Screening of isolated bacteria for their azoreductase activity was carried out in liquid molasses medium at pH 7. The cultures transferred into 100 mL of medium were incubated in a rotary shaker(New Brunswick Scientific Innova 4230) at 30 ± 1◦C and 100 rpm for 3 days. Samples taken from the incubation medium were analyzed for enzyme activities.
Cells were separated from the cultivation medium by centrifugation (Hettich Rotina 35R) at 14,000 rpm for 5 min at +4◦C. The harvested cells were washed with sodium phosphate buffer twice in microcentrifuge tubes, kept on ice and then sonicated (SonicsVibra Cell) with a power input to 60 W and a frequency of 20 kHz for 5 min. The cell debris was removed by centrifugation at 14,000 rpm for 5 min at +4◦C and the clear supernatant was used to determine the intracellular azoreductase activity.