The enhanced oxidation of sulfide minerals principally pyrite (FeS₂), by mining activities is a worldwide problem of significant environmental interest because it leads to the generation of acidic, metal-rich waters. The Richmond Mine, at Iron Mountain in northern California, USA, represents a rare opportunity to study the processes of acid mine drainage underground within an actively oxidizing pyritic body. Where many AMD sites are characterized by precipitation of iron oxyhydroxides, this site is characterized by the dissolved chemical species resulting from microbial pyrite oxidation. Specifically, this field site permits investigation of chemical and microbial factors important in the oxidation reactions that form acidic mine drainage isolated from the part of the system where precipitation of secondary oxyhydroxides occurs. An access tunnel intersects four mine tunnels within the Richmond ore deposit at a junction referred to as the "5-way" (Fig. 1). Essentially all solutions draining from the mine are collected at the 5-way, making it possible to determine and monitor the flux of metals and sulfur from the system. Previous studies of the geology, water chemistry, and microbial communities in the vicinity of the 5-way 12345 provide the basis for ongoing work at the site.

Iron Mountain is located approximately 9 miles northwest of the city of Redding, California (Fig. 1). Access to the Richmond Mine is provided by a 430 m long horizontal access tunnel that is maintained as part of the United States Government Superfund program remediation effort. The area of the 5-way is located at the edge of the main body of the Richmond deposit, a large, lenticular body that was originally over 800 m long, 60 m wide, and 60 m thick. The body contains approximately 90–95% pyrite (FeS₂), locally enriched with ore minerals. The mine is within a Kuroko-type volcanogenic massive sulfide deposit that contains chalcopyrite (CuFeS₂), sphalerite (ZnS), galena (PbS), bornite (Cu₅FeS₄), arsenopyrite (FeAsS), and tetrathedrite–tennantite (Cu₁₂Sb₄S₁₃–Cu₁₂As₄S₁₃), as well as massive pyrite and late-stage veins of fine-grained pyrite.6 The ore is hosted in Devonian-age Balaklala Rhyolite composed of quartz, albite, chlorite, and sericite,7 characterized by very limited acid-buffering capacity. Assays of the pyritic material at the Richmond Mine Complex average 1% copper and 2% zinc.8 Secondary sulfate minerals associated with the deposit include a variety of Fe²⁺, Fe²⁺–Fe³⁺, and Fe³⁺ sulfates (for details see Alpers et al.29). Several mines were operated intermittently between the 1860s and 1962 for Au, Ag, Cu, Fe, Zn, and pyrite. Of the original reserve estimated at 11.5 million tons, approximately 3.5 million tons were removed.2 Acidic effluent from the mine was blamed for significant fish kills in the Sacramento river before treatment began.2 The site has been listed under the National Priorities list for EPA Superfund since 19838 and all effluent from the mine is now collected and treated on site by addition of lime (CaO) to remove metals to below the U.S. E.P.A. drinking water standards for Cu (Joe Cobliati, private communication).

The oxidation of pyrite proceeds by a transfer of 15 moles of electrons per mole of FeS₂ (converting pyritic sulfide, S^1-, to sulfate, S⁶⁺ O₄^2- and Fe²⁺ to Fe³⁺). Redox reactions occur in increments involving 1 or 2 electrons.10 Thus, the overall pathway of sulfide oxidation must be composed of many steps. All of the electrons are lost via a series of anodic (oxidizing) half reactions, the sum of which may be represented by:

FeS₂ + 8 H₂O→Fe³⁺ + 2 + 14 H⁺ + 15e^- ,     (1)

where the product bisulfate is used to represent this reaction occurring below pH 2 [pK₂ of sulfuric acid = 1.99 (Ref. 11)] as it likely does at the site. Anodic reactions must be coupled to a cathodic reaction (in the absence of an applied potential). A subset of possible anodic and cathodic reactions describing pyrite oxidation are given in Table I.

The importance of microbial activity in pyrite dissolution and AMD formation is well documented.119232730313233 To date, the majority of microbiological studies of AMD systems have been based on physiology and characterization of cultivated organisms. Nordstrom and Southam32 and Johnson33 review the concepts that have emerged from these studies. The key finding is that microorganisms oxidize ferrous to ferric iron, increasing the rate of supply of this important oxidant to the surfaces of dissolving sulfide minerals.

