Parallel denitrification and nitrite oxidation in the unsaturated zone: Isotopic constraints from nitrate δ15N and δ18O in Tianjin, China

• Nitrate reduction drives isotope trajectory variation. • Carbon properties regulate N functional gene expression. • The Δδ 1 8 O:Δδ 1 5 N trajectory is effective for assessing denitrification performance. Denitrification plays a critical role in mitigating anthropogenic nitrate (NO 3 - ) accumulation in ecosystems. The isotopic composition of NO 3 - (δ 15 N and δ 18 O) serves as a powerful tracer for identifying N sources and transformation processes. Denitrification often superimposed on the isotope effects of NO 2 - oxidation, resulting in parallel enrichment of δ 15 N- and δ 18 O-NO 3 - (Δδ 18 O:Δδ 15 N trajectory) that causes them to be either below or above 1. This study compared the Δδ 18 O:Δδ 15 N trajectory during denitrification, functional genes ( narG, napA , and nxrA ), and carbon sources from metabolites in the Δδ 18 O:Δδ 15 N trajectories below or above 1 in unsaturated zones. The results revealed that NO 3 - reduction was more important for variation in the Δδ 18 O:Δδ 15 N trajectory because the difference in isotope effects ( 15 ɛ NO3 reduction and 18 ɛ NO3 reduction ) between the two Δδ 18 O:Δδ 15 N trajectory groups was significant, whereas the difference in isotope effects ( 15 ɛ nxr and 18 ɛ nxr ) upon NO 2 - oxidation was not. Carbon sources in the group with Δδ 18 O:Δδ 15 N trajectories below 1 facilitated more efficient electron production to promote NO 3 - reduction because of their low molecular weight and simple structure. Conversely, the lower electron production efficiency due to the high molecular weight and complex structures of carbon sources in the group with Δδ 18 O:Δδ 15 N trajectories above 1 downregulated the expression of the three functional genes ( narG, napA , and nxrA ). The group with Δδ 18 O:Δδ 15 N trajectories below 1 showed significantly higher levels of 15 ɛ NO3 reduction , 18 ɛ NO3 reduction , NO 2 - oxidation ratio, and copy numbers of narG, napA , and nxrA genes compared to the other group, revealing that NO 3 - reduction at the cellular level was more active in the former group. This study elucidated the integrated influence of isotope effects, NO 3 - reductase and NO 2 - oxidoreductase activities, and carbon sources from metabolites. These findings are significant for understanding the Δδ 18 O:Δδ 15 N trajectories of N cycling in terrestrial ecosystems and support groundwater conservation by improving carbon supplementation approaches that stimulate denitrification, with Δδ 18 O:Δδ 15 N trajectories serving as effective tracers for assessing denitrification performance in terrestrial environments.