The diversity, community structure, and novel strains of methane-oxidizing bacteria from landfill cover soil

An understanding of the methanotrophic community structure in the environment is important since the methanotrophs are the major biological sink for atmospheric methane, an important greenhouse gas. The diversity of the methanotrophic community in landfill cover soil was assessed using both culture-based and culture-independent approaches. Two primer pairs specific for the 16S rRNA gene of validly published type I and type II methanotrophs were used to amplify directly extracted soil DNA, and the products used to construct type I and type II clone libraries. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter /Methylomicrobium group, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly published Methylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. By 16S rDNA database searches, the most similar species to both type I isolates were Methylobacter spp. However, partial particulate methane monooxygenase (pMMO) sequence analysis suggested that these bacteria might be more closely related to the Methylomicrobium than the Methylobacter. Furthermore, cellular fatty acid profiles of the strains more closely resemble those of the Methylomicrobium, although the absence of significant levels of 16:1o5c argues for the uniqueness of these two strains. We have proposed that a new genus should be created, Methylosarcina gen. nov., harboring two species Methylosarcina fibrata sp. nov. (type species) and Methylosarcina quisquiliarum sp. nov. Finally, the community structure of type I and type II methanotrophic bacteria from this landfill soil was monitored with denaturing gradient gel electrophoresis (DGGE) during methane-stimulated biodegradation of trichloroethylene (TCE). Methane addition in microcosms significantly increased the TCE degradation rate. The type II DGGE profile consisted of nine distinct bands that did not change during the course of the experiment, suggesting a stable community structure. The type I profile, consisting of seven distinct bands at time 0, was altered by the appearance of at least one extra band in some of the methane-amended microcosms. The DNA sequence of the extra band showed it to be unique from any of the clones or isolates retrieved at time zero.