An overlooked microbial pathway links organic nitrogen turnover in composts to nitrous oxide formation

Biological N ₂ O production from organic nitrogen is generally assumed to require canonical nitrification, which generates oxidized nitrogen that subsequently fuel denitrification. Whether this paradigm universally applies to nitrogen-rich microbial communities remains unclear. Here, we investigated N ₂ O production across an industrial poultry manure composting process and found that substantial N ₂ O formation occurred despite the apparent absence of canonical ammonia oxidation. Neither allylthiourea inhibition nor metagenomic analyses provided evidence for ammonia-oxidizing microorganisms or their activity. Instead, metagenomic analyses identified abundant bacterial nitric oxide synthase ( bNos ) genes, many of which were phylogenetically affiliated with Bacilli, the dominant bacterial group throughout composting. Physiological experiments with Bacillus isolates demonstrated a nitrification-independent route in which L-arginine was oxidized to NO ₂ ⁻/NO ₃ ⁻, consistent with bNOS-mediated NO formation followed by abiotic oxidation. Recovery of ¹⁵ N-labelled N ₂ O following ¹⁵ NO ₂ ⁻ addition established NO ₂ ⁻ as an immediate precursor of aerobically produced N ₂ O, confirming that the oxidized nitrogen generated through this alternative route subsequently fueled denitrification. Metagenomic analyses further revealed extensive denitrification potential but comparatively low nosZ abundance. Together, these findings identify a previously overlooked route linking organic nitrogen turnover to denitrification independently of canonical nitrification, thereby expanding current models of microbial N ₂ O production in composts and potentially other protein-rich thermophilic environments.