2002 Progress Report: Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbon Compounds with Butane-Grown Microorganisms

EPA Grant Number: R828772C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R828772
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: HSRC (2001) - Western Region Hazardous Substance Research Center for Developing In-Situ Processes for VOC Remediation in Groundwater and Soils
Center Director: Semprini, Lewis
Title: Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbon Compounds with Butane-Grown Microorganisms
Investigators: Arp, Daniel J. , Bottomley, Peter , Ciuffetti, Lynda , Dolan, Mark E. , Giovannoni, Stephen , Semprini, Lewis , Williamson, Kenneth J.
Current Investigators: Arp, Daniel J. , Bottomley, Peter , Ciuffetti, Lynda , Dolan, Mark E. , Williamson, Kenneth J.
Institution: Oregon State University
EPA Project Officer: Lasat, Mitch
Project Period: June 1, 2001 through September 30, 2006
Project Period Covered by this Report: June 1, 2001 through September 30, 2002
Project Amount: Refer to main center abstract for funding details.
RFA: Hazardous Substance Research Centers - HSRC (2001) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management

Objective:

This research project aims to evaluate how to maximize the chlorinated aliphatic hydrocarbon (CAH) degrading potential of individual strains and mixed communities of hydrocarbon-degrading bacteria and fungi. Specific objectives include: (1) identifying growth conditions that maximize reductant flow to cometabolism and the cellular mechanisms that minimize the toxic effects of cometabolism and sustain the process; (2) understanding the relationship between community dynamics of hydrocarbon oxidizing bacteria and the kinetics of cometabolism in bioremediatory situations; (3) evaluating the performance of cultures in laboratory column studies; and (4) applying improved cometabolic transformation models to the results of laboratory studies.

Studies conducted under laboratory and field conditions have shown that hydrocarbon oxidizing bacteria cometabolize a wide range of CAHs. Nonetheless, there is considerable variability in the properties of cometabolism shown by different types of bacteria, both in terms of the range of CAHs degraded, and in their transformation capacities. More research is needed to better understand the microbiological reasons for the range of efficiencies observed, and to use this information to improve the biotechnology of bioremediation.

Progress Summary:

The investigators bring a range of microbiological and environmental expertise to this project. We have examined the CAH degrading properties of several individual strains of butane-oxidizing bacteria and fungi that are known to possess distinctly different butane monooxygenases (BMO). We have examined the impact of cometabolism of different CAHs on monooxygenase activity, and assessed the effect of cometabolism on cell viability and recovery from cometabolism. In one part of this research project, we focused on the cometabolic properties of the lesser chlorinated solvents such as the dichloroethenes (DCEs), because they are often persistent products of reductive dechlorination at field sites. By using molecular approaches with a reporter strain of Pseudomonas butanovora, we are conducting an examination of the ability of DCEs to induce BMO genes in P. butanovora, and the ability of various sources of electron donors to drive the cometabolism of the DCE-induced BMO activity. In another part of this research project, we are evaluating the potential for bioaugmentation of butane-utilizing cultures that are effective in transforming mixtures of 1,1,1-trichloroethane (1,1,1-TCA), 1,1-dichloroethane (1,1-DCA), and 1,1-dichloroethene. We have isolated pure cultures that effectively transform mixtures of these compounds, and that perform well under the nutrient conditions of groundwater and in the presence of indigenous microorganisms. Molecular tools have been developed to track these cultures upon their addition in subsurface remediation. A third part of this research project is to evaluate the potential of Graphium sp., a filamentous fungi, to cometabolically degrade a range of volatile organic compounds (VOCs) including CAHs, trichloromethanes, and polyaromatic hydrocarbons (PAHs). The study also aims to demonstrate that these reactions are catalyzed by an alkane inducible cytochrome P450 monooxygenase.

Decline of TCE metabolism in P. butanovora is primarily influenced by the TCE-mediated loss of BMO activity, rather than due to the loss of cell viability caused by general cellular toxicity. Distinct differences were observed, however, between the cometabolism of cis- and trans-1,2- DCE, and 1,1-DCE by P. butanovora. In particular, 1,1-DCE was shown to rapidly inactivate reductant supply to cometabolism in an acetylene-sensitive manner, implying that extremely toxic metabolites are produced during the oxidation of this compound. A better substrate for the BMO of P. butanovora is cis-1,2-DCE than is trans-1,2-DCE. BMO gene expression is induced more effectively by trans-1,2-DCE than by cis-1,2-DCE. It is well known that high concentrations of butane interfere with cooxidation of most CAHs. We have shown that lactate is an effective electron donor for driving cometabolism of CAHs. Lactate drives cometabolism more effectively than butyrate, which tends to inhibit the process as concentration is raised. Further work is in progress to characterize the role of CAHs in induction of butane oxidizing activity and the efficacy of alternate electron donors for driving cometabolism. We believe these observations might have important implications for the role of hydrocarbon oxidizing bacteria along the periphery of anaerobic plumes, where a combination of fermentative products and less chlorinated products of tetrachloroethene (PCE) or trichloroethene (TCE) degradation are available for further biodegradation.

Two pures have been isolated for the biaugmentation studies, a Rhodococcus culture and a culture that currently is being identified using 16S RNA methods. Kinetic studies have shown that both microorganisms effectively transform 1,1-DCE, 1,1-DCA, and 1,1,1-TCA. We have bioaugmented a mixture of these two cultures to the pilot-scale test zone at Moffett Air Field, as a Department of Defense (DoD) Strategic Environmental Research and Development Program (SERDP)-funded project. In the laboratory part of this program, kinetic parameters are determined for each of the individual cultures. In the center project, continous flow column studies are performed with these two cultures biaoaugmented into Moffett Field core material.

