Phylogenetically and catabolically diverse diazotrophs reside in deep-sea cold seep sediments.

Microbially mediated nitrogen cycling in carbon-dominated cold seep environments remains poorly understood. So far anaerobic methanotrophic archaea (ANME-2) and their sulfate-reducing bacterial partners (SEEP-SRB1 clade) have been identified as diazotrophs in deep sea cold seep sediments. However, it is unclear whether other microbial groups can perform nitrogen fixation in such ecosystems. To fill this gap, we analyzed 61 metagenomes, 1428 metagenome-assembled genomes, and six metatranscriptomes derived from 11 globally distributed cold seeps. These sediments contain phylogenetically diverse nitrogenase genes corresponding to an expanded diversity of diazotrophic lineages. Diverse catabolic pathways were predicted to provide ATP for nitrogen fixation, suggesting diazotrophy in cold seeps is not necessarily associated with sulfate-dependent anaerobic oxidation of methane. Nitrogen fixation genes among various diazotrophic groups in cold seeps were inferred to be genetically mobile and subject to purifying selection. Our findings extend the capacity for diazotrophy to five candidate phyla (Altarchaeia, Omnitrophota, FCPU426, Caldatribacteriota and UBA6262), and suggest that cold seep diazotrophs might contribute substantially to the global nitrogen balance.