Denitrification plays a critical role in mitigating anthropogenic nitrate (NO₃^–) accumulation in ecosystems. The isotopic composition of NO₃^– (δ¹⁵N and δ¹⁸O) serves as a powerful tracer for identifying N sources and transformation processes. Denitrification often superimposed on the isotope effects of NO₂^– oxidation, resulting in parallel enrichment of δ¹⁵N- and δ¹⁸O-NO₃^– (Δδ¹⁸O:Δδ¹⁵N trajectory) that causes them to be either below or above 1. This study compared the Δδ¹⁸O:Δδ¹⁵N trajectory during denitrification, functional genes (narG, napA, and nxrA), and carbon sources from metabolites in the Δδ¹⁸O:Δδ¹⁵N trajectories below or above 1 in unsaturated zones. The results revealed that NO₃^– reduction was more important for variation in the Δδ¹⁸O:Δδ¹⁵N trajectory because the difference in isotope effects (¹⁵ε_{NO3 reduction} and ¹⁸ε_{NO3 reduction}) between the two Δδ¹⁸O:Δδ¹⁵N trajectory groups was significant, whereas the difference in isotope effects (¹⁵ε_nxr and ¹⁸ε_nxr) upon NO₂^– oxidation was not. Carbon sources in the group with Δδ¹⁸O:Δδ¹⁵N trajectories below 1 facilitated more efficient electron production to promote NO₃^– 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 Δδ¹⁸O:Δδ¹⁵N trajectories above 1 downregulated the expression of the three functional genes (narG, napA, and nxrA). The group with Δδ¹⁸O:Δδ¹⁵N trajectories below 1 showed significantly higher levels of ¹⁵ε_{NO3 reduction}, ¹⁸ε_{NO3 reduction}, NO₂^– oxidation ratio, and copy numbers of narG, napA, and nxrA genes compared to the other group, revealing that NO₃^– reduction at the cellular level was more active in the former group. This study elucidated the integrated influence of isotope effects, NO₃^– reductase and NO₂^– oxidoreductase activities, and carbon sources from metabolites. These findings are significant for understanding the Δδ¹⁸O:Δδ¹⁵N trajectories of N cycling in terrestrial ecosystems and support groundwater conservation by improving carbon supplementation approaches that stimulate denitrification, with Δδ¹⁸O:Δδ¹⁵N trajectories serving as effective tracers for assessing denitrification performance in terrestrial environments.

Microbial denitrification and anaerobic ammonium oxidation (anammox) are two critical processes involved in nitrate ( $\mathrm{N}{\mathrm{O}}_3^{-}$) removal from riparian buffer zones. However, the responses of these processes to antibiotics used in nearby animal farms are not yet fully understood. In this study the effects of oxytetracycline (OTC) short-term exposure on microbial denitrification and anammox in riparian buffer zones were examined via slurry-based ¹⁵N tracer technique combined with network analysis. Short-term OTC exposure will significantly decrease denitrification and anammox performance by restricting related functional genes, but inhibitory upper will appear at high OTC contents. The dominant bacterial community did not significantly change, while the bacterial network became more complex to keep the network stable. Functional bacteria containing nosZ and hzsB genes underwent significant changes, manifested as inhibitory effects, promotion effects, and no significant effects. The results indicated that short-term OTC exposure seemed unable to affect all types of functional bacteria, resulting in a considerable part of bacteria maintaining certain nitrogen removal performance. In the bacteria containing nosZ gene, it was found that 22.2% are denitrification mode bacteria and 77.8% are non-denitrification mode bacteria, suggesting that the restoration of the denitrification process should rely on the maintenance of a diverse denitrifying community. These results provide scientific knowledge on the effect of antibiotics-containing wastewater on the excessive N loading in aquatic ecosystems, and also indicate that additional efforts are needed to control antibiotic pollution in riparian buffer zones and coastal ecosystems.