Genome-Resolved insights into significance of DNRA Microbes in N₂O production during manure composting

Nitrous oxide (N₂O) production during manure composting has traditionally been attributed primarily to heterotrophic denitrification (HD), while the roles of alternative pathways remain poorly resolved. Using time-resolved multi-omics across 37 samples from various manure sources, our study investigated the transcriptional landscape of N₂O-producing pathways during composting. Microorganisms associated with dissimilatory nitrate reduction to ammonium (DNRA), including Fermentimonas and JAHWKS01 lineages, accounted for 21.2-33.1% of N₂O-producing gene expression-comparable to HD-revealing DNRA as a previously underappreciated source. DNRA-associated gene expression was regulated by viral factors, predominantly through lytic Caudoviricetes phages. Expanding our analysis to 174 public metagenomic datasets revealed that DNRA-derived N₂O-producing gene abundance peaked under static and hyperthermophilic conditions, highlighting aeration and temperature as critical mitigation controls. Furthermore, our study identified a substantial proportion of microorganisms harboring both DNRA and HD pathways. These findings refine mechanistic understanding of composting N₂O emissions and inform multi-pathway mitigation strategies.