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.

在以碳为主的冷泉环境中微生物介导的氮循环仍然知之甚少。到目前为止，厌氧甲烷氧化古菌 (ANME-2) 及其硫酸盐还原细菌伙伴 (SEEP-SRB1 分支) 已被确定为深海冷泉沉积物中的固氮微生物。然而，尚不清楚其他微生物群是否可以在此类生态系统中进行固氮。为了填补这一空白，我们分析了 61 个宏基因组、1428 个宏基因组组装的基因组以及来自 11 个全球分布的冷渗漏的 6 个宏转录组。这些沉积物含有系统发育上多样化的固氮酶基因，对应于固氮营养谱系的扩大多样性。预计多种分解代谢途径可以为固氮提供 ATP，这表明冷泉中的固氮作用不一定与依赖于硫酸盐的甲烷厌氧氧化有关。冷泉中各种固氮菌群的固氮基因被推断具有遗传流动性并受到纯化选择的影响。我们的研究结果将固氮菌的能力扩展到五个候选门（Altarchaeia、Omnitropicota、FCPU426、Caldatribacteriota 和 UBA6262），并表明冷泉固氮菌可能对全球氮平衡做出了重大贡献。