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生态学杂志 ›› 2012, Vol. 31 ›› Issue (08): 2152-2157.

• 方法与技术 • 上一篇    下一篇

利用反硝化细菌法测试水体硝酸盐氮氧同位素

岳甫均1,2,李思亮1**,刘丛强1,安宁1,蔡虹明1,2   

  1. 1中国科学院地球化学研究所环境地球化学国家重点实验室, 贵阳 550002; 2中国科学院研究生院, 北京 100049)
  • 出版日期:2012-08-10 发布日期:2012-08-10

Measurement of waters nitrate dual isotopes based on denitrifier method.

YUE Fu-jun1,2, LI Si-liang1**, LIU Cong-qiang1, AN Ning1, CAI Hong-ming1,2   

  1. (1State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese Academy of Sciences, Guiyang 550002, China; 2Graduate University of Chinese Academy of Sciences, Beijing 100049, China)
  • Online:2012-08-10 Published:2012-08-10

摘要: 反硝化细菌方法作为测试硝酸盐氮氧同位素组成的最新方法,具有可测试低浓度水样、对样品无需特殊处理、不会交叉污染和需样量少等诸多优点而得到迅速发展。本研究率先在国内实验室利用反硝化细菌法成功测试了硝酸盐氮氧同位素组成,将经过5~10 d培养的反硝化细菌Pseudomonas aureofaciens离心,然后将菌液浓缩5倍,再向顶空进样瓶注入3 mL菌液,密封后利用高纯氮气吹扫3 h以上,注入50 nmol NO3-水样经过夜培养灭活后,使用Trace Gas PreconcentratorIsoprime测试N2O同位素组成,结果表明,重现性和测试精度与国际上类似研究接近。该方法的建立对于国内开展河流及湖泊(水库)、降水等氮的生物地球化学循环将起到促进作用。

关键词: 厌氧脱氯, 脱氯富集培养体, 电子供体, 变性梯度凝胶电泳, 微生物群落结构

Abstract: Denitrifier method is a novel method for nitrogen and oxygen isotopic analysis of nitrate, which has many advantages, e.g., low nitrate water sample, simple pretreatment, no crosscontamination, and small volume water, and obtained rapid popularization. In this study, the method was first developed in China for nitrate isotopic measurement in our laboratory. The denitrifier (Pseudomonas aureofaciens) was grown in an amended medium of Tryptic Soy Broth. After 5-10 days culture, the cells were harvested and concentrated 5 folds, and split into 3 mL aliquots in 20 mL headspace vials. The vials were sealed and then purged for at least 3 h with highpurity N2 to ensure anaerobic condition. A sample amount corresponding to 50 nmol of nitrate was added to the sample vials, and cultured overnight to allow the complete conversion of NO3- to N2O. On the following day, 0.1-0.2 mL of 10 mol·L-1 NaOH was injected into the vials to lyse the bacterium and to scrub any CO2 gas. Simultaneous δ15N and δ18O analysis of the produced N2O was carried out by using a Trace Gas Preconcentrator unit coupled to an IRMS instrument. The measured data showed good reproducibility and precision, in close with the similar international studies. The establishment of this method would promote our studies in nitrogen biogeochemical cycle in rivers and lakes (reservoirs) and in precipitation.

Key words: anaerobic dechlorination, dechlorination enrichment culture, electron donor, DGGE, microbial community structure.