Biomass as cell dry weight was determined by centrifugation of the culture at 8,000 rpm, 4oC for 20 min. The pellet was washed once time with hexane and twice with distilled water, placed on a pre-weighed foil-plate and dried at 105oC to obtain constant weight.
Biosurfactant activity test. After centrifugation of the culture broth, the supernatant was harvested, through roughly filtration to remove oil and examined the oil displacement test to form an oil displacement area and the ST value for its ability to reduce the surface tension. The oil displacement test was performed by measuring the diameter of the clear zone formed on the surface of an oil-water phase (15 L oil/ 153.9 cm2 surface area) after dropping 10 L of the culture supernatant and evaluating the displacement area, as previously reported (Morikawa et al., 1993). The ST value was measured by the Du Nouy Ring method (Tensiometer, K6; Kruss, Hamberg, Germany). The surface measurement was carried out at 25 ± 1 ºC after dipping the platinum ring in the solution for a while in order to attain equilibrium conditions. The measurement was repeated three times and an average value was obtained. For calibration of the instrument, the surface tension of the pure water was measured before each set of experiment.