Final Report: Application of Anaerobic and Multiple-Electron-Acceptor Bioremediation to Chlorinated Aliphatic Subsurface Contamination

EPA Grant Number: R825549C053
Subproject: this is subproject number 053 , established and managed by the Center Director under grant R825549
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: HSRC (1989) - Great Plains/Rocky Mountain HSRC
Center Director: Erickson, Larry E.
Title: Application of Anaerobic and Multiple-Electron-Acceptor Bioremediation to Chlorinated Aliphatic Subsurface Contamination
Investigators: Parkin, Gene F
Institution: University of Iowa
EPA Project Officer: Hahn, Intaek
Project Period: November 1, 1993 through June 2, 2000
Project Amount: Refer to main center abstract for funding details.
RFA: Hazardous Substance Research Centers - HSRC (1989) RFA Text |  Recipients Lists
Research Category: Organic Chemical Contamination of Soil/Water , Land and Waste Management


The goal of this project was to advance our understanding of anaerobic and mixed-electron acceptor bioremediation of chlorinated aliphatics. Research focused on the transformation of perchloroethene (PCE), 1,1,1-trichloroethane (TCA), and carbon tetrachloride (CT) in methanogenic and sequential methanogenic/methanotrophic environments. The research objectives given in the original proposal are:

1. Study the effects of mixtures of chlorinated aliphatics on transformation rates.
2. Identify organisms that are able to convert chlorinated aliphatics to non-objectionable products and determine growth requirements.
3. Investigate the potential for combined anaerobic/aerobic processes.
4. Study the effects of non-aqueous phase chlorinated aliphatics on the extent of degradation and toxicity.

Objectives 2 and 4 were to be addressed at Rice University. However, funds were not available for this part of the work. Objectives 1 and 3 were addressed by the research at Iowa.

Summary/Accomplishments (Outputs/Outcomes):

The U.S. EPA Hazardous Substance Research Centers (HSRC) and national agencies such as the Department of Defense and the Department of Energy (D.O.E.) have identified research on remediation processes for chlorinated aliphatic-contaminated subsurfaces as a high priority. A promising technique is the use of in situ bioremediation. Aerobic biodegradation of TCE and other chlorinated aliphatics has been studied intensely over the last decade. Results of these studies demonstrate severe limitations in application of aerobic bioremediation, because several of the chlorinated aliphatics of greatest concern are not degraded by aerobic bacteria. In combination with TCE, carbon tetrachloride (CT), perchloroethene (PCE), and 1,1,1-trichloroethane (TCA) are found in large volumes at D.O.E. facilities, landfills, and industrial sites. PCE and CT are not degraded aerobically. Therefore, complete remediation of a site containing these compounds by aerobic processes is not possible. Unlike aerobic biological processes, anaerobic biotransformation of all chlorinated aliphatics occur. This lack of specificity, coupled with the fact that most contaminated aquifers are anaerobic, may make anaerobic bioremediation an alternative - or supplement - to aerobic processes.

A variety of enrichment cultures were developed as a part of this project. Anaerobic enrichments were maintained on lactate or acetate and a basic inorganic, buffered nutrient medium, while an aerobic methanotrophic enrichment culture was developed for sequential anaerobic-aerobic column studies. All enrichments and experiments were maintained at 20 0C. Experiments assessing the effect of mixtures of CT, PCE, and 1,1,1-TCA were conducted primarily in 38-mL serum bottles. Sequential anaerobic-aerobic reactors were glass columns with 1.5-inch O.D. and 12 inches long, connected in series. All were filled with 3-mm borosilicate glass beads except for one anaerobic column filled with steel wool.

Compounds were analyzed by a variety of techniques (headspace analysis by GC for volatile compounds such as the chlorinated compounds, ethene, ethane, methane and H2; HPLC analysis for non-volatile compounds such as lactate; IC analysis for ionized compounds such as nitrate, acetate, and perchlorate). Standard QA/QC procedures were followed as supervised by Craig Just, Laboratory Director.

A summary of major findings follows in bullet form.

  • Due to thermodynamic and kinetic considerations, CT, PCE, 1,1,1-TCA and their daughter compounds are difficult to completely mineralize using a single anaerobic process. The capabilities of a sequential methanogenic-methanotrophic system to transform CT, PCE, and 1,1,1-TCA alone and in mixtures were investigated. Three anaerobic-aerobic column systems containing glass beads or steel wool (Fe(O)) as support media were established. Anaerobic glass-bead reactors were seeded with acetate or lactate enrichment cultures and fed acetate or lactate as an electron donor; H2 produced from Fe(0) corrosion served as an electron donor in the anaerobic steel-wool column. Methanotrophic, aerobic columns are supported by methane produced from the anaerobic columns, and fed hydrogen peroxide as an oxygen source. Abiotic controls were operated to assess volatilization and sorption losses and the effect of H2O2 on transformation of the CAHs.

