Nowadays, marine biomass is gradually considered as another utilizable material for the sustainable bioenergy development. In the present study, galactose, the main component of agar polysaccharide, was utilized for the biohydrogen production by Enterobacter sp. CN1. The highest hydrogen yield of 303.2 mL/g was obtained in the cultivation media containing 5.87 g/L of galactose, together with initial pH of 7.3 and incubation temperature of 36 degrees C, after the response surface methodology (RSM) analysis. After the saccharification process by the agarase (AgaXa) and neoagarobiose hydrolase (NH852), the agar hydrolysate obtained was further applied to generate biohydrogen by strain CN1. Under the synergistic enzymatic saccharification and fermentation process, the production of biohydrogen was obtained to be 5047 +/- 228 mL/L from 50 g/L of agar, resulting in 3.86-fold higher than the control without enzymatic pretreatment. (C) 2017 Elsevier Ltd. All rights reserved.