Final Report: The Role of Metallic Iron in the Biotransformation of Chlorinated Xenobiotics

EPA Grant Number: R825549C044
Subproject: this is subproject number 044 , 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: The Role of Metallic Iron in the Biotransformation of Chlorinated Xenobiotics
Investigators: Alvarez, Pedro J. , Parkin, Gene F , Schnoor, J. L. , Weathers, L. J.
Institution: University of Iowa
EPA Project Officer: Hahn, Intaek
Project Period: August 13, 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


This research investigates the hypothesis that both microbial and abiotic processes contribute to reductive dechlorination of xenobiotics in methanogenic incubations with elemental metals, such as iron, serving as an ultimate electron donor.

Summary/Accomplishments (Outputs/Outcomes):

Polychlorinated compounds such as carbon tetrachloride (CT) are known to be transformed via sequential reductive dechlorination by both abiotic and microbial mechanisms under anaerobic conditions. However, existing treatment processes that utilize reductive dechlorination suffer from several drawbacks including inefficient transfer of electrons from the ultimate electron donor to the chlorinated compound and slow rates of reaction, thereby resulting in possible accumulation of transformation products of equal or even greater toxicity. Elemental metals in aqueous solution can act as an energy source for methanogens via production of hydrogen. Using elemental metals as an energy source, reductive dechlorination of chlorinated compounds may proceed by three mechanisms:

1. abiotic processes whereby electrons are transferred directly from the elemental metal to the chlorinated compound,
2. microbial processes whereby electrons from hydrogen that are involved in biosynthetic processes are diverted to the chlorinated compound, and
3. microbially catalyzed abiotic processes whereby electrons from the elemental metal are transferred to the chlorinated compound via biological electron carriers.

Experiments were conducted in batch and column-reactor systems. Initial studies investigated Fe(0) and carbon tetrachloride (CT). Various chlorinated organics, nitrate, and RDX were also assayed. Hydrogen-utilizing, mixed, anaerobic cultures were developed as inoculum sources for most experiments. In addition, pure cultures of three methanogens, one denitrifier, and one iron-reducer were used in some experiments. Initial batch studies were performed to determine the general time-course that the reactions would follow. Inhibition studies using 2-bromoethanesulfonate (BES), a specific methanogenic inhibitor, addressed the role of methanogens. Analytes measured in headspace gas samples include CT, chloroform (CF), dichloromethane (DCM), chloromethane (CM), hydrogen, and methane. Subsequent batch experiments assessed a variety of conditions to better understand the versatility and limitations of bioaugmenting Fe(0), including use of pure cultures of bacteria, a wide variety of contaminants (chlorinated ethenes, nitrate, RDX, etc.), and environmental conditions (e.g., pH, redox). The stoichiometry and kinetics of pertinent reactions were determined. Electron balances were conducted to provide insight into important abiotic and biotic processes. Flow-through column experiments using adjustable-bed-length, glass chromatographic columns packed with steel wool were conducted to assess long-term performance. A limited number of column experiments used a mixture of sand and granular iron.

A summary of major findings follows in bullet form. These findings were summarized in detail in our semi-annual progress reports and in the variety of publications and presentations given under the publications listed.

  • Our initial work was conducted using CT and CF as parent compounds and a methanogenic consortium enriched on acetate. Bioaugmenting Fe(0) resulted in faster and more complete dechlorination. Pseudo-first-order rate coefficients for CF were 4 to 10 times greater than for Fe(0) alone, resting and killed cells, and media controls. Dechlorination and methanogenesis were supported by hydrogen produced from Fe(0) corrosion. The use of selective inhibitors indicated that it was primarily the methanogens responsible for CT and CF dechlorination. For systems fed CT, the primary product in the Fe(0)-alone reactor was CF (50-70% of initially fed CT) while transient production of dichloromethane (DCM) was the only volatile chlorinated product measured. Column studies with steel wool as the support medium showed complete CF removal over the 60-day experiment in a column seeded with microbes while an unseeded column showed about 70% removal of added CF.