Nordstrom1 first noted the potential importance of microorganisms at Iron Mountain. Several subsequent studies at the site have applied molecular biological methods to describe the microorganisms present and to correlate microbial community structure with the geochemistry of their environments. Rodgers et al.34 created the first small subunit riboso-mal RNA (16S rRNA) gene library from the site (see Edwards35). Subsequently, a fluorescent in-situ hydridization (FISH)-based study by Schrenk et al.3 determined the relative abundance of archaea, bacteria, and eukaryotes. Schrenk et al.3 also confirmed the findings of Rodgers et al.34 (that Acidithiobacillus ferrooxidans is not a significant organism in active AMD generating regions of the ore deposit).

Edwards et al.4 tracked microbial populations over one year and further established the restricted distribution of At. ferrooxidans. This study also suggested that another well known Fe-oxidizing acidophile, Leptospirillum ferrooxidans, could not account for the majority of microorganisms in most communities sampled. Edwards et al.29 established that archaea are abundant, especially in low-pH, high-ionic strength environments.

A novel, microaerophilic iron-oxidizing archaeon was isolated from the site by Edwards et al.36 This organism comprises up to 85% of the microbial cells in some samples. An isolate, referred to as Ferroplasma acidarmanus (strain ferl), is the subject of current detailed physiological (Bond et al.37) and genomic-based (Allen et al., in prep.) studies. Bond et al.37 analyzed 16S rRNA gene diversity of several specific microenvironments within the Richmond mine. In contrast to Rodgers et al.,34 who targeted planktonic species in AMD solutions and sediments, Bond et al.37 primarily sampled biofilms. The diversity of known archaea, specifically within the order Thermoplasmatales, was broadened by this study. Members of the Thermoplasmatales, which include clones from the public databases, have been named the "alphabet plasmas.38 Bond et al.37 also identified three distinct phylogenetic groups of Leptospirillum (groups I, II, and III). Group II was subsequently named Leptospirillum ferriphilum.39 These findings laid the groundwork for more detailed studies.

Field work at the Richmond Mine at Iron Mountain in northern California was conducted over 4 years in the present investigation, as part of a 10-year effort to study the microbial activity associated with metal-sulfide oxidation at this site. Water at the Richmond Mine offers some interesting challenges to the analytical techniques employed in conventional water analyses. Notably, pH standards require special attention to the activity coefficient of H⁺ in samples of high ionic strength25 and oxygen concentration measurements are virtually impossible using standard membrane electrodes or titration methods. On-site analysis of water samples for Eh, conductivity, and temperature were conducted routinely, after established USGS protocols.40 Standards for pH measurements were prepared from H₂SO₄ stock after the method for measuring extremely low-pH waters developed at the same location by Nordstrom et al.25 A ZoBell's solution was prepared immediately before analysis for a check of the platinum electrode. As noted by Nordstrom,23 Eh measurements were highly consistent with the Eh calculated from the Fe²⁺/Fe³⁺ couple (typically within 5%).

Water samples were collected with sterile 60 ml syringes and filtered through 0.2 μm syringe filters into several splits for later analyses. Samples were stored in 15 ml Falcon tubes, filled to occlude any headspace gas, and stored on ice and in a refrigerator until analyses were complete. Ferrous iron was measured on site using a 1,10 phenanthroline method and total iron was measured using a FerroZine method with a Hach portable digital spectrophotometer. In the lab, duplicate splits were measured for Fe²⁺ and Fe³⁺ directly, using the method of To et al.41 and an Ocean Optics UV-Visible spectrophotometer (2000S series). Iron measurements using both methods were consistent within analytical uncertainty, which is primarily due to the high level of dilution necessary for spectrophotometric analyses of these samples. Chromatographic analyses of collected samples for anion determinations were performed on a Dionex Series 500 ion chromatograph equipped with a conductivity detector and UV-Vis detector collected at 230 nm. Sulfate, sulfite, thiosulfate, dithionate, and sulfide were detectable at tens of micromolar levels using an isocratic 5 mM NaHCO₃/5 mM Na₂CO₃ eluent at a 1.0 ml/min flow rate through an IonPac AS16 column. Although chloride, phosphate, nitrate, nitrite, and fluoride are also resolvable using chromatographic methods, the extremely high sulfate concentration made quantitative analysis of these ions impossible, given the relatively low concentrations of these species compared to sulfate.

