The objective of this study was to demonstrate the feasibility of using a novel fermentation medium for butanol production by combining alfalfa juice, an agro-industrial residue from pellets production rich in nitrogen, and FHWH recovered from a pulp and paper mill rich in carbohydrates. Within this combined biorefinery system, C. acetobutylicum was able to uptake the mixture of alfalfa juice and FHWH, and to produce 3.80 g/L of ABE. However, the complexity
of the medium induced an inhibitory effect, resulting in a decrease in ABE production. Although the solvent production was lower than that obtained during the fermentation of FHWH and yeast extract, this biomass combination has opened new horizons for the future development of the integrated biorefinery concept.