To evaluate the effect of substrate concentration on competition between hydrogen producing bacteria and lactic acid bacteria during hydrogen fermentation, a series of batch tests was conducted using galactose or glucose as a carbon source. Clostridium butyricum, Lactobacillus casei, and mixture of the two species were used for inoculum, respectively. The batch tests based on single species revealed that C. butyricum utilized galactose faster than L. casei, regardless of substrate concentration (0.2-5.7 g/L), while L. casei utilized glucose faster than C. butyricum at low levels of glucose (<1 g/L). These trends were also observed in the batch test based on a mixture of the two species. L. casei outcompeted C. butyricum only when glucose was provided at low concentration. Taken together, the results suggest that failure of biohydrogen production can occur at low glucose concentration via outcompeting of lactic acid bacteria over hydrogen producing bacteria. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.