Final Report: Aerobic Cometabolism of Chloroform, 1,1,1-trichloroethane, 1,1-dichloroethylene, and Other Chlorinated Aliphatic Hydrocarbons by Microbes Grown on Butane and Propane

EPA Grant Number: R825689C019
Subproject: this is subproject number 019 , established and managed by the Center Director under grant R825689
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: HSRC (1989) - Western HSRC
Center Director: McCarty, Perry L.
Title: Aerobic Cometabolism of Chloroform, 1,1,1-trichloroethane, 1,1-dichloroethylene, and Other Chlorinated Aliphatic Hydrocarbons by Microbes Grown on Butane and Propane
Investigators: Semprini, Lewis , Arp, Daniel J.
Institution: Oregon State University
EPA Project Officer: Hahn, Intaek
Project Period: January 1, 1995 through January 1, 2001
Project Amount: Refer to main center abstract for funding details.
RFA: Hazardous Substance Research Centers - HSRC (1989) RFA Text |  Recipients Lists
Research Category: Hazardous Substance Research Centers , Land and Waste Management


The objectives of this study are: (1) to evaluate the ability of microorganisms grown on butane or propane to degrade a broad range of chlorinated aliphatic hydrocarbons (CAHs), (2) to obtain a better understanding of factors effecting the rates and extents of transformations of these compounds by these microorganisms, (3) to learn more about of biochemistry of the microorganisms and the associated cometabolic processes.

Summary/Accomplishments (Outputs/Outcomes):

In previously funded Center research, butane and propane were discovered as good cometabolic growth substrates for aerobic treatment of chloroform. Chloroform had previously been shown to be a fairly difficult substrate to degrade via aerobic cometabolism. Other contaminants such as 1,1,1-trichloroethane (TCA) and 1,1-dichlorethylene (1,1-DCE) have also been fairly resistant to aerobic cometabolism. Trichloroetheylene (TCE) can be effectively degraded by microbes grown on phenol or toluene, however, these growth substrates are regulated chemicals that may be difficult to add for subsurface remediation. This research therefore focuses on evaluating the potential of butane and propane as substrates for CAH cometabolism.

Transformation and kinetic studies evaluated the cometabolism of chlorinated methanes, chlorinated ethanes, and chlorinated ethenes by our butane grown enrichment obtained from the Hanford DOE site. Resting cell studies showed very high transformation capacities (mmole CAH/mg TSS cells) in the absence of an endogenous energy source. The chlorinated methanes transformation capacities were: chloromethane (CM), 23; dichloromethane (DCM), 5.8; and chloroform (CF), 0.6; showing transformation was more for effective for the less chlorinated methanes. The transformation of CF, however, promoted the most inactivation of butane-utilizing activity. The chlorinated ethanes transformation capacities ranged from 7.0 for chloroethane (CA) to 0.29 for 1,1,2-trichloroethane (1,1,2-TCA). The transformation and the resulting inactivation were strongly affected by the position of the chlorine substitution. 1,1-dichloroethane and 1,1,1-trichloroethane were more effectively transformed and caused less inactivation than their corresponding isomers with chlorine on both carbons, 1,2-dichloroethane and 1,1,2-trichloroethane. We suspect that the mechanism of cometabolism and the transformation products are causing these differences. The chlorinated ethenes had transformation capacities ranging from 2.7 for vinyl chloride to 0.1 for TCE. The dichloroethylene (DCE) isomers transformation capacities were: trans-DCE, 0.0; cis-DCE, 1.4; 1,1-DCE, 0.9, again showing large variations based on the position of the chlorine substitution. Butane-utilizers effectively transformed 1,1-DCE, however the transformation caused the greatest inactivation of all the compounds tested. Chloride release studies showed the chlorinated methanes and ethenes were the most completely dechlorinated (70 to 100%), while the chlorinated ethanes were the least dechlorinated (40 to 60 %).

