2005 Progress Report: Aerobic Cometabolism of Chlorinated Ethenes by Microorganisms that Grow on Organic Acids and Alcohols

EPA Grant Number: R828772C010
Subproject: this is subproject number 010 , 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 Ethenes by Microorganisms that Grow on Organic Acids and Alcohols
Investigators: Bottomley, Peter , Arp, Daniel J. , Dolan, Mark E. , Semprini, Lewis
Institution: Oregon State University
EPA Project Officer: Lasat, Mitch
Project Period: September 1, 2001 through August 31, 2006
Project Period Covered by this Report: September 1, 2004 through August 31, 2005
RFA: Hazardous Substance Research Centers - HSRC (2001) RFA Text |  Recipients Lists
Research Category: Land and Waste Management , Hazardous Waste/Remediation


The Part I research project aims to evaluate how to maximize the chloroethene degrading potential of individual strains and mixed communities of hydrocarbon-degrading bacteria. Specific sub-objectives include identifying conditions that maximize reductant flow to cometabolism, and that promote maximum expression of monooxygenase genes and enzyme activity.

A primary goal of the Part II project is to isolate and characterize pure cultures that can transform cis-DCE and VC when grown on acetate, propionate, and butyrate. Initially, the proposal was to attempt isolation of members of an enrichment culture, BA-1, known to co-oxidize cis-DCE when grown on organic acids. However, upon recommendation of the Science Advisory Committee (SAC), our initial focus has changed to isolation and metabolic evaluation of cultures able to directly metabolize cis-DCE and VC.

Progress Summary:

Part I: Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbon Compounds with Butane-Grown Microorganisms (Investigators: P.J. Bottomley and D.J. Arp)

Rationale: Studies conducted under laboratory and field conditions have shown that hydrocarbon-oxidizing bacteria cometabolize a wide range of chloroethenes. Nonetheless, there is considerable variability in the properties of cometabolism shown by different types of bacteria, both in terms of the range of chloroethenes 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 under cometabolism conditions.

Experimental Approaches:
(a) We have examined the chloroethenes degrading properties of several individual strains of butane-oxidizing bacteria (Pseudomonas butanovora, Nocardioides CF8, and Mycobacterium vaccae JOB5) that are genotypically distinct from each other, and that are known to possess distinctly different butane monooxygenases (BMO). We have examined the impact of cometabolism of different chloroethenes on monooxygenase activity, and assessed the effect of cometabolism on cell viability.

(b) We have conducted an examination of the cometabolism of the lesser-chlorinated dichloroethenes (DCEs) by P. butanovora, because they are often persistent products of reductive dechlorination at field sites. In this study, we have focused upon the abilities of different electron donors to drive DCE co-oxidation by butane and propane-grown cells, and to study why different electron donors show different efficacies in sustaining co-oxidation. In addition, we have examined the ability of DCEs to induce the alkane monooxygenase of P. butanovora.

(a) Mutant strains of P. butanovora containing single amino acid substitutions to BMO were engineered. Studies of TCE degradation using these mutants have revealed differences in rates of TCE turnover between mutant strains. One mutant strain, in particular, degraded a larger quantity of TCE, compared to the wild type strain, albeit at a slower rate. We are interested in determining if mutant strains with slower turnover rates will result in sustainable TCE degradation.

(b) Recent studies have focused upon a comparison of the efficacy of different electron donors for co-oxidation of DCEs by butane and propane-grown P. butanovora. Although propionate is an effective electron donor that supports DCE oxidation in propane-grown cells, it does not support co-oxidation in butane-grown cells. Further studies have shown propionate catabolism was inactive following growth on either butane or ethane. In contrast, propionate consumption was induced (about 80 nmoles propionate consumed x min-1 x mg protein-1) following growth on the odd chain-length alkanes, propane and pentane. Using degenerate primers, genes have been identified on a 30 Kb fragment of DNA that show close homology to propionyl-CoA carboxylase subunits, and to methylmalonyl-CoA mutase subunits. If these genes produce active protein products, then it seems reasonable to infer that propionate utilization in P. butanovora proceeds via methylmalonyl-CoA and succinyl-CoA into the TCA cycle.

