Monitoring genetic and metabolic potential for in situ bioremediation: Mass spectrometry. 1998 annual progress report
- Oak Ridge National Lab., TN (US)
- Univ. of Washington, Seattle, WA (US)
'A number of DOE sites are contaminated with dense non-aqueous phase liquids (DNAPLs) such as carbon tetrachloride and trichloroethylene. At many of these sites, microbial bioremediation is an attractive strategy for cleanup, since it has the potential to degrade DNAPLs in-situ. A rapid screening method to determine the broad range potential of a site''s microbial population for contaminant degradation would greatly facilitate assessment for in-situ bioremediation, as well as for monitoring ongoing bioremediation treatment. Current laboratory-based treatability methods are cumbersome and expensive. In this project, the authors are developing methods based on matrix-assisted laser desorption/ ionization mass-spectrometry (MALDI-MS) to rapidly and accurately detect polymerase chain reaction (PCR) products. In parallel, PCR primers to amplify DNA sequences from microbial genes involved in biodegradation of pollutants are being identified that are short enough to allow MALDI-MS detection. This work will lay the foundation for development of a field-portable MS-based technique for rapid assessment and monitoring of bioremediation processes on site. This report summarizes work after 1-1/2 years of a 3-year project. In this time, the authors have demonstrated MALDI-MS-based detection of signature bacterial PCR products (Hurst et al., 1998). A model system for interfacing MALDI-MS with PCR amplification is based on the pmoA gene for the active site subunit of particulate methane monooxygenase, a bacterial enzyme that can oxidize trichloroethylene. PCR primer pairs were designed to amplify relatively short segments (99 bases and 56 bases) of this gene in Type 1 and Type 2 methanotrophs. A rapid reverse-phase purification of the resulting PCR products allows MALDI-MS detection from a fraction of one 25-microliter PCR reaction. At this level of sensitivity, MALDI-MS has considerable potential to compete with existing electrophoresis and hybridization methods for detecting PCR products in this size range. To allow increased throughput, the PerSeptive Biosystems MALDI-TOF mass spectrometer allows automated MALDI data acquisition, and they have adapted their purification scheme to a 96-well microtiter plate format that allows parallel treatment of 96 PCR reactions in about ten minutes (Weaver et al., 1998). An in-house-constructed TOF mass spectrometer is being modified to allow more fundamental studies aimed at improving the MS detection of PCR products.'
- Research Organization:
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Sponsoring Organization:
- USDOE Office of Environmental Management (EM), Office of Science and Risk Policy
- OSTI ID:
- 13439
- Report Number(s):
- EMSP-55108-98; ON: DE00013439
- Country of Publication:
- United States
- Language:
- English
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