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

Biological N2O 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 N2O production across an industrial poultry manure composting process and found that substantial N2O 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 NO2-/NO3-, consistent with bNOS-mediated NO formation followed by abiotic oxidation. Recovery of 15N-labelled N2O following 15NO2- addition established NO2- as an immediate precursor of aerobically produced N2O, 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 N2O production in composts and potentially other protein-rich thermophilic environments.