反硝化细菌法测定土壤NO_(2)^(-)-^(15)N丰度的方法优化

Optimization of Denitrifier Method for Determination of Soil NO_(2)^(-)-^(15)N Abundance

NO_(2)^(-)是土壤中多个氮转化过程的关键中间产物,具有浓度低、转化快的特点。反硝化细菌法与质谱技术相结合,已广泛用于NO_(3)^(-)或NO_(2)^(-)的^(15)N同位素分析。本文旨在优化现有Stenotrophomonas nitritireducens反硝化细菌法的培养和反应条件,实现对土壤浸提液中NO_(2)^(-)^(15)N-丰度的专一、快速、准确测定。结果表明,使用种子液好氧摇培与单菌落微氧培养对NO_(2)^(-)样品的^(15)N同位素测定无显著差异,种子液可保证不同批次菌体的稳定性,好氧培养可将培养时间从7~8 d缩短至12~15 h。高纯N_(2)或He气吹扫0.5 h均能有效去除O_(2)和空白杂质氮,但N_(2)吹扫成本更低。转移N_(2)O气体至干燥气瓶,不影响测定结果的准确性和精密度,还能延长样品保存时间,减少碱蒸汽腐蚀仪器管路。使用处于对数生长期的菌体,调节反应体系的菌体浓度OD600值为0.3~0.9,可保证其反硝化效率,减少不同批次菌体的差异。使用不超过1 mol·L^(-1)的KCl浸提土壤,既可保证提取效率,也能减少对菌体活力的影响。优化后的方法不仅能准确测定不同类型土壤中NO_(2)^(-)的^(15)N丰度,对5~20 nmol自然丰度NO_(2)^(-)的δ^(15)N值测量精度为0.1‰~0.9‰,且大部分<0.5‰,还大幅简化了步骤、缩短了实验周期、节约了成本。

【Objective】NO_(2)^(-)is a key intermediate product of several nitrogen transformation processes in soil,with low concentration and fast transformation.The combination of denitrifier method and mass spectrometry technology has been widely used in the^(15)N isotope analysis of NO_(3)^(-)or NO_(2)^(-)in water.【Method】This paper aims to optimize the culture and reaction conditions of Stenotrophomonas nitritireducens denitrifier method,and to realize the specific,rapid and accurate determination of 15 NO_(2)^(-)N-abundance in soil extract.Thus,we will optimize the culture method and reaction conditions,and bacterial concentration of denitrifier method,then applied this method to the soil extract.【Result】There was no significant difference in the^(15)N isotopic determination of NO_(2)^(-)samples between aerobic shaking with seed solution and microaerobic incubation of single colonies.The seed solution ensures the stability of different batches of bacteria and aerobic incubation reduces the incubation time from 7-8 days to 12-15 h.High purity N_(2)or He purging for 0.5 h can effectively remove O_(2)and blank nitrogen,but the cost of N_(2)purging is lower.The transfer of N_(2)O gas to the dry gas cylinder did not affect the accuracy and precision of the determination results.However,it can prolong the sample preservation time and reduce alkali steam corrosion of the instrument pipeline.Also,using bacteria in logarithmic growth phase and adjusting the reaction system to an OD600 value of 0.3-0.9 will ensure their denitrification efficiency and reduce variation between batches.Using no more than 1 mol·L^(-1)KCl to extract the soil will ensure the extraction efficiency and reduce the impact on the viability of the bacteria.【Conclusion】The optimized method can accurately determine the^(15)N abundance of NO_(2)^(-)in different types of soil with a measurement accuracy ofδ^(15)N of 5-20 nmol and natural abundance value of NO_(2)^(-)between 0.1‰-0.9‰,with most values being less than 0.5.Also,the experimental period was greatly shortened,simplified steps and save cost.