Attempts to measure polythionates were made using an isocratic 30% acetonitrile:H₂O eluent containing 2 mM tetrabulylammonium hydroxide, buffered with 3 mM NaHCO₃/3 mM Na₂CO₃ and run through an IonPac NS1 column, modified after several advances in individual polythionate detection by chromatography.42 Tri-, tetra-, and penta-thionate were all resolvable down to 5 μM concentrations using a 25 μl sample loop. Water samples collected directly from mine effluent, supernatants of samples centri-fuged (~4000 rpm) in the field, and water samples preserved on an anion exchange resin after the method of Druschel et al.43 were all analyzed for polythionates and thiosulfate. Elemental sulfur was extracted and analyzed by HPLC using a Shimadzu system UV-Vis at 254 nm, an Alltech C18 column, and an isocratic 95% Methanol:H₂O eluent at 1.0 ml/min after the method detailed in McGuire and Hamers.44 Trace metals, cations, and silica were analyzed by ICP-MS (VG PlamsaQuad 3), operated in peak jumping mode at the University of Windsor Dept. of Geology. All standards, blanks, and samples were prepared in ultrapure (distilled) 2% HN0₃. Multiple internal standards were used to correct for machine drift that was less than 2% across the mass range.

Mineral samples from the Iron Mountain Mine site were analyzed by XRD using a Scintag PadV x-ray diffractometer (XRD). Peak matching utilizing the JCPDS Powder diffraction file was used to identify minerals in samples analyzed by XRD. Selected aliquots of cleaned minerals were also analyzed by a LEO 1530 scanning electron microscopy (SEM) and a Cameron SX50 electron microprobe (EMP). Back-scattered electron images and energy-dispersive spectrometer analyses in EMP were utilized to determine enrichments of As, Cu, and Zn in pyritic material.

Geochemical modeling using the Geochemist's Workbench suite of programs45 was used to calculate speciation in the solutions. Waters from the Richmond Mine have a very high ionic strength. Therefore, proper calculation of the activity coefficients for species of interest requires application of models able to account for interactions beyond simple bimolecular electrostatic interactions, such as the Pitzer equations or HMW models.46 Speciation calculations of the AMD solutions were carried out using the PHRQPITZ database, which is more accurate in the calculation of activity coefficients for the high-ionic strength solutions at Iron Mountain. However, this database does not include all appropriate ion pairs, especially FeS0_4(aq) species. Speciation calculations using PHRQPITZ were carried out using thermo-dynamic data including available virial coefficients. In an attempt to assess what the errors are with using a database which is missing potentially important species (especially FeS0_4(aq)), additional and separate calculations were made on these waters using the thermo.dat database provided with the React code of the GWB 3.1.

Particular attention to the total moles of iron, sulfur, and protons in the solution was paid in order to assess iron and sulfur transport and potential sinks relative to the pathways controlling pyrite oxidation and microbial activity in the mine. Analytically, the measured amounts of iron and sulfate accurately reflect the amount of each element in solution. Total H⁺ in solution generated from pyrite oxidation is derived from both the free H⁺ in solution (from pH and H⁺ activity coefficient) and the molar concentrations of proto-nated species such as bisulfate (which is strongly affected by the abundance of the FeSO_4(aq) complex). Bisulfate available to complex H⁺ is also affected strongly by iron speciation, as ferrous iron more strongly complexes SO₄² than does H⁺. Results reported for total H⁺ are calculated based on the speciation results and are functionally equivalent to the sum of free H⁺ and HSO₄^- in solution. There are other species in these solutions, but over 99% of the H⁺ is contained in these two species. As such this calculation is slightly different than would be found from an acidity titration because cations which would contain or take up OH^- are not explicitly considered.

Use of GWB 3.1 and the thermo.dat thermodynamic data will result in errors due to extrapolation of equilibrium constants well outside their useful range of ionic strength. There is no current modeling protocol to arrive at a better solution, however. We use H⁺ values resulting from these calculations in a strictly qualitative sense, as the error can be greater than 50%, based on comparison of activity coefficients for H⁺, HSO₄^-, and FeSO_4(aq). Errors in the PHRQPITZ calculations in which total H⁺ is comparable to 30–50% of the sulfur species present. Caution should be applied when attemping to use these numbers in anything but a qualitative sense.

