After 4 days, cultures of small motile rods producing
sulfide were observed. Enrichments that produced sulfide
were subsequently transferred to a sulfate-reducing bacteria
(SRB) medium that consisted of (l21 distilled water): 20 g
NaCl; 4 g Na2SO4; 3 g MgCl2.6H2O; 0?2 g KH2PO4; 0?5 g
KCl; 0?25 g NH4Cl; 3?46 g PIPES; 0?15 g CaCl2.2H2O; 1 mg
resazurin; 2 mg sodium tungstate; 0?5 mg sodium selenate;
1 ml vitamin mixture (Widdel & Bak, 1992); 1 ml thiamin
solution (Widdel & Bak, 1992); and 0?05 mg vitamin B12.
The pH of the medium was adjusted to pH 6?7 at room
temperature using 5 M HCl. After autoclaving under N2
(100 %), the pH had decreased to 6?5. Medium (10 ml) was
dispensed anaerobically in 50 ml vials sealed with butylrubber
stoppers and reduced with 0?1 ml of a 10% (w/v)
Na2S.9H2O sterile solution; H2/CO2 (80 : 20; 200 kPa) was
used as the gas phase. Cultures were incubated at 70 uC with
shaking (150 r.p.m.). The pH of the medium in uninoculated
vials checked at room temperature after incubation
at 70 uC decreased from 6?5 to 6?3.