Final Report: Anaerobic Biodegradation of 2,4,6-Trinitrotoluene and Other Nitroaromatic Compounds by Clostridium Acetobutylicum.

EPA Grant Number: R825513C006
Subproject: this is subproject number 006 , established and managed by the Center Director under grant R825513
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: HSRC (1989) - South and Southwest HSRC
Center Director: Reible, Danny D.
Title: Anaerobic Biodegradation of 2,4,6-Trinitrotoluene and Other Nitroaromatic Compounds by Clostridium Acetobutylicum.
Investigators: Hughes, Joseph B , Rudolph, F. , Bennett, G.
Institution: Louisiana State University - Baton Rouge
EPA Project Officer: Hahn, Intaek
Project Period: January 1, 1995 through January 1, 1998
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 overarching goal of this project was to delineate the metabolism of nitroaromatics by Clostridium acetobutylicum and provide fundamental information on the biotransformation of nitroaromatic compounds under anaerobic conditions. The focus on these studies was on the metabolism of TNT. Specific tasks were : 1) to analyze the TNT biotransformation pathways of TNT by Clostridium acetobutylicum and identify intermediates and end products; 2) evaluate the effect of environmental conditions on transformation rates and end products; and 3) test the ability of Clostridium acetobutylicum to transform munitions-related nitroaromatics.

Summary/Accomplishments (Outputs/Outcomes):

The experiments proposed were 1) to determine the pathway of biotransformation of TNT by C. acetobutylicum, 2) to study TNT biotransformation rates and evaluate the effect of environmental factors on rates, pathways, and final products, and 3) to screen the ability of C. acetobutylicum to transform other explosives related nitroaromatics.

To study TNT biotransformation rates and evaluate the effect of environmental factors on rates, pathways, and final productsexperiments were conducted in anaerobic serum bottles. They were inoculated with C. acetobutylicum under a range of temperatures (10 to 30 C), pH (5 to 8), contaminant levels (10 mg/L in aqueous systems to 1000 mg/kg in sediment systems), and sediment/water ratios (0% to 50%). These experiments also used radiolabeled TNT to facilitate mass balances, allows for fractionation of samples and tracking end product distribution.

Testing for the ability of Clostridium acetobutylicum to transform munitions-related nitroaromatics were conducted at conditions that demonstrate the highest rates of TNT transformation, followed by tests at conditions more representative of the natural environment. Experimental systems are identical to those described above.

2,4,6-Trinitrotoluene (TNT) was rapidly reduced (537 nM min-1 mg protein -1) to undetermined end products via monohydroxylamino derivatives. TNT reduction was more rapid than that of 2-amino-4,6-dintrotoluene, 4-amino-2,6-dintrotoluene, and 2,4-diamino-6-nitrotoluene. The metabolic phase of clostridium cultures affected rates and extents of transformation of TNT and its intermediates. Acidogenic cultures showed rapid transformation rates and the ability to transform TNT and its primary reduction products to below detection limits; solventogenic cultures did not transform TNT completely, and showed accumulation of its hydroxylamino derivatives. Carbon monoxide-induced solventogenesis was capable of slowing the transformation of TNT and intermediates. Studies employing [ring-U-14C]-TNT demonstrated that no significant mineralization occurred and that products of transformation were water-soluble.

Bamberger Rearrangement during TNT-Metabolism by Clostridium acetobutylicum
Studies conducted in anaerobic cell extracts demonstrated that a polar product formed from the transformation of 2,4-dihydroxylamino-6-notrotoluene by a mechanism known as the Bamberger rearrangement. The product was stabilized by derivatization with acetic anhydride and the structure confirmed by mass spectroscopy, 1H-NMR, and IR spectroscopy techniques. The reaction occurred in the presence of cell extract and H2, but did not occur in cell extract-free controls. From spectroscopic data the product of 2,4-dihydroxylamino-6-nitrotoluene rearrangement was identified as either 2-amino-4-hydroxylamino-5-hydroxyl-6-nitrotoluene (4-amino-6-hydroxylamino-3-methyl-2-nitrophenil) or 2-hydroxylamino-4-amino-5-hydroxyl-6-nitrotoluene (6-amino-4-hydroxylamino-3-methyl-2-nitrophenol). Acid catalyzed rearrangement of 2,4-dihydroxylamino-6-nitrotoluene resulted in a single product, which after derivatization, was identical to a dervatized product from cell extracts. Acid catalyzed Bamberger rearrangement occurs with the hydroxyl addition para to the participating hydroxylamine, indicating that the 2-amino-4-hydroylamino-5-hyroyl-6-nitrotoluene (4-amino-6hydroxylamino-3-methyl-2-nitrophenol) was the product isolated from cell extracts. This product was also confirmed in whole cell systems that had been fed TNT. Following derivatization of the culture broth, a product was isolated that was identical to those isolated from crude cell extracts and acid catalysis experiments.

