Two fermenters were operated by semi-continuous mode with suspended biomass and glycerol at concentrations of 10 and 50 g/L to evaluate the semi-continuous operational conditions to hydrogen and volatile fatty acids production. A high hydrogen production in the semi-continuous reactors at the experiment was observed, with a drop during the test, mainly in the reactor with higher substrate concentration. The yield median of the semi-continuous reactors was 0.25 and 0.01 mol H-2/mol glycerol, and the volumetric productivity median was 350 and 130 mL H-2/Ld in SCR1 and SCR2 (10 and 50 g/L), respectively. It was possible to associate the high hydrogen and volatile fatty acids production in the semi-continuous reactors with the bacterial genus Enterobacter, and the low hydrogen production to the bacterial genus Clostridium. (C) 2020 Elsevier Ltd. All rights reserved.