Biological nitrogen fixation in deep-sea and hydrothermal vent environments

Unsedimented mid-ocean ridge subseafloor environments are most likely limited by the availability of fixed nitrogen, based on the low nitrate and ammonium concentrations measured in venting fluids. Nitrogen-fixing microorganisms are able to reduce nitrogen gas, which is abundant in seawater and hydrothermal fluid, to biologically available ammonium. Nitrogen fixation was first hypothesized to occur in hydrothermal vent ecosystems in order to explain depleted 15N/14N ratios in hydrothermal vent animals compared to non-vent deep-sea animals. Genes that encode the enzyme responsible for nitrogen fixation (nifH) were recovered from diffuse hydrothermal fluids from Axial Volcano and Endeavour Segment on the Juan de Fuca Ridge, and, unexpectedly, from cold, nitrate-rich deep seawater from the axial valley of Endeavour Segment. These nifH genes are phylogenetically distinct and have dissimilar G+C content compared to those of hydrothermal vent archaea, suggesting that they belong to cold-adapted archaea that circulate through the nitrogen-poor subseafloor at the mid-ocean ridge. At the newly discovered Lost City hydrothermal field, we assessed carbonate chimneys and fluids venting from them for potential nitrogen-fixers and found significantly higher archaeal nifH diversity than expected from the extremely limited archaeal 16S rRNA diversity. In contrast to the diverse anaerobic bacterial nifH genes found in the subseafloor of the Juan de Fuca Ridge, very few bacterial nifH genes were detected at Lost City. The carbonates harbored more nifHdiversity than the fluids, and the hottest carbonate sample contained hyperthermophilic diazotrophs whereas the fluids did not. A methanogen isolated from hydrothermal fluid venting out of Axial Volcano was found to reduce N2 to NH 3 at up to 92°C, which is 28°C higher than the previous upper temperature limit of biological nitrogen fixation. The 16S rRNA gene of the hyperthermophilic nitrogen-fixer, designated FS406-22, is 99% similar to that of non-nitrogen fixing Methanocaldococcus jannaschii. At its optimal growth temperature of 90°C, FS406-22 incorporates 15N2 and expresses nifH mRNA. FS406-22 is the first reported nitrogen-fixer from a deep-sea hydrothermal vent, which expands the conditions for life in the nitrogen-limited subseafloor biosphere. Phylogenetic analysis of nitrogenase-regulating proteins from FS406-22 implies resemblance to an ancestral nitrogenase.