The development of molecular techniques for microbial population analysis in landfills

Wayne, Jonathan Mark (2001). The development of molecular techniques for microbial population analysis in landfills. PhD thesis The Open University.



Methanogens are responsible for the production of methane, which is a major component of landfill gas. Methane is a greenhouse gas, but also a potentially valuable energy source. A better understanding of the methanogens in landfill could aid the development of improved strategies for the control of landfill degradation processes.

Excavated refuse and leachate samples were obtained from five landfills. DNA was extracted from the samples and a methanogen-specific gene (mcrA) was amplified by PCR. Clone libraries were generated, and screened by PCR-RFLP. This revealed a much greater diversity of methanogens in landfill than had been detected in previous studies. Furthermore, the composition of the methanogen communities was substantially different between landfills, and within landfills. DNA sequencing and phylogenetic analysis was used to determine the phylogenetic affiliations of landfill methanogens. Members of the order Methanomicrobiales were found to be dominant in the majority of the samples. Methanomicrobiales was the second most abundant group, while Methanosarcinales appeared to be only a minor component of the methanogen population in most of the samples. Phylogenetic analyses revealed five clusters of mcrA sequences that were not closely affiliated to any described species included in the analysis, including one cluster that was not closely affiliated to any of the five methanogen orders. These unidentified clusters may represent novel methanogenic lineages. A set of nested, group-specific oligonucleotide probes for mcrA was designed to detect the groups identified by the phylogenetic analysis. The usefulness of these probes for rapidly characterising methanogen communities was demonstrated by screening clone libraries of mcrA PCR products.

The potential of molecular techniques for detecting homoacetogenic Bacteria in landfill was demonstrated by the generation of PCR products from DNA extracted from landfill using primers for the FTHFS gene, and by detection of a PCR product from landfill with a probe for FTHFS.

The results of this study have greatly increased our knowledge of the methanogen community in landfill, and the molecular techniques developed in this study should prove valuable for further investigations of the methanogen population in landfill and other environments

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