The optimal medium for butyric acid production by Clostridium thermobutyricum in a shake flask culture was studied using statistical experimental design and analysis. The optimal composition of the fermentation medium for maximum butyric acid yield, as determined on the basis of a three-level four-factor Box-Behnken design (BBD), was obtained by response surface methodology (RSM). The high correlation between the predicted and observed values indicated the validity of the model. A maximum butyric acid yield of 12.05 g/l was obtained at K(2)HPO(4) 7.2 g/l, 34.9 g/l glucose. 20 g/l yeast extract, and 15 g/l acetate, which compared well to the predicated production of 12.13 g/l. (C) 2009 Published by Elsevier Ltd.