Synergistic augmentation of the pyruvate pathway and microbial reducing power by L-theanine enhances cellulosic...

Dark fermentation hydrogen production (DFHP) is a promising route for biohydrogen generation; however, current optimization strategies primarily focus on physical and process parameters, with comparatively limited attention to metabolic regulation. Here, we investigated the effects and underlying mechanisms of L-theanine supplementation in a cellulose-based DFHP system. L-theanine significantly promoted H₂ production at an optimal concentration of 400 mg/L, increasing cumulative H₂ production by 94.82% and the substrate degradation rate by 48.60%. Mechanistically, L-theanine increased hydrogenase activity (50.41%), cellulase activity (22.85%), and electron transport system (ETS) activity (45.98%), respectively, thereby enhancing overall microbial activity. Intracellular reducing capacity was enhanced, as reflected by elevated NADH and NAD⁺ levels and a 57.26% increase in the NADH/NAD⁺ ratio. Integrated genomic and proteomic analyses further revealed that L-theanine mainly promoted H₂ synthesis by strengthening pyruvate metabolism and NADH-related electron transfer. Collectively, these findings demonstrate that L-theanine acts as an effective metabolic enhancer and provides a promising strategy for improving the efficiency of cellulose-based DFHP.