Period Covered by the Report: 08-01-00 to 07-31-02
Date of Final Report: 10-04-02
EPA Agreement Number: NP-IPEC 14-2-1201270-94836
Title: Microflora involved in phytoremediation of polyaromatic industrial contaminants.
Investigators: John S. Fletcher and David Nagle
Institution: University of Oklahoma
EPA Project Officer: Bala Krishnan
Project Period: 08/01/00 to 07/31/02
Project Amount: $88,979
Research Category: Phytoremediation
1. Examine the hypothesis that the root rhizosphere of some plant species provides an ecological habitat where polyaromatic-degrading microorganisms find a niche that favors their survival and persistence in a natural microbial community.
2. Develop molecular techniques to monitor the presence and activity of polyaromatic-degrading communities of microorganisms in the rhizosphere.
This research was accomplished by conducting a series of integrated studies starting with field work at contaminated sites, followed by laboratory studies based on the field work, and concluded with development of preliminary molecular monitoring methods tested on field samples. This field-laboratory-field approach greatly enhances the chances of developing a successful phytoremediation system, because it starts with field identification of plant species currently growing in contaminated soil, presumed to have already fostered contaminant remediation under natural conditions as shown in previous work from this laboratory (Olson et al. 2001, Environ. Sci. and Pollut. Res 4: :243-249). Following the field work, laboratory studies were conducted to gain an understanding of the biological systems responsible for field observations, thereby providing information necessary to develop molecular monitoring methods for effective evaluation of the slow but sustained implementation of phytoremediation systems under field conditions.
Tree species that successfully grew to maturity at contaminated sites included: ash, Austrian pine, birch, black locust, mulberry, and willow. Field data collected in this study have shown that the Rhodococcus genus is potentially the most important group of polyaromatic-degrading organisms under field conditions and that the root zone of certain plant species favor the presence of this taxonomic group. Elevated levels of polyaromatic- degrading bacteria in the root zone of some plant species are consistent with the laboratory-based concept that the production and release of flavonoid compounds by fine root turnover of some plant species serve, through natural substrate enrichment, to modify microbial community structure in favor of organisms that metabolize both natural and synthetic polyaromatic compounds. Degradation studies showed that some of the Rhodococcus species isolated in this work have degradative properties equal or superior to those of Burkholderia sp. LB400, an often studied bacterium with impressive degradation properties. Quantitative PCR methods were used to show that a tight correlation existed between soil extracts of DNA coding for the nah genes and numbers of naphthalene degrading bacteria isolated from duplicate soil samples. These results support the idea of quantifying the degradative properties of rhizosphere soil by quantifying gene copies of selected enzymes in catabolic pathways. Generic primers for dioxygenase coded DNA were developed from Gen Bank data and unsuccessfully tested with DNA extracted from selected organisms and soil samples. The initial disappointing performance of these theoretically sound primers is still under investigation.
The findings reported in the open literature from this investigation (see following listing of publications) support the concept of rhizosphere remediation, whereby theplanting and maintenance of selected plant species at a contaminated site will foster development of a microbial community that is conducive to contaminant degradation. Thus, the use of long-lived perennial plants provides an ecologically sound means for inexpensive, sustained remediation of contaminated soil.
1. Leigh, Mary Beth, John S. Fletcher, David P. Nagle, Martina Mackova and Thomas Macek. 2001. Vegetation and Fungi at Czech PCB-Contaminated Sites as Bioremediation Candidates. In Phytoremediation, Wetlands and Sediments, the Sixth International In Situ and On-Site Bioremediation Symposium, San Diego, Calif., June 4- 7,2001; Leeson, A., Foote, E. A., Banks, M. K. Magar, V., Eds. Battelle Press: Columbus, Ohio. 2001. P 61-68.
2. Demnerova, Katerina, Hana Stiborova, Mary Beth Leigh, Dietmar Pieper, Jarmila Pazlarova, Vladimir Brenner, Martina Mackova and Thomas Macek. 2001. Degradation of PCBs and CBs by Indigenous Bacteria Isolated from Contaminated Soil. Proceedings of the First European Bioremediation Conference, Chania, Crete, Greece, July 2-5, 2001.
3. Demnerova, K., M. Mackova, J. Pazlarova, M. Vosahlikova, H. Novakova, E. Ryslatava, T. Macek, N. Vrchotova, V. Brenner, L. Pavlu, S. Totevova, T. Kristoffer, D.D. Focht, F. Fava, D. Di Gioia, L. Marchetti, J.S. Fletcher, M.B. Leigh, P. Kucerova, H. Stiborova, V. Mateju, M. Sobotka, F. Kastanek, P. Kastanek, L. Kasak. 2001. PCBs: Approaches to Possible Removal from the Environment: Current Status of Bioremediation in the Czech Republic. Proceedings of the NATO Advanced Study Institute: Innovative Approaches to the On-Site Assessment and REMEDIATION of Contaminated Sites. May 24-June 2, 2001, Prague, Czech Republic.
