The production of a novel lipopeptide by Bacillus subtilis MO-01 was investigated in shaker flask fermentation. Response surface methodology (RSM) was applied to optimize the medium ingredients. A five-level four-factor central composite design was employed to determine the maximum lipopeptide yield at optimum levels for sucrose, ammonium chloride, ferrous sulphate and zinc sulphate. Sucrose, ferrous sulphate and zinc sulphate showed a significant linear main effect, while ammonium chloride had no significant linear effect. The production was also significantly affected by sucrose-ferrous sulphate and sucrose-zinc sulphate, as well as by the negative quadric effect of the above four variables. Optimum fermentation parameters were 22.431 g/l of sucrose, 2.781 g/l of ammonium chloride and 6.7879 mu M of 0.0377 mM of zinc sulphate. The prediction lipopeptide yield was 1.712 g/l. The actual experimental results were in agreement with the prediction. (c) 2005 Elsevier Ltd. All rights reserved.