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.