Propane-grown filter-attached Graphium sp. cultures are being used in short-term laboratory studies to determine the extent and rate of cometabolism of TCE, carbon tetrachloride (CT), chloroform (CF), and naphthalene. We also are characterizing the oxygenase responsible for the initial transformation of the target compound. We have cloned a full length genomic copy of an alkane inducible cytochrome P-450 from Graphium sp. This clone will be transformed into Saccharomycetes cerevisae AH109 and expressed under the control of a constitutive promoter. Heterologous expression in a yeast will allow us to more quantitatively describe CAH and PAH degradation because yeast cells are more amenable to laboratory manipulations. Furthermore, if S. cerevisae does not possess the other endogenous enzymes required for mineralization of the substrate, we will have the opportunity to identify intermediates in the catabolic pathway. These experiments will conclusively determine the function of the cytochrome P-450 in the cometabolic transformation of CAHs, PAHs, and trichloromethanes.

Future Activities:

The continuous column study will permit a comparison with results from the field, and also will allow a broad range of conditions to be evaluated, such as the concentration range that can be effectively treated. The distribution of the bioaugmented microorganisms in the column(s) also will be determined through destructive testing. Model simulations of the column studies and field tests also will be performed and compared.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 3 publications 3 publications in selected types All 2 journal articles
Other center views: All 158 publications 63 publications in selected types All 60 journal articles
Type Citation Sub Project Document Sources
Journal Article Kim Y, Arp DJ, Semprini L. Kinetic and inhibition studies for the aerobic cometabolism of 1,1,1-trichloroethane, 1,1-dichloroethylene, and 1,1-dichloroethane by a butane-grown mixed culture. Biotechnology and Bioengineering 2002;80(5):498-508. R828772 (2002)
R828772 (Final)
R828772C003 (2002)
  • Abstract from PubMed
  • Full-text: Oregon State University-PDF
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  • Abstract: Wiley-Abstract
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  • Journal Article Kim Y, Arp DJ, Semprini L. A combined method for determining inhibition type, kinetic parameters, and inhibition coefficients for aerobic cometabolism of 1,1,1-trichloroethane by a butane-grown mixed culture. Biotechnology and Bioengineering 2002;77(5):564-576. R828772 (2002)
    R828772 (Final)
    R828772C003 (2002)
  • Abstract from PubMed
  • Full-text: Oregon State University-PDF
    Exit
  • Abstract: Wiley-Abstract
    Exit
  • Supplemental Keywords:

    cometabolism, cooxidation, chlorinated aliphatic compounds, butane, propane, length heterogeneity, bacterial stress, cleaner production, pollution prevention, EPA Region 6, contaminated sediment, contaminated sediments, dredging, fate, fate and transport, hazardous substance disposal, outreach and education, phytoremediation, pollution prevention, EPA Region 4., RFA, Scientific Discipline, Geographic Area, Waste, TREATMENT/CONTROL, Ecosystem Protection/Environmental Exposure & Risk, Sustainable Industry/Business, Bioavailability, cleaner production/pollution prevention, Remediation, Sustainable Environment, Treatment Technologies, Technology for Sustainable Environment, Hazardous Waste, Bioremediation, New/Innovative technologies, Hazardous, Environmental Engineering, Urban and Regional Planning, EPA Region, region 4, contaminated sediments, hazardous substance disposal, fate, fate and transport, fate and transport , contaminated sediment, aliphatic compounds, aerobic cometabolism, Region 6, dredging, butane, outreach and education, pollution prevention, technology transfer, phytoremediation, contaminated aquifers, chlorinated aliphatic hydrocarbons

    Relevant Websites:

    http://wrhsrc.orst.edu/ Exit

    Progress and Final Reports:

    Original Abstract
  • 2001
  • 2003 Progress Report
  • 2004
  • 2005
  • Final

  • Main Center Abstract and Reports:

    R828772    HSRC (2001) - Western Region Hazardous Substance Research Center for Developing In-Situ Processes for VOC Remediation in Groundwater and Soils

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R828772C001 Developing and Optimizing Biotransformation Kinetics for the Bio- remediation of Trichloroethylene at NAPL Source Zone Concentrations
    R828772C002 Strategies for Cost-Effective In-situ Mixing of Contaminants and Additives in Bioremediation
    R828772C003 Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbon Compounds with Butane-Grown Microorganisms
    R828772C004 Chemical, Physical, and Biological Processes at the Surface of Palladium Catalysts Under Groundwater Treatment Conditions
    R828772C005 Effects of Sorbent Microporosity on Multicomponent Fate and Transport in Contaminated Groundwater Aquifers
    R828772C006 Development of the Push-Pull Test to Monitor Bioaugmentation with Dehalogenating Cultures
    R828772C007 Development and Evaluation of Field Sensors for Monitoring Bioaugmentation with Anaerobic Dehalogenating Cultures for In-Situ Treatment of TCE
    R828772C008 Training and Technology Transfer
    R828772C009 Technical Outreach Services for Communities (TOSC) and Technical Assistance to Brownfields Communities (TAB) Programs
    R828772C010 Aerobic Cometabolism of Chlorinated Ethenes by Microorganisms that Grow on Organic Acids and Alcohols
    R828772C011 Development and Evaluation of Field Sensors for Monitoring Anaerobic Dehalogenation after Bioaugmentation for In Situ Treatment of PCE and TCE
    R828772C012 Continuous-Flow Column Studies of Reductive Dehalogenation with Two Different Enriched Cultures: Kinetics, Inhibition, and Monitoring of Microbial Activity
    R828772C013 Novel Methods for Laboratory Measurement of Transverse Dispersion in Porous Media
    R828772C014 The Role of Micropore Structure in Contaminant Sorption and Desorption