isolate microorganisms on oligotrophic media with pH ∼ 1.5 supplemented with up to 30 mM As(III). These microorganisms were adapted to highly oligotrophic conditions which disabled long-term culturing under laboratory conditions. The extreme conditions make this environment unfavorable for intensive microbial colonization, but our first results show that certain microorganisms can adapt even to these harsh conditions.

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lakes associated with active volcanism.6,7 The principal aqueous components in the acidic hydrothermal fluids are HCl, H2SO4, and rarely also HF. Low pH values may be attained in the pore waters in tailings or in discharges from tailings and mines. The lowest measured pH was −3.6 from the abandoned mines at the Iron Mountain Superfund site.5 The acid mine drainage (AMD) waters precipitate a rich assemblage of iron sulfate and iron oxides distinctive for this environment. The principal aqueous components in these fluids are H2SO4 and FeSO4. Extreme acidic environments harbor microorganisms with a diverse position in the phylogenetic tree and variable metabolic potential.8,9 The microbial ecology of environments with low pH and heavy metal concentrations, such as natural acidic

INTRODUCTION Arsenic is a widely studied element because it contaminates numerous aquifers worldwide and has caused poisoning of communities using drinking water from these aquifers.1 Mining activities were identified as the source of some of the highest concentrations and fluxes of arsenic to the environment in either solid or dissolved form.2−4 Acid mine waters typically contain high iron concentrations and when oxidized, form ochreous precipitates of goethite [α-FeOOH], schwertmannite [ ∼ Fe8O8(OH)6(SO)4], jarosite [KFe3(SO4)2(OH)6], or other hydrous ferric oxides which can contain up to 20−30 wt % As, either as part of the mineral structure or sorbed. In high-As environments scorodite (FeAsO4·2H2O) or hydrous ferric arsenates (HFA with variable composition) can form. The current study is unusual because it concerns an extremely arsenic-rich environment, depleted in iron and sulfur. Extremely acidic aqueous fluids are known principally from two environments, namely, acid mine waters5 and acid crater © XXXX American Chemical Society

Received: May 21, 2014 Revised: October 24, 2014 Accepted: November 3, 2014

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dx.doi.org/10.1021/es5024916 | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

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rivers10 or acidic geothermal springs,11 have been investigated in detail using cultivation-based and molecular biological methods. AMD microbiology studies also focused on arsenic removal and the isolation of arsenite-oxidizing bacteria.12,13 The objective of this work is a mineralogical, geochemical, and microbiological characterization of a system of extremely arsenic-rich acid mine water (As-AMW) in the underground spaces of the Jáchymov ore deposit (Czech Republic). The strongly acidic aqueous phase has extreme arsenic concentrations, and this system represents an end-member of arsenic pollution, with high As concentrations and unusually low content of Fe and S, when compared to that of As. We identified the chemical properties of the solid and the solution phases which characterize these systems. The extreme conditions offered a unique opportunity to address the question if microorganisms can be detected and whether they are able to survive in such an environment.

