Environ. Sci. Technol. 2005, 39, 770-777
Quantification of Abiotic Reaction Rates in Mine Tailings: Evaluation of Treatment Methods for Eliminating Iron- and Sulfur-Oxidizing Bacteria ROGER B. HERBERT JR.* Department of Earth Sciences, Uppsala University, Villava¨gen 16, S-752 36, Uppsala, Sweden MARIA MALMSTRO ¨ M Department of Chemical Engineering and Technology, Royal Institute of Technology, S-100 44, Stockholm, Sweden G U S T A V E B E N A°
URSULA SALMON‡ Department of Civil and Environmental Engineering, Royal Institute of Technology, S-100 44, Stockholm, Sweden EMBAIE FERROW AND MATTHIAS FUCHS Department of Geology, Lund University, S-223 62, Lund, Sweden
Effective treatment techniques for eliminating ironoxidizing (IOB) and sulfur-oxidizing bacteria (SOB) are required for the comparison of abiotic and microbial sulfide oxidation rates and mechanisms in mine tailings. This study evaluates the effect of autoclaving, repeated heating, ethanol treatment, antibiotic treatment, γ-radiation, and washing with deionized water on tailings characteristics and concentrations of IOB and SOB. Most probable number enumeration indicates that IOB and SOB were present at very low concentrations or below detection limits following treatment with all methods except rinsing and antibiotics treatment, where higher concentrations of IOB and SOB were present. The physical, chemical, and mineralogical characterization of the tailings indicated no changes in bulk mineralogy or bulk chemical composition as a result of treatment. However, an increase in oxidized sulfur species at the tailings surface, as determined by X-ray photoelectron spectroscopy, was observed for the heating, autoclaving, and antibiotics treatments. Batch weathering experiments, used to evaluate the effect of treatment on element release rates, indicated that the final element release rates (after >30 d) were similar between treated and untreated control samples. On the basis of the results of this study, experiments over relatively long periods (>30 d) are to be recommended for the establishment of microbial and abiotic weathering rates in mill tailings samples. For the determination of abiotic reaction rates, * Corresponding author telephone: +46 18 471 2266; fax: +46 18 55 1124; e-mail:
[email protected]. † Present address: Bio-Geo-Interaktionen, Institut fu ¨ r Mikrobiologie, Universita¨t Jena, Jena, Germany. ‡ Present address: Centre for Water Research, University of Western Australia, 6009 Australia. 9
Introduction The oxidation of sulfide minerals is a potential source of environmental contamination wherever mining activities expose sulfides during the extraction of base and precious metals. Pyrite (FeS2) is the most common sulfide mineral present in sulfide ore deposits and is thus the most common source of acid mine drainage from mine workings and mine waste deposits. In the presence of dissolved molecular oxygen, pyrite oxidation can be written as follows:
FeS2(s) + 7/2O2(aq) + H2O(aq) w
Fe2+(aq) + 2SO42-(aq) + 2H+(aq) (1)
†
Department of Biology, IFM, Linko¨ping University, S-581 83, Linko¨ping, Sweden
770
treatment by γ-radiation is suggested to be the most appropriate method for sulfide-rich tailings.
