Grantee Research Project Results
2003 Progress Report: Evaluation of Monoterpene Producing Plants for Phytoremediation of PCB and PAH Contaminated Soils
EPA Grant Number: R829404Title: Evaluation of Monoterpene Producing Plants for Phytoremediation of PCB and PAH Contaminated Soils
Investigators: Crowley, David E. , Borneman, James
Institution: University of California - Riverside
EPA Project Officer: Aja, Hayley
Project Period: November 1, 2001 through October 31, 2004 (Extended to September 30, 2005)
Project Period Covered by this Report: November 1, 2002 through October 31, 2003
Project Amount: $393,135
RFA: Phytoremediation (2001) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management
Objective:
The objectives of this research are to investigate the potential application of different plant species, particularly those that produce monoterpenes, for use in phytoremediation of polychlorinated biphenyl (PCB) and polycyclic aromatic hydrocarbon (PAH) contaminated soil, and to investigate the ecology of indigenous xenobiotic degrading bacteria in the rhizosphere of monoterpene producing plants.
Progress Summary:
In previous work, we have shown that monoterpenes produced by plants are highly effective for causing the induction of genes that catabolize PCBs. Based on data in the literature indicating that dioxygenases enzymes that function for degradation of terpenes also occur in the same family as those that function for degradation of PAHs, we have since focused on the possible role of monoterpenes for biostimulation of PAH degraders. In studies addressing this question, we have primarily focused on the recalcitrant, but still degradable, lower-molecular weight PAHs, including naphthalene (easily degraded), phenanthrene, and pyrene (recalcitrant) as model compounds. To determine whether plant-derived compounds are in fact effective for inducing PAH degradation, it is first essential to have methods to quantify the gene copy number and expression of dioxygenases that function for PAH degradation. To this end, we have continued our development of realtime polymerase chain reaction (RT-PCR) methods for detection of naphthalene and pyrene degradation genes. Given the deoxyribonucleic acid (DNA) sequence variability among the different dioxygenases that are expressed by different species of soil bacteria, we have complemented this work by using a bioinformatics approach that will allow us to detect novel dioxygenases by amplification of conserved sequences found in different groups of dioxygenases. Specific details on our progress to date are provided below.
Hypothesis I: Plant and Microbial Substances in the Rhizosphere Enhance the Expression of Inducible PCB and PAH Degrading Enzymes
Although many plants have been shown to differentially affect the degradation of organic pollutants in soil, currently there is no understanding of the mechanistic basis for this effect and the reason some plants are more effective than others at stimulating the growth and activity of xenobiotic degrading microorganisms. To address these questions, we now have successfully developed a method for quantitative detection of the nahAc gene encoding naphthalene dioxygenases using RT-PCR. The method has been validated in studies in which we have followed the copy number of the gene during the biodegradation of naphthalene in artificially contaminated soil. We also have used this method to detect this gene in a PAH-contaminated soil located at Site 34 of the March Air Force Base in Riverside, CA. This work was reported at the meetings of the American Society of Microbiology this year, and is the first report of an RT-PCR method for quantitative detection of dioxygenases genes in environmental samples. Development of these methods has required careful calibration assays and protocols, such as the use of an appropriate internal standard to normalize differences in DNA extraction between samples. The detection limit for the nahAc gene using a SYBR® Green probe is extremely sensitive, allowing detection of as few as 10 cells per gram of soil, and a linear detection range between 102 and 108 cells per gram of soil (see Figure 1).
In our most recent work, we have developed and are testing RT-PCR methods for detection of genes that encode dioxygenases that are involved in phenanthrene and pyrene degradation. These currently are being tested in soils containing mixtures of PAHs to determine the degree to which each of these genes is expressed during biodegradation of mixed contaminants in soil. During the coming year, we will use these methods to quantify gene copy number and expression in rhizosphere soil from different plants that are being grown on the experimental site at March Air Field.