measurements, the samples were diluted as needed and stored in corrosion-resistant sample tubes. Density of the liquid was measured by weighing small amounts with a known volume on a microbalance. Because of the limited amount available and the corrosive nature, no other density measurements were attempted. The accuracy of the measurements was estimated by following a similar procedure with distilled water. X-ray absorption spectroscopy (XAS) experiments were carried out on the frozen (originally liquid) samples. The samples, as mentioned previously, were frozen inside the mine and driven to the synchrotron while immersed in liquid nitrogen. They were quickly transferred (within 1 min) to the end-stations of the XAS and INE beamlines at the Angströmquelle Karlsruhe (ANKA) synchrotron. The endstation of the XAS beamline was cooled with a closed cycle cryostat to ∼15 K. The end-station of the INE beamline was cooled with liquid nitrogen and maintained at ∼77 K. The XAS data were collected in transmission and fluorescence mode at the Fe K (XAS beamline) and As K and U L3 (INE beamline) edges. The spectra were inspected for beam damage, and if found unaffected, they were averaged and further processed by the software suite Athena and Artemis.14 The rock samples were fixed underground in 4% formaldehyde in 1 × phosphate-buffered saline (PBS) and later investigated by confocal laser scanning microscopy (CLSM). Samples were analyzed by CLSM using a TCS SP5X, controlled by the software LAS AF 2.4.1 (Leica, Heidelberg, Germany), equipped with an upright microscope and a supercontinuum light source. The rock samples were placed in a 5 cm diameter Petri dish, stained with the nucleic acid-specific fluorochrome SybrGreen (Molecular Probes Inc., Eugene, OR, USA) and covered with water. Images were collected with water immersible lenses (20× NA 0.5, 63× NA 0.9). The samples were examined by selecting the excitation line at 494 nm (reflection and SybrGreen). Reflection signals were recorded at 489−499 nm (mineral surface); fluorescence emission was recorded at 510−550 nm (nucleic acids). Samples were examined at 0.5 and 1.0 μm step size in the axial direction. Images were visualized by using the microscope software and Imaris, version 7.4 (Bitplane, Zürich, Switzerland). Five different rock samples were collected with an autoclaved pick and spatula which were wiped and ethanol-sterilized after each sample taking. Each of the five different samples was stored in sterile 1.5 mL Eppendorf tubes at 4 °C for DNA extraction. The DNA extraction was carried out according to eight different protocols, as outlined in detail in the Supporting Information, followed by polymerase chain reaction (PCR) amplification of the 16S rRNA gene (Supporting Information). For sequencing, purified PCR products were sent to the European branch of the Macrogen Corp. in Amsterdam, The Netherlands for DNA sequencing. Different liquid media were used for cultivation and enrichment of microorganisms present on the rock samples. Enrichment cultures were set up by adding rock samples of approximately 3 g to autoclaved 100 mL conical flasks containing 50 mL of enrichment medium sealed with cotton and tin foil on a shaker (150 rpm) at room temperature in the dark. Noninoculated media and media inoculated with autoclaved sample material served as controls. Four different types of liquid media adjusted to pH 1.5 with HCl were used (Supporting Information Table S3): R2A (see ref 15; from Carl Roth, Karlsruhe, Germany) medium to target heterotrophic microorganisms possibly in a dormant state;

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EXPERIMENTAL SECTION Samples were collected in several campaigns at the 10th level (about 450 m below the surface) of the Svornost (Einigkeit in German) mine, located in the mining town of Jáchymov in the western part of the Czech Republic. The samples were retrieved from a small side chamber which is physically separated from the rest of the mine, therefore accumulating significant concentrations of radon over time. Rock samples were collected directly from the walls of the chamber or from the debris at its floor. Samples of aqueous fluids were collected with a syringe; because of the small volume of the droplets, one sample comprised at least 10 such droplets. The aqueous samples were filtered through 0.2 μm hydrophilic polypropylene filters. Aqueous samples for chemical analyses were stored in plastic vials and brought in the liquid state to the laboratory. For the X-ray absorption experiments, the filtered samples were packed into small Kapton bags, sealed, and immersed into liquid nitrogen inside the mine. For the microbiological tests, five different rock samples with particle sizes ranging from small grains up to about 2 cm rock pieces were collected with sterile spatulas. Samples were stored and transferred into the laboratory at 4 °C in sterile Falcon tubes for cultivation purposes and in sterile 1.5 mL Eppendorf tubes for later DNA extraction. The five samples were labeled and characterized as follows: filamentous mineral phases growing vertically from ceiling plus surrounding rock material (JF), lens of native arsenic (JL), rock surrounding arsenic lens (JLM), oxidized layer of arsenic lens (JLox), and miscellaneous mineral phases taken near JF (JVM). Oxygen concentration in the As-AMW droplets was determined directly in the mine. The profiles were measured using a microelectrode attached to a microprofiling setup from Pyro Science (Aachen, Germany). The pH of the droplets was measured in the laboratory on the composite samples with a Mettler Toledo SG/2m electrode. The electrode was calibrated with a series of standardized H2SO4 solutions with variable molality according to the procedure outlined in ref 5. Minerals were identified with powder X-ray diffractometer Bruker D8 Advance, using Cu Kα radiation. Powdered samples were scanned from 5 to 60 °C 2θ, with a step of 0.01 °C 2θ and dwell time of 1 s. Elemental composition of the aqueous samples was determined by inductively coupled plasma (ICP) optical emission spectrometry (ICP-OES) with a Variant 725 ICP-OES with a charge-coupled device (CCD) detector or an ICP−-mass spectrometry (ICP-MS) instrument. For the B