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 3, 2005
This reaction can be mediated by iron-oxidizing (IOB) and sulfur-oxidizing (SOB) bacteria, such as Acidithiobacillus ferrooxidans, which is commonly identified in mine waste deposits and natural environments (e.g., refs 1-4). Laboratory experiments have indicated unequivocally that the presence of A. ferrooxidans and other bacteria can accelerate the rate of Fe2+ and sulfide mineral oxidation by up to several orders of magnitude (2, 4, 5). However, the relative contribution of abiotic and microbial processes to the overall in situ sulfide oxidation rates, under field conditions, is difficult to quantify. Instead, several studies have calculated field oxidation rates based on oxygen profiles (6) and solute discharge rates (7), but these techniques are limited to existing sites that are well-instrumented. To calculate release rates and to propose prevention and control measures (e.g., soil covers, water covers, reactive barriers) during the planning phase of mine development, a thorough understanding of sulfide oxidation and acid neutralization rates for a given mineral assemblage is required. It is therefore vital that laboratory experiments can be designed to address the relative importance of abiotic and microbial processes in mine wastes, with particular focus on sulfide oxidation. A comparison of abiotic and microbial mechanisms thus requires effective treatment techniques to eliminate ironand sulfur-oxidizing bacteria. Ideally, a treatment technique should kill microorganisms in a sample without changing the physical, chemical, mineralogical, and textural properties of the sample. In terms of sulfide oxidation, this implies that the weathering rate for the treated sample should be identical to the abiotic rate for an untreated sample. However, sterilization often requires harsh sample treatment techniques (e.g., heating or treatment with aggressive chemicals) that may alter the characteristics of the sample and may hence change the weathering behavior of the sample. This study determines if treatment to eliminate IOB and SOB results in unwanted alteration of the physical, chemical, or mineralogical characteristics of tailings. The effectiveness and impact of various treatment techniques for eliminating IOB and SOB have been investigated. Finally, the weathering rate of tailings is investigated in batch experiments in order to evaluate the effect of the treatment as well as the effect of compositional changes after treatment on element release rates.
Experimental Methods Samples. Mine tailings samples were obtained from the saturated zone of Impoundment 1 at the Kristineberg site in 10.1021/es0400537 CCC: $30.25
2005 American Chemical Society Published on Web 12/22/2004
TABLE 1. Treatment Methods for Eliminating IOB and SOB Investigated in This Study method
procedure
Phase I Autoclaving 200 g of water-saturated tailings (water content ∼25%) placed in containers and sealed under an argon atmosphere; sealed containers autoclaved at 121 °C, 1 atm over pressure, for 30 min Heating 200 g of water-saturated tailings in sealed containers placed in a 75 °C water bath for 45 min and then incubated at room temperature for 24 h; repeated 3 times Ethanol Treatment samples were treated in a 70% ethanol solution on a rotary shaker for 1 h; to de-activate the inhibitory effects of ethanol, the tailings were washed 3 times with sterile (autoclaved) N2-purged Milli-Q (Barnstead) deionized water; samples were centrifuged at 1000g for 5 min before the supernatant was decanted; this procedure was repeated 3 times Antibiotics
γ-Irradiation Water Rinsing
Phase II tailings were treated using a penicillin-streptomycin-neomycin mixture (5000 units of penicillin, 5 mg of streptomycin, and 10 mg of neomycin/mL of 0.9% NaCl; product number P3664, Sigma-Aldrich, St Louis), which was added at a concentration of 20 mL/L to a 1 L mixture of 400 g wet tailings and deionized water; slurry was incubated on a rotary shaker at 100 rpm; after 2 d of incubation the tailings were heated at 50 °C for 45 min to induce germination of spores; sand was then cooled to room temperature and placed on the rotary shaker for 24 h; a second addition of antibiotics was then performed at the same concentration of 20 mL/l; incubation of 3 d at 100 rpm followed; at the end of the incubation period, excess water was decanted after 1 h of sedimentation two sets of water-saturated tailings samples, in 50-mL sterile plastic test tubes, were treated with different γ-ray doses (10 and 25 kGy) at a dose rate of 0.7 kGy/h samples washed three times with sterile N2-purged deionized water
