1.State Key Laboratory of Soil and Sustainable Agriculture,Institute of Soil Science,Chinese Academy of Sciences;2.University of Chinese Academy of Sciences;3.Changshu Agro-ecological Experimental Station, Chinese Academy of Sciences
National Natural Science Foundation of China (Nos. U20A20113, 42177303) and Scientific Instrument and Equipment Development Project of CAS (No. YJKYYQ20170058)
反硝化是生态系统氮循环的最后一环同时也是活性氮转化为惰性氮（N2）的最主要过程。由于空气中背景N2浓度高达78%，在如此高的背景浓度N2环境中直接和准确测定反硝化过程产生的微量N2，一直是个巨大的挑战。Robot系统（Robotized incubation and analyzing system）是基于无N2背景（氦环境）的用以研究纯菌或土壤体系N2排放速率的方法，该系统平台搭建简单且测定效率高，目前应用比较广泛。但该系统在运行过程中需要频繁利用微量注射器进行取样和测定，极易造成外界N2的渗漏。为解决这一问题，本文通过使用预先置于氦（He）环境的橡胶隔垫、采用充He后的蒸馏水配制溶液及实施破坏性取样的处理，对Robot系统测定旱地N2排放速率的方法进行了优化，同时与乙炔抑制法和RoFlow系统（Robotized continuous flow incubation system）的测定结果进行了对比。研究结果表明，通过方法优化，可以大幅降低Robot系统的N2渗漏率，未优化前系统的渗漏率为5.15 μL·L-1·h-1，方法优化后系统的渗漏率在0～0.8 μL·L-1·h-1之间。优化后的Robot系统对碳源和氮源添加后N2排放速率差异的响应较好，并且对旱地土壤背景N2排放速率的测定误差最小（0.003～0.045 mg·kg-1·d-1），显著优于乙炔抑制法（0.34～3.29 mg·kg-1·d-1）和RoFlow系统（0.41～1.02 mg·kg-1·d-1）。综上，优化后的Robot系统在测定旱地N2排放速率时具有N2渗漏率低，对外源底物添加响应好及测定结果精确度高的特点，未来在研究旱地土壤背景N2排放及相关机理方面有较好的应用前景。
【Objective】The massive application of nitrogen fertilizer to agricultural soils plays an important role in ensuring the world’s food supply. However, it also leads to a large amount of reactive nitrogen (N) entering the environment, which strongly interferes with the biogeochemical cycle of N and causes a series of ecological and environmental problems. As the last step of N cycling, denitrification is the predominant pathway, converting reactive N into inert N (i.e., N2). However, measuring soil N2 production from denitrification is a major challenge in terrestrial ecosystems because of the high atmospheric background N2 concentration. Recently, direct methods for measuring N2 emission rates have been developed. Among them, robotized incubation and analyzing system (Robot system) which is based on N2 free headspace (i.e., helium environment) have been widely used for measuring N2 emission in pure denitrifying culture or soil, due to its advantage on platform construction and high throughput for N2 determination. Nevertheless, frequent sampling with the small-diameter steel needle is required during the operation and determination of the Robot system, which inevitably causes leakage of N2. This seriously interferes with the determination of low N2 emission rates (i.e., background N2 flux in upland soil). Therefore, to enable the Robot system to measure background N2 emission rate in upland soil without exogenous substrate, the leakage rate of the system must be further reduced. 【Method】In this study, helium (He)-washed rubber septa, solutions prepared by He-washed distilled water and destructive sampling treatments were explored to optimize the Robot system aiming at reducing the N2 leakage therein. Additionally, results of soil N2 emission determined by the optimized Robot system were compared with those of acetylene inhibition technique (AIT) and Robotized continuous flow incubation system (RoFlow system). 【Result】Our results showed that the N2 leakage rate of the Robot system could be remarkably reduced by optimizing with He-washed septa, solutions prepared by He-washed distilled water and destructive sampling treatments. Relative to the reported N2 leakage (5.15 μL·L-1·h-1) of the Robot system, the N2 leakage rate was reduced to 0～0.8 μL·L-1·h-1 by the aforementioned treatments. Under similar treatments, the N2 emission rate measured by the acetylene inhibition method was highest, followed by the RoFlow system, and the Robot system had the lowest results. Furthermore, the optimized Robot system was capable of determining upland soil N2 emissions in response to carbon and N addition, which also had the smallest standard error (0.003～0.045 mg·kg-1·d-1) compared with the AIT method (0.34～3.29 mg·kg-1·d-1) and RoFlow system (0.41～1.02 mg·kg-1·d-1). 【Conclusion】Overall, the optimized Robot system is characterized by low N2 leakage, effective response to substrate addition and good consistency in determining soil N2 emission. In the future, it will have a favorable application in investigating background N2 emissions and the associated mechanism of upland soil.
LI Chenglin, WEI Zhijun, WU Min, SHAN Jun, YAN Xiaoyuan. Optimization of Robot System on Determining Upland Soil N2 Emission as Compared to Other Methods[J]. Acta Pedologica Sinica,,[In Press]