The analysis of VFAs at 35 h fermentation showed that butyrate was the major fermentation by-product, with the highest production at the F/M of 7.5 (Fig. 2). This result is in line with previous work on fermentation from food waste using indigenous microbes in the feedstock [32], which suggested that the butyrate-related pathways are the main routes for H2 production that yielded 2 mol H2/mol hexose or 69% of the theoretical H2yield. Butyrate has been reported as the main metabolic product of Clostridium, which was present as the dominated microbial group at the hydrogen production stage in this study. Its production leads to a net generation of NADH, which is in turn rapidly re-oxidized to allow glycolysis to proceed [33].