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 phylogenetically and metabolically diverse organisms spanning 18 bacterial and archaeal phyla (17 within seeps). Detected NCOs included methane-oxidising ANME-2 archaea and sulfate-reducing Desulfobacteraceae, which have been shown to fix nitrogen at seeps previously, as well as Desulfuromonadales and putatively hydrocarbon-oxidising Desulfoglaeba and Candidatus Methanoliparia. We detected nifH transcripts at five of the six seeps, suggesting widespread diazotrophic activity. 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 correlated with mcrA abundance, and, when corrected, was negatively correlated with porewater ammonium < 25 μM, consistent with the inhibition of diazotrophy by ammonium. Our findings expand the known diversity of NCOs at seeps and emphasise seeps as hotspots for deep-sea diazotrophy.