Microbial community analysis of nitrifiers producing N2O in the nitrogen-saturated soils by PCR-DGGE method

日本富山吴羽山的许多溪流含有高浓度的硝酸盐。这被认为是氮饱和的结果，因为氮收支表明，过量的硝酸盐淋溶到溪流中，超过了森林流域的氮沉积。在吴羽山的百牧谷流域，尽管流域内的土壤已经被高浓度的硝酸盐酸化，但由于氮饱和，N_2O的排放也增加了。硝化菌一般在pH小于5时才能硝化，而吴羽山土壤中的硝化菌在pH为3.1时仍能硝化。为鉴定产N_2O硝化细菌的种类，采用以16 S-rDNA为靶点的聚合酶链反应-变性梯度凝胶电泳（PCR-DGGE）技术，结合DNA测序技术对产N_2O硝化细菌进行了鉴定。以培养箱培养560天的吴羽山土壤为材料，提取了硝化细菌的DNA。用针对硝化细菌的引物对提取的DNA的16 S-rDNA进行PCR扩增。DGGE法分离16 S-rDNA，在变性梯度凝胶上电泳可将不同组成的16 S-rDNA分离为一条带。结果表明，在凝胶上出现了两条不同的条带，表明两种类型的硝化细菌参与了培养土壤的硝化作用。DNA序列分析表明，其中一种硝化细菌与亚硝化螺旋菌的同源性高达98%。sp.

Many streams of Kureha Hill, Toyama, Japan, contain high concentration of nitrate. This is believed to be the result of nitrogen saturation, because the nitrogen budget shows the excess leaching of nitrate to the stream water over the nitrogen deposition on the forested watersheds. In the watershed of Hyakumakidani, one of the streams on Kureha Hill, enhanced emission of N_2O by nitrogen saturation has also been observed, even though the soils in the watersheds have been acidified by high-concentration of nitrate. Usually, nitifiers inactivate their nitrification under the pH level less than 5, while the nitrifiers living in the soils of Kureha Hill can still work under the pH of 3.1. The purpose of this study is to identify the species of the nitrifiers which produce N_2O living in the highly-acidified soils of Kureha Hill.The polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method targeting 16S-rDNA combined with DNA-sequencing analysis was applied to identify the nitrifiers. The soils of Kureha Hill, which had been incubated in an incubator for 560days to increase the population of the nitrifiers, were used for the extraction of DNA. 16S-rDNA of the extracted DNA was PCR-amplified with the primers targeting those of nitrifiers. The amplified 16S-rDNA was separated by DGGE method in which different constitution of 16S-rDNA can be separated as a band on a denaturing gradient gel by electrophoresis. As a result, two different bands were seen on the gel which meant that two types of nitrifiers participated in the nitrification in the incubated soils. DNA-sequencing analysis revealed that one of the two types of the nitrifiers showed 98% identity with that of Nitrosospira. sp.