Reagents and preparation of sulfur
Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich. Sucrose palmitate was from Alfa Aesar. SYBR Green I nucleic acid was from Molecular Probes. Colloidal sulfur referred to as c-sulfur (Sulfur, 13825 Lot #MKCC3309), and dispersed colloidal sulfur referred to as d-sulfur (Sulfur, 13825 Lot #SZBB0320VV) were the sources of sulfur used in this study.
For making defined formulations of dispersed sulfur, colloidal sulfur (c-sulfur) was used. The dispersants used were Tween 20, poly-(sodium 4-styrenesulfonate), sodium 2-naphthalenesulfonate, sucrose palmitate, lignosulfonic acid calcium salt. To prepare the 33.3 weigh by weight percent (wt %) dispersed sulfur, 1.00 g of colloidal sulfur and 0.50 g of the dispersant were added to 25 mL of ddH2O and allowed to equilibrate for 24 h. Another 25 mL of ddH2O was added at 24 h and mixed for another 24 h for a total of 48 h. The mixture was centrifuged at 5,000 x g for 3 minutes. The supernatant was removed and the dispersed sulfur was resuspended in 5 mL of ddH2O and allowed to dry at 37 °C overnight. The dried dispersed sulfur was crushed into a fine powder and rehydrated in appropriate media for use. To prepare the 20 wt % dispersed sulfur, 0.25 g of the dispersant was added instead with the same procedures above. Dispersed sulfur made with 20 wt % lignosulfonic acid calcium salt is referred to as lig-sulfur in this study.
For making dispersed sulfur using the cell lysate of A. ferrooxidans , 1 mL of an OD600 (optical density at 600 nm) 10.0 suspension of cells was sonicated on ice water for 5 minutes using a 3 second repeating pulse at a power of 4.5 W in a Misonix Sonicator 3000. Instead of adding a chemical dispersant, the cell lysate solution was added to 0.30 g of colloidal sulfur for dispersion in 25 mL of ddH2O. The same steps above for dispersing sulfur were followed for the remainder of the protocol.