Rapid Separation of Reduction Products of 2,4,6-Trinitrotoluene using TLC
Silica gel TLC methods were developed for the separation of 2,4,6-trinitrotoluene (TNT) in mixtures with possible reduction products. The methods employed repeated elutions with simple binary or ternary solvent systems in either one or two dimensional modes. The resolved analytes include TNT, selected amino derivatives (2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene) and known hydroxylamino derivatives (2-hydroxyl-amino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene and 2,4-dihydroxylamino-6-nitrotoluene).

Metabolism of 2,4,6-Trinitrotoluene by Clostridium acetobutylicum: Pathway identification and Lab-scale Evaluation of Contaminated Soil Bioremediation

The following conclusions were reached during this research:

  • when incubated with a batch culture of acetogenic Clostridium acetobutylicum, TNT is biotransformed predominately through the following pathway: TNT 4-hydroxylamino-2,6-dinitrotoluene 2,4-dihydroxylamino-6-nitrotoluene 2-amino-4-hydroxylamino-5-hydroxyl-6-nitrotoluene (4-amino-6-hydroxylamino-4-metyl-2-nitrophenol).

  • when incubated with TNT-contaminated soil, C. acetobutylicum is able to transform TNT to 2,4-dihydroxylamino-6-nitrotoluene and beyond. Initial transformation products do not irreversibly binf to soil, although later unknown metabolites appear to do so. TNT transformation appeared limited by the rate of TNT dissolution/desorption from the soil to the aqueous phase.

  • aerobic soil bacteria indigenous to TNT contaminated soil are able to mineralize a fraction of the products created by the reduction of TNT by C. acetobutylicum. Additionally, a significant fraction of such products becomes associated to the aerobic bacterial biomass.

  • the extent and rate of the transformation of TNT by C. acetobutylicum is dependent upon actively growing cells. An energy source, such a glucose or dextrose , must be available to the cells, and the culture must be in acetogenic (log growth) stage. An engineered system, must take into account the duration of this metabolic activity when modifying other parameters.

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

    Other subproject views: All 22 publications 8 publications in selected types All 7 journal articles
    Other center views: All 392 publications 154 publications in selected types All 106 journal articles
    Type Citation Sub Project Document Sources
    Journal Article Bhadra R, Spanggord RJ, Wayment DG, Hughes JB, Shanks JV. Characterization of oxidation products of TNT metabolism in aquatic phytoremediation systems of Myrilphyllum aquaticum. Environmental Science & Technology 1999;33(19):3354-3361. R825513C006 (Final)
    R825513C013 (Final)
  • Full-text: ACS Publications Full Text
  • Other: ACS Publications PDF
  • Journal Article Gerlach R, Steiof M, Zhang C, Hughes JB. Low aqueous solubility electron donors for the reduction of nitroaromatics in anaerobic sediments. Journal of Contaminant Hydrology 1999;36(1):91-104. R825513C006 (Final)
    not available
    Journal Article Hughes JB, Wang C, Yesland K, Richardson A, Bhadra R, Bennett G, Rudolph F. Bamberger rearrangement during TNT metabolism by Clostridium acetobutylicum. Environmental Science & Technology 1998;32(4):494-500. R825513C006 (Final)
  • Full-text: ACS Full Text (PDF)
  • Journal Article Hughes JB, Wang CY, Bhadra R, Richardson A, Bennett GM, Rudolph FB. Reduction of 2,4,6-Trinitrotoluene by Clostridium acetobutylicum through hydroxylamino-nitrotoluene intermediates. Environmental Toxicology and Chemistry 1998;17(3):343-348. R825513C006 (Final)
    not available
    Journal Article Hughes JB, Wang CY, Zhang C. Anaerobic biotransformation of 2,4-dinitrotoluene and 2,6-dinitrotoluene by Clostridium acetobutylicum: A pathway through dihydroxylamino intermediates. Environmental Science & Technology 1999;33(7):1065-1070. R825513C006 (Final)
  • Full-text: ACS Full Text (PDF)
  • Journal Article Khan TA, Bhadra R, Hughes J. Anaerobic transformation of 2,4,6-TNT and related nitroaromatic compounds by Clostridium acetobutylicum. Journal of Industrial Microbiology & Biotechnology 1997;18(2-3):198-203. R825513C006 (Final)
    not available
    Journal Article Pucik LE, Hughes JB. Capillar Electrophoretic Separation of TNT and its Transformation products. Journal of Capillary Electroporesis 1996;3(4):209-213. R825513C006 (Final)
    not available

