Whitin a larger project investigating ABE fermentation, a number of pre-treatment techniques (chemical, mechanical) have been applied to wheat straw prior to hydrolysis with commercial cellulases for the production of hydrolysates. After chemical analysis of sugars, organic acids and potential fermentation inhibitors (Furfural, Hydroxymethyl furfural), the hydrolysates were used as fermentation media for Clostridium acetobutylicum and Clostridium beijerinckii strains for the production of ABE. The fermentations have been followed and relevant metabolites have been determined. Furthermore, medium optimisation studies have been carried out with relevant hydrolysates in order to formulate a medium in which maximal butanol yields and productivity can be reached. Such a straw hydrolysate-based fermentation medium may be used for an efficient industrial Biobutanol process. We have observed that the fermentability of wheat straw hydrolysates depends not only on the pre-treatment and hydrolysis conditions, but also on the properties and requirements of the specific organism to be used for fermentation.