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Hydrogen (H 2 ) is one of the most promising fuels for sustainable energy production. Microbial bio-H 2 fermentation is considered to be one of the environment friendly alternatives for satisfying the demand for fossil fuels. The study has aimed to isolate the effective bio-H 2 producing microbes from rice mill wastewater (RMWW) using various pre-treatment enrichment methods.Among the 27 enriched isolates, three isolates produced higher H 2 utilizing rice mill wastewater as the substrate. Molecular level identification revealed that the three bacterial strains were Bacillus thuringiensis-RH1, Bacillus aerophilus-RH2, and Bacillus beringensis-AARAT1. Optimization studies revealed that B. thuringiensis-RH1 would be a promising bio-H 2 producing bacterium with a yield of 0.96 ± 0.06 mol H 2 / mol glucose utilizing 50% of rice mill wastewater at pH 5.5. Furthermore, the obtained optimized substrate concentration (50%) and pH (5.5) from batch fermentation were used in the 3 L batch bioreactor experiment using B. thuringiensis-RH1. A higher hydrogen yield (HY) of 1.63 ± 0.14 mol H 2 /mol glucose using B. thuringiensis-RH1 at 72 hours was obtained in the extensive scale up process.

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