Characterization of butane and chlorinated aliphatic degradation by three bacterial isolates was continued. Mycobacterium vaccae, Pseudomonas butanovora, and CF8 (an isolate from Hanford core material) all degrade chloroform when grown on butane. Studies with inactivators and inhibitors of CF degradation led to the hypothesis that each of these three bacteria produces a distinct butane monooxygenase. When cultures of each bacterium are exposed to 14C acetylene, activity is lost. When proteins from these bacteria were examined by SDS-PAGE and autoradiography, different proteins were found to be labeled. Butane oxidation is inhibited or inactivated by the same compounds which affect CF oxidation which provides further evidence that butane monooxygenase catalyzes the transformation of CF. A mixed culture (derived from Hanford core material) was grown in the presence and absence of CF. Cultures grew more slowly in the presence of chloroform. Chloroform degradation potential of resting cells taken from these cultures revealed a greater chloroform specific activity in the cultures not exposed to CF than those which were exposed to chloroform. PCR products (using a variety of primers) were similar whether the culture was grown in the presence or absence of CF, although distinct differences were also noted. These results reveal that the presence of CF during growth on butane does influence the microbial population.

In batch kinetic studies with the butane-grown enrichment the maximum degradation/ transformation rates (k), half-saturation coefficient (Ks), inhibition types (competitive, non-competitive, and mixed inhibition), and inhibition coefficients (KI and KI') of 1,1,1-TCA, 1,1-DCE, 1,1-DCA, and butane have been determined. A direct linear plot method was used to identify the types of inhibition. 1,1,1-TCA, 1,1-DCE, and 1,1-DCA all competitively inhibited each other. Competitive inhibitions kinetics were found to accurately represent the inhibition observed when all three compounds were present. However, butane (growth substrate) showed different inhibition types, that is, non-competitive inhibition on 1,1,1-TCA and 1,1-DCA and mixed inhibition on 1,1-DCE transformation. Inhibition constants were determined using a linearization method that was developed and by non-linear-least-squares-regression of the inhibition data. Good agreement was obtained for the parameters determined by both methods of analysis. When butane and two or more CAHs were present, a model, which combined both competitive and mixed inhibition kinetics, less accurately simulated our experimental results.

Strain CF8, originally isolated from a microcosm of Hanford aquifer solids, has now been brought into pure culture. Identification by 16s rDNA indicates that the bacterium is of the Nocardiodes family, the first example of an alkane oxidizer in this genus. Light sensitivity of butane oxidation and thermal aggregation of the polypeptide that labels with 14C2H2 further support a relatedness to ammonia monooxygenase and particulate methane monooxygenase.

Journal Articles on this Report : 6 Displayed | Download in RIS Format

Other subproject views: All 10 publications 7 publications in selected types All 6 journal articles
Other center views: All 385 publications 251 publications in selected types All 194 journal articles
Type Citation Sub Project Document Sources
Journal Article Hamamura N, Page C, Long T, Semprini L, Arp DJ. Chloroform cometabolism by butane-grown CF8, Pseudomonas butanovora, and Mycobacterium vaccae JOB 5, and methane-grown Methylosinus trichosporium OB3b. Applied and Environmental Microbiology 1997;63(9):3607-3613. R825689C019 (Final)
  • Full-text: AEM-Full Text PDF
  • Abstract: AEM-Abstract
  • Journal Article Hamamura N, Storfa RT, Semprini L, Arp DJ. Diversity in butane monooxygenases among butane-grown bacteria. Applied Environmental Microbiology 1999;65(10):4586-4593. R825689C019 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: AEM-Full Text PDF
  • Abstract: AEM-Abstract
  • Journal Article Hamamura N, Arp DJ. Isolation and characterization of alkane-utilizing Nocardioides sp. strain CF8. FEMS Microbiology Letters 2000;186(1):21-26. R825689C019 (Final)
  • Full-text: ScienceDirect-Full Text HTML
  • Abstract: ScienceDirect-Abstract
  • Other: ScienceDirect-Full Text PDF
  • Journal Article Kim Y, Semprini L, Arp DJ. Aerobic cometabolism of chloroform and 1,1,1-trichloroethane by butane-grown microorganisms. Bioremediation Journal 1997;1(2):135-148. R825689C019 (Final)
  • Abstract from PubMed
  • Abstract: Taylor&Francis-Abstract
  • Journal Article Kim Y, Arp DJ, Semprini L. Chlorinated solvent cometabolism by butane-grown mixed culture. Journal of Environmental Engineering-ASCE 2000;126(10):934-942. R825689C019 (Final)
  • Full-text: Research Gate-Full Text PDF
  • Abstract: ASCE-Abstract
  • Journal Article Semprini L. Strategies for the aerobic co-metabolism of chlorinated solvents. Current Opinion in Biotechnology 1997;8(3):296-308. R825689C019 (Final)
  • Abstract from PubMed
  • Abstract: ScienceDirect-Abstract
  • Supplemental Keywords:

