Grantee Research Project Results
2004 Progress Report: Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
EPA Grant Number: R829479C020Subproject: 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.
Current Investigators: Thomas, John C. , Rugh, Clayton
Institution: University of Michigan - Dearborn
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2003 through September 30, 2004 (Extended to December 31, 2007)
Project Period Covered by this Report: October 1, 2003 through September 30, 2004
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:
The objectives of this research project are to:
- isolate microbes from polycyclic aromatic hydrocarbon (PAH)-laden soils at the Miller Road site in Dearborn, Michigan;
- isolate root exudates from several plants being used for phytoremediation;
- develop a growth-stimulation screen for microbes using a microtiter plate;
- and test microbes from soils against exudates from several plants currently used in the field (Miller Road) experiment.
Our hypothesis is that effective phytoremediating plant species produce substances in the root exudates that signal specific microbes to thrive, promoting PAH destruction.
Progress Summary:
Microbial Population (Size)
On the Rouge Steel former coke oven site at the Miller Road field site, we planted a variety of Michigan native species in the mixture of coke breeze and industrial particulates containing high levels of PAHs. Plantings occurred in fall 2002. New England Aster and Boneset were identified as particularly favorable species for PAH phytoremediation (Susilawati, et al., 2004). Several samples of soil were recovered in July 2003, from plantings beneath New England Aster/Blue Stem (plot 10C), Boneset (plot 3H), and Bulrush (plot 4D). Soils were obtained from plants grown for one complete season. Microbes were recovered and planted on medium and several hundred colonies isolated to homogeneity. The results were:
- Boneset 1.07 +/- 0.23 X 105 cells/g soil
- Bulrush 7.17 +/- 0.40 X 105 cells/g soil
- New England Aster 3.37 +/- 1.38 X 106 cells/g soil
These numbers are 3 to 6 times less dense than samplings from the previous Allen Park Clay mine site reported earlier (Susilawati, et al., 2004). Both sites were prepared several years apart and with different batches and fractions of soil amendments (compost and chicken litter). Mulch used to control weeds may have differed in origin or microbial/fungal inoculums between the two sites. Furthermore, the Allan Park site was extensively fertilized the first year whereas the Miller Road site did not receive additional fertilizers.
Main Conclusion. Microbes under New England Aster/Blue Stem were 4 and 30 times more numerous than under Boneset and Bulrush (respectively) during Year 1 of the project. This supports the hypothesis that plants condition the soil to assist the microbes. Whether changes to microbial populations continue or become abated will be determined using samples from 2004 that already are in hand.
Exudates
The methodology of exudate recovery was perfected. We placed 14 hydroponically grown plants in 500 ml of water for 2 hours. The water then was lyophilized. The exudates were dissolved in 7 ml of water (0.5 ml/per plant), filtered (0.2 µm), and frozen at -80ºC. Exudates were recovered from several key plants: New England Aster (3 different sets), Boneset, Joe Pye Weed, Swamp Goldenrod, and Prairie Cord Grass, and more than 6 weeks (as the plants age). Samples were frozen for analysis. Some of this analysis is still in progress. The exudates contain 14-15 nmole of total amino acids/plants produced in 2 hours.
Methodology for rapid microtiter dish growth estimates of microbes were established by Marck Palazzo and Emily Sargent, two undergraduate students working on the project.
We surveyed 100 individual microbes from New England Aster and Boneset soils using exudates of both New England Aster and Boneset. About 9.8 percent of the microbes from New England Aster/Blue Stem soil responded to the New England Aster exudates. Soils from Boneset showed 5.7 percent response of the microbes to Aster exudate. New England Aster soil microbes are more responsive to Aster exudates than microbes from the soil of other plants (Bulrush and Boneset).
10C10 and 10C17 are microbes from beneath New England Aster/Blue Stem plants. With the addition of 3µl of exudates to 150µl total, these isolates demonstrated stimulated growth early (3-4 hours) in the growth curve. UPD7 showed enhanced growth, but only after 22 hours. New England Aster exudate may produce a fast response (10C10 and 10C17) perhaps as a result of biochemical signaling, whereas the UPD7 response may be more indicative of a metabolic adaptive growth response, where time and enzymatic adjustment may be necessary to allow delayed growth enhancement. Boneset soil microbes also respond to New England Aster/Blue Stem exudates.
Results indicate that there may be two separate responses to New England Aster exudate in this collection of microbes from soil beneath New England Aster/Blue Stem. All organisms in this experiment showed growth stimulation with the addition of exudate, the lowest level being 0.001µl/150µl or 7 X 10-6. The second response to exudates occurred at higher exudate concentrations. Comparing 10C10 and 10C68, the former microbes continued to respond to 0.01µl exudates, whereas 10C68 showed a second level of exudate stimulation at 0.1-1.0µl of exudates.
Additional Microbial Response to Exudates.
- Microbial response to exudates also may be plant-age dependent. Yet, no differences between New England Aster exudates from young or old plants were noted in microbes from soil beneath New England Aster/Blue Stem.
- Not all New England Aster exudate-responding microbes responded to exudates from Boneset, and vice versa. There must be different types, mixtures, or levels of biologically active compounds in each exudate.
- Microbes can be shown to degrade specific PAH contaminants in vitro. In one preliminary screen, none of the 10 exudate responders degraded the phenanthrene/anthracene/fluorene/fluroanthene/pyrene mixture, whereas 9 percent (1 of 11) 3H microbes were positive for degradation. The frequency of PAH degrader is similar to what was observed in an earlier study (Susilawati, et al., 2004).
Future Activities:
We will work to answer whether exudate responders are PAH degraders and, if so, which PAHs do exudate responders degrade and how well. We also will:
- identify selected microbes using DNA methods;
- characterize the exudates (amino acids and PAH-like materials);
- recover Year 2 soils from the same plots and compare microbial numbers and exudate responders;
- conduct PAH determinations from sampled soils;
- and culture PAH levels of selected microbes with and without exudate.
Journal Articles:
No journal articles submitted with this report: View all 34 publications for this subprojectSupplemental Keywords:
sustainable industry, sustainable business, waste, agricultural engineering, bioremediation, environmental engineering, geochemistry, new technology, innovative technology, bioaccumulation, biodegradation, bioenergy, bioengineering, biotechnology, phytoremediation, plant biotechnology, sustainable industry/business, environmental chemistry,, Scientific Discipline, Waste, TREATMENT/CONTROL, POLLUTANTS/TOXICS, Sustainable Industry/Business, Geochemistry, Treatment Technologies, Chemicals, Technology, Bioremediation, New/Innovative technologies, Agricultural Engineering, bioengineering, biodegradation, root exudate biostimulation, transgenic plants, biotechnology, plant biotechnology, environmental engineering, remediation, PAHs, hydrocarbons, bioacummulation, phytoremediationRelevant Websites:
Progress and Final Reports:
Original AbstractMain 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
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
2 journal articles for this subproject
Main Center: R829479
208 publications for this center
44 journal articles for this center