Rapid monitoring of intermediate states and mass balance of nitrogen during denitrification by means of cavity enhanced Raman multi-gas sensing

The comprehensive investigation of changes in N cycling has been challenging so far due to difficulties with measuring gases such as N2 and N2O simultaneously. In this study we introduce cavity enhanced Raman gas spectroscopy as a new analytical methodology for tracing the stepwise reduction of (15)N-labelled nitrate by the denitrifying bacteria Pseudomonas stutzeri. The unique capabilities of Raman multi-gas analysis enabled real-time, continuous, and non-consumptive quantification of the relevant gases ((14)N2, (14)N2O, O2, and CO2) and to trace the fate of (15)N-labeled nitrate substrate ((15)N2, (15)N2O) added to a P. stutzeri culture with one single measurement. Using this new methodology, we could quantify the kinetics of the formation and degradation for all gaseous compounds (educts and products) and thus study the reaction orders. The gas quantification was complemented with the analysis of nitrate and nitrite concentrations for the online monitoring of the total nitrogen element budget. The simultaneous quantification of all gases also enabled the contactless and sterile online acquisition of the pH changes in the P. stutzeri culture by the stoichiometry of the redox reactions during denitrification and the CO2-bicarbonate equilibrium. Continuous pH monitoring - without the need to insert an electrode into solution - elucidated e.g. an increase in the slope of the pH value coinciding with an accumulation of nitrite, which in turn led to a temporary accumulation of N2O, due to an inhibition of nitrous oxide reductase. Cavity enhanced Raman gas spectroscopy has a high potential for the assessment of denitrification processes and can contribute substantially to our understanding of nitrogen cycling in both natural and agricultural systems.