Sample collection, isolation and preparation
Fresh effluent samples obtained from two alcohol factories in Iran, were
collected in sterile glass containers, closed tightly and transferred to
the laboratory for further analysis. Filtration of effluent samples was
performed using 0.45 µm pore size filter paper. Then, filter papers
containing microorganisms of effluent were grown in YPD (yeast
extract-peptone-dextrose) medium that is a specific medium for yeast
growth. The temperature was controlled at 28 °C with shaking at 150 rpm
until OD reached 0.5 (~ 1.5 × 107cell/ml) at 600 nm, representing the middle of the logarithmic (mid-log)
phase. Grown yeasts were harvested in the mid-log phase by
centrifugation at 3000×g for 10 min and after washing the yeast pellets
with distilled water for three times, it is stored at -20 °C.