The surface colonization and dissolution of pyrite and arsenopyrite by iron- and sulfur-oxidizing micro-organisms from the Richmond Mine have been reported (e.g., Edwards et al.4748). Surface-sensitive spectroscopic studies using Raman spectroscopy revealed that elemental sulfur is an important product of the dissolution of pyrite and arsenopyrite.4950 In the case of abiotic arsenopyrite dissolution, the majority of the sulfide oxidized is converted to highly insoluble S₈, which accumulates at the mineral surface.50 Thus, in this study, we conducted experiments designed to explore the interaction between sulfur-oxidizing micro-organisms and the dissolving arsenopyrite surface. These experiments used At. caldus because this sulfur-oxidizing organism cannot impact sulfide dissolution via Fe²⁺ oxidation, and thus its primary biogeochemical effect is on the oxidation of intermediate sulfur species such as S₈. An important question to be resolved is whether microbial utilization of sulfur influences sulfide dissolution rates by removal of a diffusion-limiting S₈ coating. Cylindrical Teflon chambers were assembled for three parallel experiments. Chamber 1 was configured with a 0.22 μm pore-size polycarbonate membrane dividing the chamber into two compartments. Each end of the chamber was fitted with a port for removal and addition of material to the compartments. Chambers 2 and 3 were configured in the same manner as Chamber 1, but with no membrane partition in place.

Arsenopyrite preparations and surface-area measurements were carried out as described by McGuire et al.12 Equal portions (by mass) of crushed arsenopyrite were added such that each chamber contained approximately 0.015 m² of material. Sections of arsenopyrite polished and cut into 3 × 3 × 1 mm coupons were added to Chamber 2 for cell attachment studies. These slabs were placed in perforated 0.5 ml microcentrifuge tubes attached to a Teflon wire extending through the chamber port. The crushed arsenopyrite and the polished coupons were placed into the experiment chambers prior to sterilization by autoclaving. The sterilization techniques likely oxidized the surface of the minerals, but the long-term nature of these experiments with microbial cultures incapable of reduction should not be significantly affected by this.

Each sterilized chamber containing arsenopyrite was filled to half capacity with a total of 210 ml of growth medium. We prepared 9 K medium,51 modified by omission of FeSO₄ and amended with 0.01% (w/v) yeast extract. The medium was adjusted to pH 1.6 and was autoclaved prior to use. Cultures of At. caldus TC1 were prepared as previously described31 to serve as innoculum. The compartment of Chamber 1 isolated from the arsenopyrite material was inoculated with At. caldus cells. Chamber 2 was also inoculated with the same volume of culture (in contact with arsenopyrite material). Chamber 3 was left sterile to serve as an abiotic control. The chambers were incubated in a horizontal position at 37°C.

Samples for total dissolved iron and cell density measurements were taken from each chamber at prescribed time intervals. One arsenopyrite coupon was retrieved from Chamber 2 during each of the 23, 67, 161, 286, 384, and 477 hour sampling sessions. The coupons were fixed in 3% (w/v) paraformaldehyde for 4 h, rinsed with PBS (phosphate buffering solution), and stored in a 1:1 PBS:ethanol solution at -20°C.

Total dissolved iron was measured by the FerroZine method. Direct counts of cells in suspension were performed by light microscopy using a Petroff-Hausser counting chamber. Cells attached to arsenopyrite coupons were visualized by staining with DAPI and were viewed using an epifluores-cence microscope. Digital images were captured using a CCD camera and the NIH Image software. The area of one image at 1000× was calibrated and 15 random images were counted and averaged for cell density determinations.

In order to extend prior studies of correlations between microbial populations and geochemical habitats, samples were collected from six sites in the A-, B-, C-drifts and 5-way area (Fig. 1) in January 2001. These were taken from the following locations in the mine; a damp biofilm growing on the surface of a pyrite pile in the A-drift referred to as the "A-drift Slump" (0lIMlAslump, pH 1.1), the "A-drift Red Pool" (01IM1red pool, pH 1.4), "A-drift Slime Streamers," below the waterfall that separates the "A-drift Weir" (01IM1A1, pH 0.9) from the "5-way" (01IM1A1, pH 0.89), and biofilms on the "B-drift Weir" (01IM1B1, pH 0.75) and "C-drift Weir" (01IM1C1, pH 0.85).

