N transformations in nitrate-rich groundwaters: combined isotope and microbial approach

Abstract. This study explores nitrogen transformations in groundwater from an agricultural area utilizing organic fertilizer (wastewater from yeast production) by integrating isotope analysis, microbial gene abundance, and the isotope FRactionation And Mixing Evaluation (FRAME) model to trace and quantify nitrogen cycling pathways. Groundwater samples with elevated nitrate concentrations were subjected to controlled laboratory incubations with application of a novel low-level 15N tracing strategy to investigate microbial processes. Isotope analyses of nitrate, nitrite, and nitrous oxide (N2O), coupled with microbial gene quantification via quantitative polymerase chain reaction (qPCR), revealed a shift from archaeal-driven nitrification to bacterial denitrification in post-incubation suboxic conditions, stimulated by glucose addition. FRAME modelling further identified bacterial denitrification as the dominant pathway of N2O production, which was supported by increased nosZI, nirK, and nirS gene abundance and observed isotope effects. Simultaneously with the intensive nitrate reduction, it was observed that the majority of nitrite is likely produced through nitrification processes linked to dissolved organic nitrogen (DON) oxidation. Nitrate reduction had a minor contribution to the total nitrite pool. The results demonstrate the efficacy of integrating multi-compound isotope studies and microbial analyses to unravel nitrogen cycling mechanisms. This approach provides a robust framework for addressing nitrogen pollution in groundwater systems and improving water quality management strategies.