The aim of this research was to find out the effect of substrate concentrations (COD) of vinasse and the length of fermentation time to bio-hydrogen gas production using agent fermentation of photosynthetic bacteria, Rhodobium marinum. The production of bio-hydrogen was examined by varying COD of vinasse (10,000; 20,000; 30,000; 40,000; 50,000 mg COD/L) at certain fermentation time in the third, sixth and ninth day. The highest Hydrogen gas was obtained at ninth day of fermentation (82.66±18.6 mL). The highest Hydrogen Production Rate (HPR) and COD removal rate were obtained at concentration 50,000 mg COD/L, namely 109.98 mL H2/L/d and 1437.66 mg COD/L/d, respectively. Thus it can be concluded, the concentration of substrates (COD) from vinasse and the length of fermentation time have an effect on production of bio-hydrogen gas using Rhodobium marinum
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