Abundance, identity, and potential diazotrophic activity of nifH-containing organisms at marine cold seeps

Diazotrophic microorganisms alleviate nitrogen limitation at marine cold seeps using nitrogenase, encoded in part by the gene nifH. Here, we investigated nifH-containing organisms (NCOs) inside and outside six biogeochemically heterogeneous seeps using amplicon sequencing and quantitative real-time PCR (qPCR) of nifH genes and transcripts. We detected nifH genes affiliated with anaerobic methane-oxidizing ANME-2 archaea and sulfate-reducing Desulfobacteraceae, consistent with previous studies, but also phylogenetically and likely metabolically diverse organisms, including Desulfoglaeba, Candidatus Methanoliparia, and Desulfuromonadales. In total, we recovered 10,734 bona fide nifH sequence variants affiliated with 18 bacterial and archaeal phyla (17 within seeps), a subset of which were transcribed at nearly all seeps investigated. We corrected our qPCR data based on our amplicon results, which found that 71% of recovered sequences were not bona fide nifH, and we recommend a similar correction in future qPCR studies that use broad nifH primers. NifH abundance was up to three orders of magnitude higher within seeps, was highly correlated with mcrA gene abundance, and, when corrected, was negatively correlated with porewater ammonium <25 uM, consistent with the inhibition of diazotrophy by ammonium. Our findings significantly expand the known diversity of NCOs at seeps and emphasize seeps as hotspots for deep-sea diazotrophy.