Display omitted •Lactobacillus lactis has been employed for biohydrogen production.•NiO/Fe2O3 addition stimulates hydrogen production over 18% than control.•NiO/Fe2O3 supplementation redirects butyrate mediated hydrogen metabolism.•Dehydrogenase activity enhances after nano-materials supplementation.•NAD+/NADH and FAD+/FADH2 pool increases by 19% and 17%. The positive interaction between Clostridium sp. and lactic acid-producing bacteria (Lactobacillus sp) is commonly seen in various high-rate hydrogen production systems. However, the exact role of the hydrogen production ability of Lactobacillus sp in a dark fermentation production system is rarely studied. Lactobacillus delbrueckii was herein used for the first time, to the best of the author’s knowledge, to demonstrate biohydrogen production under anaerobic conditions. At first, the pH condition was optimized, followed by the addition of nanoparticles for enhanced biohydrogen production. Under optimized conditions of pH 6.5, substrate concentration 10 g/L, and 100 mg/L of NiO/Fe2O3, the maximum hydrogen yield (HY) of 1.94 mol/mol hexose was obtained, which is 18 % more than the control. The enhanced H2 production upon the addition of nanoparticles is supported via the external electron transfer (EET) mechanism, which regulates the metabolic pathway regulation with increased production of acetate and butyrate and reduced formation of lactate.