2003 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. , 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, 2002 through September 30, 2003
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


The overall goal of this project is to evaluate how to maximize the chlorinated aliphatic hydrocarbon (CAH)-degrading potential of individual strains and mixed communities of hydrocarbon degrading bacteria and fungi. The specific objectives of this research project are to: (1) identify growth conditions that maximize reductant flow to cometabolism and the cellular mechanisms that minimize the toxic effects of cometabolism and sustain the process; (2) understand the relationship between community dynamics of hydrocarbon oxidizing bacteria and the kinetics of cometabolism in bioremediatory situations; (3) evaluate the performance of cultures in laboratory column studies; and (4) apply improved cometabolic transformation models to laboratory study results.

Progress Summary:

Objective 1

Rationale. 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 under cometabolism conditions.

Experimental Approaches. We have examined the CAH-degrading properties of several individual strains of butane-oxidizing bacteria 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 CAHs on monooxygenase activity and assessed the effect of cometabolism on cell viability. We are focusing on a comparison of trichloroethylene (TCE) degradation in three different butane-oxidizing bacteria, Pseudomonas butanovora, Nocardioides CF8, and Mycobacterium vaccae JOB5. In addition, we are conducting an examination of the cometabolism of the lesser-chlorinated dichloroethenes (DCEs) by P. butanovora, because they often are persistent products of reductive dechlorination at field sites.

Status. Although co-oxidation of TCE by P. butanovora, and Nocardiodes CF8 results in 96 percent inactivation of the BMO, the BMO of M. vaccae was more resistant to inactivation, and 34 percent of activity remained. In P. butanovora and Nocardioides CF8, respiratory activity declined by two-thirds of the control value after TCE degradation, whereas it virtually was unaffected in M. vaccae. At high TCE concentrations (165 µM), the rates of TCE transformation by P. butanovora increased substantially relative to the other two strains, but cell viability was reduced to 17 percent of the control. Viability of M. vaccae and CF8 were unaffected because their rates of TCE transformation did not increase in response to TCE concentrations increasing greater than 22 µM. 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 toxicity might be more appropriate bioremediatory agents than strains that show high rates of TCE degradation that are transitory and accompanied by substantial loss of cell viability.

Butane-grown P. butanovora cooxidizes cis-DCE, trans-1,2-DCE (trans-DCE), and 1,1-DCE. When P. butanovora was exposed to each of the three DCEs, and residual BMO activity was measured by ethylene-dependent ethylene oxide formation, BMO activity was reduced in a time-dependent manner that varied with the specific DCE. BMO activity decreased by 50 percent after 15 minutes of exposure to cis-DCE, after 6 minutes of exposure to trans-DCE, and after 30 seconds of exposure to 1,1-DCE. In addition, co-oxidation of the DCEs had different cytotoxic effects on P. butanovora. Although co-oxidation of cis-DCE and trans-DCE inactivated the majority of BMO activity, cells retained lactate-dependent O2 consumption but were unable to grow normally after removal of DCEs. In contrast, co-oxidation of 1,1-DCE caused a rapid decrease in both BMO activity and lactate-dependent O2 consumption within 3 minutes of exposure, and cells lysed. Treating cells with acetylene to inactivate BMO could eliminate the effects of 1,1-DCE. Lactate-grown cells (in which BMO was not expressed) also were unaffected.

We compared the efficiency of induction of BMO gene expression by TCE and DCEs in wild type and in a LacZ/BMO reporter strain of P. butanovora. The relative induction characteristics of the three DCEs differed from their substrate properties. Trans-DCE induced BMO activity in both the wild type and in the LacZ reporter strain, while cis-DCE only induced enzyme activity in the wild type, implying that products of co-oxidation of cis-DCE were probably the major inducer(s). Enzyme activities in wild-type cells were induced to less than or equal to 25 and 45 percent of the butane control by cis-DCE and trans-DCE, respectively, whereas LacZ expression was induced to 80 percent of maximal by trans-DCE in the reporter strain, implying that the latter compound is an excellent inducer and that the products of its oxidation are not involved in the induction process. In the case of trans-DCE, BMO induction could be detected in the reporter strain at lower concentrations (5 to 10 µM) than could be detected by ethylene-dependent ethylene oxide formation in the wild type (30 µM). The possibility exists that 1,2 trans-DCE could be a useful model compound for gaining a better understanding of the mechanism behind gene regulation of BMO, and might serve as a surrogate inducer of BMO in the absence of butane in naturally attenuating sites downstream of zones of reductive dechlorination, where DCEs might persist.