  • Three pure cultures of methanogens were selected for additional studies with CT and CF. These cultures were selected based on their reported ability to use hydrogen: Methanosarcina barkeri can use hydrogen as a growth substrate; Methanosarcina thermophila uses hydrogen a growth substrate only after a period of about 8-10 days, and then only poorly; and Methanosaeta concillii cannot use H2. Transformation of CT and CF was more rapid for all three organisms when they were incubated with Fe(0). Enhanced transformation with M. thermophila and M. concillii indicated that H2 can serve as an electron donor for dechlorination in the absence of growth. Experiments with supematants from M. thermophila grown with and without Fe(0) indicated the presence of an excreted biomolecule(s) active in the enhanced transformation of CT and CF. Supernatants from cultures grown in the absence of Fe(0) could transform CT but not CF. Autoclaving the supernatant did not significantly alter CT transformation rate or pattern but did significantly reduce CF transformation. This extracellular factor(s) appeared to have a high transformation capacity for CT; upon dechlorinating 82 mM of CT, the rate of transformation did not decrease. Experiments with 14 CCl4 showed that mass balance recoveries were poorer in systems containing both methanogens and Fe(0). Results suggested that the unrecovered 14C may be 14CH4, 14CO, 14CS2, none of which could be recovered with the analytical methods used at the time. After 72 hours of incubation with methanogens and Fe(0), CT, CF, and dichloromethane (DCM) were below detection limits. The primary product in the Fe(0)-alone systems was CF. Statistical analyses indicated that product distributions were significantly different for the methanogen-Fe(0) system as compared to the methanogen-14CO2 was produced in the alone and Fe(0)-alone systems. For example, significantly more methanogen-Fe(0) systems.

  • Abiotic experiments with Fe(0) powder showed that CT was reduced to CF and, to a lesser degree, DCM. Dechlorination of CT was rapid and followed approximately first-order kinetics. Surface-area-normalized rate coefficients increased over time, perhaps due to an increase in reactive surface area due to cathodic depolarization and pitting of the iron surface. Dechlorination also occurred under oxic conditions (DO = 7 mg/L), although rates were significantly slower (oxygen consumption was rapid, however). A rapid pH increase was synchronous to DO consumption, and the pH remained constant after DO was depleted. Additional abiotic experiments with nitrate and an iron rotating disk electrode indicate that nitrate reduction is not mass transfer limited in the absence of an iron oxide layer(passivating the Fe(0) surface). It is likely that in the presence of an iron oxide layer, mass transfer to the Fe(0) would limit the overall rate of nitrate reduction.

  • A number of columns using steel wool as a support and acetate or lactate enrichment cultures as microbial seed have been operated for periods of up to a year to assess long-term stability. Columns were fed CT, PCE, and 1,1,1 -TCA alone and in mixtures. Results indicated that, based on extent of dechlorination, enhanced removal of CT and 1,1,1 -TCA occurs when Fe(0) is bioaugmented. Microbial seed was important as the enhancement with 1,1,1 -TCA was more dramatic with the lactate enrichment culture. This importance of microbial seed was confirmed with batch experiments with 1,1,1 -TCA. Enhancement was not observed with PCE; in fact, under the conditions tested, addition of a methanogenic enrichment may inhibit the abiotic reaction. When mixtures of CT, PCE, and 1,1,1 -TCA were fed, more complete dechlorination was observed with bioaugmented Fe(0) than with Fe(0) alone. Bioaugmentation of Fe(0) did appear to accelerate corrosion, which may decrease the lifetime of a given mass of Fe(0). This would need to be considered when designing a bioaugmented Fe(0) reactive barrier.

  • Several experiments were conducted with nitrate, a variety of different Fe(0) sources, a mixed culture of denitrifying organisms and a pure culture of Paracoccus denitrificans. Molecular hydrogen derived from Fe(0) corrosion could support autotrophic denitrification and resulted in a more favorable end-product distribution (i.e., N2 vs. NH4+). Use of less-reactive Fe(0) (i.e., steel wool vs. granular iron vs. powdered iron) gave more N2. Nitrate removal could be sustained over time in column reactors. Bacteria were found to preferentially colonize the Fe(0) surface over sand in column reactors. Even though one of the columns was seeded with P. denitrificans, native bacteria out-competed these organisms for the ecological niche provided by the sand-Fe(0) mixture. Increases in pH caused by Fe(0) corrosion inhibited the biotic reaction. Inclusion of montmorillonite, an acidic aluniinosilicate rnineral, buffered this pH increase, improved nitrate removal, and reduced the transient accumulation of nitrite. Microcosm studies showed that, while no Fe(0) treatment was needed to remove nitrate from high-TOC soils, adding Fe(0) to low-TOC soil can supplement the electron donor pool and enhance nitrate removal. These findings indicate that bioaugmentation of Fe(0) holds promise for remediating nitrate-contaminated waters.

  • RDX is an explosive found at many contaminated sites. Bioaugmentation of Fe(0) resulted in faster and more complete reduction of RDM. It was rapidly reduced in aquifer microcosms amended with Fe(0) and in flow-through columns packed with steel wool and seeded with an acetate enrichment culture. Production of H2 from Fe(0) corrosion supported microbial processes. The initial reduction products (TNX, DNX, and MNX) were observed in systems containing only microbes or only Fe(0); these heterocyclic metabolites were not found in systems containing Fe(0) and microbes. Reductive treatment of RDX with Fe(0) also reduced its toxicity to microorganisms and enhanced its subsequent biodegradability under either aerobic or anaerobic conditions. Therefore, a combined or sequential Fe(0)-biological treatment approach might improve overall treatment efficiency.