    Groundwater, chlorinated organics, TCA, TCE, biodegradation, Cometabolism, bioremediation., RFA, Scientific Discipline, Waste, Remediation, Environmental Chemistry, Chemistry, Environmental Microbiology, Hazardous Waste, Bioremediation, Hazardous, Engineering, Environmental Engineering, Groundwater remediation, degradation, biodegradation, cometabolism, aerobic cometabolism, Chloroform, chlorinated organic compounds, aerobic cometabolism of chloroform, chlorinated organics, butane, propane, TCA, groundwater, chlorinated aliphatic hydrocarbons

    Progress and Final Reports:

    Original Abstract
  • 1995
  • 1996
  • 1997
  • 1998
  • 1999

  • Main Center Abstract and Reports:

    R825689    HSRC (1989) - Western HSRC

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R825689C001 Interactions between Electron Acceptors in the Treatment of Wastewaters Containing Sulfate, Chlorophenols and Acetate
    R825689C002 Enhancing Biodegradation with Sorption and Alternating Aerobic/Anaerobic Environments
    R825689C003 Development and Verification of a Numerical Model to Predict the Fate and Transport of Chlorinated Phenols in Groundwater
    R825689C004 Redox Transformations of Inorganic Pollutants: Coupling to the Biogeochemical Matrix
    R825689C005 Hexavalent Chromium Sorption and Desorption in Natural Soils and Subsoils
    R825689C006 Biotransformation of Ordnance Wastes Using Unique Consortia of Anaerobic Bacteria
    R825689C007 The Effect of Environmental Conditions on Reductive Dechlorination Rates
    R825689C008 Lead Sorption, Transport, and Remediation in Natural Soils and Subsoils
    R825689C009 Degradation of Chlorinated Aliphatic Compounds by Nitrifying Bacteria
    R825689C010 Remediation of Contaminated Soil from the Baldock Station Maintenance Facility
    R825689C011 The Effect of Apparent EH, Compound Structure, and Electron Donor on Anaerobic Biotransformation of Trinitrotoluene and its Metabolites
    R825689C012 The "Bubble Wall": A Passive In Situ System for Treatment and/or Containment of Contaminated Groundwater
    R825689C013 Inhibition, Inactivation and Recovery: A Universal Model for Aerobic Cometabolic Degradation of Aliphatic Compounds
    R825689C014 Development, Characterization, and Performance Evaluation of Ferrous-Ferric Oxide Adsorbents for Metal Removal from Contaminated Groundwater
    R825689C015 Redox Transformations of Organic and Inorganic Contaminants in the Subsurface Environment
    R825689C016 Demonstration of a Permeable Barrier Technology for the Bioremediation of Ground Water Contaminated with Waste Mixtures
    R825689C017 Development of a Vitamin B12-Amended Bioremediation Process for the Reductive Dechlorination of Chlorobiphenyls at all Chlorine Positions
    R825689C018 An Investigation of the Chlorinated Hydrocarbon Substrate Range of the Filamentous Fungus, Graphium sp.
    R825689C019 Aerobic Cometabolism of Chloroform, 1,1,1-trichloroethane, 1,1-dichloroethylene, and Other Chlorinated Aliphatic Hydrocarbons by Microbes Grown on Butane and Propane
    R825689C020 Cytochrome P-450: An Emerging Catalyst for the Cometabolism of Chlorinated Aliphatic Hydrocarbons and Methyl tert-butyl Ether?
    R825689C021 In-Situ Measurement of TCE Degradation Using a Single-Well "Push-Pull" Test
    R825689C022 Development and Characterization of Redox Sensors for Environmental Monitoring
    R825689C023 Assessing Metal Speciation in the Subsurface Environment
    R825689C024 Simultaneous Removal of the Adsorbable and Electroactive Metals from Contaminated Soils and Groundwater
