Isolation of hydrogenotrophic methanogenic archaea from a subtropical paddy field

A total of 347 blue-autofluorescent colonies on roll tube cultures were transferred individually for isolation in order to examine methanogenic flora in a subtropical paddy field after application of organic matter with three treatments (control soil, straw-amended soil, and cellulose-amended soil). Two hundred and three strains showed a positive reaction for methane production. However, only 36 strains were found to be pure. Among the 36 pure methanogenic isolates, 19 utilized H2-CO2 as sole source for energy and carbon for growth, 17 utilized H2-CO2 and formate, and none utilized methanol and acetate. All 36 isolates were rods distinguishable into three groups by cell morphology: group 1, irregularly crooked long rods; group 2, gently curved long (or very long) rods; and group 3, relatively straight long (or very long) rods. Three isolates, strains 3544-4 and 6544-9 of group 2 and 5544-3 of group 3, were identified as Methanobacterium spp. by partial sequencing analysis of 16S rRNA. The partial 16S rRNA sequence of strain 6544-9 was identical to that of Methanobacterium bryantii. The other three isolates which belonged to group 1, strains 3543-10, 3544-9, and 6132-1, seemed to represent a new species within the genus Methanobacterium or probably Methanobrevibacter. Each of the pure isolates from control and straw-amended soil belonged to group 1. It is suggested that the group 1 methanogens are characterized as a geographically interesting indigenous type in the subtropical paddy field.