In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46-0.53 g reducing sugar/g starch. Five H2-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H2 from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H2 production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H2 yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H2/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H2 production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H2 production rate increased from 250 to 534 ml/g VSS/h, whereas the H2 yield decreased from 2.03 to 1.50 mol H2/mol glucose. While operating at 2 h HRT, the volumetric H2 production rate reached a high level of 1.5 l/h/l.
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