Identifying active diazotrophs in deep-sea sediment

Recent molecular and isotopic evidence suggests that diazotrophs are present and active in deep-sea sediment. However, their diversity and identity remain uncharacterized. Here, we analyzed ¹⁵N₂-incubated sediment collected at 2,893 m water depth off the coast of California, USA, using a combination of nifH amplicon sequencing and density gradient-based stable isotope probing (¹⁵N-SIP) to 1) assess the diversity of deep-sea nifH sequences with greater sequencing depth than previously employed, 2) identify nifH-containing microorganisms, and 3) identify the active diazotrophs. We detected diverse nifH sequences – 434 unique sequence variants from a single sediment core – and inferred host identity using a novel pipeline for nifH-host assignment. Deltaproteobacteria were the most abundant nifH-containing microbes and also comprised the majority of the active diazotrophs. Many lower abundance, highly diverse nifH sequences were also recovered, and included sequences closely related to those from Acidobacteria and Firmicutes. ¹⁵N-SIP indicated that many of these lower abundance nifH-containing groups were also actively fixing nitrogen. The putative diazotrophs’ closest characterized relatives suggests diverse catabolisms might fuel diazotrophy in the deep sea, with sulfur, iron, nitrate, and oxygen all serving as potential terminal electron acceptors. Such catabolic diversity may confer resilience during environmental perturbations, and enable diazotrophy to serve as a robust source of fixed nitrogen in the deep seafloor.