Nitrogen bioavailability, governed by the balance of fixation and loss processes, is a key factor regulating oceanic productivity, ecosystem functions, and global biogeochemical cycles. The key nitrogen loss organisms—denitrifiers and anaerobic ammonium-oxidizing (anammox) bacteria—are not well understood in marine seafloor environments, especially in deep-sea cold seeps. In this study, we combined geochemical measurements, ¹⁵N stable isotope tracer analysis, metagenomics, metatranscriptomics, and three-dimensional protein structural simulations to investigate the diversity of denitrifying and anammox microbial communities and their biogeochemical roles in these habitats. Geochemical evidence from 301 sediment samples shows significantly higher nitrogen loss rates in cold seeps compared to typical deep-sea sediments, with an estimated annual nitrogen loss of 6.16 Tg from seafloor surface sediments. Examination of a total of 147 million non-redundant genes reveals a high prevalence and active expression of nitrogen loss genes, including nitrous-oxide reductase (NosZ; 6.88 genes per million or GPM on average), nitric oxide dismutase (Nod; 1.29 GPM), and hydrazine synthase (HzsA; 3.35 GPM) in surface sediments. Analysis of 3,164 metagenome-assembled genomes from this habitat has expanded the known diversity of nitrous-oxide reducers to three phyla and nitric oxide-dismutating organisms to one phylum and two new orders, while ten phyla host anammox bacteria going beyond Planctomycetota. These microbes show diverse structural adaptations and complex gene cluster arrangements that potentially enable survival in the cold seeps. These findings suggest that cold seeps, despite their low temperatures, are significant, previously underestimated hotspots of nitrogen loss, potentially contribute substantially to the global nitrogen cycle.

Cold seeps; nitrogen-loss; nitrous-oxide reductase; nitric oxide dismutase; hydrazine synthase