Final Report: Gene probes for detecting anaerobic alkylbenzene-degrading bacteria

EPA Grant Number: R825689C092
Subproject: this is subproject number 092 , 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: Gene probes for detecting anaerobic alkylbenzene-degrading bacteria
Investigators: Spormann, Alfred M.
Institution: Stanford University
EPA Project Officer: Hahn, Intaek
Project Period: January 1, 1999 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 objective of this project is to develop molecular tools for detecting anaerobic alkybenzene-degrading bacteria in natural samples. This includes the identification of the genes involved, as well as the fabrication of a DNA microarray carrying probes for the key enzymes involved in anaerobic alkylbenzene degradation. We and others have cloned the genes encoding the key enzyme for anaerobic toluene mineralization, benzylsuccinate synthase, however, the genes facilitating anaerobic ethylbenzene degradation to benzoate are unknown.

Summary/Accomplishments (Outputs/Outcomes):

Remediation of the many fuel hydrocarbon-contaminated sites by traditional physical-chemical processes amounts to enormous costs to the industry and society. Because aromatic hydrocarbons can be degraded biologically, it is in principle possible to predict whether or not these compounds can be mineralized at a given site and what the estimated rate would be. Furthermore, knowledge of whether a site has the potential for rapid or slow intrinsic degradation of aromatic hydrocarbons is crucial for taking action to enhance the intrinsic rate. Traditionally, microcosm studies with samples obtained from the contaminated site have been employed to determine the potential and the rate of biodegradation. These studies are time and labor intensive. With knowledge of the biochemical pathways and the genes involved, more rapid and less expensive tests can be developed. The principle of a molecular test that we envision is based on whether the genes encoding the key enzymes involved in these processes are present in contaminated environments. Using those genes that encode the key enzymes will provide highly specific probes, which unequivocally will query for the presence of the first unique biochemical steps involved in anaerobic degradation of the alkylbenzenes.

The specific objectives of this research proposal are:

    I. To identify and isolate the genes involved in anaerobic ethylbenzene oxidation to benzoate
    II. To develop a DNA microarray-based test system to detect genes encoding key enzymes in anaerobic alkylbenzene degradation in field samples

We are studying anaerobic toluene, m-xylene, and ethylbenzene-degrading denitrifying bacteria (Azoarcus strain T and Azoarcus strain EB1, respectively), and have so far identified the genes encoding the key enzymes involved in anaerobic toluene and, most likely, in m-xylene mineralization. These genes will provide the basis for developing probes for detecting the presence of these enzymes. However, the genes encoding the key enzymes of ethylbenzene degradation are unknown. We therefore will isolate those genes. Preliminary data obtained from 2-D protein gel electrophoresis indicated several polypeptides that are specifically expressed in cells of Azoarcus strain EB1 when grown on ethylbenzene but not when grown on benzoate. Using reverse genetics, the corresponding genes will be cloned, and probes derived from these genes will be printed on a DNA microarray together with the benzylsuccinate synthase genes.

The anaerobic degradation of ethylbenzene in strain EB1 is initiated by a novel enzyme, ethylbenzene dehydrogenase, that converts ethylbenzene to (S)-(-)-1-phenylethanol (Johnson and Spormann 1999 J.Bacteriol. 181). The ethylbenzene dehydrogenase activity has recently been purified and characterized in our laboratory. Biochemical data indicates the enzyme contains molybdenum, iron and sulfur. The N-terminal amino acid sequences of this heterotrimeric enzyme was used to identify and clone the three structural genes of ethylbenzene dehydrogenase. A 6 kb region of the EB1 chromosome was cloned and sequenced. The nucleotide sequence contains three predicted open reading frames (ebdA, ebdB and ebdC). The predicted EbdA polypeptide (100 kDa) has homology to several polypeptides known to bind a molybdopterin cofactor, selenate reductase (SerA), nitrate reductase(NarG) and DMSO reductase (DmsA). The EbdB subunit (35 kDa) contains a number of proposed 4Fe-4S binding motifs. The EbdC subunit (23 kDa) has homology to only one sequence in the protein data bases, SerC of selenate reductase. SerC has been proposed to be a cytoplasmic membrane anchor subunit, suggesting a role in anchoring the soluble A and B subunits to the cell membrane. These results are all consistant with the biochemical data obtained with purified ethylbenzene dehydrogenase. Further experiments are in progress to identify the other genes facilitating ethylbenzene degradation to benzoate.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other subproject views: All 2 publications 1 publications in selected types All 1 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 Johnson HA, Pelletier DA, Spormann AM. Isolation and characterization of anaerobic ethylbenzene dehydrogenase, a novel Mo-Fe-S enzyme. Journal of Bacteriology 2001;183(15):4536-4542. R825689C092 (Final)
R828772 (2002)
R828772 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: Journal of Bacteriology HTML
  • Abstract: Journal of Bacteriology
  • Other: Journal of Bacteriology PDF
  • Supplemental Keywords:

    Groundwater remediation, BTEX, anaerobic biodegradation, gene probes, bioremediation, genetics, chemistry, hazardous waste., RFA, Scientific Discipline, Waste, Ecosystem Protection/Environmental Exposure & Risk, exploratory research environmental biology, Remediation, Environmental Chemistry, Ecosystem/Assessment/Indicators, Ecosystem Protection, Chemistry, Microbiology, Monitoring/Modeling, Ecological Effects - Environmental Exposure & Risk, Environmental Microbiology, Hazardous Waste, Biochemistry, Bioremediation, Molecular Biology/Genetics, Groundwater remediation, Hazardous, Ecological Indicators, degradation, contaminated sites, genetics, mineralization, contaminated waste sites, bacteria, aromatic pollutants, biodegradation, enzymes, anaerobic bacteria, genes, detect, anaerobic biodegradation, BTEX, detection system, ethylbenzene oxidation, anaerobic microbial communities, gene probes, anaerobic degradation, anaerobic bioremediation, anaerobic ethylbenzene oxidation, bacterial degradation, groundwater

    Progress and Final Reports:

    Original Abstract
  • 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