College of Resources and Environmental Sciences, China Agricultural University, 100193 Beijing, China.
Environ Microbiol Rep. 2012 Feb;4(1):113-22. doi: 10.1111/j.1758-2229.2011.00311.x. Epub 2011 Nov 27.
Denitrification occurs actively in rice field soils. In the present study, the responses of nirK and nirS denitrifier communities to nitrate addition in the anoxic rice soil were determined through molecular analyses of nitrite reductase genes nirK and nirS and 16S rRNA genes. Denitrification occurred rapidly when nitrate was added at the beginning of anoxic incubation (experiment I). The structure of nirK-type denitrifiers did not change; but their abundance as determined by quantitative (real-time) PCR increased in nitrate treatments compared with control. Both the structure and abundance of nirS denitrifiers remained unaffected in experiment I. The rate of denitrification was slowed down when nitrate was added 20 days after the onset of anoxic incubation (experiment II). The structure and abundance of nirK-type denitrifier community did not respond to nitrate addition; but the nirS community changed substantially in this experiment. The copy number of nirS genes increased by an order of magnitude in the treatments of 5 mM and 10 mM nitrate compared with control. The terminal restriction fragment length polymorphism (T-RFLP) analysis of nirS genes revealed that the 100 bp T-RF substantially increased in the nitrate treatments. Cloning and sequence analysis indicated that this T-RF had similarity of up to 90% with Herbaspirillum sp. T-RFLP profiles of the bacterial 16S rRNA genes also showed that Herbaspirillum sp. increased after nitrate amendments. Collectively, the nirK-type denitrifiers were probably active at the beginning of anaerobic incubation, while the nirS denitrifiers, especially those related with Herbaspirillum sp. probably were more active when anaerobic condition was fully developed.
反硝化作用在稻田土壤中活跃进行。本研究通过分子分析亚硝酸盐还原酶基因 nirK 和 nirS 以及 16S rRNA 基因，确定了缺氧稻田土壤中硝酸盐添加对 nirK 和 nirS 型反硝化菌群落的响应。在缺氧培养开始时添加硝酸盐会迅速引发反硝化作用（实验 I）。nirK 型反硝化菌的结构没有变化；但与对照相比，硝酸盐处理中 nirK 型反硝化菌的丰度通过定量（实时）PCR 增加。在实验 I 中，nirS 型反硝化菌的结构和丰度都没有受到影响。当硝酸盐在缺氧培养开始 20 天后添加时，反硝化作用的速度会减慢（实验 II）。nirK 型反硝化菌群落的结构和丰度对硝酸盐添加没有反应；但在这个实验中，nirS 群落发生了很大的变化。与对照相比，5 mM 和 10 mM 硝酸盐处理中 nirS 基因的拷贝数增加了一个数量级。nirS 基因的末端限制性片段长度多态性（T-RFLP）分析显示，硝酸盐处理中 100 bp T-RF 显著增加。克隆和序列分析表明，该 T-RF 与 Herbaspirillum sp. 的相似度高达 90%。T-RFLP 图谱的细菌 16S rRNA 基因也表明，硝酸盐添加后 Herbaspirillum sp. 增加。总的来说，nirK 型反硝化菌可能在厌氧培养开始时活跃，而 nirS 型反硝化菌，特别是与 Herbaspirillum sp. 有关的反硝化菌，在厌氧条件充分发展时可能更活跃。