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Table 1. Chemical Composition of the Liquid Samples from Jáchymova composition of major elements, mg·L−1 As

Ca

Cu

J-1 J-2 J-3

108179 136452 86306

113.0 69.7 71.9

37.3 32.9 10.9

U

La

J-1 J-2 J-3

17467 2834 10317

not measured 7.0 6.3

J-2 J-3

Mo

Na

P

S

51.6 20.1 1048 8.2 24.5 23.4 762 12.5 19.0 composition of minor and trace elements, μg·L−1

Mg

Fe

K

75.6 63.7

2.2 24.2

9625 16333

Sm

Eu

Tb

21 34.6

4.9 7.1

9.0 14.2

Ce

Pr

Nd

not measured not measured 6.9 1.7 14.9 17.3 4.2 30.2 composition of minor and trace elements, μg·L−1

Gd

Dy

Ho

Er

Tm

Yb

Lu

44.2 68.0

55.1 85.0

8.7 13.6

21.32 34.9

2.61 4.24

15.3 24.86

2.03 3.07

a

The droplets were saturated with O2 to 96−98%, according to the in situ measurements in the mine. The pH was measured for sample J-2 where sufficient amount of liquid was available and found to be pH 0.15. The density of this sample was measured as 1.65 ± 0.02 g·cm−3 at room temperature.

Figure 1. X-ray absorption near-edge structure (XANES) spectra of the frozen aqueous solution and standards from Jáchymov, measured at the Fe K (left) and As K (right) absorption edges. The spectra from the sample are shown by thick dashed lines.

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mineral medium16 with low organic carbon content (0.6 g·L−1 Na3-citrate and 0.1 g·L−1 yeast extract) for microorganisms adapted to a limited carbon supply; two media based on the mineral medium described earlier with an additional source of As(III), ferrous iron, and S(IV) [FeSO4 (15 mM) and Na2S2O5 (0.16 mM, with respect to S(IV); corresponds to 10 mg·L−1 10 mM S(IV) and 30 mM As2O3, respectively)] for the enrichment of Fe-, As-, or S-oxidizing microorganisms. Media were filter-sterilized before inoculation. Each of the five different samples was added to the following enrichment medium: R2A medium, pH 1.5; mineral medium, pH 1.5; mineral medium with Fe(II), S(IV), and 10 mM As(III), pH 1.5; mineral medium with Fe(II), S(IV), and 30 mM As(III), pH 1.5. Enrichment cultures were incubated on a shaker at room temperature and ventilated manually under a clean bench every 6 days. All enrichments were plated after 16 days in order to obtain colonies. For plating, 300 μL of the enrichment culture were transferred onto an agar plate containing the same medium as the original enrichment in a solid form. For isolation, plates were prepared with the same media solidified with agar (20 g· L−1) and 300 μL of enrichment culture was plated and incubated at room temperature in the dark. Noninoculated media and media inoculated with autoclaved sample material served as controls. After a further 35 days of incubation at room temperature, colonies were picked and plated once more onto the solid medium (same composition as enrichment medium) to obtain single colonies. The procedure for a low-pH solid medium is based on17 and is described in the Supporting Information.

