Phylogenetic assembly of methanogenesis regulates methane yield in food-waste anaerobic digestion

Anaerobic digestion (AD) of food waste (FW) is a key wate-to-energy strategy, yet daily biogas yield is often challenging to sustain, partly due to a limited understanding of the internal methanogens and their functional divergence. Here, we investigated seven full-scale mesophilic FW-AD systems distributed across China along a broad latitudinal gradient (>2,800 km), linking methane production variations (0.38-2.11 m3/m3•d-1) with the phylogenetic distributions of methanogens and their methanogenic genes. We found that hydrogenotrophic and aceticlastic pathways were ubiquitous, whereas methylotrophic methanogenesis showed regional enrichment in warmer regions, reflecting persistent influences of climate-associated upstream conditions on downstream methanogenic communities. Gene-level phylogeny of methanogenesis-related alleles, rather than species-level phylogeny, closely tracked biogas yield variation (Mantel's P < 0.05) and showed consistently stronger associations than gene-level compositions (mean standardized total effect: 0.491 vs. 0.298, P < 0.01). Higher methane yields (1.61 vs. 0.61 m3/m3•d-1 in high- vs. low-performing systems, P < 0.01) were significantly associated with reduced Faith's phylogenetic diversity (1.82 vs. 2.30, P < 0.01) and tighter clustering (mean pairwise phylogenetic distance, MPD: 0.25 vs. 0.30, P < 0.01) of methanogenic gene variants, suggesting that phylogenetic coherence may reflect ecological filtering favoring efficient methanogenesis, albeit at the expense of functional redundancy. These findings highlight gene-level trait phylogeny as a potential proxy for functional robustness, offering a framework for ecological design of AD microbiomes.