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 cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including for former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4.8 versus 6.8), air/CH{sub 4}/CO{sub 2} 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 1x NMS selected for type I methanotrophs, which the nutrient-poor 0.2x NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types.
- Research Organization:
- Univ. of Georgia, Athens, GA (US)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FC09-96SR18546
- OSTI ID:
- 20005473
- Journal Information:
- Applied and Environmental Microbiology, Vol. 65, Issue 11; Other Information: PBD: Nov 1999; ISSN 0099-2240
- Country of Publication:
- United States
- Language:
- English
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