Subsurface biogeochemical cycling of nitrogen in the actively serpentinizing Samail Ophiolite, Oman

Nitrogen (N) is an essential element for life. N compounds such as ammonium ( $$ ) may act as electron donors, while nitrate ( $$ ) and nitrite ( $$ ) may serve as electron acceptors to support energy metabolism. However, little is known regarding the availability and forms of N in subsurface ecosystems, particularly in serpentinite-hosted settings where hydrogen (H₂) generated through water-rock reactions promotes habitable conditions for microbial life. Here, we analyzed N and oxygen (O) isotope composition to investigate the source, abundance, and cycling of N species within the Samail Ophiolite of Oman. The dominant dissolved N species was dependent on the fluid type, with Mg²⁺- $$ type fluids comprised mostly of $$ , and Ca²⁺-OH^- fluids comprised primarily of ammonia (NH₃). We infer that fixed N is introduced to the serpentinite aquifer as $$ . High concentrations of $$ (>100 μM) with a relict meteoric oxygen isotopic composition (δ¹⁸O ~ 22‰, Δ¹⁷O ~ 6‰) were observed in shallow aquifer fluids, indicative of $$ sourced from atmospheric deposition (rainwater $$ : δ¹⁸O of 53.7‰, Δ¹⁷O of 16.8‰) mixed with $$ produced in situ through nitrification (estimated endmember δ¹⁸O and Δ¹⁷O of ~0‰). Conversely, highly reacted hyperalkaline fluids had high concentrations of NH₃ (>100 μM) with little $$ detectable. We interpret that NH₃ in hyperalkaline fluids is a product of $$ reduction. The proportionality of the O and N isotope fractionation (¹⁸ε / ¹⁵ε) measured in Samail Ophiolite $$ was close to unity (¹⁸ε / ¹⁵ε ~ 1), which is consistent with dissimilatory $$ reduction with a membrane-bound reductase (NarG); however, abiotic reduction processes may also be occurring. The presence of genes commonly involved in N reduction processes (narG, napA, nrfA) in the metagenomes of biomass sourced from aquifer fluids supports potential biological involvement in the consumption of $$ . Production of $$ as the end-product of $$ reduction via dissimilatory nitrate reduction to ammonium (DNRA) could retain N in the subsurface and fuel nitrification in the oxygenated near surface. Elevated bioavailable N in all sampled fluids indicates that N is not likely limiting as a nutrient in serpentinites of the Samail Ophiolite.