Enhanced isotopic approach combined with microbiological analyses for more precise distinction of various N-transformation processes in contaminated aquifer – a groundwater incubation study

Abstract. This study explores nitrogen transformations in groundwater from an agricultural area utilizing organic fertilizer (wastewaters from yeast production) integrating isotopic analysis, microbial gene abundance, and the FRAME (isotope FRactionation And Mixing Evaluation) 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 PCR (qPCR), revealed a shift from archaeal-driven nitrification to bacterial denitrification in post-incubation suboxic conditions, stimulated by glucose addition. FRAME modeling further identified bacterial denitrification (bD) as the dominant pathway of N₂O production, which was supported by increased nosZI, nirK and nirS gene abundance and observed isotope effects.

Simultaneously to 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 minor contribution in 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.