Highly efficient butanol-producing bacterial microflora were isolated from hydrogen-producing sludge of a sewage treatment plant. Based on denaturing gradient gel electrophoresis (DGGE) analysis and 16s rDNA comparison, four strains from the butanol-producing microflora were identified as Clostridium saccharoperbutylacetonicum, Clostridium butylicum, Clostridium beijernckii, and Clostridium acetobutylicum. The effects of glucose. FeSO4 center dot 7H(2)O and yeast extract concentrations on the butanol production by the mixture culture were investigated on batch mode. The medium composition for bio-butanol production was optimized using the Box-Behnken design and response surface methodology (RSM). The maximum butanol production rate (0.25 +/- 0.02 g/L-h) and concentration (12.4 g/L) were obtained under the condition of glucose concentration, 60 g/L; FeSO4 center dot 7H(2)O, 0.516 g/L; yeast extract concentration, 5.13 g/L. Addition of 6.0 g/L butyric acid significantly increased the butanol titer to 17.51 +/- 0.49 g/L. Pressurized fermentation strategy (employed with a 5 L fermentor) further enhanced the butanol concentration to 21.35 g/L, along with a maximum butanol rate of 1.25 g/L-h. (C) 2011 Elsevier Ltd. All rights reserved.