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Title: Monitoring genetic and metabolic potential for in situ bioremediation: Mass spectrometry. 1997 annual progress report

Abstract

'A number of US Department of Energy (DOE) sites are contaminated with mixtures of dense non-aqueous phase liquids (DNAPLs) such as carbon tetrachloride, chloroform,. perchloroethylene, and trichloroethylene. At many of these sites, in situ microbial bioremediation is an attractive strategy for cleanup because it has the potential to degrade DNAPLs in situ without producing toxic byproducts. A rapid screening method to determine the broad range metabolic and genetic potential for contaminant degradation would greatly reduce the cost and time involved in assessment for in situ bioremediation as well as for monitoring ongoing bioremediation treatment. In this project, the ORNL Organic Mass Spectrometry (OMS) group is developing mass-spectrometry-based methods to screen for the genetic and metabolic potential for assessment and monitoring of in situ bioremediation of DNAPLs. In close collaboration, Professor Mary Lidstrom''s group at the University of Washington is identifying short DNA sequences related to microbial processes involved in the biodegradation of pollutants. This work will lay the foundation for development of a field-portable mass-spectrometry-based technique for rapid assessment and monitoring of bioremediation processes on site.'

Authors:
; ; ; ;
Publication Date:
Research Org.:
Oak Ridge National Lab., TN (US)
Sponsoring Org.:
USDOE Office of Environmental Management (EM), Office of Science and Risk Policy
OSTI Identifier:
13438
Report Number(s):
EMSP-55108-97
ON: DE00013438
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
44; 55; 54; Progress Report; Measuring Instruments; Medicine; Microorganisms; Genetics; Remedial Action; PROGRESS REPORT; MEASURING INSTRUMENTS; MEDICINE; MICROORGANISMS; GENETICS; REMEDIAL ACTION

Citation Formats

Buchanan, M.V., Hurst, G.B., Britt, P.F., McLuckey, S.A., and Doktycz, M.J.. Monitoring genetic and metabolic potential for in situ bioremediation: Mass spectrometry. 1997 annual progress report. United States: N. p., 1997. Web. doi:10.2172/13438.
Buchanan, M.V., Hurst, G.B., Britt, P.F., McLuckey, S.A., & Doktycz, M.J.. Monitoring genetic and metabolic potential for in situ bioremediation: Mass spectrometry. 1997 annual progress report. United States. doi:10.2172/13438.
Buchanan, M.V., Hurst, G.B., Britt, P.F., McLuckey, S.A., and Doktycz, M.J.. 1997. "Monitoring genetic and metabolic potential for in situ bioremediation: Mass spectrometry. 1997 annual progress report". United States. doi:10.2172/13438. https://www.osti.gov/servlets/purl/13438.
@article{osti_13438,
title = {Monitoring genetic and metabolic potential for in situ bioremediation: Mass spectrometry. 1997 annual progress report},
author = {Buchanan, M.V. and Hurst, G.B. and Britt, P.F. and McLuckey, S.A. and Doktycz, M.J.},
abstractNote = {'A number of US Department of Energy (DOE) sites are contaminated with mixtures of dense non-aqueous phase liquids (DNAPLs) such as carbon tetrachloride, chloroform,. perchloroethylene, and trichloroethylene. At many of these sites, in situ microbial bioremediation is an attractive strategy for cleanup because it has the potential to degrade DNAPLs in situ without producing toxic byproducts. A rapid screening method to determine the broad range metabolic and genetic potential for contaminant degradation would greatly reduce the cost and time involved in assessment for in situ bioremediation as well as for monitoring ongoing bioremediation treatment. In this project, the ORNL Organic Mass Spectrometry (OMS) group is developing mass-spectrometry-based methods to screen for the genetic and metabolic potential for assessment and monitoring of in situ bioremediation of DNAPLs. In close collaboration, Professor Mary Lidstrom''s group at the University of Washington is identifying short DNA sequences related to microbial processes involved in the biodegradation of pollutants. This work will lay the foundation for development of a field-portable mass-spectrometry-based technique for rapid assessment and monitoring of bioremediation processes on site.'},
doi = {10.2172/13438},
journal = {},
number = ,
volume = ,
place = {United States},
year = 1997,
month = 9
}

Technical Report:

