Trichloroethylene (TCE) is a major groundwater contaminant and is a cause of serious health concern. Methanotrophic TCE degradation is very promising compared with other treatments. Methanotrophs produce methane monooxygenases (MMOs) which catalyze methane oxidation and cometabolize chlorinated and aromatic compounds. High rate of TCE degradation is attributed to only soluble MMO (sMMO) expressed mainly by type II methanotrophs under copper-deficient conditions. To make methanotrophic TCE degradation practical, high density methanotrophic biomass with high sMMO activity is required. Methane is the primary substrate for methanotrophs and sufficient quantities must be supplied to support biomass growth. Because of the poor water solubility of methane, mass transfer limitation essentially restricts high biomass production. When methanol was used as the growth substrate, biomass concentration of 7.4 g l-1 Methylosinus trichosporium OB3b was achieved in a 160-h fermentation using an exponential feeding strategy based on pre-determined . Even higher biomass density of 19 and 29 g l-1 biomass were obtained by a modified feeding strategy based on carbon dioxide production. It is concluded that methanol is a promising substrate for the production of large amounts of M. trichosporium OB3bbiomass. In addition, allylthiourea was applied to methanotrophs growth medium to circumvent the inhibitory effect of copper, which inhibits sMMO activity but not particulate MMO (pMMO). We successfully retained sMMO activity by supplementing allylthiourea. Even when M. trichosporium OB3b was grown with 4.5 M copper, which would completely block sMMO expression, addition of 15 M allylthiourea preserved half of the sMMO activity. It was also observed that switching the growth substrate from methane to methanol did not significantly affect sMMO activity. An immobilized soil bioreactor was developed to examine the efficiency of methanotrophic TCE degradation by combining the knowledge obtained on high biomass production and applying allylthiourea for sMMO expression. In a batch TCE degradation experiment, about 63% of TCE was removed in 5.75 h. The maximal TCE degradation rate of 1.40 mg l-1 h-1 was obtained in a continuous TCE degradation at a dilution rate of 0.15 h-1. This study demonstrated the effectiveness of a novel bioreactor system for methanotrophic TCE degradation. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2008-09-30 14:51:26.59

The obligate methanotroph, Methylosinus trichosporium 0B3b synthesised up to 20% of its dry weight as poly-beta-hydroxybutyrate when grown in batch culture or nitrogen limited continuous culture on a methanol/ammonium salts medium. Four soluble enzymes associated with polymer metabolism, D(-)-3-hydroxybutyrate dehydrogenase (3-HBD), acetoacetyl-CoA synthetase, beta-ketothiolase and acetoacetyl-CoA reductase have been purified from the crude extract of this organism. Detailed kinetic studies were conducted on each enzyme and their role in the regulation of polymer metabolism was assessed. Cont/d.