    R825689C025 Multisolute Sorption and Transport Model for Copper, Chromium, and Arsenic Sorption on an Iron-Coated Sand, Synthetic Groundwater System
    R825689C026 Development of Alkoxysilanes as Slow Release Substrates for the Anaerobic/Aerobic Transformation of Chlorinated Solvents
    R825689C027 Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbons by Toluene-Oxidizing Bacteria
    R825689C028 Development and Characterization of Sensors and Field Instrumentation for Monitoring of Environmental Redox Conditions
    R825689C029 Aerobic Cometabolism of Methyl tert-butyl Ether by Microorganisms Grown on Aliphatic Hydrocarbons
    R825689C030 Biotransformation of Lead and Chromate by Bacteria
    R825689C031 Magnetic Resonance Studies of Heavy Metals in Clays, Zeolites and Ceramics
    R825689C032 Probing the Redox Properties of Environmental Systems: Natural Phenolic Materials
    R825689C033 Reductive Dehalogenation at Carbon and Derivatized Carbon Electrodes
    R825689C034 Detection of Microorganisms Capable of Anaerobic Degradation of Hazardous Substances in Natural Environments
    R825689C035 Treatment of Complex Mixtures
    R825689C036 Oxidation of Chlorinated Solvents by Methanotrophs
    R825689C037 Detection and Assessment of Subsurface Contamination
    R825689C038 Design of Reliable and Cost-Effective Mitigation Schemes
    R825689C039 Gaseous Stripping of Nonaqueous Phase Liquids from the Vadose Zone
    R825689C040 Anaerobic Microbial Transformation of Homocyclic and Heterocyclic Polynuclear Aromatic Hydrocarbons
    R825689C041 Effects of Sorption on Biodegradation of Halogenated Organics
    R825689C042 Trace Metal Removal Processes
    R825689C043 FASTCHEM Applications and Sensitivity Analysis
    R825689C044 Long-term Chemical Transformation of 1,1,1-Trichloroethane (TCA) and Freon 113 under Aquifer Conditions
    R825689C045 In-Situ Anaerobic Biological Treatment of Aromatics in Groundwater
    R825689C046 Use of Starvation and Stress Promoters for Biodegradation of Hazardous Wastes
    R825689C047 Determining and Modeling Diffusion-Limited Sorption and Desorption Rates of Organic Contaminants in Heterogeneous Soils
    R825689C048 Dispersion Modeling of Volatile Organic Emissions from Ground-Level Treatment Systems
    R825689C049 Subsurface Mixing of Nutrients and Groundwater for in-Situ Bioremediation
    R825689C050 Test-Bed Evaluation of In-Situ Bioremediation of Chlorinated Aliphatic Compounds by Toluene Oxygenase Microorganisms
    R825689C051 Demonstration of in-Situ Bioremediation of Chlorinated Aliphatics by Methanotrophs at St. Joseph
    R825689C052 Aquifer Remediation Design in the Presence of Kinetic Limitations
    R825689C053 Determination of Macroscopic Transport Parameters for Biologically Reacting Solutes in Aquifers
    R825689C054 Transformation of Chlorinated Hydrocarbons by Reduced Metallocoenzymes--Kinetic Model Development and Applications to Environmental Systems
    R825689C055 Microbial Degradation of Toluene Under Sulfate-Reducing Conditions--The Role of Iron
    R825689C056 Transformation of TCE by Methanotrophic Biofilms
    R825689C057 Heavy Metals in Ceramic Matrix: Heavy Metals/Clay Interactions in Ceramic Processing
    R825689C058 Radon-222 Method for Locating and Quantifying Contamination by Residual Non-Aqueous Phase Liquids in the Subsurface
    R825689C059 Process Submodel Formulation and Parameter Estimation for Simulation of Bioremediation
    R825689C060 Enhancement of Biodegradation through the Use of Substituted Porphyrins to Treat Groundwater Contaminated with Halogenated Aliphatics
