The existence of life in the deep terrestrial subsurface is established, yet few studies have investigated the origin of nitrogen that supports deep life. Previously, 16S rRNA gene surveys cataloged a diverse microbial community in subsurface fluids draining from boreholes 3000 feet deep at Henderson Mine, CO, USA (Sahl et al., 2008). The prior characterization of the fluid chemistry and microbial community forms the basis for the further investigation here of the source of NH(4) (+). The reported fluid chemistry included N(2), NH(4) (+) (5-112 μM), NO(2) (-) (27-48 μM), and NO(3) (-) (17-72 μM). In this study, the correlation between low NH(4) (+) concentrations in dominantly meteoric fluids and higher NH(4) (+) in rock-reacted fluids is used to hypothesize that NH(4) (+) is sourced from NH(4) (+)-bearing biotite. However, biotite samples from the host rocks and ore-body minerals were analyzed by Fourier transform infrared (FTIR) microscopy and none-contained NH(4) (+). However, the nitrogenase-encoding gene nifH was successfully amplified from DNA of the fluid sample with high NH(4) (+), suggesting that subsurface microbes have the capability to fix N(2). If so, unregulated nitrogen fixation may account for the relatively high NH(4) (+) concentrations in the fluids. Additionally, the amoA and nxrB genes for archaeal ammonium monooxygenase and nitrite oxidoreductase, respectively, were amplified from the high NH(4) (+) fluid DNA, while bacterial amoA genes were not. Putative nitrifying organisms are closely related to ammonium-oxidizing Crenarchaeota and nitrite-oxidizing Nitrospira detected in other subsurface sites based upon 16S rRNA sequence analysis. Thermodynamic calculations underscore the importance of NH(4) (+) as an energy source in a subsurface nitrification pathway. These results suggest that the subsurface microbial community at Henderson is adapted to the low nutrient and energy environment by their capability of fixing nitrogen, and that fixed nitrogen may support subsurface biomass via nitrification.
Keywords: Crenarchaeota; Nitrospira; archaeal ammonia oxidation; nitrite oxidation; nitrogen fixation; subsurface.