Spatial distribution of nitrogen fixation in methane seep sediment and the role of the ANME archaea

Nitrogen (N₂) fixation was investigated at Mound 12, Costa Rica, to determine its spatial distribution and biogeochemical controls in deep‐sea methane seep sediment. Using ¹⁵N₂ tracer experiments and isotope ratio mass spectrometry analysis, we observed that seep N₂ fixation is methane‐dependent, and that N₂ fixation rates peak in a narrow sediment depth horizon corresponding to increased abundance of aggregates of anaerobic methanotrophic archaea (ANME‐2) and sulfate‐reducing bacteria (SRB). Using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH‐NanoSIMS), we directly measured ¹⁵N₂ uptake by ANME‐2/SRB aggregates (n = 26) and observed maximum ¹⁵N incorporation within ANME‐2‐dominated areas of the aggregates, consistent with previous analyses. NanoSIMS analysis of single cells (n = 34) from the same microcosm experiment revealed no ¹⁵N₂ uptake. Together, these observations suggest that ANME‐2, and possibly physically associated SRB, mediate the majority of new nitrogen production within the seep ecosystem. ANME‐2 diazotrophy was observed while in association with members of two distinct orders of SRB: Desulfobacteraceae and Desulfobulbaceae. The rate of N₂ fixation per unit volume biomass was independent of the identity of the associated SRB, aggregate size and morphology. Our results show that the distribution of seep N₂ fixation is heterogeneous, laterally and with depth in the sediment, and is likely influenced by chemical gradients affecting the abundance and activity of ANME‐2/SRB aggregates.