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[Display omitted] • From glucose, S. flexneri SPD1 showed production of 3.20 mol of H 2 /mol of hexose. • Green solvents-based pretreatment waste biomass yields up to 36.0 g/L of sugars. • The H 2 yield achieved up to 80 % of theoretic production under dark-fermentation. • Upscaled bioprocess yielded 3.06 mol of H 2 /mol of hexose from cotton hydrolysate. This study evaluated the dark-fermentative hydrogen (H 2) production potential of isolated and identified Shigella flexneri SPD1 from various pure (glucose, fructose, sucrose, lactose, and galactose) and biowastes (coconut coir, cotton fiber, groundnut shells, rice-, and wheat-straws)-derived sugars. Among sugars, S. flexneri SPD1 exhibited high H 2 production of up to 3.20 mol/mole of hexose using glucose (5.0 g/L). The pre-treatment of various biowastes using green solvents (choline chloride and lactic acid mixture) and enzymatic hydrolysis resulted in the generation of up to 36.0 g/L of sugars. The maximum H 2 production is achieved up to 2.92 mol/mol of hexose using cotton-hydrolysate. Further, the upscaling of bioprocess up to 5 L of capacity resulted in a maximum yield of up to 3.06 mol/mol of hexose. These findings suggested that S. flexneri SPD1, a novel H 2 -producer, can be employed to develop a circular economy-based approach to produce clean energy. [ABSTRACT FROM AUTHOR]