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Experimental methods
- The effect of adsorbed lipid on pyrite oxidation under biotic conditions

Pyrite pretreatment followed protocols, described in earlier work, [15] to generate clean pyrite (~10 μm average diameter) powder with a surface area of 0.75 m2/g. In brief, pyrite samples were crushed and subsequently acid washed using 0.1 M HCl. Acid washing of the pyrite was carried out by flowing pH 2-deoxygenated water over the mineral powder [14].

A pure culture of A. ferrooxidans (ATTC 23270) was obtained from the American Type Culture Collection (Manassas, VA). The culture was grown and maintained in ATCC #2039 broth as suggested by the supplier. Briefly, the medium consisted of two separately prepared solutions: Solution A contained 0.8 g (NH4)2SO4, 2.0 g MgSO4·7H2O, 0.4 g K2HPO4, 5.0 mL Wolfe's Mineral Solution, and 800 mL distilled water, adjusted to pH 2.3 with H2SO4 and then filter-sterilized. Solution B contained 20.0 g FeSO4·7H2O and 200.0 ml distilled water, which was filter-sterilized and then mixed with Solution A (above) to produce the complete medium. Cultures (9 ml) for experiments were grown unshaken using 100 ml of the medium in 250 ml Erlenmeyer flasks (autoclaved at 126°C and 20 psi for 0.5 h prior to use) at 25°C. The cultures were stoppered with nylon wool wrapped with aluminum foil.

For the experiments, A. ferrooxidans was grown to the early stationary phase of growth and harvested by a series of filtrations. First, the bacteria were separated from particulate material, which are presumably iron hydroxide particles in the media, by filtration through a 5-μm pore size polycarbonate filter (Millipore™). Second, the bacteria in the filtrate were collected on a 0.2 μm polycarbonate filter by vacuum filtration and resuspended in sterile water [pH 2.3] by vortexing. The amount of nutrients in this cell suspension (carried over from the culture) was 1% of the original concentration. This low nutrient concentration was necessary to minimize the impact of aqueous phosphate on pyrite oxidation [17]. Nevertheless, the concentration of nutrients present was sufficient to support growth of the bacteria in the presence of pyrite.

Lipid solutions were prepared by techniques described elsewhere [14]. In the current study, 0.1 g of acid washed pyrite was added to lipid solution (20 ml). The lipid solution contained 7.5 μmol of 23:2 Diyne PC lipid (Avanti Polar Lipids), which has the following structure:


This particular concentration of lipid was chosen on the basis of prior research in our laboratory, and it was sufficient to cover the majority of the pyrite surface [14]. After 1 hour of mixing, the pyrite slurry was allowed to settle and the supernatant, which contained free lipid, was subsequently removed by decanting. The remaining fraction of lipid-coated pyrite slurry was used in the experiments.

The experiment was performed on two separate dates (from here on referred to as Experiment 1 and 2). The experimental conditions, treatments and analyses for both experiments were identical with the exception of the starting bacterial densities (see below). The treatments consisted of: 1) pyrite, 2) lipid-coated pyrite, 3) pyrite + bacteria, 4) lipid-coated pyrite + bacteria. All the treatments were prepared, in duplicate, in distilled water (pH 2.0) for a total volume of 50 ml. The treatments with pyrite contained 0.1 g of pyrite. Approximately the same amount of pyrite was present in the pyrite/lipid treatments (see preparation of lipid-coated pyrite slurry described above). The final density of A. ferrooxidans in the treatments with bacteria was approximately 3.3 × 107cells/ml for Experiment 1 and 1.7 × 107cells/ml for Experiment 2. Each treatment mixture was stirred continuously (using a teflon coated stir bar) throughout the experiment, which lasted a maximum of 25 days. Aliquots (1 ml) were withdrawn every 2–3 days for cell count analysis and preserved in 3.7% formaldehyde. In addition, 0.1 ml aliquots were withdrawn to determine the iron concentration in solution, which was used as a reaction progress variable. Total iron solution concentration determinations (including ferrous and ferric iron) were determined spectrophotometrically by using the ferrozine technique (UV method 8008). Since this technique is intrinsically sensitive to the concentration of ferrous, ascorbic acid was added to the solution prior to analysis to reduce any ferric to ferrous. All the sample aliquots for the ferrozine test were filtered through a 0.2 μm polycarbonate filter before analysis. Solution pH measurements were carried out with a PHM82 standard meter with a resolution of 0.01 pH units.

Enumeration of bacterial cell densities in solution and on pyrite powder were performed by epifluorescence microscopy following staining with the fluorochrome dye, acridine orange (AO) [18,19]. Briefly, preserved water samples were vacuum filtered onto 0.2-μm-pore-size blackened polycarbonate filters (Nuclepore) and the cells collected on the filter were stained with AO at a final concentration of 0.01% for 3 min. The volume of sample filtered for cell counts was chosen such that the layer of pyrite particles that accumulated on the filter would not obscure the bacteria. Cells were observed and counted with a Zeiss standard microscope equipped for epifluorescence microscopy. Filter sets used for AO fluorescence observations are as follows: a BP450-490 exciter filter, and FT510 chromatic beam splitter, and an LP520 barrier filter. For each sample, the number of bacteria on 10 to 15 randomly chosen fields distributed over the filter (or approximately 300 to 600 cells in total per filter) was counted. To determine the number of cells bound to pyrite, 0.1 ml aliquots (initial total volume was 50 ml) of the bacteria/pyrite solutions were collected on a 5 μm polycarbonate membrane filter (Millipore). The particles were subsequently washed so that any bacteria in solution would pass through the filter. The density of cells on the particulate material remaining on the filter was then determined using the staining method described above.

Surfaces for AFM were prepared from pyrite cubes (Navajun, Spain) obtained from Ward's Natural Science. Growth surfaces were cut from the cube and were subsequently polished. Prior to an experiment, the samples were exposed to pH 2 water (by HCl addition) to remove oxidized layers prior to exposing to lipid solution. To prepare the lipid/pyrite samples the pyrite platelet was submerged into the lipid solution in a vertical orientation (via a leading edge). The sample was then immediately removed, washed with pH 2 water to remove any loosely bound lipid, and then reintroduced to pH 2 water for imaging with a PicoSPM II (Molecular Imaging) microscope. The probes used in all the AFM measurements [NSC15, μMasch] had a nominal spring constant of 40 N/m and a resonant frequency of 325 kHz.

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