2005 Progress Report: Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation

EPA Grant Number: R829479C020
Subproject: this is subproject number 020 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
Investigators: Thomas, John C. , Rugh, Clayton
Institution: University of Michigan - Dearborn
EPA Project Officer: Lasat, Mitch
Project Period: October 1, 2003 through September 30, 2004 (Extended to December 31, 2007)
Project Period Covered by this Report: October 1, 2004 through September 30,2005
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research

Objective:

This research project was divided into two components, one carried out at the University of Michigan at Dearborn and one carried out at Michigan State University. The objectives for the research project at the University of Michigan are to: (1) isolate microbes from promoting polycyclic aromatic hydrocarbon (PAH) laden soils at the Miller Road site in Dearborn, Michigan; (2) isolate root exudates from several plants being used for phytoremediation; (3) develop a growth-stimulation screen for microbes using a microtiter plate; and (4) test microbes from soils against exudates from several plants currently used in the field (Miller Road) experiment.

The objectives of the research project at Michigan State University are to: (1) identify plant species exhibiting enhanced PAH biodegradation in contaminated soils; (2) quantify biodegrading bacterial community response in rhizosphere soils and purified root exudates of superior phytoremediation plant species; and (3) develop DNA-based tools for rapid identification and quantification of PAH biodegrader bacteria in rhizosphere and root exudate-stimulated microbial consortia.

Progress Summary:

University of Michigan Component

Hypothesis. Effective phytoremediating plant species produce substances in the root exudates that signal specific microbes to thrive, promoting PAH destruction.

Microbial Population. On the Rouge Steel former coke oven site at the Miller Road field site, we have planted a variety of Michigan native species in the mixture of coke breeze and industrial particulates containing high levels of PAHs. Plantings occurred in the Fall 2002. New England Aster and Boneset were identified as particularly favorable species for PAH phytoremediation (Rugh, et al., 2005). Several samples of soil were again recovered in July 2004 and August 2005 from plantings beneath NE Aster (plot 3F) NE Aster/Blue Stem (plot 10C), Boneset (plot 3H) and Bulrush (plot 4D). Control soil was obtained from underneath the tarp beneath the gravel road near plot 3. This soil was identical to that placed in the plots with the exception that it was not planted. Microbes were recovered, plated on medium, and several hundred colonies isolated to homogeneity. The colony forming units were as depicted in Figure 1 below.

Plants and the Microbes They Support


Year

Figure 1. Colony Forming Units Per Gram of Soil From Control (Unplanted) and Planted Plots at Miller Road. Lines indicate standard deviations and data are expressed as the mean of at least 6 plating counts.

These colony-forming units are about 5 times less dense than samplings from the previous Allen Park Clay Mine site reported (Rugh, et al., 2005). This is largely as a result of the use of fresh weight of soil, rather than dry weight for the calculation. Currently, soil samples are being dried and adjustments to the data will allow a more direct comparison to the previous study (Rugh, et al., 2005).

Main Conclusion. Microbes under Boneset and Green Bulrush increased significantly from 2003 to 2004. Current data suggest an equally precipitous bacterial population decline, owing to a dry summer and, perhaps, nutrient depletion. Microbial populations under New England Aster seemed more constant from year to year (Figure 1, data not shown). Because the soil in this experiment began as a mixture of compost (10% v/v), chicken manure (5% v/v), and Rouge Coke Oven “soil” (85% v/v), changes in microbial populations over time are likely the direct result of particular plant/microbe interaction(s).

Characterization of PAH Degradation by Individual Microbes from Unplanted Controls and Beneath Phytoremediating Plants. In the previous report, we determined 9.8 percent of the culturable microbes from New England Aster/Blue Stem soil responded to the New England Aster exudates, while 5.7 percent of the microbes beneath Boneset responded to Aster exudate. Not all exudates were equally capable of stimulating microbial growth. Exudate levels were from 3 µL/200 µL of culture. Because population surveys were made, extensive growth stimulation by exudates was not continued.

To measure PAH degradation, a color-change assay was adapted for the use of root exudates. Color-change assays used a mixture of phenanthrene, fluorene, and dibenzothiophene. PAHs were dissolved as a concentrate in pentane and added to microtiter wells. Microbes were placed in medium, medium + 2 µL/mL of exudate, PAH alone, or exudate and PAH. Known PAH degrading organisms (UPD 1, 5, and 6) showed a great deal of pigment formation, whereas the other bacteria were either uncolored after 4 weeks, or colored in red/brown and/or yellow. Figure 2 demonstrates the testing of the same bacteria from New England Aster/Blue Stem soils (2004) in two different exudates. Each bacterium was replicated in four consecutive horizontal wells. At least 100 individual microbes from each of the above-mentioned 5 plots were screened using root exudates from Boneset, New England Aster, and Swamp Goldenrod. Exudates (3 µL/200 µL medium) from older plants tended to produce more color in PAH-degrading bacteria. Swamp Goldenrod was the most active root exudates compared to others including Boneset.

