Abstract: In this study, we use the two-step method to produce hydrogen by the anaerobic bacteria in dark fermentation and bacteria of photosynthetic fermentation, which have co-advantages and complementary synergy. The influences of different substrate concentrations at the anaerobic fermentation stage, the anaerobic fermentation time and photosynthetic microbial fermentation with fermentation time on hydrogen productin are investigated. The experiment showed that glucose concentration had a great influence on the production of anaerobic bio-hydrogen,e.g., glucose concentration of 15 g/L has better hydrogen production. The glucose utilization and total volatile fatty acid with the change of anaerobic fermentation time showed that during anaerobic fermentation using glucose as the substrate,the optimal glucose concentration is 15 g/L.Glucose utilization can reach up to 72.08% in 37 h, the total amount of volatie fatty acids reach up to 9 326.3 mg/L, and 182 mL/g of the cumulative hydrogen production can be obtained. After 37 h of anaerobic fermentation, the product of anaerobic fermentation is moved to photosynthetic fermentation reactor and inoculated with photosynthetic bacteria of growing logarithmic phase under the suitable pH value of the medium and the light sources for photosynthetic hydrogen production. 352 mL/g of the cumulative hydrogen production was obtained in 88 h. The total amount of hydrogen production reached 534 mL/g with this two-step method.

Key words: anaerobic hydrogen-producing was obtained bacteria, photosynthetic hydrogen-producing bacteria, two-step method combined with hydrogen production