RESULTS AND DISCUSSION Primary Mineralogy and Geochemistry. The As-AMW is spatially and genetically linked to an arsenic lens of ∼5 m length and ∼2 m width. The height of the lens could be roughly judged at 2−3 m, of which large portions have been already mined out. A number of smaller veins emanate from the lens and follow the general strike and dip of the lens and the associated fractures in the country rocks. The material of the lens and the veins consists mostly of native arsenic with locally abundant proustite (Ag3AsS3) and traces of pyrite (FeS2), argentopyrite (AgFe2S3), and arsenopyrite (FeAsS). The primary chemical composition of the studied system is therefore very simple, being strongly dominated by As, with minor Fe, Ag, and S. The lens has been exposed for more than 40 years and remained in contact with moist air underground. The temperature of 18.4 °C and 100% relative humidity are constant in the chamber. The radon concentration in the air was measured as 42,300 Bq·m−3, a value that exceeds many times the limit allowed for human health. Composition and Properties of the Aqueous Phase in AsAMW. Water vapor from the air condenses on the arsenic lens and the country rocks. Numerous droplets hang from the ceiling and harvest the elements present at their specific site on the rocks. Under the high humidity in the underground, they do not evaporate but either drip down and interact with the rock fragments on the mine floor or slowly creep downward along the walls. From visual observation, the droplets have viscosity similar to water; i.e., they do not behave as a viscous liquid, similar to, for example, concentrated sulfuric acid. C



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The droplets have an unusual composition (Table 1). The dominant solute is arsenic, with up to 136 g of As/(L of solution), a rather high concentration. The As K edge XAS spectra collected at ∼77 K show progressive beam-induced damage of the samples, with the edge position shifting to lower energies (Supporting Information Figure S1). When the spectra collected as first at each measured spot are evaluated, they hint that arsenic is present in both trivalent and pentavalent state (Figure 1); linear combination fitting gave 86.7% As(III) and 14.5% As(V) (sum 101.2%; neither sum nor individual percentages were constrained during fitting). The first feature in the Fourier transform extended X-ray absorption fine structure (FT EXAFS) spectra of the individual scans can be fit with 3.3(2) oxygen atoms at a mean As−O distance of 1.69(1) Å (Supporting Information Figure S2 and Table S1). The mean As−O distance, however, is closer to the average As−O distances in crystalline arsenates of 1.685 Å than that in crystalline arsenites of 1.782 Å.18 A weak second-shell feature (Supporting Information Figure S2 and Table S1) can be fit by a combination of As−As single scattering paths and multiple (As−O−O−As) scattering paths. The presence and persistence of As(III) in the droplets is interesting because, as shown by our in situ measurements, the droplets are saturated with oxygen. The microelectrode was driven into the droplets using a micromanipulator in order to check for heterogeneity within the droplets. None was found; the oxygen saturation near the surface of the droplets and inside them is the same. Therefore, the entire volume of the droplets is saturated with oxygen but As(III) prevails over As(V). Using the measured density of 1.65 ± 0.02 g·cm−3, the total As molality in the droplets is 1.22 m (calculated for sample J-2; see Table 1). Applying the results of the linear combination of the XAS spectra, the molalities are 1.05 m As(III) and 0.18 m As(V). The molalities of As(III) and As(V), calculated previously, may be challenged based on the available thermodynamic data for As2O3 (arsenolite) and As2O5. The equilibrium As(III) molality of a solution saturated with respect to arsenolite is 0.207 m,19 much lower than the calculated As(III) molality for the droplets. Solubility of As2O5 is very high and not precisely known, reported as ∼260 g of As·L−1.20 Hence, either the XAS spectra yield erroneous information because of beam-induced damage already in the first measurement at each spot or the droplets are, for unknown reasons, grossly supersaturated with respect to As(III). The droplets are extremely acidic, with pH of 0.15 measured in one sample where the amount of fluid was sufficient. The droplets are essentially a mixture of arsenious and arsenic acid, with a minor component of sulfuric acid. The measured pH value may contain a systematic error because the electrode was calibrated with standardized H2SO4 solutions. A similar calibration with H3AsO4 solutions is not possible because the thermodynamic speciation models for such solutions are not available. We have tested the response of the electrode in concentrated H2SO4 solutions upon addition of As2O5/As2O3 and found that the addition of the As oxides caused negligible drift in the millivolt reading (≤1 mV) of the electrode. A comparison of the arsenic concentration in the droplets with literature data shows that we were studying the most arsenic-rich aqueous fluid reported to date from a field setting (Figure 2). This figure compares water at a number of sites polluted by arsenic, either naturally or by human action. The natural pollution, aggravated by water extraction from