northern Sweden (8). The field site and the geochemistry and mineralogy of Impoundment 1 have been previously investigated (e.g., refs 9 and 10). The acquired tailings were unoxidized, as oxygen concentrations in the pore water below the water table are extremely low. After recovery of the tailings from the field site, the tailings were stored under water in sealed plastic buckets. Bucket contents were thoroughly mixed prior to removing subsamples for treatment. Treatment Techniques. Treatment methods chosen in this study are shown in Table 1. Washing with water is included as to study the effects of washing per se. The project was carried out in two phases, where Phase I included autoclaving, repeated heating, and ethanol sterilization; Phase II included treatment with antibiotics, γ-irradiation and water rinsing. Control samples associated with the particular tailings used in each phase were analyzed in parallel with the treated samples. Post-treatment Analysis of Tailings. As the main emphasis of this study was on treatment techniques effective in killing IOB and SOB, the content of these groups of bacteria was determined by the most probable number (MPN) technique. The groups of bacteria were investigated by preparing appropriate slurries of tailings and media. The medium used for the IOB contained 0.4 g of (NH4)2SO4, 0.1 g of K2HPO4, 0.4 g of MgSO4‚7H2O, and 33.3 g of FeSO4‚7H2O per liter, adjusted to pH 2.3 with H2SO4 (11). The medium used for SOB contained 3.5 g of K2HPO4, 0.3 g of (NH4)2SO4‚7H2O, 0.018 g of FeSO4‚7H2O, 0.25 g of CaCl2, and 5 g of S per liter, adjusted to pH 4.5 with H2SO4 (12). Both these media have been successfully used for the enumeration of IOB and SOB in studies of sulfide oxidation (11, 12). The MPN setup consisted of five parallel replicates at five different initial tailings concentrations, with the highest and lowest concentrations corresponding to 0.75 g and 0.75 µg tailings (dw) per 5 mL of medium, respectively. To avoid excessive heterogeneity between MPN series, the field samples were well-mixed prior to removing these small sample masses. MPN cultures were allowed to grow for 30 d at room temperature. As growth indicators, the formation of a rustcolored precipitate was used for IOB, and a decline in pH to one unit lower than the sterile control medium was used for the SOB. The number of bacteria in the resulting slurries was then estimated by the MPN technique (13, 14). From the weathering experiments (see below), bacterial content was determined by staining with 4,6-diamidino-2-
phenylidole (DAPI; 15). Samples for staining with DAPI were occasionally withdrawn from the reactors. Crystalline and amorphous mineral phases were characterized using a combination of powder X-ray diffractometry (XRD), optical microscopy, Mo¨ssbauer spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. The surface chemistry of treated and untreated tailings samples was investigated using X-ray photoelectron spectroscopy (XPS). The chemical composition of treated and untreated samples was determined in terms of ascorbate-extractable metals (16) and total chemical composition. Specific surface area was determined by nitrogen gas adsorption evaluated with the BET equation, and the grain-size distribution of treated and untreated tailings was determined by granulometry. Details on analytical methodology are presented in Supporting Information. Batch weathering experiments (further detailed in the Supporting Information) were performed on tailings samples at constant temperature (22 °C) and at pH ∼2.3 (adjusted with HNO3). Reactors were continuously purged with sterile filtered air. During the weathering experiments, aqueous samples were removed on 13-14 occasions over ∼100 d. Samples were immediately filtered, and the Eh and pH were measured. Filtered samples were then acidified and analyzed for aqueous components using ICP-AES. The accumulated amount (n) of element i released to solution up to sampling occasion k was calculated from the measured concentration (Cmeas) using the following equation: k-1
k k nik ) [C i,meas V total +
∑C
s i,meas
s V sample ] (mol)
(2)
s)1
where Vtotalk is the total volume of the aqueous phase in the reactor immediately before removal of the kth sample, and Vsamples is the volume of sample removed on sampling occasion s (cf. ref 17). The cumulative release was normalized to the dry weight of tailings in each reactor. Interpretation of cumulative release data versus time in terms of element release rates through linear regression of data is detailed in Supporting Information; Figure S1 (Supporting Information) shows a typical example of cumulative release of some elements versus time. VOL. 39, NO. 3, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Microbiological and Physical Properties of Treated and Untreated Tailingsa MPNb IOB (no. g-1) method
MPN SOB (no. g-1)
mean
max
min
Control 1 Autoclaving Heating Ethanol Treatment
213 000 7 11 31
613 000 19 29 88
53 000
