Characterization of methanotrophic bacteria isolated from a subtropical paddy field

Isolation and characterization of methane-oxidizing bacteria from a subtropical paddy field of Ishigaki, Japan, were performed. Two strains, namely R16 and R18, of Methylosinus-like organisms were isolated from methane enrichment cultures of rice rhizosphere. The isolates were motile, and stained Gram-negative. Cells of strains R16 and R18 were short rods, while slightly curved cells and coccobacillus-like cells were also observed for strains R16 and R18, respectively. Strain R16 was slightly smaller in size than R18. The organisms had an optimal pH range of 6.3–6.8 and a temperature optimum of 29–30°C. Methane and methanol were used as sole carbon and energy sources. Nitrate and ammonium sulfate could serve as nitrogen sources. The acetylene reduction method showed that the strains were able to fix nitrogen. On the basis of morphological and physiological properties, and 16S rRNA gene sequence analysis, the organisms were identified as genus Methylosinus. Strain R16 shows the highest 16S rRNA gene similarity of 95.5% with Methylosinus trichosporium, whereas R18 shows the highest 16S rRNA gene similarity with Methylosinus sporium (95.6%).