Figure 2. Overview of arsenic concentrations in waters polluted by geogenic or anthropogenic sources. Data from this work shown by diamonds at the top of the diagram, and other data,21−35 as box-andwhiskers diagrams where the boxes represent the 25% and 75% percentiles; the short line inside the boxes is the median. The whiskers show the minima and maxima. In a few cases, when no data were given in the original source (only statistical values listed), extrema are shown by the whiskers and an average by a cross. The vertical solid line represents the concentration of 10 ppb As, the maximum permissible As concentration in drinking water (recommended by the World Health Organization). The numbers in the square brackets correspond to the entries in the reference list. The locations are specified in the Supporting Information. [*]: Thuringian basin, Germany (M. Lonschinski, personal communication).

moderately shallow or deep wells (e.g., in Bangladesh), reaches maxima between 1 and 10 mg·L−1, with median values around 100 μg·L−1. Higher values were observed in systems polluted by mining waste where the maxima can be invariably tracked to pore waters in the tailings or other waste forms. Here, the extremes reach hundreds or thousands of milligrams per liter.36,37 The droplets from Jáchymov are still more arsenicrich, with As concentrations of 80,000−136,000 mg·L−1. The Fe XAS spectra measured at 15 K show no beaminduced damage and document the trivalent oxidation state of iron in the droplets (Figure 1 and Supporting Information Figure S3). A shell-by-shell analysis of the EXAFS spectra is reported in Supporting Information Table S2. The results also agree with clear predominance of Fe(III), as witnessed by the Fe−O distance of 2.01(1) Å, and they also reveal complexation of Fe(III) with As. The distinction of As(III) or As(V) is in this case impossible but it could be assumed, based on simple bondvalence estimates, that Fe(III) associates preferentially with As(V). Mineralogical and Geochemical Evolution of the AsAMW. The oxidative dissolution of the crystalline arsenic begins with oxidation of native arsenic to As2O3. This oxidation reaction is thermodynamically driven, and the arsenic lens is D



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Figure 3. Scanning electron microphotographs of (a) octahedral crystals of arsenolite and acicular crystals of kaatialaite, (b) crystals of běhounekite, and (c) crystals of štěpite.

mineral rhomboclase [(H5O2)Fe(SO4)2·2H2O] from the ironsulfate AMD systems dominated by sulfuric acid. Both minerals are ferric salts which crystallize from extremely acidic solutions and are able to store protons (i.e., acidity) in their crystal structure. Precipitation of one or both of these minerals from concentrated Fe(III)−As(V)−S(VI) solutions was confirmed experimentally.44 An intervening minor phase in some of their syntheses was sarmientite [Fe2(AsO4)(SO4)(OH)·5H2O], not identified at the site studied here. It is remarkable that, despite an ample supply of As(III) to the droplets, tooeleite (an Fe(III)−As(III) phase) does not precipitate. We have no means of deciding whether the precipitation of tooeleite is hampered kinetically or disfavored thermodynamically. The second most abundant aqueous component is S. The source of S is probably rare sulfides in the lens, proustite being, locally, the most abundant one. The S concentration in the droplets is 15−40 times higher (on a molar basis) than the Fe concentrations. Proustite contains no Fe and supplies the droplets only with S and As. Fe from weathering of sparse pyrite and arsenopyrite is drawn out of the fluid by the precipitation of kaatialaite, leaving sulfur, which has no major sink, behind. Apart from rare native sulfur and unusual sulfates (see below) which certainly do not constitute a large sink for sulfur, the distal parts of the system contain melanterite (FeSO4·7H2O) and gypsum (CaSO4·2H2O). Gypsum is ubiquitous on the walls beside the arsenic lens but never grows directly on arsenic or arsenolite. Melanterite occurs as massive aggregates and crusts in the debris on the floor under the lens where it forms by weathering of pyrite. These pyrite aggregates are spatially separated from the arsenic lens; some of them produce massive melanterite, some of them massive scorodite, probably by interaction of iron-sulfate-rich solutions with the dripping As-AMW. Native sulfur is rare and was usually found in the small cavities in arsenic as pseudomorphs after argentopyrite. The behavior of uranium at the studied site is most peculiar. There is no evidence of primary uranium minerals within the arsenic lens, either by mineralogical studies or by inspection with a Geiger counter. The Jáchymov deposit was, however, exploited for uranium between 1945 and 1964, and traces of uraninite remain certainly in the mineralized zones above the arsenic lens. The fluids circulating through the rocks thus could accumulate a minor load of uranium and precipitate secondary minerals locally. At the studied site, but unlike most of the occurrence reported worldwide, the secondary uranium minerals contain mostly U4+. These are the sulfate mineral běhounekite [U4+(SO4 )2 ·4H2 O]45 and arsenates štěp ite [U4+(AsO3OH)2·4H2O]46 and vysokýite [U4+[AsO2(OH)2]4· 4H2O]47 (Figure 3b,c). The two arsenates contain doubly protonated arsenate tetrahedra and are able to store acidity.

