Characterization of cultivable methanotrophs from paddy soils and rice roots

Abstract

Characterization of methanotrophs isolated from paddy soils and rice (Oryza sativa) roots was investigated in the present study. The number of methanotrophs in root homogenates of the rice cultivar Mutsuhomare was 4.9 × 107 most-probable-number (MPN) g−1 dry roots, in Yumeakari it was 2.0 × 108 MPN g−1 dry roots and in Kirara it was 4.6 × 107 MPN g−1 dry roots. Although bacterial cells were observed infrequently on the surface and in the interiors of roots before incubation, a large number of colonies, measuring 0.5–5 mm in diameter, were observed on the sterilized roots after incubation on nitrate mineral agar plates under methane in air. In particular, a large number of colonies were observed at the emergence sites of lateral roots and root hairs. Strains MD5-1 and M1 were isolated from the roots of Mutsuhomare and strain R62 was isolated from the root homogenate of Yumeakari. All isolates were catalase-positive and oxidase-positive, Gram-negative, straight-rod-shaped and curved-rod-shaped bacteria, and formed exospores. The isolates were able to fix nitrogen and grew in the absence of copper. In addition, all were found to be positive for naphthalene-oxidizing activities (corresponding to soluble methane mono-oxygenase activities). Strains MD5-1, M1 and R62 were closely related to Methylosinus sporium. Methanotrophic strains W3-6, SD3-5 and 2-19, isolated previously from paddy field soils, were classified into Methylosinus (W3-6) and Methylocystis (SD3-5 and 2-19) type II methanotrophs. Isolates from the rice roots (MD5-1, R62 and M1) grew logarithmically when casamino acid was used as the nitrogen source; however, the growth of these strains was reduced on the nitrate medium. These strains preferred amino acids over inorganic nitrogen as a nitrogen source for growth.