This study examined the mesophilic continuous biohydrogen fermentation from galactose and glucose mixture with an initial substrate concentration of 15 g/L (galactose 12 g/L and glucose 3 g/L) as a resembling carbon source of pre-treated red algal hydrolyzate. A fixed bed reactor was fed with the sugar mixture at various hydraulic retention times (HRTs) ranging 12 to 1.5 h. The maximum hydrogen production rate of 52.6 L/L-d was found at 2 h HRT, while the maximum hydrogen yield of 2.3 +/- 0.1 mol/mol hexose(added), was achieved at 3 h HRT. Microbial communities and species distribution were analyzed via quantitative polymerase chain reaction (qPCR) and the dominant bacterial population was found as Clostridia followed by Lactobacillus sp. Packing material retained higher 16S rRNA gene copy numbers of total bacteria and Clostridium butyricum fraction compared to fermentation liquor. The finding of the study has demonstrated that H-2 production from galactose and glucose mixture could be a viable approach for hydrogen production. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.