covered by a crust of glittering, colorless, octahedral crystals of arsenolite (As2O3) (Figure 3a) and the less common dimorph claudetite. The evolution of As-AMW is afterward controlled by the sluggish reactions of As2O3 dissolution and arsenic oxidation. Laboratory experiments with arsenolite dissolution reported that the system was after 1 month “far from equilibrium”.38 From their data, it seems that the system may reach equilibrium after several months or perhaps even after a year. Oxidation of As(III) to As(V) in an acidic aqueous phase is also slow, much slower than at circumneutral pH.39 Hence, the prevalence of As(III) over As(V) is not surprising even though the droplets are saturated with oxygen. Interestingly, the dissolution of arsenolite can be self-sustained or selfcatalyzed to a certain extent, as reported by ref 40 and assumed by ref 38. If so, this would be an important analogy between the As-AMW and iron-sulfate AMD. The As-AMW would then be catalyzed by enhanced dissolution of As2O3 in the presence of arsenite oligomers in the liquid phase. The iron-sulfate AMD is also self-catalyzed, in this case by redox cycling of iron between the aqueous phase and dissolving pyrite or pyrrhotite. The As(V) generated within droplets is to a certain extent removed by the precipitation with the most abundant metal in the droplets, that is, iron. Hence, kaatialaite [Fe3+(H2AsO4)3· 5H2O] is the most abundant arsenate at the studied site. We observed all stages of kaatialaite precipitation, from initial bundles of acicular crystals inside the droplets to crusts which cover the arsenic lens or country rocks (Supporting Information Figure S4). The aqueous Fe−As complexes, as determined by the analysis of EXAFS spectra with the assumption that As in the complexes is pentavalent, consist of a central Fe(III) octahedron and two arsenate tetrahedra with mononuclear monodentate geometry (Supporting Information Figure S3 and Table S2). Such clusters can be seen as a building unit of the crystal structure of kaatialaite, and if they truly exist, they may easily condense into the structure of this mineral. Kaatialaite is a rare mineral, reported only from a few occurrences. The first description of kaatialaite mixed with arsenolite came from a weathering product of löllingite (FeAs2) in Kaatiala pegmatite in Finland.41 Subsequent studies described the mineral as an oxidation product of As−Ag ore paragenesis in Nieder-Beerbach (Germany)42 and a weathering product of löllingite-arsenopyrite (FeAsS) concentrate near Přebuz (Czech Republic).43 Experimental study44 confirmed that kaatialaite precipitated from concentrated Fe(III) and As(V) solutions. The sparse literature on kaatialaite and our observations in Jáchymov imply that this mineral indicates the conditions of the extreme As-AMW, given that iron is present at least in trace amounts. Kaatialaite therefore fulfills the function of the E



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Figure 4. (a, b) Confocal laser scanning microscopy (CLSM) of mineral samples taken in the Jáchymov mine. Maximum intensity projection, showing the distribution of (a) bacteria on the surface (rock sample, JL) after staining with SybrGreen for nucleic acids and (b) hyphae-like structures (rock sample, JVM). Color allocation: green = nucleic acids; white = reflection signal. (c) Light microscopy of the isolated FK1. The arrows show two different cell morphologies, small cocci and filamentous structures.

