Porewater nitrate isotope dynamics in selected benthic habitats: Modulation by sediment reactivity and process coupling via nitrite

Denitrification is the primary pathway for fixed nitrogen (N) removal from aquatic systems. Its occurrence in sediments is promoted by suboxic conditions, high organic matter reactivity, sharp redox transitions, and the co-occurrence of multiple N-transformation processes. Benthic denitrification can be investigated through natural abundance nitrate N and oxygen (O) isotope ratio measurements to trace nitrate reduction and related N transformation processes within the sediment. Yet, while the biological and apparent (i.e., in the overlying water) isotope effects for nitrate reduction, ε Den , have been widely investigated, the controls on their expression at the sediment porewater level remain underexplored, particularly in lacustrine systems. Additionally, it is unclear how simultaneously occurring N-cycle processes may modify the resulting nitrate isotope signatures. Since apparent ε Den values are commonly used to estimate benthic denitrification, it is also crucial to identify how these processes affect their expression. Here, we measured nitrate isotope ratios in sediment porewaters from multiple lacustrine and marine settings and quantified denitrification, dissimilatory nitrate reduction to ammonium, and anammox rates using 15 N-tracer incubations. We further employed a novel diagenetic N isotope model to estimate ε Den under different sedimentary reactivity and redox conditions. In most investigated environments, Rayleigh-derived N and O isotope effects are strongly reduced (mostly < 4‰) in porewaters compared to reported biological isotope effects, whereas model-based estimates provide intermediate fractionations. We attribute the generally low isotope fractionation to high sediment reactivity and insufficient nitrate diffusion to the nitrate-reducing zone, leading to near-complete nitrate consumption at the microbial level (i.e., before the cellular level effect is expressed at the porewater level). Consistent with this interpretation, environments characterized by higher organic matter reactivity exhibited the lowest isotope fractionation. The N and O isotope values in nitrate exhibited partial decoupling, resulting in both negative and positive Δδ 18 O:Δδ 15 N deviations from 1:1. Negative deviations may reflect an enhanced contribution of the periplasmatic nitrate reductase, particularly in lacustrine environments with fluctuating O 2 concentrations. Positive deviations likely indicate nitrate (re)generation via nitrification or anammox, and tight nitrate-nitrite redox cycling (i.e., nitrate reduction coupled to nitrite oxidation). The spatial overlap of N transformations in our model results (e.g., nitrification–denitrification) increased the coupling of processes through nitrite, which strongly influences nitrate isotope dynamics. Model simulations indicate that nitrite-reducing processes generate 15 N-enriched nitrite due to N isotopic fractionation; subsequent nitrite oxidation, associated with an inverse kinetic isotope effect, produces highly 15 N-enriched nitrate. Such 15 N enrichment of nitrate should therefore not be interpreted as evidence for nitrate reduction and fixed-N loss. Our findings demonstrate that explicit consideration of nitrite dynamics is essential for correctly interpreting nitrate isotope data in sediment porewaters and for avoiding inaccurate estimation of benthic N transformation pathways and N-loss rates.