Microflora Involved in Phytoremediation of Polyaromatic Hydrocarbons

EPA Grant Number: R827015C005
Subproject: this is subproject number 005 , established and managed by the Center Director under grant R827015
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: IPEC University of Tulsa (TU)
Center Director: Sublette, Kerry L.
Title: Microflora Involved in Phytoremediation of Polyaromatic Hydrocarbons
Investigators: Nagle, David P. , Fletcher, John S.
Institution: University of Oklahoma
EPA Project Officer: Lasat, Mitch
Project Period: August 1, 2000 through July 31, 2001 (Extended to July 31, 2002)
Project Amount: Refer to main center abstract for funding details.
RFA: Integrated Petroleum Environmental Consortium (IPEC) (1999) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research

Objective:

Polyaromatic hydrocarbons (PAHs) comprise a major class of recalcitrant industrial pollutants and are a significant byproduct of coal, chemical, and petroleum processing and refining. PAHs are concentrated in food chains, are toxic, and some (e.g. benzo[a]pyrene) are recognized mutagens and carcinogens. The EPA guidelines for containment and control of PAH compounds have become more and more strict. Older disposal methods, such as lagooning, are no longer acceptable in the long term. Phytoremediation has the potential to be applied m a "living cap" of plants and associated microorganisms which will alleviate pollution in the medium term (tens of years). The costs of such waste treatment are far below those required for installation of an impermeable cap or incineration of contaminated soil. In this project data from a Texas City Union Carbide Corp. PAH site will be used in evaluating the potential for use of phytoremediation of PAHs. It should be possible to predict what plants will be favorable for stimulating microbial biodegradation of PAHs, and to design test protocols for additional contaminated sites. The proposed research also encompasses many basic questions, such as what influences the interactions between roots and microorganisms. The answers to such questions may be exploited in the long term in other bioremediation schemes.

Union Carbide Corporation is committed to environmentally-friendly remediation of its plants. The Texas City PAH site is a remnant of old containment practices. When it became apparent that the vegetation contributed to improvement of the site (conversion of waste to soil and decrease in PAH levels in the soil), Union Carbide began to support basic research into the processes that had occurred. They provided historical data, aerial photographs, and other information, such that the history of the site could be reconstructed over the 20-year period in which the vegetation developed, and provided unlimited access to the site for research purposes. Carbide has been very pro-active in implementing bioremediation schemes for PAH contamination. In the spring of 1997 they planted two 17-acre PAH-impacted sites at the Texas City plant as a test of different plant species as potential phytoremediators. These sites will also be very useful in studies of the microflora that develops in a newly begun phytoremediation systems. In fact, they may be a model of the original PAH-contaminated site during its early years of vegetation and microflora development.

The proposed research is a two-year study of microorganisms from an industrial research area which is contaminated with polyaromatic hydrocarbons (PAHs). The site has been populated with native plants through a process of natural succession over a period of 15 to 20 years. Chemical characterization of samples shows that PAH concentrations increase with depth, and in areas of the site which now contain mature trees the regions of lower concentration are deeper than in nearby grassy areas. The results lead to a hypothesis that trees and microflora have acted in concert to bioremediate soil contaminants in the root region, suggesting that it is critical to study what has happened and what is happening in to the pollutants in the field. This contrasts to the approach of a pure laboratory study followed by attempts to introduce new technology to the field. If it is true that the plant-microbe system has accelerated the biodegradation of PAHs, then analysis of a field site should be the most rapid route to planned implementation of phytoremediation.

Studies on the microflora from the PAH-impacted site and their relationship to plants growing there will utilize both classical microbiology and molecular biology methods. Plant and chemical analysis work is now being complemented by an extensive quantitative and qualitative characterization of bacteria in different vegetated regions and at different depths of the field site. To date, counts of total bacteria, as well as of bacteria apparently capable of using PAHs (naphthalene, phenanthrene, or pyrene) as carbon/energy source have been performed. Analysis of the data is not yet complete, but it is already clear that the number of bacteria decreases dramatically at the interface between soil and PAH-containing sludge. In bulk samples of soil beneath grassy or tree-containing regions there is no significant difference in bacterial numbers between samples taken at corresponding depths. What has not been tested is the local region around the roots themselves.