Figure 1. Detection Limit of nahAc Gene in 0.5 Gram of Soil by SYBR® Green Real-Time PCR
Hypothesis II: Monoterpene-Producing Plants Selectively Enrich for Diverse Populations of Xenobiotic Degrading Microorganisms That Will Occur at Higher Population Densities in the Rhizosphere Than the Plants That Do Not Produce These Substances
In our prior work with PCB degradation, we observed that the monoterpenes carvone and limonene were highly effective for inducing gram-positive bacteria to degrade PCBs. Last year, we extended this research to PAHs to determine if monoterpenes also might have a similar function for enhancing degradation of these substances. Initial studies revealed that some plants were much more effective than others in stimulating degradation of PAH, and that celeriac, a plant species that contains very high amounts of limonene, can greatly stimulate the degradation of the low-molecular weight PAHs, anthracene, phenanthrene, and pyrene. Initially, we believed that limonene was the active agent in this plant that was causing induction of PAH dioxygenases, especially since degradation stimulated by this plant was limited to gram-positive bacteria. This belief reflected results we had obtained for terpenes and PCBs. Several subsequent, detailed, and carefully controlled experiments, however, have shown that monoterpenes alone do not cause enhanced degradation of PAHs when applied to soil, either as a pure chemical, or in a mixture with a supplemental sugar carbon source. This indicates that there is either another component in the celeriac that is a potent enhancer of PAH degradation, or that a mixture of chemicals contained in the plant is responsible for this effect. Our ongoing studies related to this question are focused on identifying the active components, following defined chemical fractionation procedures. Identification of the active compound will allow us to screen and select for other plant species that contain these substances for use in phytoremediation of PAHs. We also will be able to better characterize the genes responding to the presence of the active compound, and then quantify their expression in the rhizosphere.
Another research issue that we are exploring is the possible induction of multiple dioxygenase genes within the same bacterial species. In a recent publication, we showed that there were different enantiomer selectivity patterns for selected PCB congenors when bacteria were induced with either biphenyl or with a monoterpene. This strongly suggests that bacteria contain more than one dioxygenase that may function for degradation of aromatic pollutants. To follow up on this important finding, we are examining the protein expression patterns of the PCB degrader Arthrobacter B1B cells grown in the presence of biphenyl or the monoterpene carvone. Our preliminary results show different protein banding patterns related to growth on these substrates. To identify the differentially expressed proteins, we currently are identifying the protein bands, using nuclear magnetic resonance methods at the University of California–Riverside genomics facility.
Along with fundamental research on understanding the mechanisms by which plants enhance organopollutant degradation, one of the main goals of our research is to develop a practical phytoremediation technology that optimizes the selection of plant species for use in cleanup of contaminated soils. This past year, we selected 20 different native and weed species for screening to see which plants may promote degradation of PAHs. For the screening assay, the plant roots of each species were pulverized under liquid nitrogen and were added to soil containing 100 parts per million of pyrene. The soils were incubated for 30 days, after which they were extracted to determine pyrene degradation by gas chromatograph-flame ionization detector analysis. Results of this study will be available in the next few months following this report.
Future Activities:
Based on our success in developing methods to quantify PAH degrading genes in environmental samples, we now are poised to conduct our planned research on rhizosphere systems of different plants grown under field conditions in a PAH contaminated soil from a U.S. Environmental Protection Agency site located at March Airfield. During the past 2 years, two Ph.D. students have been working on this project and have focused on the genetics aspects of the research. This year, two new students have joined the project, both of whom will emphasize field research in their dissertation research projects on phytoremediation.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 29 publications | 5 publications in selected types | All 5 journal articles |
---|
Type | Citation | ||
---|---|---|---|
|
Singer AC, Wong CS, Crowley DE. Differential enantioselective transformation of atropisomeric polychlorinated biphenyls by multiple bacterial strains with different inducing compounds. Applied and Environmental Microbiology 2002;68(11):5756-5759. |
R829404 (2003) R829404 (Final) |
not available |
|
Singer AC, Smith D, Jury WA, Hathuc K, Crowley DE. Impact of the plant rhizosphere and augmentation on remediation of polychlorinated biphenyl contaminated soil. Environmental Toxicology and Chemistry 2003;22(9):1998-2004. |
R829404 (2003) R829404 (Final) |
not available |
|
Singer AC, Crowley DE, Thompson IP. Secondary plant metabolites in phytoremediation and biotransformation. Trends in Biotechnology 2003b;21(3):123-130. |
R829404 (2003) R829404 (Final) |
not available |
Supplemental Keywords:
brownfield, cometabolism, phytodecontamination, rhizoremediation., Scientific Discipline, Toxics, Waste, National Recommended Water Quality, Remediation, Environmental Chemistry, Contaminant Candidate List, Environmental Microbiology, Bioremediation, Ecological Risk Assessment, Environmental Engineering, plant-based remediation, degradation, microbial degradation, biodegradation, PCBs, microflora, PAH, contaminated soil, Polychlorinated Biphenyls PCBs:, contaminants in soil, recalcitrant hydrocarbons, PCB, earthworm, phytoremediation, plant-microbe system, bacterial degradationRelevant Websites:
http://envisci.ucr.edu/Faculty/crowley/default.htm Exit
http://www.facultydirectory.ucr.edu/cgi-bin/pub/public_individual.pl?faculty=9 Exit
Progress and Final Reports:
Original AbstractThe 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.