Limosilactobacillus Regulating Microbial Communities to Overcome the Hydrolysis Bottleneck with Efficient One‐Step Co‐Production of H₂ and CH₄

Abstract The efficient co‐production of H 2 and CH 4 via anaerobic digestion (AD) requires separate stages, as it cannot yet be achieved in one step. Lactic acid bacteria (LAB) ( Limosilactobacillus ) release H 2 and acetate by enhancing hydrolysis, potentially increasing CH 4 production with simultaneous H 2 accumulation. This study investigated the enhanced effect of one‐step co‐production of H 2 and CH 4 in AD by LAB and elucidated its enhancement mechanisms. The results showed that 236.3 times increase in H 2 production and 7.1 times increase in CH 4 production are achieved, resulting in profits of 469.39 USD. Model substrates lignocellulosic straw, sodium acetate, and H 2 confirmes LAB work on the hydrolysis stage and subsequent sustainable volatile fatty acid production during the first 6 days of AD. In this stage, the enrichment of Limosilactobacillus carrying bglB and xynB , the glycolysis pathway, and the high activity of protease, acetate kinase, and [FeFe] hydrogenase, jointly achieved rapid acetate and H 2 accumulation, driving hydrogenotrophic methanogenesis dominated. From day 7 to 24, with enriched Methanosarcina , and increased methenyltetrahydromethanopterin hydrogenase activity, continuously produced acetate led to the mainly acetoclastic methanogenesis shift from hydrogenotrophic methanogenesis. The power generation capacity of LAB‐enhanced AD is 333.33 times that of China's 24,000 m 3 biogas plant.

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