Enrichment of a marine methanotrophic population and its kinetics of methane and TCE oxidation

Citation

Smith, Kelly S. (1996) Enrichment of a marine methanotrophic population and its kinetics of methane and TCE oxidation. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/vh4c-mr17. https://resolver.caltech.edu/CaltechETD:etd-01032008-090734

Abstract

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Bacteria that grow on methane as their sole carbon and energy source (methanotrophs) are capable of degrading a variety of halogenated methanes and ethylenes, including trichloroethylene (TCE). These compounds are common pollutants in a variety of coastal and nearshore marine environments, and therefore, marine methanotrophs are attractive candidates for bioremediation of these contaminants. The enzyme which degrades TCE, the methane monooxygenase, occurs in two forms, the soluble, or cytoplasmic form (sMMO) and the particulate, or membrane-bound form (pMMO). These enzymes have different kinetics of methane and TCE degradation, and while all known methanotrophs produce the pMMO, only a few species of the Type II group produce the sMMO. The sMMO is only produced under conditions of copper limitation in these strains. No Type II methanotrophs have been isolated from the marine environment. Detection and identification of marine methanotrophs, as well as characterization of their rates of oxidation of methane and TCE, are important to our understanding of their response to attempts to stimulate them for in-situ bioremediation of TCE. Additionally, this information is necessary to the development of predictive models of bioremediation processes. The rate of methane oxidation by the native methanotrophic population of an estuarine sediment was measured, and the [...] and [...] values for methane of this population were determined from the Michaelis-Menten equation. The [...] values (8-26 [...]) suggest that the particulate methane monooxygenase is being used for methane oxidation. Several methanotrophs were isolated from this estuarine sediment after enriching it, and gene probes based on 16S rRNA sequences were used to identify the genus groups of these isolates. No Type II methanotrophs were isolated. The kinetics of methane and TCE oxidation by one of the methanotrophic isolates were measured, and it was found that the amount of copper added to the growth medium affects the kinetics of methane and TCE oxidation by this strain. When copper was added, the [...] values were 10-13 [...] but when no copper was added the results varied from a slightly elevated [...] (16[...]) to a high [...] for which saturation with methane was not observed. When copper was added to cultures used for TCE degradation, [...] values were 8-10 [...], but when no copper was added, TCE degradation was not observed. The kinetics of methane and TCE oxidation in enriched estuary sediment were measured, and based on the elevated [...] values (48-53[...]), the limited increase in the [...] values (an indicator of a relatively small increase in the population), and the lack of TCE degradation in the enriched sediment, it is suggested that the methanotrophs may become limited for copper during enrichment. The data suggest that the sMMO is not being produced, even under copper limitation, consistent with the fact that no Type II methanotrophs were isolated. Therefore simple enrichment of marine methanotrophic populations may not result in enhanced TCE degradation rates. Nutrient addition, including bioavailable copper, will be necessary for the use of marine methanotrophs for in-situ TCE bioremediation. Gene probing was used to detect the presence of Type I methanotrophs in the sediment by probing slot blots of DNA extracted directly from the sediment. These blots were also used to estimate the number of methanotrophs present in the enriched sediment and this value was compared to estimates of methanotroph numbers based on [...] values. These estimates were found to agree within the expected error of each estimation method, suggesting that gene probing will be a useful method for estimation of methantroph numbers in environmental samples.

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