Experimental Design, Cell Growth, and Counting

In order to jointly assess both gene expression and changes in sulfur speciation, the experimental design included collection of samples for cell counts, gene expression, microscopy, S speciation and pH for A. thiooxidans grown in both S⁰ and S2⁢O32- treatments over 12 days to ensure both exponential and stationary phases were encompassed in the characterization. Greater details on collection and analyses of samples for each of these variables are provided subsequently.

Acidithiobacillus thiooxidans ATCC 19377 cells were grown in liquid elemental sulfur or thiosulfate media (Staley et al., 1989). The media contained two components, the salt medium and the sulfur source. Elemental sulfur salt medium: (NH₄)₂SO₄, 0.2 g; MgSO₄ × 7 H₂O, 0.5 g; CaCl₂ × 2 H₂O, 0.331 g; KH₂PO₄, 3.0 g; FeSO₄ × 7 H₂O, 9.15 mg; distilled water, 1,000 ml. The salt medium was sterilized by passing through a 0.22 μm filter. Elemental sulfur powder was heated in an oven at 100°C for 30 min and the cycle was repeated three times. The salt medium was then added to the culture flasks and the final sulfur concentration was 1% (m/v). Thiosulfate medium: salt medium as above and Na₂S₂O₃ was added at 0.2% (m/v), followed by filter sterilization (0.22 μm filter). For both cultures, the total volume of medium corresponded to a fifth of the total volume of the Erlenmeyer flask. All cultures were initially inoculated at 5% v/v with cultures pre-grown in the corresponding media; the inoculant bacteria were washed with sterile 1% NaCl solution prior to inoculation. All cultures were grown under aerobic conditions at 30°C and flasks were shaken at 120 rpm.

Cells were harvested at the desired time points (days 1, 2, 3, 4, 5, 8, 10, and 12) and washed with 1% NaCl. Optical density (O.D.) values were determined to generate cell counts; however errors introduced by S⁰ clumping precluded their use for these experiments. Thus, for the growth curves, 2 μl of the Live/Dead marker mixture of component A and component B at a ratio of 1:1 (L7012 LIVE/DEAD^® BacLight, Bacterial Viability Kit, Thermo Fisher Scientific) were added to 1.5 ml of bacterial suspension. The rationale behind the Live/Dead stain is that all cells will be stained green, because SYTO 9 penetrates into live and dead cells and stains their DNA, whereas propidium iodide (red stain) penetrates only into dead or damaged cells with leaky membranes staining their DNA. For the negative control (dead cells), the cells were first washed with 1% NaCl and then incubated in 70% ethanol for 1 h, followed by washing with 1% NaCl. Propidium iodide (Component B) was added (0.66 μl for 1 ml of bacterial suspension). For the positive control, 0.66 μl of SYTO 9 (Component A) was added to 1 ml of bacterial suspension. All samples were incubated in the dark at room temperature for 15 min, followed by counting in a FACS BD Canto II instrument. Experiments were conducted in triplicates.