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  • '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 rapidlymore » 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.'« less
  • 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, we are developing methods based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)more » for rapid and accurate detection of polymerase chain reaction (PCR) products from microbial genes involved in biodegradation of pollutants. PCR primers are being developed to amplify DNA sequences that are amenable to MALDI-MS detection. This work will lay the foundation for development of a field-portable MS-based technique for rapid on site assessment and monitoring of bioremediation processes.« less
  • 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, we are developing methods based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) formore » rapid and accurate detection of polymerase chain reaction (PCR) products from microbial genes involved in biodegradation of pollutants. PCR primers are being developed to amplify DNA sequences that are amenable to MALDI-MS detection. This work will lay the foundation for development of a field-portable MS-based technique for rapid on site assessment and monitoring of bioremediation processes.« less
  • A number of DOE sites are contaminated with mixtures of dense non-aqueous phase liquids (DNAPLs) such as carbon tetrachloride, chloroform, perchloroethylene, and trichloroethylene. At many of these sites, in situ microbial bioremediation is an attractive strategy for cleanup, since it has the potential to degrade DNAPLs in situ without the need for pump-and-treat or soil removal procedures, and without producing toxic byproducts. A rapid screening method to determine broad range metabolic and genetic potential for contaminant degradation would greatly reduce the cost and time involved in assessment for in situ bioremediation, as well as for monitoring ongoing bioremediation treatment. Themore » objective of this project was the development of mass-spectrometry-based methods to screen for genetic potential for both assessment and monitoring of in situ bioremediation of DNAPLs. These methods were designed to provide more robust and routine methods for DNA based characterization of th e genetic potential of subsurface microbes for degrading pollutants. Specifically, we sought to (1) Develop gene probes that yield information equivalent to conventional probes, but in a smaller size that is more amenable to mass spectrometric detection, (2) Pursue improvements to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) methodology in order to allow its more general application to gene probe detection, (3) Increase the throughput of microbial characterization by integrating gene probe preparation, purification, and MALDI-MS analysis. Effective decision-making regarding remediation strategies requires information on the contaminants present and the relevant hydrogeology. However, it also should include information on the relevant bacterial populations present and the biodegradative processes they carry out. For each site at which bioremediation is considered, it is necessary to determine whether sufficient intrinsic degradative capability is present to suggest intrinsic bioremediation as a viable option, or whether a strategy involving addition of specific nutrients is more likely to be successful. In addition, if the existing genetic potential does not include the desired processes, it may be necessary to add external organisms as well as nutrients, which would negatively impact cost and feasibility scenarios. Once a bioremediation strategy is decided upon and initiated, it is important to carry out monitoring of the bacteria and their activities. Real-time data of this type during the treatment process can allow ongoing evaluation to optimize biodegradation, reducing cost and avoiding possible toxic byproducts. Clearly, the development of novel bioremediation technologies and informed decision-making regarding bioremediation as a treatment option will require in-depth information on the bacteria present at each site and the processes they carry out. Currently such information is generated by labor- and time-intensive treatability tests in the laboratory, and these do not generally assess a broad range of metabolic processes. We undertook this project because a rapid screening method to evaluate genetic potential is an important development to reduce costs for implementing in situ bioremediation strategies at DOE sites. At the outset of this project, it was clear that the explosion of information in the DNA sequence database raised the possibility of developing diagnostic DNA signatures for key microbial processes, as a means for assessing genetic potential. The methods developed in our project would be able to take advantage of the growing information on sequences from environmental samples as well as from microbial genome sequencing projects. An increasing number of metabolic functions could be screened as the depth of information available for designing diagnostic sequences increased.« less
  • A number of DOE sites are contaminated with mixtures of dense non-aqueous phase liquids (DNAPLs) such as carbon tetrachloride, chloroform, perchloroethylene, and trichloroethylene. At many of these sites, in situ microbial bioremediation is an attractive strategy for cleanup, since it has the potential to degrade DNAPLs in situ without the need for pump-and-treat or soil removal procedures, and without producing toxic byproducts. A rapid screening method to determine broad range metabolic and genetic potential for contaminant degradation would greatly reduce the cost and time involved in assessment for in situ bioremediation, as well as for monitoring ongoing bioremediation treatment. Themore » objective of this project was the development of mass-spectrometry-based methods to screen for genetic potential for both assessment and monitoring of in situ bioremediation of DNAPLs. These methods were designed to provide more robust and routine methods for DNA-based characterization of the genetic potential of subsurface microbes for degrading pollutants. Specifically, we sought to (1) Develop gene probes that yield information equivalent to conventional probes, but in a smaller size that is more amenable to mass spectrometric detection, (2) Pursue improvements to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) methodology in order to allow its more general application to gene probe detection, and (3) Increase the throughput of microbial characterization by integrating gene probe preparation, purification, and MALDI-MS analysis.« less