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

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.