The effects of substrate concentration on fermentative hydrogen production from galactose at a fixed hydraulic retention time of 12 h were investigated in an immobilized continuously stirred tank reactor. Peak hydrogen production rate and hydrogen yield of 9.57 L/L-d and 1.10 mol/mol galactose(added), respectively, were obtained at a feed substrate concentration of 30 g/L and an organic loading rate of 60 L/L-d. Quantitative polymerase chain reaction analysis showed that the variations in the performance resulted primarily from metabolic alterations within the metabolism of the established microbial community rather than modifications in the population. The results obtained showed that optimal substrate concentration is essential for the efficient, continuous production of hydrogen from galactose. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.