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

doi:10.1016/j.jia.2026.01.010

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

Dongmei Xue^1*, Jinglei Wang^1*, Lanxin Xiang¹, Xiaoxian Peng¹, Ke Jin^2#, Yunting Fang^3#, Xiangzhen Li⁴, Yidong Wang¹, Zhongliang Wang⁵

¹Tianjin Key Laboratory of Water Resources and Environment, Tianjin Normal University, Tianjin 300387, China

²Institute of Grassland Research, Chinese Academy of Agricultural Sciences, Huhhot 010010, China

³Key Laboratory of Forest Ecology and Management, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang110016, China

⁴College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China

⁵Tianjin Key Laboratory of Aquatic Science and Technology, Tianjin Chengjian University, Tianjin 300384, China

Highlight

l Nitrate reduction drives isotope trajectory variation.

l Carbon properties regulate N functional gene expression.

l The Δδ¹⁸O:Δδ¹⁵N trajectory is effective for assessing denitrification performance.

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摘要

反硝化作用是缓解生态系统人为硝酸盐（NO₃^-）累积的关键过程，而NO₃^-同位素组成（δ¹⁵N 与 δ¹⁸O）是识别氮来源与转化过程的有效示踪手段。反硝化过程常叠加亚硝酸盐（NO₂^-）氧化产生的同位素效应，导致δ¹⁵N-NO₃⁻与δ¹⁸O-NO₃⁻同步富集，形成低于或高于1的Δδ¹⁸O:Δδ¹⁵N轨迹。本研究以非饱和带为研究对象，比较了Δδ¹⁸O:Δδ¹⁵N轨迹低于或高于1两组条件下，反硝化过程中的Δδ¹⁸O:Δδ¹⁵N轨迹变化、功能基因（narG、napA、nxrA）及代谢碳源特征。结果表明，NO₃^-还原是导致Δδ¹⁸O:Δδ¹⁵N轨迹变异的主要因素，两组轨迹间NO₃^-还原同位素效应（¹⁵ε_{NO3 reduction}与¹⁵ε_{NO3 reduction}）差异显著，而NO₂^-氧化过程的同位素效应（¹⁵ε_nxr和 ¹⁸ε_nxr）无显著差异。在Δδ¹⁸O:Δδ¹⁵N轨迹低于1的组别中，碳源因其低分子量与简单结构，有助于更高效的电子产率，从而促进NO₃^-还原；相反，在轨迹高于1的组别中，碳源的高分子量与复杂结构导致电子产率降低，进而下调三个功能基因（narG、napA、nxrA）的表达。轨迹低于1的组别在¹⁵ε_{NO3 reduction}、¹⁵ε_{NO3 reduction}、亚硝酸盐氧化比例以及narG、napA与nxrA基因拷贝数方面均显著高于另一组，表明该组在细胞水平的NO₃^-还原活性更强。本研究系统阐明了同位素效应、NO₃^-还原酶与NO₂^-氧化还原酶活性以及代谢碳源对反硝化过程的综合影响。该发现对理解陆地生态系统氮循环中Δδ¹⁸O:Δδ¹⁵N轨迹的形成机制具有重要意义，并为通过优化碳源添加策略以强化反硝化、实现地下水保护提供了理论依据，同时证明Δδ¹⁸O:Δδ¹⁵N轨迹可作为评估陆地环境反硝化效能的有效示踪指标。

Abstract

Denitrification plays a critical role in mitigating anthropogenic nitrate (NO₃^-) accumulation in JIA-2025-1634 Jinke slj ZR.docxecosystems. 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_nxrand 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.

Keywords: denitrification NO²-oxidation Δδ¹⁸O:Δδ¹⁵N trajectory NO³-reductase NO²-oxidoreductase carbon sources

Online: 13 January 2026

Fund:

This work was financially supported by the National Natural Science Foundation of China (42361144860, 41973017 and 32071861).

About author: Dongmei Xue, E-mail: xuedongmei@tjnu.edu.cn; Jinglei Wang, E-mail: 17861007620@163.com; #Correspondence Ke Jin, E-mail: jinke@caas.cn; Yunting Fang, E-mail: fangyt@iae.ac.cn * These authors contributed equally to this study.

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Dongmei Xue, Jinglei Wang, Lanxin Xiang, Xiaoxian Peng, Ke Jin, Yunting Fang, Xiangzhen Li, Yidong Wang, Zhongliang Wang. 2026. Parallel denitrification and nitrite oxidation in the unsaturated zone: isotopic constraints from nitrate δ¹⁵N and δ¹⁸O in Tianjin, China. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2026.01.010

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