  • Preliminary work was done with perchlorate, a highly oxidized component of solid rocket fuel. Abiotic reduction of ClO4- was thermodynamically favorable and we hypothesized that the H2 produced from Fe(0) corrosion could support microbes that would reduce ClO4-. We have developed enrichment cultures that can degrade ClO4-. However, when these organisms are incubated with Fe(0), no ClO4- removal occurs. Additionally, ClO4- is not abiotically reduced by Fe(0). Experiments have shown that soluble Fe(II) species is not causing the inhibition and that high pH is not likely a problem.

  • Among the related studies recently completed are the effect of surface area concentration and mixtures of CT, Cr(VI), and nitrate on removal of these redox sensitive contaminants. The preferential degradation order of these contaminants in abiotic reactors was: Cr(VI) > CT > nitrate. Results indicated that at low surface area concentration (11 m2/L) significant competitive effects were observed. Yet, no inhibition was observed at high concentrations (1140 m2/L). It is hypothesized that inhibition is due to competition for a limited number of reactive surface sites at a low Fe(0) dose. We have recently begun work assessing the hypothesis that biogeochemical interactions between iron-reducing bacteria (i.e., Geobacter metallireducens GS-15) could enhance the removal of redox-sensitive pollutants such as RDX. Results suggest that iron-reducing bacteria could enhance the long-term performance of Fe(0)-reactive-barriers, not only through reductive dissolution of passivating layers of Fe(III) oxide, but also by the production of (as of yet undetermined biogenic reactive iron species. In addition, GS-15 was shown to degrade RDX, suggesting that iron-reducers can also participate directly in the remediation process. Analyses of samples from field Fe(0) barriers have shown that indigenous microbes are colonizing the Fe(0). Techniques used include scanning electron microscopy and fluorescent in situ hybridization with 16S rDNA probes.

    A patent application has been filed for Fe(0)-based remediation. Investigators have made numerous presentations of this research at technical conferences. Results have been published in peer-reviewed journals.

  • Journal Articles on this Report : 9 Displayed | Download in RIS Format

    Other subproject views: All 46 publications 10 publications in selected types All 9 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 Dejournett TD, Alvarez PJJ. Combined microbial-Fe(0) treatment system to remove nitrate from contaminated groundwater. Bioremediation Journal 2000;4(2):149-154. R825549C044 (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 Helland BR, Alvarez PJJ, Schnoor JL. "Reductive dechlorination of carbon tetrachloride with elemental iron. Journal of Hazardous Materials 1995;41(2-3):205-216. R825549C044 (Final)
    not available
    Journal Article Novak PJ, Daniels L, Parkin GF. Enhanced dechlorination of carbon tetrachloride and chloroform in the presence of elemental iron and Methanosarcina barkeri, Methanosarcina thermophila, or methanosaeta concillii. Environmental Science & Technology 1998;32(10:1438-1443. R825549C044 (Final)
    not available
    Journal Article Novak PJ, Daniels L, Parkin GF. Rapid dechlorination of carbon tetrachloride and chloroform by extracellular agents in cultures of Methanosarcina thermophila. Environmental Science & Technology 1998;32(20):3132-3136. R825549C044 (Final)
  • Full-text: ACS Full Text
  • Other: ACS PDF
  • 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 Till BA, Weathers LJ, Alvarez PJJ. Fe(0)-supported autotrophic denitrification. Environmental Science & Technology 1998;32(5):634-639. R825549C044 (Final)
    not available
    Journal Article Weathers LJ, Parkin GF, Alvarez PJJ. Utilization of cathodic hydrogen as electron donor for chloroform cometabolism by a mixed, methanogenic culture. Environmental Science & Technology 1997;31(3):880-885. R825549C044 (Final)
    not available
    Journal Article Wildman MJ, Alvarez PJJ. RDX degradation using an integrated Fe(0)-microbial treatment approach. Water Science and Technology 2001;43(2):25-33. R825549C044 (Final)
    not available

    Supplemental Keywords:

    dechlorination, xenobiotics, heavy metals, iron., Scientific Discipline, Waste, Water, Environmental Chemistry, Contaminated Sediments, Remediation, Analytical Chemistry, dechlorination, contaminants, xenobiotics, contaminated sediment, chemical contaminants, metallic ion, groundwater remediation, biotechnology, biotransformation, contaminated groundwater, extraction of metals, heavy metal contamination, metal contamination

    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