Table 2. Agar Plates with Microbial Growth after 35 days of Incubation.a

a

sample

medium

description of grown colonies

JVM* JVM JVM* JVM JL JF*

MMAs30 MMAs10 MM R2A R2A MM

two pale yellow colonies, no hyphae one yellow colony, no hyphae eight pale green, hyphae-producing colonies with a white corona (diameter up to 1.5 cm) white, hyphae-producing colonies (smaller in comparison to colonies in mineral medium (MM), diameter up to 1 cm) one pale green, hyphae-producing colony with big (diameter approximately 3 cm) white corona up to 10 small white, hyphae-producing colonies

Asterisks (*) mark samples where colonies were plated once more for further culturing and possible DNA sequencing.

Another very rare phase able to store acidity, an arsenate of hexavalent uranium [(H3O)+2(UO2)2(AsO4)2·nH2O], was found on a few samples. Our attempts to measure the oxidation state of U directly in the droplets by XAS in fluorescence mode failed because the signal was too weak. Ca and Mg are stored in the abundant gypsum (see above) and minor picropharmacolite, Ca4Mg(AsO4)2[AsO3(OH)]2· 12H2O. No other arsenates of Ca or Mg were identified. Detection of Life on Rocks of As-AMW and Adjacent Areas. Extremophilic microorganisms are known to colonize a wide range of different habitats,48 e.g., extreme acidic biofilms, so-called “snottites” reported to produce water droplets with pH between 0 and 1 leading to the dissolution of carbonate host rock and precipitation of gypsum.49 Although arsenic is generally toxic to life, a range of microorganisms can use arsenite as an electron donor or arsenate as an electron acceptor,50 whereas others have adapted to high arsenic concentrations and possess arsenic detoxification mechanisms. To determine whether microorganisms are able to survive under the harsh conditions of As-AMW, we applied CLSM as a noninvasive method to directly investigate the rock surfaces of selected JL, JF, and JVM samples. Nucleic acid-stained samples revealed low cell densities on all samples, without continuous coverage by microbial cells or extended biofilm formation. Nonetheless, the JL sample often shows small microcolonies of cells on the rock surface (Figure 4a), whereas scattered cells on JVM and JF samples appeared to be more randomly distributed, and we detected up to 50 cells·cm−2 on the studied JL and JVM rock surfaces (Figure 4a,b). In comparison, biofilms made of extracellular polymeric substance infused with spirillum-shaped cells and cocci were reported from extremely acidic (pH between 0.77 and 1.31) slime samples developed on a slump of finely disseminated pyrite ore in Iron Mountain, CA, USA.51

In addition to the bacterial cells, numerous hyphae-like structures were found on the surface of the sampled rocks, especially on JVM samples. These hyphae-like structures probably derived from fungi appeared to cover preferentially overhanging parts of the rock (Figure 4b). Long filamentous structures have been also reported in the acidic river Rio Tinto in Spain that has a pH of up to 2.19.52 Fungal members of Sporidiobolales belonging to the Basidiomycota and of the Dothideomycetes have also been detected in Richmond mine biofilms.53 The latter are common to sites with low pH and high metal concentrations54 suggesting a selective advantage for survival in extremely acidic environments, such as the presence of metal resistance genes. Fungal hyphae can contribute to the establishment and stabilization of biofilms on sediments and provide surfaces for the attachment of prokaryotes.8 The low microbial biomass detected on the rock samples may have led to insufficient amounts of DNA for downstream PCR amplification. Different attempts to extract DNA using eight different protocols failed. Thus, phylogenetic identification of the different microorganisms using molecular tools such as clone libraries construction or pyrosequencing were not possible. Extraction of whole microbial community DNA from extreme environments is challenging due to the low nucleic acid concentrations, the adsorption of cells to rock particles, and the frequent coextraction of enzymatic inhibitors such as heavy metals.55 Several studies investigated microbial communities of extremely acidic environments using nucleic acid- and/or cultivation-based methods. Those environments, however, are characterized by higher cell densities.56 The comparable low densities of microbial cells detected in this study may be explained by the special characteristics of the former Jáchymov mining site: extremely high As concentrations in solid and liquid phases, high Rn concentration in the air, the depths of the mine, and the lack of light. F



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and Fe (Figure S3, Table S3) EXAFS spectra, macrophotographs of kaatialaite precipitates from the studied site (Figure S4), geographic locations of the aqueous samples whose As content is shown in Figure 2 (Figure S5), macrophotographs of colonies after 35 days of plating (pH ≈ 2; Figure S6), and detailed description of DNA extraction protocols and of PCR amplification of the 16S rRNA gene. This material is available free of charge via the Internet at http://pubs.acs.org.

