Grantee Research Project Results
Final 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 Amount: $393,135
RFA: Phytoremediation (2001) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management
Objective:
The primary objective of this research project was to investigate the mechanisms by which plants are able to enhance the biodegradation of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) in contaminated soils. Our studies focused in particular on plant-produced monoterpenes and other phytochemicals that affect the ecology and activity of indigenous aromatic hydrocarbon-degrading bacteria in the plant rhizosphere. A second objective of this research project was to study the expression of genes encoding enzymes for PAH and PCB degradation when induced by monoterpenes. This objective further entailed the development of methods for detection and quantification of key genes encoding degradative enzymes that can be used to monitor the influence of phytochemicals on the population size and activity of degrader organisms during bioremediation. A third objective of this research project was to examine the influence of plant roots and earthworms on oxygen diffusion into soil, which is required for the aerobic degradation of aromatic hydrocarbons.
Summary/Accomplishments (Outputs/Outcomes):
Plants have been shown to enhance the biodegradation of many common xenobiotic pollutants of soils including pesticides, PCBs and PAHs. Although this technology is now being used commercially for clean up of contaminated sites, and certain plants have been recognized as particularly effective for promoting degradation, there is still little mechanistic understanding of the processes by which different plant species affect the population size and activity of xenobiotic-degrading microorganisms in soil. This knowledge is needed to develop criteria for selection of plant species that can be used for phytoremediation and may also lead to new technologies that directly employ phytochemicals and soil organic amendments for clean up of contaminated soils. This technology is particularly relevant to the clean up of brownfields and large land areas with low levels of contamination that cannot be treated cost-effectively using excavation or thermal desorption technologies.
Prior to this research, we demonstrated that certain chemical components of plants, termed monoterpenes, were highly effective for increasing cometabolism of PCBs in soils. Based on the general induction of dioxygenase enzymes that occurs when microbes degrade monoterpenes, we hypothesized that plants producing high amounts of these chemicals also should promote biodegradation of PAHs, which are degraded by similar enzymes by an aerobic process in which oxygen is introduced into the aromatic ring structures of the PAHs, leading ultimately to complete mineralization of the contaminant. Similarly to PCBs, another controlling factor is the low bioavailability of these substances which are strongly adsorbed to clay and organic matter. Achieving biodegradation of both classes of these recalcitrant substances thus requires consideration of four criteria: (1) solubilization of the pollutant to increase its bioavailability; (2) the presence of a large and active degrader population; (3) enzyme induction either by the chemical itself or by a cometabolite that is degraded by the same types of enzymes; and (4) availability of oxygen for aerobic degradation by the dioxygenase enzymes.
A summary of key findings reported in technical journal articles follows below. Supporting data and discussion of the results of manuscripts follow in the final technical report that describes our research in the context of our original hypotheses.
Summary of Key Findings
- Confirmation of monoterpenes as inducing substances for promoting PCB degradation (Singer, et al., 2003a; Singer, et al., 2003b).
- Demonstration of soil aeration effects of plant roots and earthworms in promoting aerobic degradation of PCBs and evaluation of earthworms for facilitating soil mixing and promoting the rate and extent of PCB degradation at different soil depths (Singer, et al., 2003).
- Characterization of differential induction of dioxygenases by different types of monoterpenes and other inducing substances by individual microbial strains (Singer, et al., 2002).
- Elucidation of key differences in the effects of monoterpenes on PCB and PAH degradation, in which we show bioavailability, and not induction, is the key factor that limits PAH degradation (Yi and Crowley, manuscript in preparation, 2006).
- Discovery of the role of linoleic acid as an effector of PAH degradation and demonstration of ways in which plant cultivation and soil amendments with materials containing high amounts of linoleic acid can be used to achieve clean up of PAH-contaminated soils (Yi and Crowley, manuscript in preparation, 2006).
- Development and application of molecular techniques using real-time polymerase chain reaction for quantification of genes encoding PAH-degrading dioxygenases in soils during treatment to clean up soil contamination (Park and Crowley, 2005; Park and Crowley, 2006).
- Identification of bacterial species that are responsible for PAH degradation in soils treated with linoleic acid (Yi and Crowley, manuscript in preparation, 2006).
- Identification of PAH-degrading bacteria in the rhizospheres of plant species that promote the removal and degradation of PAH from contaminated soils (Balcom and Crowley, manuscript in preparation, 2006).
Practical Applications
There are several ways in which plants can influence the biodegradation of organic pollutants in soils. These include both general effects and plant species-specific effects that increase the population of degrader organisms and their activity. General effects that occur with almost any plant likely are caused by the increased availability of carbon to the rhizosphere, which promotes microbial growth and activity through “growth-linked metabolism.” In contrast, more specific effects on degrader populations are speculated to involve fortuitous enrichment of degrader organisms through the release of certain phytochemicals that either serve as analogs of the target contaminant or that influence the bioavailability of the contaminant so that an effective population size of active degrader organisms can be maintained in the soil. A particular example of this is the cometabolism of PCBs, which can be stimulated by monoterpenes that serve as chemical analogs of PCBs and, thereby, induce the requisite enzymes for biodegradation of these substances. In addition to growth-linked metabolism and gene induction, plants also may fortuitously select for degrader organisms and increase the bioavailability of hydrophobic compounds through the production of surfactants. This latter aspect has been ignored almost completely in prior research on phytoremediation.
