Methanotroph diversity in landfill soil: Isolation of novel type I and type II methanotrophs whose presence was suggested by culture-independent 16S ribosomal DNA analysis

The diversity of the methanotrophic community in mildly acidic landfill cov er soil was assessed by three methods: two culture-independent molecular ap proaches and a traditional culture-based approach. For the first of the mol ecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs we re identified and tested. These primers were used to amplify directly extra cted soil DNA, and the products were used to construct type I and type LT c lone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic commun ity members as distinguished by sequence differences in variable region 3 o f the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant str ains. The key variables of the series of enrichment conditions were initial pH (4.8 versus 6.8), air/CH4/CO2 headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1x nitrate minimal salts [NMS] versus 0.2x NMS). Screening of the isolates showed that the nutrient-rich Ix NMS selec ted for type I methanotrophs, while the nutrient-poor 0.2 x NMS tended to e nrich for type II methanotrophs. Partial sequencing of the 16S rRNri gene f rom selected clones and isolates revealed some of the same novel sequence t ypes. Phylogenetic analysis of the type I clone library suggested the prese nce 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 s pecies distinct from the validly published Methylosinus and Methylocystis g enera, and two members of this cluster were also successfully cultured. Par tial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placeme nt of the four isolates. Finally, not all of the bands separated by DGGE co uld be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.