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.