4. K. Demnerova, M. Mackova, P. Kucerova, M.B. Leigh H. Novakova, J. Burkhard and T. Macek. 2000. Practical use of bioremediation for PCB removal from contaminated soil. Focus on Biotechnology, (M. Hofman and J. Anne, series eds.), Vol. 3, Kluwer Academic Publishers, Dordrecht.
b. Platform and Poster Presentations
1. Fletcher, John 5., Mary Beth Leigh, David P. Nagle, Petra Kucerova, Martina Mackova, Tomas Macek. Rhizosphere Remediation: A Field Study in the Czech Republic. 8th FECS Conference on Chemistry and the Environment. Sept. 1-4, 2002, Athens, Greece.
2. Fletcher, John S. Phytoremediation from a Biologists Perspective. 12th International Biodeterioration and Biodegradation Symposium. July 14- 18,2002, Prague, Czech Republic (Keynote Talk).
3. Leigh, Mary Beth, John S. Fletcher, David P. Nagle. Petra Kucerova, Martina Mackova and Tomas Macek. Rhizoremediation of PCBs Based on Field Studies in the Czech Republic. 12th International Biodeterioration and Biodegradation Symposium. July 14-18, 2002, Prague, Czech Republic.
4. Fletcher, John S. Phytoremediation of Organic Contaminants. Annual Meeting of the Japanese Society of Plant Physiologists. Mar 28-30, 2002. Okayama, Japan (Symposium Talk).
5. Leigh, Mary Beth, John S. Fletcher, David P. Nagle, Petra Kucerova, Martina Mackova and Tomas Macek. PCB-degrading bacteria in the rhizosphere: a field study in the Czech Republic. Joint Annual Meeting of the American Society for Microbiology, Missouri and Missouri Valley Branches. April 5-6, 2002, Kansas City, MO.
6. Leigh, Mary Beth, John S. Fletcher, David P. Nagle, Martina Mackova and Tomas Macek. Rhizoremediation of PCBs in the Czech Republic. 8th International Petroleum Environmental Conference: Issues and Solutions in Exploration, Production and Refining. November 6-9,2001, Houston, Texas, USA.
7. Kyle, Michael D., J. Fletcher, and D. Nagle. Apparent Enrichment for PAH- Degrading Bacteria in the mulberry Rhizozone. 8th International Petroleum Environmental Conference: Issues and Solutions in Exploration Production and Refining. Nov. 6-9, 2001, Houston, Texas, USA.
8. Leigh, Mary Beth, John S. Fletcher, David P. Nagle, Martina Mackova and Thomas Macek. Vegetation and Fungi at Czech PCB-Contaminated Sites as Bioremediation Candidates. Platform Presentation. The Sixth International Symposium on In-Situ and On-Site Bioremediation. July 4-7, 2001, San Diego, California, USA.
9. Fletcher, John 5., Mary Beth Leigh, Martina Mackova and Thomas Macek. Enhanced Microbial Degradation of Recalcitrant Soil Contaminants Through the Action of Plant Roots, Nature's Injection System. NATO Advanced Study Institute: Innovative Approaches to the On-Site Assessment and Remediation of Contaminated Sites. May 24-June 2, 2001, Prague, Czech Republic.
10. Leigh, Mary Beth, J.S. Fletcher, D.P. Nagle, M. Mackova, T. Macek. Field Studies in Rhizosphere Bioremediation of PCBs. ISEB 2001 Meeting on Phytoremediation. May 15-17, 2001, Leipzig, Germany.
11. Fletcher, John 5., Paul Olson, Mary Beth Leigh. The Plant Root: A Natural Injection System to Stimulate Microbial Degradation of Recalcitrant Soil Contaminants. ISEB 2001 Meeting on Phytoremediation. May 15-17, 2001, Leipzig, Germany.
12. Fletcher, John 5., Mary Beth Leigh, Paul Olson. Remediation of recalcitrant soil contaminants through the action of nature's injection system, the root. Organic Soil Contaminants Conference, Sept. 2-5, 2001. Copenhagen, Denmark.
13. Leigh, May Beth, John S. Fletcher, David P. Nagle, Martina Mackova and Thomas Macek. Field Studies Examining Rhizosphere-enhanced PCB Degradation in the Czech Republic. Seventh Annual International petroleum Environmental Conference, November 7-10, 2000. Albuquerque, New Mexico, USA.
14. Leigh, Mary Beth and John Fletcher. Mulberry root flavones as stimulators of rhizosphere remediation. Seventh International Petroleum Environmental Conference, November 7-10,2000. Albuquerque, New Mexico, USA.
15. Leigh, Mary Beth, John S. Fletcher, David P. Nagle, Martina Mackova and Thomas Macek. 2000. Field Studies Examining Rhizosphere-enhanced PCB Degradation in the Czech Republic. Prague 2000: Fifth International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe. Sept. 12-14,2000. Prague, Czech Republic.
16. Leigh, Mary Beth, John S. Fletcher, David P. Nagle, Martina Mackova and Thomas Macek. 2000. Field Studies Examining Rhizosphere-enhanced PCB Degradation in the Czech Republic. Abstract published in The Proceedings of the Czech and Slovak Societies of Biochemistry and Molecular Biology 17th Biochemical Congress, Sept. 7-10, 2000, Prague, Czech Republic. Chemicke Listy 94 (8): 732.
Bacteria, Bioremediation, Ecology, Microbial Monitoring, PAHs, PCBs, Plants, Rhizosphere, Roots, Soil, Terrestrial