Molecular ecology studies of methanotrophs in a freshwater lake sediment.

摘要

Major advances have been made recently in the application of molecular biological techniques to environmental settings. The methane oxidizing bacteria, methanotrophs, are ubiquitous in nature and grow on methane as their sole source of carbon and energy. They have important roles in both the global carbon cycle and the degradation of hazardous compounds. Methanotrophs are capable of degrading a number of halogenated compounds including the common groundwater contaminant, trichloroethylene (TCE). In spite of their environmental importance, genetic tools for the methanotrophs have not been widely developed or applied for studying natural populations of these organisms in situ.; In this thesis, genes for the particulate methane monooxygenase (pMMO) were cloned from pure cultures of methanotrophs. Using these data and others, robust molecular tools were developed for use in the methanotrophs. These include PCR primers and oligonucleotide probes designed for the 16S rRNA and pmoA genes in methanotrophs. The tools were utilized in this study to examine the diversity of methanotrophs in the sediments of Lake Washington, a freshwater lake habitat. The data obtained suggest that the methanotrophs detectable using the newly developed genetic tools demonstrate a diversity as broad as the known methanotrophs from all mesophilic environments. These results are in contrast to other environments, such as peat and marine environments, that appear to be dominated by a limited diversity of methanotrophs.; The capacity of the Lake Washington methanotrophic populations for TCE degradation under conditions that mimic intrinsic and enhanced bioremediation protocols was investigated. The changes in the populations were followed by observing methane and TCE oxidation rates in addition to hybridization with methanotroph specific oligonucleotide probes. The data obtained suggest that the soluble methane monooxygenase (sMMO) is not expressed under methane enrichment conditions typical of enhanced bioremediation protocols. Thus, in situ bioremediation protocols involving methanotrophs will probably involve degradation by the pMMO and not the sMMO, as is currently believed. These results have strong implications for modeling solvent bioremediation by methanotrophs and must be addressed in future designs.