All enrichments contained a basal media consisting of 0.8 g/l (NH₄)₂SO₄, 0.4 g/l KH₂PO₄, and 0.16 g/1 MgSO₄. As carbon sources 0.25 g/1 trypcase soy broth (TSB) and 0.2 g/1 yeast extract were supplemented to 20 g/1 FeSO₄ and incubated in 250 ml flasks with vented covers. To enrich for members that can utilize intermediate sulfur compounds 5 mM, thiosulfate, tetrathionate, and S° with 10 mM glucose were added to screw-cap tubes and sealed (with air head-space). The enrichments were incubated at a range of temperatures commonly seen in the mine (24°C, 37°C, 45°C, and 60°C). All media were pH adjusted to 0.8–1.0. Once growth was observed, < 1010 dilution series were made to isolate novel organisms.

In January 2001, samples from six sites were collected into sterile containers, placed on ice, and transported back to the laboratory. Samples stored frozen in 25% glycerol were washed in PBS buffer (at pH 1.2 to prevent lysis of obligate acidophiles) to remove extracellular ions. The samples were then resuspended in pH 7.0 PBS, and DNA extractions were carried out as previously described37 with an additional chloroform wash to remove residual phenol that might inhibit PCR reactions. A suite of domain level primers (27F5' -AGAGTTTGATCCTGGCTCAG-3'; 21Fa5' -TTCC GGTTGATCCYGCCGGAA-3'; A16F235' -TCYGGTTGAT CCTGCC-3'; and 1492R5'-GGWDACCTTGTTACG ACTT-3') were utilized to amplify 16S rRNA genes from total genomic DNA extract of each of the samples. Specifics of 16S rRNA gene amplification, cloning, sequencing, and analyses of the libraries are provided elsewhere.52 A few chi-meric sequences were found via dual tree comparisons and not included in the final phylogenetic analyses. Sequences were deposited in GenBank under the accession numbers AF543496 through AF543512. Phylogenetic trees were generated using ARB software package and the topologies were confirmed using bayesian inferences as described previously.52

Samples collected from the same sites, and at the same time as those used for clone library construction, were washed with pH 1.2 PBS buffer and fixed using 4% paraformaldehyde within 12 hours of collection. Hybridization was done following protocols previously reported.53 Details of all probe synthesis, sequences, and specificity are described elsewhere.53 Background staining was done to all FISH slides with a DNA stain, DAPI (4',6-Diamidino-2-Phenylindole Dihydrochloride). Slides were viewed using a Leica LEITZ DMRX epifluorecence microscope. Images were captured with a Hamamatsu digital charge-coupled-device camera using Axiovision software.

Much of the pyrite ore is fine grained, thus readily liberated from the ore deposit by dissolution. This fine-grained material accumulates as sediment in slumps and on the tunnel floors in layers up to several meters thick in some areas. Analysis by optical and scanning electron microscopy and electron microprobe analyses indicated the sediment is predominantly pyrite with an average particle diameter of ~10- 1000 μM. The sediment also contains a few percent quartz. Particle surfaces are roughened by dissolution (Fig. 2). Sul-fate minerals (including blue-green melanterite, yellow-brownish jarosite, copiapite, green-blue chalcanthite, pinkish coquimbite, and a compound with cubic crystal form and composition consistent with voltaite) form by evaporative concentration and are locally abundant (inset in Fig. 2).8 Electron microprobe data indicate that the pyrite particles contain regions that are locally enriched in Zn, Cu, and As. These impurities may account for several percent of the cations in pyrite. No discrete metal sulfide inclusions were observed and no other sulfides were detected in this sediment (although other sulfide phases do occur in the deposit). It is conceivable, however, that the zones of observed Zn, Cu, and As enrichment were small inclusions of ZnS, CuFeS, and FeAsS too small to be imaged in the electron microprobe.