Although the aliphatic organic acids, propionate and butyrate, initially enhanced the rate of ethene and chloroethene co-oxidation by sBMO, these initial rates of ethene co-oxidation could not be sustained for greater than 10 min. Preliminary in vivo studies indicate both propionate and butyrate irreversibly inactivated the sBMO enzyme. The rate of ethene co-oxidation by BMO was reduced to one -half of the initial rate following 15 min. of exposure to either 10 mM butyrate or propionate. Furthermore, the physiological substrate, butane, was able to protect the sBMO enzyme from the effect of either propionate or butyrate. The possibility that organic acids act as mechanism -based inactivators of the sBMO enzyme is currently being explored. This project will characterize the effects of a range of organic acids on sBMO and to extend our observations of organic acid-dependent inactivation to other monooxygenase enzymes. The long range plans for this project will be to explore the mechanism of inactivation. Both a bacterial aromatic oxygenase and a peroxidase were irreversibly inactivated during the catalytic oxidation of either phenylpropionaldehyde and phenylbutyraldehyde (Raner, et al., 2000) or by aliphatic fatty acids, in general (Huang, et al., 2004). If a similar phenomenon occurs in sBMO, it would provide a mechanism to explain why the aliphatic fatty acids, in particular, did not sustain BMO activity.

Because the potential of P. butanovora to degrade chlorinated ethenes is intimately linked to the expression of the sBMO enzyme, we have studied the regulation of sBMO. Preliminary experiments showed that cells of P. butanovora grown on alkanes C2 through C5 achieved similar maximum levels of sBMO activity (~160 nmoles ethene oxide x min-1 x mg protein-1), the up-regulation of sBMO activity in lactate-grown, sBMO repressed cells was consistently delayed in propane-exposed cells relative to butane. Furthermore, when lactate-grown cells were exposed to butane and propane simultaneously, the presence of propane reduced the ability of butane to induce sBMO activity, indicating that the lag in sBMO induction during exposure to propane is due to repression by propane rather than to its inability to induce sBMO. The repressive behavior of propane was extended to other odd chain alkanes when it was shown that butane induction of sBMO activity could be aborted by additions of either propane, or pentane to cultures already actively inducing sBMO activity. In contrast, the increase in sBMO activity was unaffected by additions of ethane or more butane. These data indicate that propane and pentane were capable of suppressing sBMO activity in P. butanovora, despite their ability to promote sBMO activity when provided as sole growth substrates. As mentioned above, the pathway of propane and pentane consumption is blocked at propionate during growth on either ethane or butane. We believe sBMO expression, in P. butanovora, is repressed in situations where propionic acid accumulates. This need to have the propionate catabolic pathway induced led to the striking disparity in the ability of even chain-length vs. odd chain-length alkanes and alcohols to induce BMO expression.

Part II. Isolation and Investigation of Cultures Capable of Direct Metabolism of VC and cis DCE (Investigators: M. Dolan and L. Semprini)

Rationale: The recent identification of microorganisms capable of aerobic metabolic growth on cis-DCE and VC illustrates the potential for these organisms in the aerobic remediation of distal areas of chlorinated ethene contaminated plumes. Unlike the VC-utilizing organisms, to date, no organisms capable of direct metabolism of cis-DCE have been isolated from aquifer solids or groundwater samples. Two recent field studies on co-metabolic transformation of chlorinated ethenes performed in contaminated zones at Ft. Lewis, WA, and McClellan AFB, CA, showed effective TCE and cis-DCE transformation upon stimulation of the microbial population with toluene or propane. However, after terminating substrate addition, TCE concentrations were observed to rebound to near pre-treatment levels while cis-DCE concentrations remained very low over extended periods. Therefore, organisms may exist at the sites capable of direct metabolism of cis-DCE, which may have been directly or indirectly stimulated by the addition of toluene or propane.

Experimental Approaches: Groundwater samples were obtained at Ft. Lewis, WA, from stimulated and control monitoring wells used in a push-pull experiment to investigate cometabolic TCE and cis-DCE transformation upon stimulation with toluene. Microbial community composition as measured by terminal restriction fragment length polymorphism (T-RFLP) analysis showed considerable community shift as a result of toluene stimulation. Groundwater samples from non-perturbed wells and from toluene-stimulated wells were amended with mineral salts medium (MSM) and a single (carbonaceous) substrate of cis-DCE, ethene, or toluene and monitored for substrate depletion and increased turbidity as an indication of microbial growth. After repeated cycles of enrichment, the cultures were again analyzed for microbial community composition and efforts were begun to isolate cultures from these enrichments. Also, sub-samples of the ethene-enriched systems were used to test for the ability of the enrichments to grow on either VC or fluoroethene (FE), a fluorine-substituted analog of VC.

