This study evaluates how fermentation inhibitors derived from biomass, namely 5-hydroxymethylfurfural (HMF), levulinic acid (LA), and formic acid (FA), affect H2 production by a Clostridium beijerinckii strain. The specific fermentative H2 production rate (mu H2), bacterial cell growth rate (mu), and substrate (glucose) consumption rate (mu S) during fermentation helped to estimate which HMF, LA, and FA concentrations inhibited 50% of the rates (IC50). IC50 for mu was 2.4, 2.7, and 1.4 g/L for HMF, LA, and FA, respectively. HMF inhibited H2 production the most potently and favored the lactate and ethanol pathways. Butyric acid was the only metabolite to be detected in the presence of LA or FA, which attested that these inhibitors completely inhibited the acetate pathway. The glucose consumption rate was the least affected by the inhibitors, and FA was more potent than HMF and LA. This information should be useful for more appropriate biomass feedstock application in fermentative H2 production. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.