Some surfaces of pyrite from the sediments contain deep euhedral pits (Fig. 3). Occasionally very thin, elongated cells occur on the pit base and pit walls. These pitmicrobe associations are very similar to those noted by Edwards et al.3554 in a SEM study of polished pyrite surfaces of in-situ experiments retrieved from the field. In general, the sediment pyrite surfaces are colonized by cells with a limited morphological diversity, suggesting direct interactions involve only a few species.

Acidithiobacillus caldus cells were allowed to directly contact the arsenopyrite in some experiments and were kept separate from the mineral substrate by a 0.2 μM filter in others. As shown by total soluble iron measurements, arsenopyrite dissolution rates were only slightly higher with cells present relative to the abiotic controls [Fig. 4(a)]. Dissolution was most rapid in Chamber 2 where cells were allowed to contact the mineral surfaces. However, the difference in rates between the two biologic and the abiotic reactions is minimal. Given that previous experiments have shown that α-S₈ builds up on the surface during arsenopyrite dissolution,1214 the similarity between the abiotic and biotic rates (measured with Fe_T as the reaction progress variable) indicates the absence of dissolution-inhibiting surface products.

At. caldus cells were observed to grow regardless of attachment to the mineral surface [Fig. 4(b)]. This observation suggests that a microbial product may be involved in converting elemental sulfur, which is extremely insoluble under the experimental conditions,55 into a bioavailable form. This compound may be a shuttle that moves electrons from elemental sulfur to the cell surface. It is unlikely that iron is the electron shuttle as At. caldus is unable to oxidize Fe²⁺ and inorganic iron oxidation kinetics are extremely slow under the experimental conditions.

Biofilms, and subaqueous and water surface streamer occur in the 5way area (Fig. 5). Microscopic observations indicate variable numbers of micro-organisms associated with the pyritic sediment. No simple trend between cell numbers and depth of sample site within the sediment was noted.

Tables II and III list the Fe²⁺, Fe³⁺, SO₄², Cu²⁺, Zn²⁺, Al³⁺, , Cd²⁺, K⁺, Mg²⁺, Mn_T, Na⁺, Pb_T, and SiO₂ concentrations at the sites where microbial samples were collected. Insufficient fluid was available for analysis at the A-drift slump in 2001, and only one sample was collected from the A-drift samples because they are separated by only a few meters of flow path.

Because of PCR bias, clone abundances cannot be used as a proxy for species abundance. Consequently, in-situ hybridization studies using probes designed to bind specifically to RNA of organisms detected via clone library analysis were conducted. Due to the high cell density of most samples, the probe-based studies are only semiquantitative. A representative image is shown in Fig. 6, which highlights the Sulfobacillus cells in a biofilm. Results confirm earlier deductions5 that the number of distinct organism types (a type being a phylotype with generally >97% homology in the sequence of the 16S rRNA) is small.

After several months of incubation on selected intermediate sulfur compounds (thiosulfate, tetrathionate, and S°) no growth was detected. Enrichments containing yeast extract and TSB often resulted in growth. Dilutions of these cultures largely resulted in isolation of Ferroplasma. Some Sulfobacillus isolates were obtained on yeast extract and FeSO₄. Figure 7 shows the phylogenetic placement of three isolates obtained on heterotrophic (yeast extract as a carbon source) media at 37°C from the mine (SB6 and SB37) that are very closely related to previously characterized organisms (S. thermosulfidooxidans VKM and S. disulfidooxidans) and clones (see below, BW7). Attempts to grow isolate SB37 on S° were unsuccessful.

As shown in Table IV, 232 clones from six libraries were obtained from spatially separate microenvironments sampled in January 19, 2001. No more than six distinct organism lineages were detected in any sample. Previous studies have also shown that AMD communities are characterized by a limited number of taxonomic groups.38 Clones belonging to Nitrospira, Bacillales, Alphaproteobacteria, Deltaproteobac-teria, Gammaproteobacteria, Actinobacteria, Acidobacteria, and the archaeal "alphabet plasma" groups were identified and near complete 16S rRNA gene sequences were obtained (Fig. 7). Some of these lineages are closely related to sequences that were previously published from the Richmond mine37 and a forested wetland site (U.S. Dept. of Energy's Savannah River Site) impacted by acid solutions derived from coal56 (see coal refuse clones in Fig. 7).