A lipopeptide biosurfactant, C9-BS produced by Bacillus (B.) subtilis C9, emulsified hydrocarbons, vegetable oil and crude oil. A high yield of C9-BS was obtained from a culture of B. subtilis C9 using a carbohydrate substrate, while a hydrocarbon substrate inhibited the production of the biosurfactant. The optimum medium for the production of C9-BS was as follows (g/l) : glucose, 40; NH4HCO3, 13.5; K2HPO4, 10.5; NaH2PO4, 1.5; MgSO4 . 7H(2)O, 0.5; MnSO4 . 4H(2)O, 0.05; yeast extract, 0.5. The initial pH was 8.0 and the culture temperature was 30 degrees C. The biosurfactant C9-BS was obtained by collection of the foam that overflowed in the fermenter culture. The production of the biosurfactant by B. subtilis C9 was growth-associated. Under O-2-limited conditions, C9-BS was produced, in a 3-fold higher yield compared to that under O-2-sufficient conditions. C9-BS is soluble in ethanol, acetone, methanol, butanol, chloroform dichloromethane and alkaline water. The biosurfactant C9-BS lowered the surface tension of water to 28.5 dyne/cm, and the CMC (critical micelle concentration) was 40 mu M. C9-BS was stable from pH 5.0 to pH 9.5, in incubation at 100 degrees C for 1h, and at a salt concentration of 1,000 mM for NaCl and 10 mM for CaCl2.