Two hundred bacterial strains were isolated, with PAH as sole carbon and energy source. Partial characterization (staining, physiological tests) indicates that these PAH-degraders are diverse: both Gram-negative and Gram-positive bacteria, representing many genera were found. A subset of these strains was tested for the presence of plasmids (extrachromosomal genetic elements). Certain plasmids provide metabolic abilities on their hosts, and some are known to permit metabolism of PAHs. of our strains, some but not all contain plasmids, whose functions are not yet known. It is possible to conclude, however, that PAH utilization is not exclusively plasmid-coded.

In the first year of this project, we propose to: 1) thoroughly characterize the diversity of the PAH-degrading microorganisms isolated from the contaminated soil (This will be done by classical microbiological tests as well as by molecular biology techniques (quantitative polymerase chain reactions, PCR), which can be a powerful test of diversity, including that of organisms that can not be cultured. Probes will include ribosomal RNA genes. As more information is obtained about metabolic plasmids and other biodegradation genes in organisms from the site, probes will be developed for these genes as well.), 2) study the spatial organization of PAH-degrading organisms (This requires microsampling of soil adjacent to plant roots, to compare the numbers of PAH-degraders in close proximity to growing roots to the numbers far removed from roots. Initial studies will be carried out by counting colonies, but quantitative PCR and plasmid characterizations will be done as well. In years two and beyond it is hoped that fluorescence microscopy techniques can be used as a powerful test to localize PAH degraders and their relationship to plant roots.), 3) begin to characterize microflora found in a PAH phytoremediation site which has been planted for only 10 months (Using techniques similar to those listed under aim no. I, microorganisms found in soil of this new site will be studied. This may be a functional equivalent to the early years of the Texas City site.), and 4) study the interactions of organisms from the site with plant roots in normal and PAH-contaminated soils (This requires information from aims 1 and 2, and is not likely to be a major research area in the first year of the project.).

Publications and Presentations:

Publications have been submitted on this subproject: View all 20 publications for this subprojectView all 120 publications for this center

Supplemental Keywords:

RFA, Scientific Discipline, Toxics, INTERNATIONAL COOPERATION, Geographic Area, Waste, Water, National Recommended Water Quality, Contaminated Sediments, Remediation, Chemistry, Contaminant Candidate List, State, Microbiology, Environmental Microbiology, Hazardous Waste, Bioremediation, Molecular Biology/Genetics, Biology, Engineering, Hazardous, Environmental Engineering, hazardous waste treatment, degradation, waste treatment, petroleum, contaminated sites, microbial degradation, biodegradation, decontamination of soil, field studies, Naphthalene, Pyrene, PAH, microbes, microflora, contaminated soil, soils, Texas (TX), contaminants in soil, bioremediation of soils, soil, hydrocarbons, water quality, phytoremediation, soil microbes, plant-microbe system

Progress and Final Reports:

  • 2001 Progress Report
  • Final Report

  • Main Center Abstract and Reports:

