Simplified bacterial denitrification method with Stenotrophomonas nitritireducens for nitrite dual isotope analysis in low concentration environmental samples

SUSHMITA DEB ^* Dominika Lewicka-Szczebak

• Institute of Geological Sciences, University of Wrocław, Wrocław, Silesian, Poland

This study presents a simplified and optimized bacterial denitrification method using Stenotrophomonas nitritireducens for the precise nitrite isotope analysis in low-concentration environmental samples. The improved method reduces the bacterial cultivation period from approximately 3–4 weeks to just 24 hours. Additionally, it allows for reliable nitrite analyses at concentrations as low as 150 nmol NO2- L-1, enabling analyses at concentrations of about one order of magnitude lower than previous methods.Three treatments were tested to evaluate their impact on isotopic precision and accuracy: Treatment 1 used a direct incubation approach, Treatment 2 incorporated an additional growth step of re-inoculation of the bacterial culture into fresh medium, and Treatment 3 included a 24-hour stabilization step at 4°C after the initial incubation. The method was validated using internal standards and applied to environmental samples, achieving good precision. Isotope ratio mass spectrometry (IRMS) measurements demonstrated superior accuracy for Treatment 1, with mean accuracies of ±0.7‰ for δ¹⁵N and ±0.4‰ for δ¹⁸O, while Treatment 2 (±2.0‰ for δ¹⁵N and ±1.7‰ for δ¹⁸O) and Treatment 3 (±1.8‰ for δ¹⁵N and ±4.3‰ for δ¹⁸O) showed lower precision. Among the treatments, Treatment 1 delivered the most accurate and reproducible results, showing minimal deviations of the measured N2O values from the true nitrite values. The oxygen isotope exchange between water and NO₂⁻ during bacterial conversion was in the range from 7 to 16%, which is similar to previous methods. This study integrates advanced analytical tools, such as laser spectroscopy and isotope ratio mass spectrometry, enabling reliable isotopic measurements even at trace nitrite concentrations. IRMS offered higher precision for high concentrations, while laser spectroscopy was better suited for repeated measurements at trace levels in low-concentration samples. The enhancements in cultivation efficiency, detection sensitivity, and precision make this approach highly valuable for environmental studies, especially in tracing nitrogen transformations in soil and water systems.