University of California, Riverside, Department of Botany and Plant Sciences, 900 University Avenue, Riverside, CA, 92521, USA.
Rapid Commun Mass Spectrom. 2014 Mar 15;28(5):520-6. doi: 10.1002/rcm.6824.
Previous research has shown that the denitrifying bacteria Pseudomonas chlororaphis ssp. aureofaciens (P. aureofaciens) can be used to measure the δ(15)N and δ(18)O values of extracted soil nitrate (NO3(-)) by isotope ratio mass spectrometry. We discovered that N2O production from reference blanks made in 1 M KCl increased relative to blanks made of deionized water (DIW). Further investigation showed that isotopic standards made in KCl yielded δ(15)N and δ(18)O values different from the standards prepared in DIW.
Three grades of crystalline KCl were dissolved in DIW to create solutions of increasing molarity (0.1 M to 2 M), which were added to P. aureofaciens broth and measured as blanks. Reference standards USGS-32, USGS-34, and USGS-35 were then dissolved in a range of KCl concentrations to measure isotopic responses to changing KCl molarity. Reference blanks and standards created in DIW were analyzed as controls to measure the impact of KCl on the δ(15)N and δ(18)O values.
The amount of N2O in the KCl blanks increased linearly with increasing molarity, but at different rates for each KCl grade. The isotopic values of the reference standards measured in KCl were systematically different from those measured in DIW, suggesting contamination by background NO3(-) in the KCl reagents. However, we also noted reduced conversion of NO3(-) into N2O as the KCl molarity increased, suggesting there is a physiological response of P. aureofaciens to KCl.
There is a small amount of NO3(-) present in crystalline KCl, which can bias isotopic measurement of NO3(-) at low sample concentrations. This can be minimized by making standards and blanks in the same KCl as is used in samples, diluting all samples and standards to the appropriate NO3(-) concentration using matched KCl solutions, and adding samples and standards to the broth at a constant volume to standardize the KCl molarity in the reaction vial.
先前的研究表明，脱氮假单胞菌亚种铜绿假单胞菌（P. aureofaciens）可用于通过同位素比质谱测量提取土壤硝酸盐（NO3(-)）的 δ(15)N 和 δ(18)O 值。我们发现，相对于用去离子水（DIW）制成的空白对照，用 1 M KCl 制成的参考空白的 N2O 产量增加。进一步的研究表明，在 KCl 中制备的同位素标准品产生的 δ(15)N 和 δ(18)O 值与在 DIW 中制备的标准品不同。
将三种等级的结晶 KCl 溶解在 DIW 中，制成摩尔浓度逐渐增加的溶液（0.1 M 至 2 M），然后将其加入 P. aureofaciens 肉汤中并作为空白对照进行测量。然后将 USGS-32、USGS-34 和 USGS-35 参考标准品溶解在一系列 KCl 浓度中，以测量对 KCl 摩尔浓度变化的同位素响应。用 DIW 制成的参考空白和标准品作为对照进行分析，以测量 KCl 对 δ(15)N 和 δ(18)O 值的影响。
KCl 空白对照中的 N2O 量随摩尔浓度的增加呈线性增加，但每种 KCl 等级的增加速率不同。在 KCl 中测量的参考标准品的同位素值与在 DIW 中测量的同位素值系统不同，这表明 KCl 试剂中的背景 NO3(-)存在污染。然而，我们还注意到，随着 KCl 摩尔浓度的增加，NO3(-)转化为 N2O 的量减少，这表明 P. aureofaciens 对 KCl 存在生理反应。
结晶 KCl 中存在少量的 NO3(-)，这可能会在低样品浓度下对 NO3(-)的同位素测量产生偏差。通过在与样品相同的 KCl 中制备标准品和空白对照、使用匹配的 KCl 溶液将所有样品和标准品稀释至适当的 NO3(-)浓度以及向肉汤中以恒定体积添加样品和标准品以标准化反应小瓶中的 KCl 摩尔浓度，可以将这种偏差降到最低。
