Diversity and structure of the methanogenic community in anoxic rice paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval

Abstract

A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with Methanobacterium bryantii and Methanobacterium formicicum, six were closely related but not identical to those of strains of Methanosaeta concilii, two grouped with members of the genus Methanosarcina, and two were related to the methanogenic endosymbiont of Plagiopyla nasuta. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 10(7) per g of dry soil for the H2-CO2-utilizing methanogens and of up to 10(6) for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H2-CO2, grouped within the phylogenetic radiation characterized by M. bryantii and M. formicicum and the environmental sequences of the Methanobacterium-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel Methanosaeta-like group and the Methanobacterium-like group. Methanosarcina spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 microM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known Methanosarcina spp. but 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 Methanosarcina spp. appeared to be less abundant.

FIG. 1

Time course of the total cell counts determined with various substrates, showing the increase in the MPN index with increasing incubation time of the MPN dilution series. Symbols: ▪, count on H₂-CO₂; □, count on formate; •, count on acetate; ○, count on methanol.

FIG. 2

Phase-contrast photomicrographs of dominant cell forms observed in liquid serial dilutions. (a) Mainly fluorescent rod-shaped cells in a culture on H₂-CO₂, from a dilution level corresponding to a population size of about 10⁶ cells per g of dry soil. (b) Fluorescent Methanosarcina-like pseudosarcinae in a culture on acetate, from a dilution level corresponding to a population size of 10 to 100 cells or aggregates per g of dry soil. (c) Nonfluorescent Methanosaeta-like filaments in a culture on acetate, from a dilution level corresponding to a population size of 10⁵ to 10⁶ cells per g of dry soil. (d) M. concilii VeAc9 for comparison. Scale bar = 10 μm for all panels.

FIG. 3

16S rRNA-based dendrogram showing the phylogenetic relationship between the cultured organisms (strains VeA23 and VeH52 as well as culture A5.1) and environmental sequences (ABS clones) obtained in this study and a representative selection of members of the Euryarchaeota. Members of the Crenarchaeota and Fervidobacterium islandicum were used as outgroup references. The tree topology was derived by performing a distance matrix analysis (calculation of the distance matrix was done with the Jukes-Cantor equation [27]; tree reconstruction was done with the neighbor-joining method [53]). The branching pattern within defined lineages, i.e., within the Methanosaeta cluster, the Methanosarcina cluster, and the Methanobacterium cluster, was corrected in accordance with maximum-likelihood analyses. The scale bar represents a 10% estimated difference in nucleotide sequences.