    CT was fed alone or with 1,1,1 -TCA or with PCE and 1,1,1 -TCA. Concentrations were 1, 2, or 4 mM. Results showed complete anaerobic removal of CT at 1 and 2 mM when fed alone, or in mixtures with PCE and 1,1,1-TCA. Removal of 1,1,1-TCA was essentially complete. Very little transformation of PCE was observed with the acetate enrichment culture; however, complete removal of PCE was observed with the lactate enrichment culture. When CT was fed at 2 mM with 1,1,1-TCA to the reactor containing the acetate enrichment culture, there were some inhibitory effects as chloroform (CF) and 1,1,-dichloroethane accumulated. Similarly, when CT was fed at 2 mM with PCE and 1,1,1-TCA to reactors containing acetate or lactate enrichment cultures, CF and 1,1-DCA accumulated along with vinyl chloride (VC for lactate enrichment only and PCE was not transformed with acetate enrichment culture). Most of these transformation products were completely removed in the aerobic columns. Anaerobic cultures supported by Fe(0) appeared to give more complete removal of parent compounds and their reductive dechlorination products. When parent compounds were fed at 2 or 4 mM, accumulation of dichloromethane, 1,1-dichloroethane, and VC were transiently observed. These compounds were completely removed in the aerobic column.

    For the conditions studied (concentrations of 1, 2, or 4 mM), sequential anaerobic-aerobic treatment demonstrated the ability to achieve effluent concentrations near or below the MCLs for the parent compounds and their reductive dechlorination products.

  • Batch experiments were conducted with an unacclimated acetate enrichment culture and CT, PCE, and 1,1,1-TCA fed alone and in mixtures. CT was transformed most rapidly, followed by 1,1,1-TCA and PCE. Rate coefficients for the transformation of CT or 1,1,1-TCA were significantly lower when the concentration of either compound was increased (alone and in mixtures). CT and 1,1,1-TCA were detrimental to the transformation of each other. PCE transformation was limited, and its presence did not significantly impact degradation of CT or 1,1,1-TCA. In once-fed batch reactors used to assess the potential for toxicity, a transformation limit (analogous to a transformation capacity) was observed for both PCE (1.91 ? 0.21 mg of PCE per mg of cells) and 1,1,1-TCA (7.84 ? 0.34 mg of 1,1,1-TCA per mg of cells), but not for CT. The low observed limit of transformation for PCE, combined with its lack of effect on the transformation of the other two compounds, suggests that the number of PCE-degrading organisms in the enrichment culture was low. Transformation of repeated spikes of CT was maintained for over 400 days without supplementation of acetate, indicating that inhibition, rather than potential inactivation, was responsible for the negative impact observed when CT was present in mixtures.

  • Several anaerobic enrichment cultures were developed as part of this project. Three showed halorespiring activity on PCE (that is, high rates of PCE transformation with conversion to ethene at concentrations as high as 200 mM). Two were enriched on lactate; one was fed PCE alone (LEC1) and one was fed PCE with CT and 1,1,1-TCA (LEC3). A third halorespiring culture was enriched on acetate and fed PCE alone (AEC1). Within these cultures, CT and 1,1,1-TCA were transformed as fortuitous substrates (i.e., no observable benefit to the organisms). Cultures enriched on lactate and acetate without CAH addition were termed LEC0 and AEC0, respectively. In addition, a stream-sediment sample from a PCE-contaminated site (Burlington Northern Railroad in Burlington, IA) was enriched on lactate and transformed PCE in a manner similar to that of LEC 1. Preliminary phylogenic analysis using PCR techniques and a 16S rDNA-based probe specific for Dehalococcoides ethenogenes strain 195 suggested that LEC1 contains an organism with "high similarity" to strain 195. Similar analysis of LEC3 and AEC1 also indicated the presence of a "highly similar" organism, although the evidence wasn't as strong as for LEC1. This work was done by colleagues at Cornell University. LEC1 demonstrated the ability to transform higher concentrations of PCE at higher rates than did LEC3 or AEC1.

