Hydrogen (H
2) is one of the most promising renewable energy sources, anaerobic bacterial H
2 fermentation is considered as one of the most environmentally sustainable alternatives to meet the potential fossil fuel demand. Bio-H
2 is the cleanest and most effective source of energy provided by the dark fermentation utilizing organic substrates and different wastewaters. In this study, the bio-H
2 production was achieved by using the bacteria
Acinetobacter junii-AH4. Further, optimization was carried out at different pH (5.0–8.0) in the presence of wastewaters as substrates (Rice mill wastewater (RMWW), Food wastewater (FWW) and Sugar wastewater (SWW). In this way, the optimized experiments excelled with the maximum cumulative H
2 production of 566.44 ± 3.5 mL/L (100% FWW at pH 7.5) in the presence of
Acinetobacter junii-AH4. To achieve this, a bioreactor (3 L) was employed for the effective production of H
2 and
Acinetobacter junii-AH4 has shown the highest cumulative H
2 of 613.2 ± 3.0 mL/L, HPR of 8.5 ± 0.4 mL/L/h, HY of 1.8 ± 0.09 mol H
2/mol glucose. Altogether, the present study showed a COD removal efficiency of 79.9 ± 3.5% by utilizing 100% food wastewater at pH 7.5. The modeled data established a batch fermentation system for sustainable H
2 production. This study has aided to achieve an ecofriendly approach using specific wastewaters for the production of bio-H
2.
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