Macroalgae, commonly known as seaweed, are rich in carbohydrates which makes them a potential feedstock for biohydrogen production via dark fermentation. In this study, the green macroalgal biomass Ulva sp. was subjected to mild acid-thermal combined pretreatment for the effective release of fermentable sugars. Among the H2SO4 acid concentrations tested, 4% H2SO4 and 121 °C for 40 min attained the highest hydrolysis efficiency with a reducing sugar yield of 0.21 g RS/g biomass. The concentration of fermentation inhibitors furfural and 5-hydroxymethyl furfural were below 1 g/L. Using an initial reducing sugar concentration of 12 g/L and pH 5.5, Clostridium butyricum CGS5 achieved the highest cumulative hydrogen production (2340 mL/L), maximum hydrogen productivity (208.3 mL/L/h), and hydrogen yield (1.53 mol H2/mole RS). In continuous fermentation with 6 h hydraulic retention time, maximum hydrogen productivity increased to 782.45 mL/L/h with a hydrogen yield of 1.52 mol H2/mol hexose. To the best of our knowledge, we report for the first time, biohydrogen production via dark fermentation from green macroalgal biomass Ulva sp. with better yield and productivity.
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