We performed cultivation experiments to confirm that microorganisms observed by CLSM on the rock surfaces could tolerate the extreme conditions, especially low pH, low organic carbon availability, and high As(III) concentrations. When pieces of rocks were added to the different media with a pH of 1.5, clouding of all media except the one containing rock samples from JL occurred, which inhibited microbial growth measurements that are based on increased turbidity. Therefore, all enrichments were plated after 16 days in order to verify growth. A small number of colonies with different morphologies were observed from all rock samples after 35 days of incubation at room temperature in the dark (Table 2 and Supporting Information Figure S5). Pigmentation of different colonies suggested their light tolerance. Only microorganisms derived from JVM samples could grow on all media. Some pale yellow no-hyphae-forming colonies appeared even on oligotrophic media supplemented with 10 or 30 mM As(III). Based on media composition, we cannot differentiate if the microbes really used arsenite as electron donor or if they grow as chemoheterotrophes, just tolerating the presence of high concentrations of arsenic species. Research conducted at an arsenic-contaminated site in New Zealand showed that no chemoautotrophs could be isolated that could utilize As(III) as an electron donor but arsenic-resistant heterotrophs were able to tolerate up to 20 mM arsenite.57 Most colonies were observed on media designed for slow-growing heterotrophic bacteria (Supporting Information Table S1). A colony obtained from the JVM sample could be transferred several times. When DNA was extracted from a single colony, sequencing results of the 16SrRNA gene showed that FK1 was not a pure culture but apparently a coculture of two different microorganisms, which was supported by microscopic investigations. Two different cell morphologies with small rods and hyphae-like structures could be observed (Figure 4c). However, since all colonies could not be maintained long-term under laboratory conditions, further phylogenetic identification was excluded. Environmental Implications. The mineralogy associated with the extremely arsenic-rich acid mine water (As-AMW) includes arsenolite, claudetite, and kaatialaite. These minerals may be used to identify this type of pollution even if the pollution source has been exhausted or removed. In addition, such extreme environment causes deviation of the oxidation state and speciation of some elements from the commonly encountered state, for example, uranium. Our study suggests that microbes present in low densities in the As-AMW habitat were adapted to very specific environmental conditions which could not be simulated in the laboratory. They might be growing extremely slowly on available organic and inorganic nutrients in situ, making them microscopically viable but their cultivation difficult. Studies with positive cultivation results had less extreme cultivation conditions, such as lower arsenic concentrations57 or higher pH.12 The arsenite-oxidizing Hydrogenobaculum strain is sensitive to both arsenite and arsenate and was isolated from a geothermal spring with a pH of 3.1 where As(III) concentrations are less than 50 μM.43

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +49-3641-948700; fax: +49-3641-948602; e mail: Juraj. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank three anonymous reviewers for their comments and suggestions. We are most grateful to mineral collectors who captured our attention by reporting that the droplets itch when in contact with skin; our special thanks go to Jan Hloušek (Jáchymov) and František Veselovský (Praha). We thank D. Merten for the ICP analyses and S. Richter for the density measurements of the dangerous and toxic fluids. We are grateful to the Karlsruhe Institute of Technology for the beam time at the Angströmquelle Karlsruhe (ANKA) at the XAS and INE beamlines. F.K., A.R. and K.K. were financially supported by the research project AquaDiv@Jena funded by the ProExcellence Initiative of the federal state of Thuringia, Germany. This research was funded by the DFG Research Training Group GRK 1257/1 within the Jena School of Microbial Communications (JSMC) and a postdoctoral grant of the GAČ R No. 13-31276P to J.P.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Brief history and description of the mines in Jáchymov, documentation of the beam-induced damage in the As XAS spectra (Figure S1), summary of media used for enrichments (Table S1), results of fitting of the As (Figure S2, Table S2) G



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