Temperature-driven resistome and functional profiling in AD reactors using metagenome-assembled genomes

Abstract

Anaerobic digestion (AD) is a widely implemented biotechnological process for organic waste treatment and renewable energy recovery, yet temperature-specific, genome-resolved links among AD microbiomes, resistance traits, and metabolic functions remain poorly understood. This study contrasted how mesophilic (37℃) and thermophilic conditions (55℃) shape microbial community composition, functional capacity, and antibiotic resistance genes (ARGs). In total, 524 high-quality metagenome-assembled genomes (MAGs) (≥90% completeness, ≤10% contamination) were reconstructed from 21 metagenomic datasets spanning both temperature regimes. Mesophilic conditions supported greater taxonomic diversity across 40 phyla and yielded significantly more MAGs per sample (mean: 39.1 vs 17.9), with Proteobacteria particularly Enterobacter dominating as the primary carriers of multidrug resistance, β-lactam, and aminoglycoside resistance genes. Thermophilic MAGs harbored substantially more SOS-related proteins (305 vs 118 in mesophilic MAGs), indicating stronger responses to DNA damage under thermal stress. They also encoded more specialized carbohydrate degrading enzymes linked to efficient biomass hydrolysis. KEGG pathway analysis showed that core AD metabolic pathways were conserved across both temperature regimes. However, the dominant microbial hosts differed, with Clostridium and Methanosaeta enriched under mesophilic conditions and Thermoanaerobacter and Methanothermobacter enriched under thermophilic conditions. Overall, mesophilic conditions favored broader resistome diversity and metabolic redundancy, while thermophilic conditions favored specialized stress tolerance functions. These findings demonstrate that temperature acts as a key ecological driver of AD microbiome structure and function, with direct implications for reactor performance optimization, digestate biosafety assessment, and the management of ARGs dissemination in wastewater treatment systems.