Redox-centric metabolic rewiring for dark-fermentative hydrogen production in Enterobacter aerogenes

Dark-fermentative hydrogen is promising but constrained by acidification, mixed-acid by-products, and competition for reducing equivalents. Enterobacter aerogenes IAM1183, a rapidly growing and substrate-flexible facultative anaerobe, was used as the H₂-producing chassis. A unified benchmark was then established to systematically redirect flux along three levers: deleting the pyruvate formate-lyase-associated pflAB locus (ΔpflAB), enlarging reducing power/electron transfer by overexpressing pntA or fdx, and deleting phosphoenolpyruvate carboxylase ppc (Δppc) to probe pyruvate-node pressure. In 20 h glucose fermentations, H₂ yield increased by 38.0% in Ea/pntA, 32.8% in Ea/fdx, and 31.3% in ΔpflAB, whereas Δppc lowered H₂ yield by 10.5%. Intracellular NADH/NAD⁺ increased by 137% in Ea/pntA and by approximately 51% in ΔpflAB. In ΔpflAB, formate fell below HPLC detection, endpoint pH was 6.06 versus 4.70 in WT, and final biomass increased by approximately 50%, consistent with a strong apparent shift away from detectable formate-associated H₂ production under the present batch condition. Shifts toward acetoin and 2,3-butanediol across engineered strains indicate residual NADH sinks accompanying redox gains. Together, these side-by-side data distill actionable rules: alleviate acid load, expand reducing capacity, and constrain NADH sinks. They also nominate ΔpflAB combined with pntA or fdx overexpression as a promising design for future validation under pH-controlled and gas-removal conditions. This integrated evaluation clarifies how de-acidification, redox reinforcement, and carbon redistribution jointly reshape endpoint H₂ phenotypes in E. aerogenes, providing a coherent platform for future strain-and-process co-optimization of dark-fermentative biohydrogen production.