Article pubs.acs.org/est
Anaerobic, Nitrate-Dependent Oxidation of Pyrite Nanoparticles by Thiobacillus denitrif icans Julian Bosch,† Keun-Young Lee,‡,§ Guntram Jordan,∥ Kyoung-Woong Kim,‡ and Rainer U. Meckenstock*,† †
Institute of Groundwater Ecology, Helmholtz Zentrum München, 85764 Neuherberg, Germany School of Environmental Science and Engineering,, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea § Korea Atomic Energy Research Institute, Daejeon, Republic of Korea ∥ Department für Geo- und Umweltwissenschaften, Ludwig-Maximilians-Universität, Theresienstrasse 41, 80333 München, Germany ‡
S Supporting Information *
ABSTRACT: Pyrite is a key mineral in the global biogeochemical cycles of sulfur and iron, yet its anaerobic microbial oxidation has eluded geochemists and microbiologists for decades. Recent reports indicated that anaerobic oxidation of pyrite is occurring, but the mechanism remains unclear. Here, we provide evidence for the capability of Thiobacillus denitrif icans to anaerobically oxidize a putatively nanosized pyrite particle fraction with nitrate as electron acceptor. Nanosized pyrite was readily oxidized to ferric iron and sulfate with a rate of 10.1 μM h−1. The mass balance of pyrite oxidation and nitrate reduction revealed a closed recovery of the electrons. This substantiates a further “missing lithotrophy” in the global cycles of sulfur and iron and emphasizes the high reactivity of nanominerals in the environment.
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FeS2(s) + 3NO3−(aq) + 2H2O → Fe(OH)3(s) INTRODUCTION
+ 2SO4 2 −(aq) + 1.5N2(g) + H+
Pyrite (FeS2) is the most abundant sulfide mineral in the earth’s crust and constitutes a major reservoir in the global cycles of sulfur and iron.1,2 Yet to date, pyrite cycling in the environment and especially its role as an electron donor for microbial respiration are only partially understood. When molecular oxygen and water are available, iron and sulfur in pyrite can be oxidized by aerobic chemolithoautotrophic microorganisms such as Acidithiobacillus ferrooxidans.3,4 The microbial reactions are exploited by the mining industry to produce precious elements such as copper from inferior ores.5,6 However, pyrite oxidation generates sulfuric acid leading to enormous environmental problems in mining areas due to the formation of acid mine drainage.7,8 Under anoxic conditions, ferric iron is a potent oxidant for pyrite.9−11 Nevertheless, pyrite in anoxic geological confinements like e.g. ore and sedimentary deposits has been considered stable.12 Indeed, many sediments contain pyrite in significant amounts as long as they remain anoxic, and pyritecontaining conglomerates have been used for dating the evolution of redox zones.13 Yet, anaerobic pyrite oxidation should be thermodynamically feasible involving an electron acceptor with a positive redox potential such as nitrate (eq 1):12 © 2011 American Chemical Society
(ΔG0 ′ = − 2439 kJ/mol)
(1)
If the reduction of nitrate is incomplete nitrite accumulates. In this case, the reaction proceeds as shown in eq 2:14
FeS2(s) + 7.5NO3−(aq) + 3.5H2O → Fe (OH)3(s) + 2SO4 2 −(aq) + 7.5NO2−(aq) + 4H+ (ΔG0 ′ = − 1954 kJ/mol)
(2)
There have been several field observations indicating anaerobic pyrite oxidation with nitrate as electron acceptor.15−18 Anaerobic, nitrate-dependent microbial pyrite oxidation would be an enormous, yet not unlimited sedimentary source of denitrification. Such oxidation of pyrite would protect shallow aquifers against the detrimental effects of anthropogenic nitrate influx into groundwater. Denitrifying chemolithotrophic bacteria like Thiobacillus denitrif icans19 are well-known to use ferrous iron20 and FeS12 Received: Revised: Accepted: Published: 2095
June 29, 2011 November 30, 2011 December 5, 2011 December 5, 2011 dx.doi.org/10.1021/es2022329 | Environ. Sci. Technol. 2012, 46, 2095−2101
Environmental Science & Technology
Article
immediately after synthesis for further analysis by e.g. XRD and SEM. Washing in HCl was omitted to preserve the nanoparticulate fraction within our preparation. Oxidation Experiments. Batch experiments were conducted in glass serum bottles sealed with butyl rubber stoppers and flushed with CO2/N2 (20/80%). The bicarbonate-buffered (30 mM, pH 6.8) experimental reaction medium contained 0.85 mM Na 2 HPO 4 , 1.32 mM KH 2 PO 4 , 0.04 mM MgSO4•7H2O, and the precultivation supplements described above but diluted in a 1:10 ratio. Five mM NaNO3 was used as electron acceptor, and nanopyrite was added to the medium as electron donor. Sterile controls were performed by adding 0.22 μm-filtered cell suspensions to exclude abiotic oxidation of the pyrite. Furthermore, individual controls containing only Thiobacillus denitrificans, and no nanopyrite were performed to monitor a potential background metabolic reaction of the bacteria (see the Supporting Information). Experiments and controls were performed in independent triple biological replicates. To proof the viability of the cell suspension, positive control experiments were done with FeS (Sigma) as electron acceptor (see Figure S3, Supporting Information). The main experiment was completely reproduced using similar material (see the Supporting Information). X-ray Diffractometry (XRD). X-ray diffraction patterns were recorded in transmission mode using a STOE STADI-P diffractometer (Stoe, Darmstadt, Germany) equipped with a curved Ge(111) primary monochromator producing monochromatic Mo Kα-radiation (λ = 0.07093 nm). Rietveldrefinement was performed with FullProf. Flow-Cytometry. Cell densities within the oxidation experiments were determined by flow-cytometry using a LSRII (Becton Dickson Bioscience, Franklin Lakes, NJ, USA). Paraformaldehyde-fixed cells from the cell suspensions were stained by SYBR Green I nucleic acid stain (Molecular Probes, Eugene, OR, USA), diluted in 0.22 μm-filtered Dulbecco’s-PBS, and counted at a wavelength of 510 nm in Trucount bead (Becton Dickson) calibrated measurements. Definition of Nanoparticulate Pyrite Fraction. ICP-AES data included total dissolution of the material with aqua regia. By definition, large pyrite crystals are insoluble in weak acids. However, we observed that the nanoparticulate fraction dissolved in 1 M HCl, and we used a standard photometric assay to measure the ferrous iron content of nanoparticulate pyrite via the ferrozine and phenantroline assays29,30 in these incubated samples. Our comparison of the ICP-AES data to the photometric results after incubation in 1 M HCl revealed that about 10% of the material was soluble in 1 M HCl. We termed this fraction the nanoparticulate fraction, as we could rule out any contamination such as e.g. FeS (see Results). Iron Determination. Aliquots of 0.1 mL were anoxically withdrawn from the experiment, diluted 1:10 in 1 M hydrochloric acid (HCl), and shaken at 1.400 rpm for 24 h in Eppendorf tubes to dissolve the nanoparticulate pyrite fraction. Absorbance at 560 nm (ferrozine) and 430 nm (phenantroline) was measured using a Wallac 1420 Viktor3 plate reader (Perkin-Elmer, MA, USA). Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES). ICP-AES was applied to analyze the chemical compositions of the pyrite nanoparticles and to determine total iron concentrations. Pyrite was totally dissolved with aqua regia at 70 °C for 1 h. Total Fe and S concentrations
as electron donors with nitrate as electron acceptor. However, initial attempts to directly show anaerobic microbial pyrite oxidation under defined laboratory conditions have not been successful so far.12,21 Recent studies on pyrite-containing sedimentary material in a flow-through reactor setup22 and in laboratory batch experiments23 tentatively confirmed the positive field observations. Both studies presented evidence on nitrate reduction and sulfate release from pyritic material upon microbial incubation. However, a clear description of the oxidized material is still not available (e.g., XRD data). Contamination of the pyrite FeS2 with FeS, ferrous sulfate minerals, marcasite (a polymorph of FeS2), or ferric iron need to be excluded a priori to present an unbiased reaction. A thorough characterization is even of more importance if the pyrite is achieved from oxic milling procedures, as milling can chemically modify pyrite surfaces.24 A direct proof of the reaction is needed to close a gap in our understanding of the global turnover of iron, sulfur, and nitrogen. Here, we report on experiments to obtain direct evidence for anaerobic, nitrate-dependent oxidation of pure pyrite by Thiobacillus denitrif icans. We applied pyrite nanoparticles to study pyrite reactivity, as recent research emphasized the generally increased reactivity of nanoscale minerals toward microbial processes.25,26 Large effort was taken to exclude the presence of easily oxidizable pyrite contaminating minerals like e.g. FeS or ferrous sulfates, which would lead to unclear results, and to prove pyrite oxidation through closed electron and mass balances.
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MATERIALS AND METHODS Cultivation of Microorganisms. Thiobacillus denitrificans DSMZ 73927 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) Braunschweig, Germany. The strain was cultivated using standard anaerobic techniques at 30 °C in the dark. The bicarbonate-buffered (30 mM, pH 6.8) medium consisted of 8.5 mM Na2HPO4, 13.2 mM KH2PO4, 0.4 mM MgSO4•7H2O, 0.8 mM (NH4)2SO4, 0.2 mM CaCl2•2H2O, 0.1 mM FeCl3•6H2O, and 0.1 mM MnSO4•xH2O. The medium was supplemented with trace elements solution SL10, selenite-tungsten, vitamins,28 and 10 μM of adenosine 3′,5′-cyclic monophosphate as mediator for anaerobic growth. The medium contained 5 mM NaNO3 as electron acceptor and 5 mM Na2S2O3•5H2O as electron donor. Growth was monitored via ion chromatography by measuring the turnover of thiosulfate to sulfate and the depletion of nitrate. After growth to the late exponential phase (3−4 days) and complete consumption of thiosulfate and nitrate, the cell suspension was harvested by centrifugation (20 min at 2000 g at 20 °C), resuspended in experimental reaction medium without electron acceptor, and immediately injected to the oxidation experiments in a 1:10 ratio. Residual sulfate caused an initial sulfate concentration in the reaction medium of ∼0.3 mM. Preparation of Nanopyrite. Natural pyrite crystals (3−4 mm large) acquired from Georg Maisch Import, Freising, Germany, were preground under ambient atmosphere in an agate mortar to a particle size of