Mycobacterium and Nocardioides strains capable of growth on VC have been obtained from the researchers that isolated the cultures (Coleman, et al., 2000a), as well as the culture JS666 (Coleman, et al., 2000b), the only known organism capable of growth on cis-DCE. Studies were conducted on these strains to determine their substrate range and possible ability to grow on natural fermentation products such as organic acids or alcohols and whether they retain their ability to utilize VC or cis-DCE. Additionally, the VC utilizers were screened for their ability to grow on the VC surrogate compound, FE, to determine if FE could be a useful surrogate to investigate the potential for VC metabolism at VC-contaminated sites. Mycobacterium strain JS60 is able to cometabolize FE and direct growth on ethene and VC. Currently, no bacteria have been described in the literature that are capable of utilizing FE as a sole carbon and energy source, but preliminary work with Nocardioides strain JS614 shows growth on FE along with ethene and VC.

Status: Attempts to isolate organisms out of the ethene, VC, and FE amended cultures began with streak plating on tryptic soy agar. Representative colonies were back transferred into MSM with either ethene, FE, or VC as growth substrates. Resumption of utilization and growth on these substrates was slow and, in all but one case, the cultures that retained their ability to utilize ethene, FE, or VC were mixed. One isolate, EE13A, which grows on ethene and cometabolizes VC and FE, was obtained. The mixed cultures were streaked on MSM plates and incubated in jars with ethene, VC, or FE in the headspace. Colonies that grew under these conditions were streaked to tryptic soy agar plates to check for purity, and representative colonies inoculated into MSM with either ethene, FE, or VC as growth substrates. Isolated cultures will be checked for purity and phylogeny will be established based on their 16S rDNA sequence and compared to known cultures of VC utilizing organisms. Further study to determine their substrate range and possible ability to grow on natural fermentation products such as organic acids or alcohols and whether they retain their ability to utilize VC will be conducted.

Further studies are being conducted with three phenotypes of Eth utilizing bacteria that will be used in assessing the potential for FE to serve as a surrogate of VC in the subsurface. EE13A was isolated from Ft. Lewis groundwater, utilizes Eth as a growth substrate, and will cometabolize VC and FE. JS60 utilizes Eth and VC as growth substrates and cometabolically degrades FE, and JS614 utilizes ethene, VC, and FE as growth and energy substrates. Preliminary kinetic studies with JS60, JS614, and EE13A show that FE and VC are transformed at approximately the same rate regardless of whether degradation is achieved by direct or co-metabolism. Additionally, significant halogen release was observed for both direct and cometabolic degradation of FE and VC. Degradation intermediates during cometabolism are being determined to allow for a mass balance of halide-associated compounds, and to assist in the development of a hypothetical pathway for degradation during cometabolism.

Mycobacterium strain JS60 was also found to grow on acetate, propionate, and butyrate, but could not grow on formate or lactate. Acetate was chosen for further study because strain JS60 consumed acetate the most rapidly of all the organic acids tested, and acetate is a common product of fermentation reactions in the subsurface. Comparatively, strain JS60’s rate of growth on VC is much slower than that of ethylene. With acetate as an augmenting growth substrate, ethylene and VC utilization rates increased by 30% and 48%, respectively. When strain JS60 was exposed to the isomers of DCE (trans-1,2-dichloroethylene (t-DCE), cis-1,2-dichloroethylene (c-DCE), and 1,1-dichloroethylene (1,1-DCE)), the cells were unable to grow on these compounds. However, when growing on acetate, strain JS60 cometabolized c-DCE and t-DCE, but not 1,1-DCE, with c-DCE transformed more rapidly than t-DCE.

Field Projects

Development of Effective Aerobic Cometabolic Systems for the In-situ Transformation of Problematic Chlorinated Solvent Mixtures, Department of Defense (DoD) Strategic Environmental Research and Development Program (SERDP).