    Supplemental Keywords:

    bioremediation, TNT, and microbial transformation., RFA, Scientific Discipline, Waste, Water, Ecosystem Protection/Environmental Exposure & Risk, Chemical Engineering, Contaminated Sediments, Environmental Chemistry, Fate & Transport, Analytical Chemistry, Hazardous Waste, Ecology and Ecosystems, Hazardous, Environmental Engineering, environmental technology, sediment treatment, hazardous waste management, hazardous waste treatment, risk assessment, fate and transport, contaminated marine sediment, soil and groundwater remediation, clostridium acetobutylicum, biodegradation, contaminated sediment, kinetics, chemical contaminants, contaminated soil, bioremediation of soils, marine sediments, remediation, chemical kinetics, hydrology, biotransformation, anaerobic biotransformation, extraction of metals, technology transfer, nitroaromatic compounds, heavy metals, bioremediation, CERCLA, aquifer fate and treatment, technical outreach

    Progress and Final Reports:

    Original Abstract
  • 1995
  • 1996

  • Main Center Abstract and Reports:

    R825513    HSRC (1989) - South and Southwest HSRC

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R825513C001 Sediment Resuspension and Contaminant Transport in an Estuary.
    R825513C002 Contaminant Transport Across Cohesive Sediment Interfaces.
    R825513C003 Mobilization and Fate of Inorganic Contaminant due to Resuspension of Cohesive Sediment.
    R825513C004 Source Identification, Transformation, and Transport Processes of N-, O- and S- Containing Organic Chemicals in Wetland and Upland Sediments.
    R825513C005 Mobility and Transport of Radium from Sediment and Waste Pits.
    R825513C006 Anaerobic Biodegradation of 2,4,6-Trinitrotoluene and Other Nitroaromatic Compounds by Clostridium Acetobutylicum.
    R825513C007 Investigation on the Fate and Biotransformation of Hexachlorobutadiene and Chlorobenzenes in a Sediment-Water Estuarine System
    R825513C008 An Investigation of Chemical Transport from Contaminated Sediments through Porous Containment Structures
    R825513C009 Evaluation of Placement and Effectiveness of Sediment Caps
    R825513C010 Coupled Biological and Physicochemical Bed-Sediment Processes
    R825513C011 Pollutant Fluxes to Aquatic Systems via Coupled Biological and Physicochemical Bed-Sediment Processes
    R825513C012 Controls on Metals Partitioning in Contaminated Sediments
    R825513C013 Phytoremediation of TNT Contaminated Soil and Groundwaters
    R825513C014 Sediment-Based Remediation of Hazardous Substances at a Contaminated Military Base
    R825513C015 Effect of Natural Dynamic Changes on Pollutant-Sediment Interaction
    R825513C016 Desorption of Nonpolar Organic Pollutants from Historically Contaminated Sediments and Dredged Materials
    R825513C017 Modeling Air Emissions of Organic Compounds from Contaminated Sediments and Dredged Materials title change in last year to "Long-term Release of Pollutants from Contaminated Sediment Dredged Material"
    R825513C018 Development of an Integrated Optic Interferometer for In-Situ Monitoring of Volatile Hydrocarbons
    R825513C019 Bioremediation of Contaminated Sediments and Dredged Material
    R825513C020 Bioremediation of Sediments Contaminated with Polyaromatic Hydrocarbons
    R825513C021 Role of Particles in Mobilizing Hazardous Chemicals in Urban Runoff
    R825513C022 Particle Transport and Deposit Morphology at the Sediment/Water Interface
    R825513C023 Uptake of Metal Ions from Aqueous Solutions by Sediments
    R825513C024 Bioavailability of Desorption Resistant Hydrocarbons in Sediment-Water Systems.
    R825513C025 Interactive Roles of Microbial and Spartina Populations in Mercury Methylation Processes in Bioremediation of Contaminated Sediments in Salt-Marsh Systems
    R825513C026 Evaluation of Physical-Chemical Methods for Rapid Assessment of the Bioavailability of Moderately Polar Compounds in Sediments
    R825513C027 Freshwater Bioturbators in Riverine Sediments as Enhancers of Contaminant Release
    R825513C028 Characterization of Laguna Madre Contaminated Sediments.
    R825513C029 The Role of Competitive Adsorption of Suspended Sediments in Determining Partitioning and Colloidal Stability.
    R825513C030 Remediation of TNT-Contaminated Soil by Cyanobacterial Mat.
    R825513C031 Experimental and Detailed Mathematical Modeling of Diffusion of Contaminants in Fluids
    R825513C033 Application of Biotechnology in Bioremediation of Contaminated Sediments
    R825513C034 Characterization of PAH's Degrading Bacteria in Coastal Sediments
    R825513C035 Dynamic Aspects of Metal Speciation in the Miami River Sediments in Relation to Particle Size Distribution of Chemical Heterogeneity