Bacillus subtilis MUV4 produced biosurfactant in shake-flask culture (200 rpm) at 30°C with modified Mckeen medium containing 1% glucose as a carbon source, 1% monosodium glutamate and 0.3% yeast extract as nitrogen sources. The supernatant of B. subtilis MUV4 reduced the surface tension of the medium from 53.50 mN/m to 33.50 mN/m after 48 h of cultivation. The yield of crude biosurfactant from B. subtilis MUV4 after precipitating the supernatant with 6N HCl was 0.652 g/L. Growth kinetics studies showed the specific growth rate (µ) of 0.14 h-1, yield of biomass to substrate (Yx/s) of 0.713, yield of product to substrate (Yp/s) of 0.072 and yield of product to biomass (Yp/x) of 0.101. Moreover, B. subtilis MUV4 produced 0.30 g/L crude biosurfactant after 96 h of cultivation in the fermentor with agitation rate of 200 rpm without aeration and uncontrolled pH condition. The crude biosurfactant was dissolved in methanol and dried by vacuum evaporator (crude methanol). The supernatant, the crude biosurfactant and the crude methanol retained the biosurfactant activity over the pH range of 1-6, 7-10 and 4-10, respectively and the emulsion stability at 24 h (E24) at pH 7 were 66.67%, 33.33% and 33.33%, respectively. The supernatant and the crude biosurfactant showed surface tension activity at 4°C, room temperature (30±2oC) and 50oC after incubation for 5 h. However, only crude methanol still retained surface tension activity after 100oC for 5 h. The surface tension activity of the supernatant and the crude biosurfactant was stable in 3-10% (w/v) NaCl while crude methanol showed stability in 3-20% (w/v) NaCl. However, all samples lost emulsion stability when NaCl concentration was higher than 5% (w/v). With sand pack column technique, crude methanol enhanced the recovery of crude oil and kerosene oil by 41.85% and 75.00%, respectively. In hydrocarbon degradation application study, the crude biosurfactant was added to the culture medium containing 0.3% crude oil as carbon source and the microorganism consortium from oil contaminated soil. The result showed that the saturated hydrocarbon was reduced by 96.63% after 7 day cultivation, while the control without crude biosurfactant showed only 19.96% reduction.