Main Conclusion. According to Figure 2, the percentage of PAH degraders was greater for Boneset, Bulrush, and New England Aster microbes than unplanted controls. Generally, about 10-30 percent of the microbes grown on YEPG medium turned the medium red/brown/yellow in the presence of Swamp Goldenrod exudate plus PAHs. Only a small minority (0.1%) of bacteria were able to produce a color change in PAH wells without exudate. This indicates that the exudate may indeed play a major role in PAH destruction. In degrader estimation using the color-assay, the percentage of PAH degraders was almost twice that observed in an earlier study (Rugh, et al., 2005).




Plot of Microbe Origin
unplanted aster/bs boneset aster bullrush
Very few microbes were color + in the absence of exudates

Figure 2. Percentage Culture Medium Color Change Positive Bacteria From the Total Isolated Under Different Plants

To confirm the color-change PAH assay, we grew bacteria in PAH, and after 1 to 2 weeks, cultures were freeze-dried, the remaining PAH dissolved, and was analyzed using a C-18 column in 70 percent acetonitrile, 30 percent water. PAHs were detected at 280 nm. PAH-alone tubes were used as 100 percent control. Generally, UPD 6 and 7 were able to reduce phenanthrene levels. These positive controls also were capable of changing the PAH medium color in the presence of exudates. Furthermore, these same bacterial cultures were able to digest PAHs (and leave a clearing zone) in an agar plate spray assay (Rugh, et al., 2005). Future analysis will include phenanthrene degradation assays of selected PAH degrader bacteria from phytoremediating plants already identified by the color-change assay. LBA4404 Agrobacterium negative controls were able to remove only a small percentage of PAH (Figure 2).

Soil samples were collected in August and are being sent to Kemron, a commercial company that will use gas chromatography/mass spectrometry to fractionate and analyze the remaining PAHS. These data will provide a relative effect of different plant species collaborating with the microbial community.

Michigan State University Component

Our initial field trials indicated that individual plant species affect PAH biodegradation rates to different extents. Most treatments achieved 20 percent reduction of soil PAH after the first season, though a limited number of planted treatments displayed continued reduction to approximately 40 percent over the 3-year field study (e.g., cardinal flower, Lobelia cardinalis; New England aster, Aster novae-anglicae; green bulrush, Scirus atrovirens; and big bluestem, Andropogon gerardii).

Numerous laboratory studies have examined the mechanistic basis of PAH phytoremediation. As a result of the demonstrated plant-microbe interaction, this process is termed “rhizosphere-assisted bioremediation” and is based upon the fundamental hypothesis: Effective phytoremediation plant species produce substances in root exudates that induce bacterial biodegrader activity, promoting PAH destruction.

To examine this hypothesis with the ultimate goal of optimizing management of root-­microbe processes for enhanced PAH bioremediation, we are studying microbial community responses to specific rhizosphere environments and products. To develop a mechanistic understanding of the PAH phytoremediation process, we tested various analytical methods to identify the PAH Spray Metabolism Assay as a reproducible, PAH biodegrader indicator, allowing isolation of confirmed degrader bacterial strains for further study. Soil bacterial extracts were cultured on Petri plates oversprayed with a cloudy residue of phenanthrene, which is the most common “tester” PAH compound, to quantify PAH biodegrader abundance among the different planted soil treatments by formation of cleared zones around the bacterial colonies. We obtained approximately 2,100 phenanthrene biodegrader 1° isolates representing rhizosphere bacterial community subsamples from each of the 20 different planted field treatments for further study. For broad-spectrum analysis, spray assays were repeated using phenanthrene and the additional PAH compounds—anthracene, fluoranthene, and pyrene—on separate replica plates. The multiple PAH compound study demonstrated that soil bacterial communities from phytoremediation treatments typically had much broader-spectrum metabolic capabilities for a wider range of PAH compounds than unplanted or untreated soils.

Note that several of the more metabolism-enriching treatments also are shown to enhance PAH degradation in field trials: AND GER (Andropogon gerardii), AST NOV (Aster novaeanglicae), LOB CAR (Lobelia cardinalis), and SCI ATR (Scirus atrovirens). This novel observation indicates that a critical aspect of the rhizosphere-effect in PAH phytoremediation may not be only increased total bacterial or degrader cell densities, but more importantly for mixed chemical contaminants such as PAHs, enhanced bacterial metabolic competence against a broader range of target compounds (Rugh, et al., 2005).

