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 andMethanobacterium 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 107 per g of dry soil for the H2-CO2-utilizing methanogens and of up to 106 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 theMethanobacterium-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 theMethanobacterium-like group. Methanosarcinaspp. 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 μM in the pore water of this rice paddy soil. These concentrations are too low for the growth of knownMethanosarcina spp. but are at the acetate utilization threshold of Methanosaeta spp. Thus, our data indicatedMethanosaeta spp. and Methanobacterium spp. to be the dominant methanogenic groups in the anoxic rice soil, whereasMethanosarcina spp. appeared to be less abundant.

Dynamics of the methanogenic archaeal community in anoxic rice soil upon addition of straw

“…Triplicate samples were used to extract DNA and analyse the terminal restriction length polymorphism (T-RFLP) of archaeal 16S rRNA genes (see Chin et al, 1999) using 109f/ 915r as the primer pair (Grosskopf et al, 1998). T-RFLP of archaeal 16S rRNA genes is a fingerprinting technique, which determines the lengths (base pairs: bp) and relative abundance of particular terminal restriction fragments (T-RF).…”

“…In rice-field soil, H 2 plus CO 2 can be utilized by several groups of methanogens, i.e. Methanobacteriales, Methanomicrobiales, Methanosarcinaceae and Rice Cluster I (RC-I) (Grosskopf et al, 1998), that were found to occur in such soils from various geographic locations (Ramakrishnan et al, 2001). RC-I methanogens are a deeply branching group of methanogens, which have so far not been isolated into pure culture but have recently been characterized genomically (Erkel et al, 2005).…”

Methanogenic archaea and CO2‐dependent methanogenesis on washed rice roots

“…DNA extraction from Methanobacterium sp. Bab-1 and the methanogenic consortia enriched on H 2 and ethanol, respectively, was performed as described by Großkopf et al (1998b). Partial SSU rDNA from strain Bab-1 was amplified via PCR using the forward primer 109F (5Ј-GCTCAGTAA-CACGTGG-3Ј; Großkopf et al, 1998b) and the reverse primer 1391R (5Ј-GACGGGCGGTGTGTRCA-3Ј; Lane, 1991).…”

“…Whereas the methanogenic community in the bulk soil is dominated by Methanosaeta spp. (Großkopf et al, 1998b), that on the roots of the rice plants is dominated by other methanogenic taxa (Großkopf et al, 1998a).…”

Thermophilic methanogens in rice field soil

“…The SSU rDNA fraction of the DNA samples was amplified by PCR with the archaeal-specific primers described by Groûkopf et al (1998a), which amplifies from position 109± 934 (Escherichia coli 16S rRNA numbering; Brosius et al, 1978) as described previously (Chin et al, 1999). The thermal profile used for amplification of slurry±DNA included 20 (for cloning, see below) and 28 (for T-RFLP) cycles of denaturation (30 s), primer annealing at 528C (1 min) and elongation at 728C (2 min).…”