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The biological conversion of into bioenergy offers a dual advantage of sustainable energy generation and effective waste management. Until now, the efficiency of dark fermentation processes has depended critically on optimized pre‑treatment strategies that unlock fermentable substrates from complex biowaste feedstocks. This study aims to evaluate and optimize multiple pre‑treatment approaches to enhance the simultaneous production of biohydrogen and bioethanol from biowaste. Among the tested methods, the integrated acid–enzymatic hydrolysis pathway released the highest reducing sugar content, increasing it by 39% by effectively depolymerizing lignocellulosic and polysaccharide‑rich fractions. The resulting hydrolysate produced the maximum biohydrogen and minimum bioethanol yields, reaching 97 mL/g and 5.12 mg/g, respectively, under dark fermentation conditions. Comparative assessments showed that ViL enzymatic hydrolysis and acid hydrolysis alone yielded lower biohydrogen yields of 88 mL/g and 75 mL/g, respectively. Overall, the findings highlight that integrating strategic pre-treatment with dark fermentation can serve as an efficient and sustainable route for biohydrogen production from biowaste.
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