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

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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.