From saccharides to methane: substrate-driven microbial diversity and methanogenesis in anaerobic digestion revealed by 390 MAGs

ABSTRACT Anaerobic digestion (AD) is a key technology for organic waste treatment and renewable energy production, yet its microbial processes remain poorly understood. This study employed genome-centric metagenomics to investigate synthetic AD systems fed with five substrates: starch, glucose, butyrate, propionate-valerate, and acetate. A total of 390 high-quality metagenome-assembled genomes (MAGs) were reconstructed, 64.36% of which represent potentially novel species. Substrate-driven selection shaped distinct functional consortia, with Bifidobacterium enriched in starch hydrolysis and Syntrophomonas enriched in butyrate oxidation. A complete set of acetoclastic methanogenesis-associated genes was identified in a bacterial MAG ( Armatimonadota UBA5829). A complete CO₂ reduction pathway was also identified in Methanothrix soehngenii . Glycine cleavage systems were prevalent in high-abundance MAGs, while key Wood-Ljungdahl pathway enzymes were absent. These findings provide genomic insights into substrate-driven assembly and methanogenic pathway distribution in synthetic AD systems. IMPORTANCE Anaerobic digestion is a key technology for converting organic waste into renewable energy, yet the microbial processes underlying its efficiency remain largely hidden. By reconstructing 390 microbial genomes from digestion systems fed with different sugar-derived substrates, this study unveils how specific substrates shape microbial communities and reveals unexpected metabolic pathways for methane production. These findings challenge long-standing assumptions about which organisms perform key steps in the process, provide a valuable genomic resource for the field, and offer concrete targets for improving waste-to-energy systems through microbiome engineering.