DIVERSITY AND STRUCTURE OF THE METHANOGENIC COMMUNITY IN ANOXIC RICE PADDY SOIL MICROCOSMS AS EXAMINED BY CULTIVATION AND DIRECT 16S RIBOSOMAL-RNA GENE SEQUENCE RETRIEVAL

A dual approach consisting of cultivation and molecular retrieval of p artial archaeal 16S rRNA genes was carried out to characterize the div ersity and structure of the methanogenic community inhabiting the anox ic bulk soil of flooded rice microcosms, The molecular approach identi fied four groups of known methanogens. Three environmental sequences c lustered with Methanobacterium bryantii and Methanobacterium formicicu m, six were closely related but not identical to those of strains of M ethanosaeta concilii, two grouped with members of the genus Methanosar cina, and two were related to the methanogenic endosymbiont of Plagiop yla nasuta. The cultivation approach via most-probable-number counts w ith a subsample of the same soil as an inoculum yielded cell numbers o f up to 10(7) per g of dry soil for the H-2-CO2-utilizing methanogens and of up to 10(6) for the acetate-utilizing methanogens, Strain VeH52 , isolated from the terminal positive dilution on H-2-CO2, grouped wit hin the phylogenetic radiation characterized by M. bryantii and M. for micicum and the environmental sequences of the Methanobacterium-like g roup. A consortium of two distinct methanogens grew in the terminal po sitive culture on acetate. These two organisms showed absolute 16S rRN A gene identities with environmental sequences of the novel Methanosae ta-like group and the Methanobacterium-like group. Methanosarcina spp, were identified only in the less-dilute levels of the same dilution s eries on acetate, These data correlate well with acetate concentration s of about 11 mu M in the pore water of this rice paddy soil, These co ncentrations are too low for the growth of known Methanosarcina spp, b ut are at the acetate utilization threshold of Methanosaeta spp, Thus, our data indicated Methanosaeta spp, and Methanobacterium spp, to be the dominant methanogenic groups in the anoxic rice soil, whereas Meth anosarcina spp, appeared to be less abundant.