| Methanotroph-mediated in situ bioremediation of halogenated hydrocarbons has great potential. Its successful application, however, has been complicated by the existence of two forms of the enzyme responsible for pollutant degradation, the methane monooxygenase (MMO). These two forms, one found in the cytoplasm, or soluble methane monooxygenase (sMMO) and the other in membranes, or particulate methane monooxygenase (pMMO), exhibit very different ranges and rates of pollutant degradation. A great deal is known about the ability of sMMO-expressing cells to degrade halogenated hydrocarbons, while relatively little is known in this area on pMMO-expressing cells. In this thesis, the range and rates of pollutant degradation by pMMO-expressing cells is discussed, as well as the development of a modern molecular based technique to determine which form of MMO is expressed by methanotrophic populations.; From analysis of halogenated hydrocarbon degradation kinetics and the inhibitory effects of these compounds on microbial growth and pMMO activity, it was found that these compounds could be separated into four general categories when degraded by pMMO-expressing cells, namely (1) biodegradable with minimal inactivation, (2) biodegradable with substantial inactivation, (3) not biodegradable with minimal inactivation, and (4) not biodegradable but substantial inactivation. Among these halogenated hydrocarbons, chloromethane was found to support growth in the presence of methanol, although it could not serve as a sole growth substrate. This result suggests that cometabolic reactions may actually provide some benefit to methanotrophs and that these cells can use multiple compounds to enhance growth.; To better monitor methanotrophic activity, a competitive reverse transcription polymerase chain reaction (RT-PCR) methodology was developed. Expression of both sMMO and pMMO could be quantified by means of the competitive RT-PCR and capillary electrophoresis. Using this approach, it was found that pmoA is constitutively expressed and the level increases dramatically at high copper concentration. For this technique to be useful in monitoring microbial activity in situ, a new method for soil RNA extraction was also developed based on a conceptual change to consider soil as a binding matrix for nucleic acids. This methodology may prove useful in support of natural or engineered in situ bioremediation applications using methanotrophs. |
