The industrial culture media for butanol-ethanol-acetone fermentation (ABE) was optimized by experimental design. A butanol resistant mutant isolated from Clostridium acetobutylicum DSM 1732 was used. This mutant produced 15.5 g/1 of total solvents, 30% more than the wild strain solvent production. Mutant strain resists a concentration of 2,5% v/v meanwhile the type strain resists 1 % v/v butanol concentration. Molasses of sugar cane as carbon source were used. The molasses concentration was

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... ermined based on the necessary glucose concentration for producing 15 g/1 of butanol as limit product in the ABE fermentation. The nutrients were calculated in according lo literature reports and lo highest biomasse production on vegetative medium 3.8g/l. For determining which variables have significant effect on the total solvent production, the PLAKET-BURMAN method was used. The final concentrations of the culture medium were determined by EVOP-Simplex method. A liter of optimized industrial medium is composed by: molasses 130 g, biotin 4.0 mg, PAB A 3.0 mg, KH2PO41.8 g, yeast extract 3.0 g, minerals stock 4 ml and distilled water lo complete 1 liter; pH 6.1 before sterilization. Using this medium the total solvents production was 24,6 g/1. The production increment is equivalent lo 58,7%, compared lo the mutant strain before the medium was optimized.