  • A number of experiments were conducted assessing the impact of mixtures of CT, PCE, and 1,1,1-TCA on transformation of these compounds by LEC1. This culture was able to rapidly transform PCE to ethene in conjunction with methanogenesis and to degrade both CT and 1,1,1-TCA despite no previous exposure to these compounds. While the presence of less than 20 mM of 1,1,1-TCA had little effect on PCE conversion to ethene, the addition of 10-15 mM of CT negatively impacted both the PCE and VC transformation steps. CT and 1,1,1-TCA primarily inhibited methanogenesis before each compound was completely transformed. They served to further inhibit methanogenesis as well as utilization of acetate and propionate in PCE-containing treatments via increased persistence of metabolites such as VC. The inclusion of CT with PCE increased peak hydrogen concentrations from 2 to 20 mM, suggesting CT disrupted the ability of dechlorinating and other hydrogenotrophic organisms to maintain low hydrogen thresholds. Despite the negative impacts on multiple populations as a result of the addition of CT and 1,1,1-TCA, transformation proceeded in treatments containing all three parent compounds. This indicates that the specific dechlorinating organisms within LEC1 were capable of either transforming these cocontaminants or remaining functional while non-specific organisms mediated their removal. These findings are promising with respect to the feasibility of remediating complex sites. They suggest that natural attenuation and enhanced bioremediation based on the assumed presence of halorespiring organisms are still reasonable strategies even if sites contain a variety of CAHs.

  • The role served by the acetoclastic methanogens within LEC1 was investigated through a series of supplementation experiments. LEC1 was supplemented with a 20% dilution (v/v) of one of three other mixed enrichment cultures (LEC0, AEC0, or AEC1 ) or with filtered supernatants from each of these cultures. When this set of experiments was conducted during a period of inhibited acetoclastic activity within LEC1, whole-culture amendments significantly decreased chlorinated ethene removal times (8 to 78%) while increasing methane production rates (92 to 275%). Supplementation with filtered supernatant improved dechlorination rates relative to undiluted LEC1 without significantly impacting methanogenesis, indicating that the benefit was provided directly to dechlorinating organisms via secretion of extracellular components from the added culture. AEC0 performed the best of the three cultures used. This culture would be expected to be dominated by acetoclastic methanogens. Because no benefit was observed in subsequent experiments when acetoclastic methanogenesis was not inhibited in LEC1, it is apparent that the catalytic or nutritional needs of specific dechlorinating organisms were satisfied when the acetoclastic methanogens were healthy. These results suggest that survival of different members of a microbial community may be necessary to ensure effective remediation of PCE with a minimum of external amendments. That is, perhaps maintaining a healthy population of acetoclastic methanogens can insure PCE removal by halorespirers.

  • Radiolabeled PCE (1,2-14C-PCE) and CT (14CCI4) were added to aliquots of LEC1 to assess the effect of mixtures of these two compounds on product distribution. Both PCE and CT were readily dechlorinated, although significant carbon disulfide formation was observed during CT degradation. The level produced could be reduced significantly by reducing the sulfide content of the nutrient medium. Calculated mass-balance recoveries were typically greater than 100% due to probable underestimation of initial activity. While the majority of the 14C-CT was recovered in the volatile fraction, 14CO2 increased significantly over time. The majority of 14C from PCE was recovered in the volatile and non-strippable fractions; however, a significant increase in 14CO2 was observed relative to cell-free controls. This suggests a pathway other than reductive dechlorination of PCE to ethene. We believe this represents the first evidence of these two types of pathways within the same enrichment culture. The addition of CT and PCE together inhibited the transformation of each other and reduced the percent of 14C recovered as 14CO2. However, the magnitude of these reductions was not severe and appeared to be the result of slower overall transformation rather than a complete inhibition of mineralization pathways. In addition, the presence of CT and PCE together did not seem to result in a change in product distribution, but rather the aforementioned reduction in rates of formation and degradation.

    The results of this research have been presented at several professional meetings and published in widely read journals. The principal investigator has responded to many requests for information on the project.

