RESUMO
Nitrogen (N) is an essential element for life. N compounds such as ammonium ( NH 4 + ) may act as electron donors, while nitrate ( NO 3 - ) and nitrite ( NO 2 - ) 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+- HCO 3 - type fluids comprised mostly of NO 3 - , and Ca2+-OH- fluids comprised primarily of ammonia (NH3). We infer that fixed N is introduced to the serpentinite aquifer as NO 3 - . High concentrations of NO 3 - (>100 µM) with a relict meteoric oxygen isotopic composition (δ18O ~ 22, Δ17O ~ 6) were observed in shallow aquifer fluids, indicative of NO 3 - sourced from atmospheric deposition (rainwater NO 3 - : δ18O of 53.7, Δ17O of 16.8) mixed with NO 3 - 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 NO 3 - detectable. We interpret that NH3 in hyperalkaline fluids is a product of NO 3 - reduction. The proportionality of the O and N isotope fractionation (18ε / 15ε) measured in Samail Ophiolite NO 3 - was close to unity (18ε / 15ε ~ 1), which is consistent with dissimilatory NO 3 - 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 NO 3 - . Production of NH 4 + as the end-product of NO 3 - 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.
RESUMO
Dissimilatory nitrate reduction (DNR) to nitrite is the first step in denitrification, the main process through which bioavailable nitrogen is removed from ecosystems. DNR is catalyzed by both cytosolic (Nar) and periplasmic (Nap) nitrate reductases and fractionates the stable isotopes of nitrogen (14N, 15N) and oxygen (16O, 18O), which is reflected in residual environmental nitrate pools. Data on the relationship between the pattern in oxygen vs nitrogen isotope fractionation (18ε/15ε) suggests that systematic differences exist between marine and terrestrial ecosystems that are not fully understood. We examined the 18ε/15ε of nitrate-reducing microorganisms that encode Nar, Nap, or both enzymes, as well as gene deletion mutants of Nar and Nap to test the hypothesis that enzymatic differences alone could explain the environmental observations. We find that the distribution of 18ε/15ε fractionation ratios of all examined nitrate reductases forms two distinct peaks centered around an 18ε/15ε proportionality of 0.55 (Nap) and 0.91 (Nar), with the notable exception of the Bacillus Nar reductases, which cluster isotopically with the Nap reductases. Our findings may explain differences in 18ε/15ε fractionation between marine and terrestrial systems and challenge current knowledge about Nar 18ε/15ε signatures.