In continuation of our Consortium Plant Biotechnology Research, Inc.-Ford funded efforts, we have initiated collaborative studies with researchers in the Michigan State University Center for Microbial Ecology to develop DNA-based methods of bacterial quantification (e.g., Real-Time PCR) for characterization of the differential effects observed in varied plant species rhizospheres. We hypothesize that rhizosecretions may play an additional role for biostimulation of broad-spectrum degrader bacterial strains by promoting enhanced competency and exchange of mobile dioxygenase-encoding elements among bacterial populations in the contaminated root zone. We currently are testing various bacterial strain-specific and generic dioxygenase gene sequence targeted DNA primer sets for amplification of PAH degrading elements from phytoremediation treated soil bacterial community and isolate preparations. Effective dioxygenase amplifying primer sets will be used for analysis of biodegrader cell density, PAH substrate specificity among isolates and consortia, and PAH metabolism gene exchange among peripheral bacterial populations. Elucidation of these complementary processes will each contribute to our understanding and more effective management of plant-based cleanup of PAH-contaminated soil and sediment media.

Future Activities:

University of Michigan

Future activities are to: (1) confirm color change positive bacteria degrade phenanthrene with HPLC; (2) confirm color change positive bacteria degrade greater levels of phenanthrene with exudates present (HPLC); (3) characterize the exudates (amino acids and sugars); and (4) make PAH determinations from sampled soils (Kemron commercial laboratory uses GC/MS) using GC/MS or HPLC methods.

Michigan State University

Future activities will include: (1) maintain and monitor phytoremediation field installation through Year 3 (and final) growing season; (2) complete PAH analysis of phytoremediation treated soils; (3) characterize bacterial biodegrader community structure of different phytoremediation treated soils; and (4) evaluate DNA-based tools for phytoremediation treated soil bacterial isolate and community characterization.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 34 publications 4 publications in selected types All 2 journal articles
Other center views: All 211 publications 48 publications in selected types All 44 journal articles
Type Citation Sub Project Document Sources
Journal Article Rugh CL. Genetically engineered phytoremediation: one man's trash is another man's transgene. Trends in Biotechnology 2004;22(10):496-498. R829479C020 (2005)
R829479C020 (Final)
  • Abstract from PubMed
  • Full-text: Science Direct Full Text
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  • Journal Article Rugh CL, Susilawati E, Kravchenko AN, Thomas JC. Biodegrader metabolic expansion during polyaromatic hydrocarbons rhizoremediation. Zeitschrift fur Naturforschung C 2005;60(3-4):331-339. R829479C020 (2005)
    R829479C020 (Final)
  • Abstract from PubMed
  • Abstract: Z. Naturforsch
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  • Supplemental Keywords:

    sustainable industry, waste, agricultural engineering, bioremediation, environmental engineering, new technology, innovative technology, bioaccumulation, biodegradation, bioenergy, bioengineering, biotechnology, phytoremediation, plant biotechnology, bioremediation, carcinogen, contamination, phytodegradation, pollutant, toxicity,, Scientific Discipline, Waste, TREATMENT/CONTROL, POLLUTANTS/TOXICS, Sustainable Industry/Business, Treatment Technologies, Geochemistry, Technology, Chemicals, New/Innovative technologies, Bioremediation, Agricultural Engineering, bioengineering, biodegradation, root exudate biostimulation, transgenic plants, biotechnology, plant biotechnology, environmental engineering, remediation, PAHs, hydrocarbons, bioacummulation, phytoremediation

    Relevant Websites:

    http://www.cpbr.org exit EPA

    Progress and Final Reports:

    Original Abstract
  • 2004 Progress Report
  • 2006
  • 2007
  • Final Report

  • Main Center Abstract and Reports:

    R829479    The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R829479C001 Plant Genes and Agrobacterium T-DNA Integration
    R829479C002 Designing Promoters for Precision Targeting of Gene Expression
    R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
    R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
    R829479C005 Development of Novel Plastics From Agricultural Oils
    R829479C006 Conversion of Paper Sludge to Ethanol
    R829479C007 Enhanced Production of Biodegradable Plastics in Plants
    R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
    R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
    R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
    R829479C011 Biological Effects of Epoxy Fatty Acids
    R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
    R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
    R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
    R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
    R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
    R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
    R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
    R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
    R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
    R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
    R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
    R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
    R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
    R829479C025 Chemical Induction of Disease Resistance in Trees
    R829479C026 Development of Herbicide-Tolerant Hardwoods
    R829479C027 Environmentally Superior Soybean Genome Development
    R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
    R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
    R829479C030 Engineered Plant Virus Proteins for Biotechnology