Objective 2

To achieve this objective we will: (1) evaluate the potential for bioaugmentation of butane-utilizing culture that is effective in transforming mixtures of 1,1,1-trichloroethane (1,1,1-TCA), 1,1-dichloroethane (1,1-DCA), and 1,1-DCE; (2) use molecular tools that have been developed to track the culture upon its addition to a continuous flow laboratory column; (3) develop kinetic parameters for butane-utilization and CAH transformation for use in modeling analyses of the column tests; and (4) model the results of the column experiment with a numerical model that includes kinetic terms for the microbial processes that have been independently determined in the laboratory.

Rationale. Laboratory and microcosm studies have shown different abilities of butane-utilizing microorganisms to cometabolize CAHs. Of particular interest are 1,1,1-TCA, 1,1-DCE, and 1,1-DCA. A Rhodocococcus sp. culture has been sequenced and obtained in pure culture that effectively transforms these compounds. This culture has been bioaugmented to the subsurface at the Moffett Field test zone in a collaborative research grant funded through the Department of Defense's Strategic Environmental Research and Development Program. The Center project is performing continuous flow column experiments similar to those experiments performed in the field, for direct comparison with the field results, and to permit more detailed evaluation of the processes and conditions that cannot be performed in the field. Transformation rate parameters for the Rhodocococcus culture, including maximum utilization rate (kmax) and half-saturation coefficient (Ks) values, also are being determined and compared with previously determined values obtained with the mixed culture from which the pure culture was derived.

Experimental Approach. Continuous flow column studies are being performed with groundwater and aquifer material from the Moffett Airfield experimental test site. The 30 cm long column is being operated with an average groundwater velocity of 2.6 x 10-3 cm/s to yield a hydraulic residence time of 3.2 hours. The transport time is in the range of that achieved in the field for the first monitoring well observation point. The column system has been constructed to permit the feed of two separate groundwater solutions that are amended with either oxygenated groundwater or groundwater containing butane. Both groundwater solutions contain the CAHs of interest. A clock timing system permits the alternate pulsed injection of two different groundwater amended solutions, consistent with the operation of the field tests. Bromide also has been added as a conservative tracer to determine transport characteristics of the column.

Resting cell transformation kinetic tests also have been performed with the Rhodocococcus sp. culture for butane-utilization, and 1,1,1-TCA and 1,1-DCA transformation. Studies also are being performed in a constant injection batch reactor to compare kinetic constants generated at constant butane and contaminant concentrations. Modeling analysis is being performed and compared with the results obtained from the reactor experiments.

Status. Transport, bioaugmentation, and biotransformation experiments have been performed in the continuous flow column experiment. A porosity of 0.26 and a dispersion coefficient of 1.96 x 10-3 cm2/s were obtained from a one-dimensional transport model analysis of bromide transport tests. The transport of 1,1,1-TCA was observed to be retarded, with a retardation coefficient of 3.1. No evidence of 1,1,1-TCA transformation was observed prior to bioaugmentation and biostimulation of the column. Oxygen and butane were transported through the column prior to biostimulation, and butane mass balances indicated that no butane was consumed. Upon bioaugmentation of 0.3 mg (dry-mass) of the Rhodocococcus culture to the column, rapid biostimulation was achieved as indicated by decreases in dissolved oxygen, butane, and 1,1,1-TCA concentrations. Decreases in 1,1,1-TCA concentrations were correlated with decreases in oxygen and butane concentrations. After 10 days of bioaugmentation, 1,1,1-TCA concentrations in the column effluent gradually decreased to 20 from 30 µg/L, representing a > 85 percent reduction in the injection concentrations of 200 µg/L. Steady-state removals of more than 85 percent were achieved over a 15-day period. When 1,1,1-TCA concentrations were increased to 500 µg/L, about 40 percent removal was achieved. Upon decreasing the influent concentration to 200 µg/L, effluent concentrations reached steady-state levels of 80 µg/L, representing about 70 percent removal. Transient tests were performed where butane addition was stopped and 1,1,1-TCA continued. Upon the removal of butane, 1,1,1-TCA concentrations increased to the influent value, indicating that transformation quickly ceased upon the removal of butane. Upon starting butane addition, 1,1,1-TCA removals of about 70 percent were achieved. Thus far, 1,1,1-TCA removal has been maintained for a period of 80 days since the column was bioaugmented. The results are consistent with those obtained in the field demonstration. Some of the loss of activity of 85 percent removal to 70 percent removal may be related to the stimulation of indigenous microorganisms.

Molecular-based polymerase chain reaction (PCR) probes have detected the bioaugmented microorganism in the column groundwater effluent after bioaugmentation and throughout the column test. Currently, a real-time PCR method is being developed to quantify the concentration of microorganisms in the column effluent. At the end of the column experiments, we also will determine the concentrations of the bioaugmented microorganisms distributed spatially throughout the column.