The most important practical application of our research was the discovery of linoleic acid as a strong effector of PAH degradation. Linoleic acid is a common fatty acid that is present in all plant and fungal tissues but varies greatly in concentration among different plant species and within different plant tissues. In this research project, we showed that a one-time application of 1,000 ppm of linoleic acid resulted in a 90 percent disappearance of the model PAH pyrene from a contaminated soil. This effect also can be achieved by growing plant species that contain high amounts of this chemical for in situ phytoremediation. It also should be possible to use plant materials and agricultural waste products that contain linoleic acid as soil amendments to facilitate degradation of PAHs. For example, one of the main plants studied here, celeriac, can be purchased for $2 per kg. When added to soil at 20 tons per hectare, the cost would be $40,000 per hectare for soil treatment. This cost is much less than that required for excavation or other intensive treatments. Treatment cost could be decreased further by directly cultivating plants that contain high amounts of linoleic acid and turning these under as green manures or by incorporating agricultural waste products that contain this substance. Our most recent studies since have shown that linoleic acid also is effective for promoting degradation of the five-carbon PAH, benzo[a]pyrene. A caveat at this time is that our study used pyrene-spiked soils in which these contaminants are more bioavailable than in soils containing aged contaminants. Additional studies now are necessary to extend these results to the field.
Two other practical applications of this research are the identification of the predominant bacteria that degrade PAHs in soils and the development of methods for quantification of key genes that encode dioxygenase enzymes that function for PAH degradation. Marker genes for these bacteria and degradative genes provide biomarkers that can be used to track the population size and potential activity of PAH degraders in different soils. These marker genes also will help to determine whether microbial communities within contaminated soils contain the necessary degradative genes and what their potential is for manipulation through phytoremediation or other soil treatments. This is an especially important consideration in soils containing mixed contaminants, where the growth and activity of PAH degraders may be constrained by metal toxicity, pH, or other environmental factors.
Future Research Needs
Although there is considerable evidence that many secondary plant metabolites can stimulate microbial degradation of xenobiotics and broaden the spectrum of their activity, we still know very little about how they control regulatory mechanisms for expression of degradative enzymes by microorganisms. Nonetheless, plant phytochemicals have many advantages for bioremediation that merit further study. Monoterpenes and compounds such as salicylic acid are often effective at very low concentrations, are viewed as being environmentally friendly, and there are few concerns about introducing these compounds into the environment. Given that they are effective at or below their solubility in water, they can be applied easily to poorly accessible sites in the subsoil or deep aquifers. With an increased understanding, it should be possible to identify more precisely which inducers or classes of compounds are most effective at stimulating microbial degradation of specific contaminants or classes of chemicals. By introducing plants to the site that naturally produce appropriate secondary metabolites, a readily available and cheap barrier could be established for long-term site protection.
There are several new questions that arise from the present research related to the mode of action of linoleic acid as an effector of PAH degradation and also whether this compound might influence PCB degradation. Among the possible ways by which linoleic acid may affect degradation, this fatty acid may act as a surfactant, thereby making PAHs more accessible for degradation. Alternatively, this fatty acid may fortuitously enrich for PAH-degrader organisms, and/or induce enzymes that function for PAH degradation. The role of soil fungi, which represent a large component of the soil biomass and also are represented by species that are known PAH degraders, still is understood poorly. Future research should examine the relative importance of fungal and bacterial populations in the degradation of PAHs, and their responses and interactions during phytoremediation and other in-situ treatments for clean up of contaminated soils.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 29 publications | 5 publications in selected types | All 5 journal articles |
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Type | Citation | ||
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Luepromchai E, Singer AC, Yang C-H, Crowley DE. Interactions of earthworms with indigenous and bioaugmented PCB-degrading bacteria. FEMS Microbiology Ecology 2002;41(3):191-197. |
R829404 (Final) |
not available |
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Park J-W, Crowley DE. Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE. BioTechniques 2005;38(4):579-586. |
R829404 (Final) |
not available |
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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 |
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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 |
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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:
bioremediation, biodegradation, rhizosphere, microbial communities, polycyclic aromatic hydrocarbons, soil contamination, toxics, waste, environmental chemistry, environmental engineering, environmental microbiology, remediation, PAH, PCB, PCBs, polychlorinated biphenyls, bacterial degradation, contaminants in soil, contaminated soil, degradation, earthworm, microbial degradation, microflora, organic contaminants, phytoremediation, plant-based remediation, plant-microbe system,, 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://www.envisci.ucr.edu/index.php?file=faculty/crowley/crowley.html Exit
http://www.facultydirectory.ucr.edu/cgi-bin/pub/public_individual.pl?faculty=9 Exit
http://www.nhm.org/research/RanchoLaBrea/RLBMicrobes.html 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.