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.

phosphorus acquisition strategy  /  soil zymography  /  microbial strategies  /  root exudate  /  hotspot areas