a b s t r a c tEnzymatically liquefied sweet sorghum stalks and beet molasses were evaluated for butyrate production using Clostridium tyrobutyricum in 1 L scale fed-batch fermentations. The hydrolysates used for the fermentations were prepared separately by liquefying the sorghum stalks at 50 • C, pH 5.0 for 18 h, with 30% (w/v) DM content using the enzyme preparation Cellic ® CTec2 at an activity of 26.5 FPU/g DM. To enhance sucrose consumption, the fermentations were supplemented with invertase at an activity equivalent to 8.3 U/g DM. With the hydrolysate as the feedstock, a butyrate concentration of 37.2 ± 0.8 g/L, a productivity of 0.86 ± 0.02 g/L h and a yield of 0.39 ± 0.02 g/g (p = 0.05) consumed sugars were obtained. Finally, a butyrate concentration of 58.8 g/L, a productivity of 1.9 g/L h, a butyrate yield of 0.52 g/g consumed sugars and a dry cell mass concentration of 15.7 g/L were obtained with fed-batch cultivation and a constant feed consisting of 64% sorghum hydrolysate juice and 36% molasses. Evidence for inducible saccharolytic activity was also proven, as the cellulase activity in the culture supernatant was found more than double during feed with limiting sugar concentrations. The present study clearly demonstrates that combinations of low cost raw materials can be used for efficient butyrate production, also without cell immobilization.