Graduate Students Supported on this Project

Kimberley Halsey, Ph.D. candidate, Molecular and Cellular Biology Program.

David M. Doughty, Ph.D. candidate, Microbiology Graduate Program.

Anne Taylor, Ph.D. candidate, Civil Construction and Environmental Engineering.

Cecilia Razzetti, Visiting Ph.D. Scholar, Ph.D. candidate, University of Bologna, Civil Construction and Environmental Engineering.

Christina Blatchford, M.S. candidate, Civil Construction and Environmental Engineering.


Coleman NV, Mattes TE, Gossett JM, Spain JC. Phylogenetic and kinetic diversity of aerobic vinyl chloride-assimilating bacteria from contaminated sites. Applied and Environmental Microbiology 2002a;68(12):6162-6171.

Coleman NV, Mattes TE, Gossett JM, Spain JC. Biodegradation of cis-dichloroethene as the sole carbon source by a ß-proteobacterium. Applied and Environmental Microbiology 2002b;68(6):2726-2730.

Coleman NV, Spain JC. Distribution of the coenzyme M pathway of epoxide metabolism among ethene-and vinyl chloride-degrading Mycobacterium strains. Applied and Environmental Microbiology 2003;69(10):6041-6046.

Huang L, Colas C, Ortiz de Monetellano PR. Oxidation of carboxylic acids by horseradish peroxidase results in prosthetic heme modification and inactivation. Journal of the American Chemical Society 2004;126(40):12865-12873.

Raner GM, Hatchell AJ, Morton PE, Ballou DP, Coon MJ. Stopped-flow spectrophotometric analysis of intermediates in the peroxo-dependent inactivation of cytochrome P450 by aldehydes. Journal of Inorganic Biochemistry 2000;81:153-160

Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other subproject views: All 18 publications 5 publications in selected types All 5 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 Doughty DM, Sayavedra-Soto LA, Arp DJ, Bottomley PJ. Effects of dichloroethene isomers on the induction and activity of butane monooxygenase in the alkane-oxidizing bacterium “Pseudomonas butanovora.” Applied and Environmental Microbiology 2005;71(10):6054-6059. R828772 (2003)
R828772 (Final)
R828772C010 (2005)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: AEM Full Text
  • Abstract: AEM
  • Other: AEM PDF
  • Journal Article Doughty DM, Sayavedra-Soto LA, Arp DJ, Bottomley PJ. Product repression of alkane monooxygenase expression in Pseudomonas butanovora. Journal of Bacteriology 2006;188(7):2586-2592. R828772 (Final)
    R828772C010 (2005)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: Journal of Bacteriology Full Text
  • Abstract: Journal of Bacteriology
  • Other: Journal of Bacteriology PDF
  • Journal Article Halsey KH, Sayavedra-Soto LA, Bottomley PJ, Arp DJ. Trichloroethylene degradation by butane-oxidizing bacteria causes a spectrum of toxic effects. Applied Microbiology and Biotechnology 2005;68(6):794-801. R828772 (2003)
    R828772 (Final)
    R828772C010 (2005)
  • Abstract from PubMed
  • Abstract: SpringerLink
  • Journal Article Kim Y, Semprini L. Cometabolic transformation of cis-1,2-dichloroethylene and cis-1,2-dichloroethylene epoxide by a butane-grown mixed culture. Water Science & Technology 2005;52(8):125-131. R828772 (Final)
    R828772C010 (2005)
  • Abstract from PubMed
  • Full-text: Oregon State University PDF
  • Abstract: IWA Publishing
  • Other: Oregon State University PDF
  • Journal Article Yeager CM, Arthur KM, Bottomley PJ, Arp DJ. Trichloroethylene degradation by toluene-oxidizing bacteria grown on non-aromatic substrates. Biodegradation 2004;15(1):19-28. R828772 (2003)
    R828772 (2004)
    R828772 (Final)
    R828772C010 (2005)
  • Abstract from PubMed
  • Abstract: SpringerLink
  • Relevant Websites:

    http://wrhsrc.oregonstate.edu/ Exit

    Progress and Final Reports:

    Original Abstract
  • 2002
  • 2003
  • 2004 Progress Report
  • 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