  • Journal Articles on this Report : 7 Displayed | Download in RIS Format

    Other subproject views: All 28 publications 7 publications in selected types All 7 journal articles
    Other center views: All 904 publications 230 publications in selected types All 182 journal articles
    Type Citation Sub Project Document Sources
    Journal Article Adamson DT, Parkin GF. Impact of mixtures of chlorinated aliphatic hydrocarbons on a high-rate, tetrachloroethene-dechlorinating enrichment culture. Environmental Science & Technology 2000;34(10):1959-1965. R825549C053 (Final)
    not available
    Journal Article Adamson DT, Parkin GF. Dependence of a high-rate PCE-dechlorinating enrichment culture on methanogenic activity. Bioremediation Journal 2001;5(1):51-62. R825549C053 (Final)
    not available
    Journal Article Adamson DT, Parkin GF. Product distribution during transformation of multiple contaminants by a high-rate, etrchlorothene-dechlorinating enrichment culture. Biodegradation 2001;12(5):337-348. R825549C053 (Final)
    not available
    Journal Article Adamson DT, Parkin GF. Biotransformation of mixtures of chlorinated aliphatic hydrocarbons by an acetate-grown methanogenic enrichment culture. Water Research 1999;33(6):1482-1494 R825549C053 (Final)
    not available
    Journal Article Gregory KB, Mason MG, Picken HD, Weathers LJ, Parkin GF. Bioaugmentation of Fe(0) for the remediation of chlorinated aliphatic hydrocarbons. Environmental Engineering Science 2000;17(3):169-181. R825549C044 (Final)
    R825549C053 (Final)
    not available
    Journal Article Parkin GF. Anaerobic biotransformation of chlorinated aliphatic hydrocarbons: Ugly duckling to beautiful swan. Water Environment Research 1999;71(6):1158-1164. R825549C044 (Final)
    R825549C053 (Final)
    not available
    Journal Article Weathers LJ, Parkin GF. Toxicity of chloroform biotransformation to methanogenic bacteria. Environmental Science & Technology 2000;34(13):2764-2767. R825549C053 (Final)
    not available

    Supplemental Keywords:

    anaerobic, biodegradation, chlorinated hydrocarbons, mineralization., Scientific Discipline, Toxics, Waste, Ecosystem Protection/Environmental Exposure & Risk, Contaminated Sediments, Environmental Chemistry, Geochemistry, Analytical Chemistry, Fate & Transport, Bioremediation, Ecology and Ecosystems, 33/50, carbon tetrachloride, fate and transport, contaminant transport, migration, biodegradation, contaminated sediment, adsorption, electron acceptors, biotechnology, contaminants in soil, bioremediation of soils, chemical kinetics, 1, 1, 1-Trichloroethane, humification

    Relevant Websites: Exit

    Progress and Final Reports:

    Original Abstract
  • 1994
  • 1995
  • 1996
  • 1997
  • 1998
  • 1999

  • Main Center Abstract and Reports:

    R825549    HSRC (1989) - Great Plains/Rocky Mountain HSRC

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R825549C006 Fate of Trichloroethylene (TCE) in Plant/Soil Systems
    R825549C007 Experimental Study of Stabilization/Solidification of Hazardous Wastes
    R825549C008 Modeling Dissolved Oxygen, Nitrate and Pesticide Contamination in the Subsurface Environment
    R825549C009 Vadose Zone Decontamination by Air Venting
    R825549C010 Thermochemical Treatment of Hazardous Wastes
    R825549C011 Development, Characterization and Evaluation of Adsorbent Regeneration Processes for Treament of Hazardous Waste
    R825549C012 Computer Method to Estimate Safe Level Water Quality Concentrations for Organic Chemicals
    R825549C013 Removal of Nitrogenous Pesticides from Rural Well-Water Supplies by Enzymatic Ozonation Process
    R825549C014 The Characterization and Treatment of Hazardous Materials from Metal/Mineral Processing Wastes
    R825549C015 Adsorption of Hazardous Substances onto Soil Constituents
    R825549C016 Reclamation of Metal and Mining Contaminated Superfund Sites using Sewage Sludge/Fly Ash Amendment
    R825549C017 Metal Recovery and Reuse Using an Integrated Vermiculite Ion Exchange - Acid Recovery System
    R825549C018 Removal of Heavy Metals from Hazardous Wastes by Protein Complexation for their Ultimate Recovery and Reuse
    R825549C019 Development of In-situ Biodegradation Technology
    R825549C020 Migration and Biodegradation of Pentachlorophenol in Soil Environment
    R825549C021 Deep-Rooted Poplar Trees as an Innovative Treatment Technology for Pesticide and Toxic Organics Removal from Soil and Groundwater
    R825549C022 In-situ Soil and Aquifer Decontaminaiton using Hydrogen Peroxide and Fenton's Reagent
    R825549C023 Simulation of Three-Dimensional Transport of Hazardous Chemicals in Heterogeneous Soil Cores Using X-ray Computed Tomography
    R825549C024 The Response of Natural Groundwater Bacteria to Groundwater Contamination by Gasoline in a Karst Region
    R825549C025 An Electrochemical Method for Acid Mine Drainage Remediation and Metals Recovery
    R825549C026 Sulfide Size and Morphology Identificaiton for Remediation of Acid Producing Mine Wastes
    R825549C027 Heavy Metals Removal from Dilute Aqueous Solutions using Biopolymers
    R825549C028 Neutron Activation Analysis for Heavy Metal Contaminants in the Environment
    R825549C029 Reducing Heavy Metal Availability to Perennial Grasses and Row-Crops Grown on Contaminated Soils and Mine Spoils
    R825549C030 Alachlor and Atrazine Losses from Runoff and Erosion in the Blue River Basin
    R825549C031 Biodetoxification of Mixed Solid and Hazardous Wastes by Staged Anaerobic Fermentation Conducted at Separate Redox and pH Environments
    R825549C032 Time Dependent Movement of Dioxin and Related Compounds in Soil
    R825549C033 Impact of Soil Microflora on Revegetation Efforts in Southeast Kansas
    R825549C034 Modeling the use of Plants in Remediation of Soil and Groundwater Contaminated by Hazardous Organic Substances
    R825549C035 Development of Electrochemical Processes for Improved Treatment of Lead Wastes
    R825549C036 Innovative Treatment and Bank Stabilization of Metals-Contaminated Soils and Tailings along Whitewood Creek, South Dakota
    R825549C037 Formation and Transformation of Pesticide Degradation Products Under Various Electron Acceptor Conditions
    R825549C038 The Effect of Redox Conditions on Transformations of Carbon Tetrachloride
    R825549C039 Remediation of Soil Contaminated with an Organic Phase
    R825549C040 Intelligent Process Design and Control for the Minimization of Waste Production and Treatment of Hazardous Waste
    R825549C041 Heavy Metals Removal from Contaminated Water Solutions
    R825549C042 Metals Soil Pollution and Vegetative Remediation
    R825549C043 Fate and Transport of Munitions Residues in Contaminated Soil
    R825549C044 The Role of Metallic Iron in the Biotransformation of Chlorinated Xenobiotics
    R825549C045 Use of Vegetation to Enhance Bioremediation of Surface Soils Contaminated with Pesticide Wastes
    R825549C046 Fate and Transport of Heavy Metals and Radionuclides in Soil: The Impacts of Vegetation
    R825549C047 Vegetative Interceptor Zones for Containment of Heavy Metal Pollutants
    R825549C048 Acid-Producing Metalliferous Waste Reclamation by Material Reprocessing and Vegetative Stabilization
    R825549C049 Laboratory and Field Evaluation of Upward Mobilization and Photodegradation of Polychlorinated Dibenzo-P-Dioxins and Furans in Soil
    R825549C050 Evaluation of Biosparging Performance and Process Fundamentals for Site Remediation
    R825549C051 Field Scale Bioremediation: Relationship of Parent Compound Disappearance to Humification, Mineralization, Leaching, Volatilization of Transformaiton Intermediates
    R825549C052 Chelating Extraction of Heavy Metals from Contaminated Soils
    R825549C053 Application of Anaerobic and Multiple-Electron-Acceptor Bioremediation to Chlorinated Aliphatic Subsurface Contamination
    R825549C054 Application of PGNAA Remote Sensing Methods to Real-Time, Non-Intrusive Determination of Contaminant Profiles in Soils
    R825549C055 Design and Development of an Innovative Industrial Scale Process to Economically Treat Waste Zinc Residues
    R825549C056 Remediation of Soils Contaminated with Wood-Treatment Chemicals (PCP and Creosote)
    R825549C057 Effects of Surfactants on the Bioavailability and Biodegradation of Contaminants in Soils
    R825549C058 Contaminant Binding to the Humin Fraction of Soil Organic Matter
    R825549C059 Identifying Ground-Water Threats from Improperly Abandoned Boreholes
    R825549C060 Uptake of BTEX Compounds by Hybrid Poplar Trees in Hazardous Waste Remediation
    R825549C061 Biofilm Barriers for Waste Containment
    R825549C062 Plant Assisted Remediation of Soil and Groundwater Contaminated by Hazardous Organic Substances: Experimental and Modeling Studies
    R825549C063 Extension of Laboratory Validated Treatment and Remediation Technologies to Field Problems in Aquifer Soil and Water Contamination by Organic Waste Chemicals