Analysis of Microbial Community in Soil to Evaluate Greenhouse Effective Gas, Methane from Siberia.

分析土壤中的微生物群落以评估温室有效气体，来自西伯利亚的甲烷。

• 批准号: 13680640
• 负责人: OHASHI Akiyoshi
• 金额: $ 0.83万
• 依托单位: Nagaoka University of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680640/
• 关键词: greenhouse effect; FISH technique; high sensitive detection; methane; microorganisms; Siberia; CARD-FISH; soil

It was difficult to quantify methanogens in Siberia marshland by Fluorescence in situ hybridization (FISH). To overcome problems of FISH with mono fluorescent dye labeled oligonucleotide, we studied methanogens' cell-wall treatments technique to apply high sensitive FISH methods. Targeted four types of methanogenetic cell wall, psudomurein, S-layer, methanochondroitin and sheath, were tested using pure methanogens. Cell wall structure of s-layer and methanochondroitin did not require any further treatments. For cell wall structure specific to pseudomurein and sheath, we found enzymatic treatment is the most effective among all tested treatment methods such various physical and/or chemical treatments. ECF-FISH, HNPP-FISH, and CARD-FISH were tested to select the high sensitive FISH. CARD-FISH has the highest intensity in tested and fluorescent signal was up to 15-folds. The application of cell wall treatment techniques with CARD-FISH for UASB granule sludge and anaerobic digested sludge was successful, indicating the application for other environmental samples.

利用荧光原位杂交技术对西伯利亚沼泽地产甲烷菌进行定量分析存在一定的困难。为了克服单荧光标记寡核苷酸荧光原位杂交技术存在的问题，我们研究了甲烷菌细胞壁处理技术，以实现高灵敏度的荧光原位杂交技术。有针对性的四种类型的产甲烷细胞壁，psudomurein，S-层，methanochondroitin和鞘，进行了测试，使用纯产甲烷菌。细胞壁结构的s-层和甲烷软骨素不需要任何进一步的处理。对于细胞壁结构特异性的假刀豆素和鞘，我们发现酶处理是最有效的所有测试的处理方法，如各种物理和/或化学处理。通过ECF-FISH、HNPP-FISH和CARD-FISH检测，筛选出高灵敏度的FISH。CARD-FISH在所测试的样品中具有最高的荧光强度，荧光信号高达15倍。CARD-FISH细胞壁处理技术在UASB颗粒污泥和厌氧消化污泥中的应用是成功的，为其它环境样品的处理提供了借鉴。

项目成果

久保田健吾, 大橋晶良, 原田秀樹: "Tyramide Signal Amplificationによるメタン生成古細菌の検出"土木学会年次講演概要集. No.7 57. 280 (2002)

Kengo Kubota、Akira Ohashi、Hideki Harada：“通过酪酰胺信号放大检测产甲烷古菌”日本土木工程师学会年度摘要第 7 期 57. 280 (2002)。

久保田健吾, 大橋晶良, 原田秀樹: "高感度FISH法による低16S rRNA含量微生物の検出"水環境学会年会講演概要集. 36. 302 (2002)

Kengo Kubota、Akiyoshi Ohashi、Hideki Harada：“通过高灵敏度 FISH 方法检测低 16S rRNA 含量的微生物”日本水环境学会年会摘要 36. 302 (2002)。

A. Ohashi, E. Fukuda, H. Harada: "Graphical Estimation of Specific Biological Activity on Sludge with Low Biodegradability by Batch Test and New Analytical Method"Journal of Japan Society on Water environment. 26. 167-170 (2003)

A. Ohashi、E. Fukuda、H. Harada：“通过批量试验和新分析方法对低生物降解性污泥的比生物活性进行图形估计”日本水环境学会杂志。

K. Kubota, A. Ohashi, H. Harada: "Identification of Low 16S rRNA Content Microbes in Environments by Fluorescence in situ Hybridization Combined with Catalyzed Reporter Deposition."Proceeding of Annual Conference of Japan Society on Water environment. Kum

K. Kubota、A. Ohashi、H. Harada：“通过荧光原位杂交结合催化报告基因沉积鉴定环境中低 16S rRNA 含量的微生物。”日本水环境学会年会论文集。

大橋晶良, 福田江津子, 原田秀樹: "低活性な汚泥における回分試験からの比活性値評価図解法"水環境学会誌. 26, No.3. 167-170 (2003)

Akiyoshi Ohashi、Etsuko Fukuda、Hideki Harada：《低活性污泥批量试验评估比活性值的说明方法》日本水环境学会杂志 26，第 3. 167-170 期（2003 年）。