In this study, a new fermentation medium combining a flocculated hemicellulosic wood hydrolysate(FHWH), rich in carbohydrates, obtained from a pulp and paper mill and an alfalfa juice, rich in nitrogen was used to produce butanol by Clostridium acetobutylicum ATCC 842. It was demonstrated that pH control enhances solvent production. The culture containing the two renewable biomass resources resulted in the production of 3.80 g/L of acetone-butanol-ethanol (ABE) with a productivity of 0.03 g/L h and a yield of 0.14 g/g. The obtained results demonstrated that the proposed combination can be used as a new fermentation medium for ABE production. However, the complexity of the medium induced an inhibitory
effect and a decrease in the production of ABE, compared to the fermentation of FHWH and yeast extract.