Grantee Research Project Results

2004 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: National Research Program on Design-Based/Model-Assisted Survey Methodology for Aquatic Resources
Center Director: Stevens, Don L.
Title: Aerobic Cometabolism of Chlorinated Ethenes by Microorganisms that Grow on Organic Acids and Alcohols
Investigators: Bottomley, Peter , Dolan, Mark E. , Arp, Daniel J. , Semprini, Lewis
Institution: Oregon State University
EPA Project Officer: Aja, Hayley
Project Period: September 1, 2001 through August 31, 2006
Project Period Covered by this Report: September 1, 2003 through August 31, 2004
RFA: Hazardous Substance Research Centers - HSRC (2001) Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management

Objective:

The Part I project aims to evaluate how to maximize the chloroethene degrading potential of individual strains and mixed communities of hydrocarbon-degrading bacteria. Specific subobjectives 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-dichloroethene (cis-DCE) and vinyl chloride (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 (P. J. Bottomley, PI; D.J. Arp, Co-PI)

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 (BMOs). 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.

Status:
(a) While co- oxidation of TCE by P. butanovora, and Nocardiodes sp. CF8 ultimately results in 96% inactivation of the butane monooxygenases, the BMO of M. vaccae is more resistant to inactivation. Although the rates of TCE transformation by P. butanovora increase with increasing TCE concentrations (up to 165 μM), cell viability is concomitantly reduced to 17% of the control. In the case of M. vaccae and Nocardioides CF8, rates of TCE transformation do not increase in response to TCE concentrations > 22 μM, and viability is unaffected. These findings indicate that situations might be identified where the use of strains (such as M. vaccae) possessing slower rates of CAH degradation without cell death, might be more appropriate bioremediatory agents than strains that show high transitory rates of TCE degradation that are accompanied by substantial loss of cell viability.

(b) Although co-oxidation of DCEs in P. butanovora can be driven by a variety of organic acids provided as exogenous electron donors, lactate supports the highest initial rates, and also sustains rates for the longest period of time. Using lactate as electron donor, butane-grown P. butanovora co- oxidize cis-DCE, 1,2, -trans DCE, and 1,1-DCE at distinctly different rates, with 1,1-DCE being the best and 1,2-trans DCE the worst substrates. After exposure of P. butanovora to DCEs, BMO activity was reduced in a time-dependent manner that varied with the specific DCE. BMO activity decreased by 50% after 15 min exposure to cis-DCE, after 6 min exposure to trans-DCE, and after only 30 sec exposure to 1,1-DCE. However, because the velocity of 1,1-DCE oxidation was ~10x faster than that of 1,2-trans DCE, the cells actually consumed about equal amounts of the two DCEs to achieve similar inactivation of sBMO. We conclude that the products of oxidation of both 1,1- and 1,2 -trans DCEs were equally toxic to sBMO activity despite being generated at different rates. Although the same amounts of 1,1, -DCE and 1, 2-trans DCE were consumed by P. butanovora in 30 sec and 6 min, respectively, the declines in lactate-dependent O₂ consumption was delayed to follow the same kinetics of decline as the 1,2-trans DCE treatment. This observation indicates that, in contrast to effects on sBMO activity, the products of 1,2-trans DCE oxidation are more toxic to general respiratory activity than are those of 1,1-DCE.

Recent studies have been 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. Propane is a slower inducer of sBMO than butane, and also negatively interferes with the induction of sBMO by butane. The products of propane metabolism, propanol, propanaldehyde and propionate, are potent repressors of BMO induction. We hypothesize that the pathway for metabolizing propionate is repressed during growth on even chain length alkanes, organic acids and alcohols, and that addition of odd chain length hydrocarbons, alcohols and organic acids causes the accumulation of propionate and prevents expression of sBMO. This work is being further investigated.

Not only are the organic acids, butyrate and propionate, less efficient electron donors than lactate, they have been shown to inactivate sBMO when turning over poorer substrates like 1,2-trans DCE. Although the aliphatic organic acids provide reductant to sBMO, they seem to compete with substrates for the active site of the enzyme. The mechanism of BMO inactivation by aliphatic organic acids is currently being studied.

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

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 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 know organism capable of growth on cis-DCE. Studies will be 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 will be 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. A proposal was submitted to the Environmental Security Technology Certification Program (ESTCP) program that would incorporate the findings from this research for use in field tests to evaluate the potential for direct VC or cis-DCE metabolism in contaminated aquifers.

Status: T-RFLP analyses of the enrichment cultures produced from the same field groundwater sample showed that each substrate yielded distinct microbial communities. Although there was much diversity and many common TFLs between the enrichments, the dominant fragments produced from each enrichment substrate were different. Similarities in dominant fragments were observed in enrichments of different groundwater samples with the same substrate. Microcosms enriched with toluene or ethene produced turbid cultures upon repeated feeding. The groundwater microcosms enriched with cis-DCE did not grow dense cultures, but slow transformation of the cis-DCE was observed over time. Back transfers of the cis-DCE enriched cultures into MSM media have not successfully retained cis-DCE transformation ability. To ensure that ammonia oxidizers were not being enriched in the microcosms, the ammonium in the MSM was replaced with nitrate for further attempts.

Groundwater microcosms enriched with ethene were combined and subdivided for incubation with either ethene, VC, or FE as the sole carbon source to test for evidence of direct VC metabolism and the ability to transform or grow on FE. In some of the tests, the enrichment culture was diluted so that VC or FE disappearance would likely indicate growth on these compounds. Compound disappearance and concomitant increases in optical density were observed with all three compounds over repeated feedings with similar rates of utilization for VC and FE and ethene rates about 5 times greater. After about 30 days of exposure to the respective substrates, T-RFLP analyses showed the VC and FE communities had shifted relative to the ethene-fed cultures. Genomic DNA from the ethene enrichment culture was probed with CoM transferase primers after growing on Eth, VC or FE for 30d. CoM transferase, which is expected to be involved with the metabolism of the epoxides (Coleman, et al., 2003) that were formed, was detected with growth on all three substrates.

Attempts to isolate organisms out of the ethene, VC, and FE amended cultures began with streak plating on tryptic soy agar. Isolates were obtained from the ethene enriched microcosms, were back transferred into MSM and retained their ability to utilize ethene. They are now being tested in MSM for the ability to grow on VC or FE, a fluorinated VC surrogate. 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.

Highlights

David Doughty received the Janet Ford studentship award for outstanding graduate research in the Department of Microbiology.

Anne Taylor received a National Science Foundation (NSF) IGERT Fellowship from the OSU Subsurface Biosphere Grant.

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).

Students Working on the Project

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

David M. Doughty, Ph.D. candidate, Microbiology Graduate Program, Oregon State University.

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

Cecilia Razzetti, visiting Ph.D. scholar, Ph.D. candidate, University of Bologna, Civil Construction and Environmental Engineering.

References:

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- p roteobacterium. 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.

Journal Articles:

No journal articles submitted with this report: View all 18 publications for this subproject

Relevant Websites:

http://wrhsrc.oregonstate.edu/ Exit

Progress and Final Reports:

Original Abstract

2002

2003

2005 Progress Report

Final

Main Center Abstract and Reports:

R828772    National Research Program on Design-Based/Model-Assisted Survey Methodology for Aquatic Resources

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
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