Unraveling the biological mechanisms of biohydrogen production through dark fermentation using assembled genomes from metagenomic data.

Dark fermentation represents a sustainable and promising approach for biohydrogen generation. However, achieving high yields depends on understanding the complex microbial interactions driving the process. This study used genome-centric metagenomics to analyze microbial communities from 11 hydrogen-producing reactors. In total, 44 metagenome-assembled genomes (MAGs) were analyzed in detail. High-yield reactors demonstrated a strong synergy between hydrogen-producing bacteria (HPB) and lactic acid bacteria (LAB), particularly Clostridium butyricum and Clostridium beijerinckii. These species encode the electron-transferring flavoprotein-lactate dehydrogenase complex (EtfAB-ldh complex), enabling hydrogen production from lactic acid. In contrast, reactors with lower hydrogen yields exhibited a higher prevalence of hydrogenotrophic microorganisms, including homoacetogens and methanogens, which redirected electron flow toward competing pathways, thereby decreasing hydrogen output. These results emphasize the importance of promoting HPB while suppressing hydrogen consumers to maintain an optimal microbial community. By linking community composition with metabolic potential, this study provides a framework for improving reactor performance, increasing hydrogen yields, and advancing sustainable hydrogen production from organic waste streams.