Laboratory batch kinetic tests are being performed to determine maximum degradation rates and the half-saturation coefficients of butane, 1,1,1-TCA and 1,1-DCA with resting cells of the Rhodocococcus culture. A cell yield (Y) for growth on butane of 0.01 mg protein/µmol of butane was obtained. The kmax and Ks values obtained thus far are as follows: Butane: 4.3 µmol/hour/mg protein and 7.9 µM; 1,1,1-TCA: 0.44 µmol/hour/mg protein and 9 µM; 1,1-DCA: 0.46 µmol/hour/mg protein and 15 µM. The kinetic parameters were in the range of values previously determined for the mixed culture from which the culture was isolated.

Objective 3

To achieve this objective we will: (1) describe the ability of Graphium sp. to degrade a range of volatile organic compounds, including CAHs, trichloromethanes, and polyaromatic hydrocarbons (PAHs); and (2) demonstrate that these reactions are catalyzed by an alkane inducible cytochrome P450 monooxygenase through heterologous expression assays with yeast.

Rationale. Volatile organic compounds, including TCE, 1,1-DCE, 1,2-DCE, carbon tetrachloride (CT), and chloroform (CF), a trichloromethane, are important soil and groundwater contaminants. The ability of microorganisms to degrade these compounds represents a promising avenue for the attenuation of polluted sites.

Status. Graphium sp., a filamentous fungus, is one of the few eukaryotes known to grow on gaseous n-alkanes. The initial enzymatic step by which Graphium sp. oxidizes n-alkanes for energy and growth is initiated by a highly nonspecific and alkane-inducible cytochrome P450 monooxygenase. Previous studies have suggested that this enzyme also enables Graphium sp. to cometabolically degrade CAHs, trihalomethanes, and PAHs. More specifically, evidence suggests that Graphium sp. can degrade numerous CAHs, including all four trihalomethanes, chloromethane, dichloromethane, chloroethane, 1,2-DCE, and 1,1,2,2-tetrachloroethane. This fungus also can reductively dechlorinate CT to CF in the absence of oxygen, and then consume CF when aerobic conditions are re-established. Neither the substrate range nor the rates of these Graphium sp. mediated reactions have been determined. The goal of this research is to more quantitatively describe both the substrate range and the rate of these reactions. Although preliminary evidence suggests that a cytochrome P450 monooxygenase catalyzes the initial steps of these reactions, the role of this enzyme has not been conclusively established. The study also aims to demonstrate the role of this enzyme in cometabolic degradation of environmentally significant pollutants.

Progress. The cytochrome P450 alkane monooxygenase (GRSPALK1) cDNA was amplified from propane-grown Graphium sp. mRNA and cloned into the expression vector, ppiczß. The resulting plasmid was sequenced and used to transform Pichia pastoris, a methylotrophic yeast. Sequence analyses revealed that PCR amplification introduced various errors into the cDNA cassette, which resulted in key amino acid mutations. Therefore, colorimetric assays used to monitor for GRSPALK1 activity were negative. This study will be repeated with a higher fidelity copy of GRSPALK1.

Future Activities:

Future studies in the continuous column for Objective 2 will evaluate the transformation of CAH mixtures, including 1,1-DCE and 1,1,1-TCA. Modeling analyses will be performed on the results from the column test using independently measured kinetic parameters. At the end of the column test, molecular analyses of the aquifer solids will be performed to determine the spatial distribution of the bioaugmented culture.

In support of Objective 3, we will demonstrate the role of the P450 monooxygenase enzyme in cometabolic degradation of environmentally significant pollutants, and repeat the study of GRSPALK1 using a higher fidelity copy of the cytochrome.

Journal Articles:

No journal articles submitted with this report: View all 3 publications for this subproject

Supplemental Keywords:

cometabolism, cooxidation, chlorinated aliphatic compounds, CAH, butane, BMO, butane monooxygenase, propane, length heterogeneity, bacterial stress, sustainable industry, bioavailability, bioremediation, civil/environmental engineering, hazardous, hazardous waste, new/innovative technologies, remediation, sustainable environment, treatment technologies, urban and regional planning, cleaner production, aerobic cometabolism, aliphatic compounds, contaminated aquifers, contaminated sediment, dredging, fate, fate and transport, hazardous substance disposal, outreach and education, phytoremediation, pollution prevention, TCE, trichloroethylene, TCA, trichloroethane, DCE, dichloroethene, DCA, dichloroethane, CT, carbon tetrachloride, CF, chloroform., 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.oregonstate.edu/ Exit

Progress and Final Reports:

Original Abstract
  • 2001
  • 2002 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