    R825689C061 Field Test of In-Situ Vapor Stripping for Removal of VOCS from Groundwater
    R825689C062 System Design for Enhanced In-Situ Biotransformation of Carbon Tetrachloride: Application to DOE's Arid Site Integrated Demonstration
    R825689C063 Modeling Strategies for Optimizing In-Situ Bioremediation
    R825689C064 Anaerobic Treatment of Chlorinated Solvent Contaminated Groundwater
    R825689C065 In Situ Treatment of Chlorinated Solvents
    R825689C066 Moffett Field In-Situ Bioremediation Study in Support of Full Scale Application
    R825689C067 Full-Scale Evaluation of In Situ Bioremediation of Chlorinated Solvent Groundwater Contamination
    R825689C068 Upscaling Pore-Scale Hydrodynamics and the Transport of Reactive Solutes
    R825689C069 Pathways of Anaerobic Toluene Metabolism by a Sulfate-Reducing Bacterium, Strain PRTOL1
    R825689C070 Anaerobic Ethylbenzene Oxidation in Denitrifying Strain EB1
    R825689C071 Molecular Approaches to Optimize Starvation Promoter Dricen TCE Bioremediation in Pseudomonas
    R825689C072 Modeling VOC Emissions from Hazardous Waste Sites
    R825689C073 Reductive Transformation of Chlorinated Hydrocarbons by Reduced Ethenes Catalyzed by Vitamin B12 - Mechanistic and Kinetic Studies
    R825689C074 Evaluation of Strategies for Full Scale Bioremediation of the Seal Beach Site Using Anaerobic Microbial Processes
    R825689C075 Trace Element Adsorption in Porous Particle Packed Beds
    R825689C076 Hydrologic and Biological Factors Affecting Aquifer Clogging During In-Situ Bioremediation
    R825689C077 Full-Scale Evaluation of an Apparatus for Down-well Oxygen Transfer to Implement In situ Bioremediation at Edwards AFB
    R825689C078 Field Testing of Palladium-Catalyzed Hydrodehalogenation for Chlorinated Hydrocarbon Removal from Groundwater
    R825689C079 Physics of Dissolution of Nonaqueous Phase Liquids: Pore Networks and Field Simulations
    R825689C080 Three-Phase Flow in Fractured Media
    R825689C081 Effects of Redox Zones on the Fate and Transport of Contaminants in the Saturated Subsurface; Characterization and Simulation
    R825689C082 Biochemical Mechanisms of PCE Dehalogenation by Strain MS-1, and its Potential for In-situ Bioaugmentation
    R825689C083 A Large Scale Model for Anaerobic Bioremediation at the Seal Beach Site
    R825689C084 Mechanisms, Chemistry, and Kinetics of Anaerobic Degradation of cDCE and Vinyl Chloride
    R825689C085 Bioenhanced In-Well Vapor Stripping to Treat Trichloroethylene (TCE)
    R825689C086 Effect of Chemical Structure on the Biodegradability of Halogenated Hydro-carbons
    R825689C087 Trace Element Adsorption in Porous Particle Packed Beds
    R825689C088 Arsenic Removal in High Capacity Porous Alumina Packed-Bed Reactors
    R825689C089 Measurement of Interfacial Areas and Mass Transfer Coefficients Between Residual PCE and Water During Surfactant Enhanced Aquifer Remediation
    R825689C090 Proof of Gene Expression During Bioaugmentation
    R825689C091 Experimental and Mathematical Study of Biomass Growth in Pore Networks and its Consequences in Bioremediation
    R825689C092 Gene probes for detecting anaerobic alkylbenzene-degrading bacteria
    R825689C093 Investigation of Palladium Catalyzed Hydrodehalogenation for the Removal of Chlorinated Groundwater Contaminants: Surface Chemistry of Catalyst Deactivation and Regeneration
    R825689C094 Aerobic Methanotrophic Transformation of Biphenyl, Monochlorobiphenyls, and Dichlorobiphenyls