The extensive use of tetrachloroethene (PCE) and trichloroethene (TCE) as cleaning solvents has resulted in widespread contamination of groundwater systems with vinyl chloride (VC). VC, a known human carcinogen, is primarily formed in groundwater via incomplete anaerobic reductive dechlorination of PCE and TCE. Aerobic, methane-degrading bacteria (methanotrophs), which are capable of VC cometabolism while growing on methane, could be important in natural attenuation of VC plumes that escape anaerobic treatment. Real-time PCR (qPCR) represents an innovative approach for detecting and quantifying the presence and activity of these VC-degrading microbes. Immediate applications of this technique include use in a laboratory setting to help elucidate the potential bacterial-substrate interactions occurring in the subsurface environments at these contaminated sites; interactions that could ultimately affect the role of methanotrophs in VC degradation. This technique could also provide lines of evidence for natural attenuation of VC, thus support existing anaerobic bioremediation technologies that generate VC as a metabolic intermediate. In this work, we evaluated several PCR primer sets from the literature for use in methanotroph qPCR assays of groundwater samples. PCR primers targeting two functional genes involved in VC cometabolism, pmoA (sub-unit of particulate methane monooxygenase (pMMO)) and mmoX (sub-unit of soluble MMO (sMMO)), as well as 16S rRNA gene primers that targeted Bacteria, and Type I and Type II methanotrophs were tested. These assays were made quantitative by constructing standard curves with DNA from Methylococcus capsulatus (Type I) and Methylocystis sp. strain Rockwell (Type II). Primer sets were evaluated by comparing gene abundance estimated against known amounts of Type I and Type II methanotroph DNA. After primer validation, an effort to substantiate this methanotroph qPCR method was made by attempting to investigate methanotroph populations in groundwater samples from VC-contaminated sites. Some samples studied were also subjected to 16S rRNA gene pyrosequencing, allowing for relative abundance comparisons with qPCR analyses. Following our primer assessment experiments, effective primer sets were used to estimate the presence of methanotrophs at environmental sites in Soldotna, Alaska; Naval Air Station Oceana, Virginia Beach, Virginia; and Carver, Massachusetts. Results showed that methanotrophs were present in nearly all wells sampled from all environmental sites. Estimations of methanotroph relative abundance in environmental samples were determined by comparing the Type I and Type II primer estimates to those of the 16S universal primers. Methanotrophs in these groundwater samples ranged from 0.2% to 6.6% of the total bacterial population. Pyrosequencing analysis of the same samples showed methanotroph relative abundances that ranged from 1.7% to 54%. In groundwater samples where both DNA and RNA was extracted, the quantities of functional gene transcripts per gene copy was compared, revealing that the transcripts/gene ratio for both pmoA and mmoX was less than one, implying relatively low methanotroph activity. Analysis of mmoX environmental sample dissociation curves revealed a double peak, indicating possible non-specific PCR products. Our data suggests that most of the qPCR primer sets used in the environmental samples adequately detect methanotrophs, though the mmoX primers need to be further validated. These primer sets will be useful for supporting VC bioremediation strategies by providing a rapid, convincing, and cost effective alternative the enrichment culture technique currently employed. Comparison of qPCR and pyrosequencing analysis revealed biases in either one, or both techniques. Finally, our preliminary transcripts/gene data suggests that the methanotrophs at the Carver site are not actively expressing pMMO and sMMO genes above basal levels.

The long-term cattle impact on soil methanotrophs was investigated at cattle overwintering area located at the ecofarm in the South Bohemia. Four sets of soil samples were sampled in spring and fall of 2009 and 2010. Differences in methanotrophic community structure among experimental sites and were investigated by DGGE and MISA. Real-Time PCR was used for estimation of quantity of Type I methanotrophs. The CARD-FISH method was used to estimate the percentage shared and cell counts of Type I and Type II methanotrophs among all prokaryotic cells. Substantial part of the thesis was focused on optimization of methods used.

This Master’s degree study examined concentration and isotopic trends of dissolved methane, isotopic trends of phospholipid fatty acids (PLFA), and generated 1st order flux calculations to identify and assess biogeochemical cycling of dissolved methane in the first full-scale demonstration of EPL technology in the Alberta Oil Sands Region (AOSR). Base Mine Lake (BML) was commissioned by Syncrude Canada Ltd. in 2012 to facilitate the long-term storage and remediation of Fluid Fine Tailings (FFT) that are generated as a result of bitumen extraction via the Clark Hot Water Extraction (CHWE) processes. The results of this project provide evidence of methane oxidation by type I methanotrophs in BML, reducing dissolved oxygen concentrations in the hypolimnion layer. The FFT layer is identified as a source zone of fermentative methanogenesis, creating saturated conditions of dissolved gases. Dissolved methane is transferred to the water column primarily by advective processes related to FFT consolidation, while diffusion is a significant secondary transfer mechanism. Dissolved methane concentrations decrease significantly across the FFT-water interface where diffusive flux rates decrease by several orders of magnitude. Concentrations decreased linearly through the hypolimnion to trace concentrations by the metalimnion, resulting in a minor enrichment of δ13C of the residual dissolved methane pool. A minor enrichment of δ13C in C14:0, C16:0, and C16:1 PLFA coincided across the same interval, indicating utilization of a less depleted carbon source further away from the FFT-water interface where dissolved methane concentrations were lower. PLFA δ13C signatures were depleted relative to expected values of typical DOC substrates, further supporting the incorporation of a depleted signature by transfer of depleted carbon from dissolved methane. / Thesis / Master of Science (MSc)

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