Abstract

Nitrogen fixation, the microbial conversion of N₂ to NH₃ (i.e., diazotrophy), is the largest natural source of bioavailable nitrogen to the biosphere. Previous work has demonstrated the unexpected occurrence of N₂ fixation in deep-sea sediments, particularly at sites of elevated carbon loading, including methane seeps, whale falls, and oxygen minimum zones.  However, the organisms responsible for this diazotrophic activity are largely unknown. Here, we investigated diazotroph identity, diversity and activity within marine sediment (0-3 and 9-12 cmbsf) collected at 2893 m water depth within Monterey Canyon off the coast of San Francisco, CA. Through an analysis of nifH sequences, a key gene in nitrogen fixation, and a density-gradient stable-isotope-probing experiment (¹⁵N-DNA-SIP), we found evidence for a diverse assemblage of functional diazotrophs spanning multiple phylogenetic groups, including Deltaproteobacteria, Gammaproteobacteria, Planctomycetes, and Acidobacteria. Comparison to closest cultured representatives based on 16S rRNA identities suggests these putative diazotrophs are catabolically diverse. Such diversity may increase their collective resilience and ability to provide a sustained source of new nitrogen to the ecosystem. Additionally, we quantified the effect of sample preparation on isotopically-labelled archaeal cells for analysis by nanoscale secondary ion mass spectrometry (nanoSIMS), a technique to investigate activity of uncultured cells. This analysis revealed a greater effect of sample preparation than was previously reported, suggesting that previous estimates of microbial activity assessed by this method are underestimates. Together, our results reveal the identity of diverse diazotrophs in deep marine sediment, and improve our ability to quantify rates of activity in uncultured microbial cells.