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

PCR-DGGE法分析氮饱和土壤中产N2O硝化菌的微生物群落

• 批准号: 18510018
• 负责人: KAWAKAMI Tomonori
• 金额: $ 2.01万
• 依托单位: Toyama Prefectual University Junior College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18510018/
• 关键词: Nitrogen Saturation; Nitrous oxide; Microbial community analysis; Nitrosospira sp.; PCR-DGGE; AOB; Nitrification; Acidbactor sp.; 富山市呉羽山; Acidbactor; Nitrosospira

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.

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.

项目成果

耐酸性硝化細菌の含アンモニア廃水処理への適用と菌種の特定,2008

耐酸硝化细菌在含氨废水处理中的应用及细菌种类鉴定，2008年

• 发表时间: 2008
• 期刊: 富山県立大学紀要 18巻
• 作者: Kaneko;N;金子 信博;川上 智規
• 通讯作者: 川上 智規

Identifying nitriflers working in the highly acidified soils by PCR-DGGE method

PCR-DGGE 方法鉴定高酸化土壤中的硝基化合物

• 发表时间: 2007
• 期刊: Journal of Ecotechnology Research Vol.13
• 作者: Kaneko;N;金子 信博;川上 智規;Tomonori Kawakami;KAWAKAMI Tomonori;川上 智規・加藤 康夫・岩本 千鶴・出口 真裕子・中島 由理;KAWAKAMI Tomonori;KAWAKAMI Tomonori
• 通讯作者: KAWAKAMI Tomonori

An application of acid-tolerant nitrifiers to ammonia-contaminated waste water and identification of the nitrifiers by PCR-DGGE method

耐酸硝化菌在氨污染废水中的应用及PCR-DGGE法鉴定

• 发表时间: 2008
• 作者: KAWAKAMI Tomonori;KATO Yasuo;IWAMOTO Chizuru;UENO Hiromi and NAKASHIMA Yuri
• 通讯作者: UENO Hiromi and NAKASHIMA Yuri

The application of acid-tolerant nitriflers to the waste water containing high concentration of ammonium and identification of the ammonium-oxidizing bacteria participating in nitrification.

耐酸硝化细菌在高浓度铵废水中的应用及参与硝化作用的铵氧化细菌的鉴定

• 发表时间: 2008
• 期刊: Bulletin of Toyama Prefectural University Vol.18
• 作者: Kaneko;N;金子 信博;川上 智規;Tomonori Kawakami
• 通讯作者: Tomonori Kawakami

Identifying nitrifiers working in the highly acidified soils by PCR-DGGE method

PCR-DGGE 方法识别高酸化土壤中的硝化菌

• 发表时间: 2007
• 期刊: Journal of Ecotechnology Research Vol.13,No.2
• 作者: Kaneko;N;金子 信博;川上 智規;Tomonori Kawakami;KAWAKAMI Tomonori;川上 智規・加藤 康夫・岩本 千鶴・出口 真裕子・中島 由理;KAWAKAMI Tomonori
• 通讯作者: KAWAKAMI Tomonori