Final Progress Report: Coupled Biogeochemical Process Evaluation for Conceptualizing Trichloroethylene Cometabolism

doi:10.2172/972214

GSTDTAP > 地球科学

DOI	10.2172/972214
报告编号	Final Report
来源ID	OSTI ID: 972214
	Final Progress Report: Coupled Biogeochemical Process Evaluation for Conceptualizing Trichloroethylene Cometabolism
	Crawford, Ronald L; Paszczynski, Andrzej J
	2010-02-19
出版年	2010
语种	英语
国家	美国
领域	地球科学
英文摘要	Our goal within the overall project is to demonstrate the presence and abundance of methane monooxygenases (MMOs) enzymes and their genes within the microbial community of the Idaho National Laboratory (INL) Test Area North (TAN) site. MMOs are thought to be the primary catalysts of natural attenuation of trichloroethylene (TCE) in contaminated groundwater at this location. The actual presence of the proteins making up MMO complexes would provide direct evidence for its participation in TCE degradation. The quantitative estimation of MMO genes and their translation products (sMMO and pMMO proteins) and the knowledge about kinetics and substrate specificity of MMOs will be used to develop mathematical models of the natural attenuation process in the TAN aquifer. The model will be particularly useful in prediction of TCE degradation rate in TAN and possibly in the other DOE sites. Bacteria known as methanotrophs produce a set of proteins that assemble to form methane monooxygenase complexes (MMOs), enzymes that oxidize methane as their natural substrate, thereby providing a carbon and energy source for the organisms. MMOs are also capable of co-metabolically transforming chlorinated solvents like TCE into nontoxic end products such as carbon dioxide and chloride. There are two known forms of methane monooxygenase, a membrane-bound particulate form (pMMO) and a cytoplasmic soluble form (sMMO). pMMO consists of two components, pMMOH (a hydroxylase comprised of 47-, 27-, and 24-kDa subunits) and pMMOR (a reductase comprised of 63 and 8-kDa subunits). sMMO consists of three components: a hydroxylase (protein A-250 kDa), a dimer of three subunits (Îą2Î˛2Îł2), a regulatory protein (protein B-15.8 kDa), and a reductase (protein C-38.6 kDa). All methanotrophs will produce a methanol dehydrogenase to channel the product of methane oxidation (methanol) into the central metabolite formaldehyde. University of Idaho (UI) efforts focused on proteomic analyses using mass spectrometry and genomic analyses using RT-PCR to characterize these enzyme systems. UIâs specific objectives were to develop the proteomics and genomic tools to assess the presence of the methane monooxygenase (MMO) proteins in the aquifers under study and relate this to the enumeration of methanotrophic microorganisms. We targeted the identification of both sMMO and pMMO. We believe that the copper level in the TAN aquifer is most likely suppressing the expression of sMMO and mediates the higher levels of pMMO expression. Hence our investigations included the identification of both forms of MMOs, and we expected a higher concentration of pMMO proteins in TAN samples. The amounts of these proteins present were correlated with numbers of methanotrophs determined by us and other members of the research team using PCR-based methods. In summary, to accomplish our objectives we applied environmental proteomics techniques to monitor proteins that are involved in the co-metabolic degradation of trichloroethylene (TCE) in groundwater of the INL TAN site on Department of Energy ands of near Idaho Falls, ID USA. To acquire peptides sequences information we used an ultra performance chromatography (UPLC) system coupled with QToF Premiere nano-electrospray tandem quadropole-time of flight mass spectrometer. Our goal was to identify signature peptides of methane monooxygenases (MMOs) within methanotrophic bacteria that are active in cometabolic degradation of TCE. We developed a new method for extracting total proteins from environmental planktonic and/or biofilm samples that involve a new time course cell lysis and protein extraction method in combination with chromatographic separation of peptide and tandem mass spectrometry sequencing. The techniques resulted in successful extraction and identification of MMO-based peptides from both pure cultures and TAN site samples. The work confirmed the importance of mathonotrophs in the co-metabolic removal of TCE from the TAN site aquifer.
英文关键词	biogeochemistry co-metabolism trichloroethylene groundwater microbiology bioremediation
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来源平台	US Department of Energy (DOE)
引用统计
文献类型	科技报告
条目标识符	http://119.78.100.173/C666/handle/2XK7JSWQ/4900
专题	地球科学
推荐引用方式 GB/T 7714	Crawford, Ronald L,Paszczynski, Andrzej J. Final Progress Report: Coupled Biogeochemical Process Evaluation for Conceptualizing Trichloroethylene Cometabolism,2010.