Keiner Robert, Herrmann Martina, Küsel Kirsten, Popp Jürgen, Frosch Torsten
Institute of Physical Chemistry, Friedrich Schiller University Jena, Jena 07743, Germany; Leibniz Institute of Photonic Technology, Jena 07745, Germany.
Institute of Ecology, Friedrich Schiller University Jena, Jena 07743, Germany; German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Leipzig 04103, Germany.
Anal Chim Acta. 2015 Mar 15;864:39-47. doi: 10.1016/j.aca.2015.02.007. Epub 2015 Feb 12.
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.
到目前为止，由于同时测量诸如N₂和N₂O等气体存在困难，对氮循环变化进行全面研究一直具有挑战性。在本研究中，我们引入了腔增强拉曼气体光谱技术，作为一种新的分析方法，用于追踪反硝化细菌斯氏假单胞菌对¹⁵N标记硝酸盐的逐步还原过程。拉曼多气体分析的独特能力能够对相关气体（¹⁴N₂、¹⁴N₂O、O₂和CO₂）进行实时、连续且非消耗性的定量，并通过单次测量追踪添加到斯氏假单胞菌培养物中的¹⁵N标记硝酸盐底物（¹⁵N₂、¹⁵N₂O）的去向。使用这种新方法，我们能够量化所有气态化合物（反应物和产物）的形成和降解动力学，从而研究反应级数。气体定量分析辅以硝酸盐和亚硝酸盐浓度分析，以在线监测总氮元素预算。所有气体的同时定量分析还能够通过反硝化过程中的氧化还原反应化学计量和CO₂ - 碳酸氢盐平衡，非接触式且无菌地在线获取斯氏假单胞菌培养物中的pH变化。连续的pH监测（无需将电极插入溶液）揭示了例如pH值斜率的增加与亚硝酸盐的积累相吻合，这进而由于一氧化二氮还原酶的抑制导致了N₂O的暂时积累。腔增强拉曼气体光谱技术在评估反硝化过程方面具有很高的潜力，并且可以极大地有助于我们理解自然和农业系统中的氮循环。