    R827015    IPEC University of Tulsa (TU)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R827015C001 Evaluation of Road Base Material Derived from Tank Bottom Sludges
    R827015C002 Passive Sampling Devices (PSDs) for Bioavailability Screening of Soils Containing Petrochemicals
    R827015C003 Demonstration of a Subsurface Drainage System for the Remediation of Brine-Impacted Soil
    R827015C004 Anaerobic Intrinsic Bioremediation of Whole Gasoline
    R827015C005 Microflora Involved in Phytoremediation of Polyaromatic Hydrocarbons
    R827015C006 Microbial Treatment of Naturally Occurring Radioactive Material (NORM)
    R827015C007 Using Plants to Remediate Petroleum-Contaminated Soil
    R827015C008 The Use of Nitrate for the Control of Sulfide Formation in Oklahoma Oil Fields
    R827015C009 Surfactant-Enhanced Treatment of Oil-Contaminated Soils and Oil-Based Drill Cuttings
    R827015C010 Novel Materials for Facile Separation of Petroleum Products from Aqueous Mixtures Via Magnetic Filtration
    R827015C011 Development of Relevant Ecological Screening Criteria (RESC) for Petroleum Hydrocarbon-Contaminated Exploration and Production Sites
    R827015C012 Humate-Induced Remediation of Petroleum Contaminated Surface Soils
    R827015C013 New Process for Plugging Abandoned Wells
    R827015C014 Enhancement of Microbial Sulfate Reduction for the Remediation of Hydrocarbon Contaminated Aquifers - A Laboratory and Field Scale Demonstration
    R827015C015 Locating Oil-Water Interfaces in Process Vessels
    R827015C016 Remediation of Brine Spills with Hay
    R827015C017 Continuation of an Investigation into the Anaerobic Intrinsic Bioremediation of Whole Gasoline
    R827015C018 Using Plants to Remediate Petroleum-Contaminated Soil
    R827015C019 Biodegradation of Petroleum Hydrocarbons in Salt-Impacted Soil by Native Halophiles or Halotolerants and Strategies for Enhanced Degradation
    R827015C020 Anaerobic Intrinsic Bioremediation of MTBE
    R827015C021 Evaluation of Commercial, Microbial-Based Products to Treat Paraffin Deposition in Tank Bottoms and Oil Production Equipment
    R827015C022 A Continuation: Humate-Induced Remediation of Petroleum Contaminated Surface Soils
    R827015C023 Data for Design of Vapor Recovery Units for Crude Oil Stock Tank Emissions
    R827015C024 Development of an Environmentally Friendly and Economical Process for Plugging Abandoned Wells
    R827015C025 A Continuation of Remediation of Brine Spills with Hay
    R827015C026 Identifying the Signature of the Natural Attenuation of MTBE in Goundwater Using Molecular Methods and "Bug Traps"
    R827015C027 Identifying the Signature of Natural Attenuation in the Microbial Ecology of Hydrocarbon Contaminated Groundwater Using Molecular Methods and "Bug Traps"
    R827015C028 Using Plants to Remediate Petroleum-Contaminated Soil: Project Continuation
    R827015C030 Effective Stormwater and Sediment Control During Pipeline Construction Using a New Filter Fence Concept
    R827015C031 Evaluation of Sub-micellar Synthetic Surfactants versus Biosurfactants for Enhanced LNAPL Recovery
    R827015C032 Utilization of the Carbon and Hydrogen Isotopic Composition of Individual Compounds in Refined Hydrocarbon Products To Monitor Their Fate in the Environment
    R830633 Integrated Petroleum Environmental Consortium (IPEC)
    R830633C001 Development of an Environmentally Friendly and Economical Process for Plugging Abandoned Wells (Phase II)
    R830633C002 A Continuation of Remediation of Brine Spills with Hay
    R830633C003 Effective Stormwater and Sediment Control During Pipeline Construction Using a New Filter Fence Concept
    R830633C004 Evaluation of Sub-micellar Synthetic Surfactants versus Biosurfactants for Enhanced LNAPL Recovery
    R830633C005 Utilization of the Carbon and Hydrogen Isotopic Composition of Individual Compounds in Refined Hydrocarbon Products To Monitor Their Fate in the Environment
    R830633C006 Evaluation of Commercial, Microbial-Based Products to Treat Paraffin Deposition in Tank Bottoms and Oil Production Equipment
    R830633C007 Identifying the Signature of the Natural Attenuation in the Microbial Ecology of Hydrocarbon Contaminated Groundwater Using Molecular Methods and “Bug Traps”
    R830633C008 Using Plants to Remediate Petroleum-Contaminated Soil: Project Continuation
    R830633C009 Use of Earthworms to Accelerate the Restoration of Oil and Brine Impacted Sites