Grantee Research Project Results
2002 Progress Report: Mechanistic Role of Plant Root Exudates in the Phytoremediation of Persistent Organic Pollutants
EPA Grant Number: R829405Title: Mechanistic Role of Plant Root Exudates in the Phytoremediation of Persistent Organic Pollutants
Investigators: White, Jason C. , Smets, Barth F. , Mattina, MaryJane Incorvia , Gage, Daniel J. , Gent, Martin P.N.
Institution: University of Connecticut
Current Institution: Connecticut Agricultural Experiment Station , University of Connecticut
EPA Project Officer: Aja, Hayley
Project Period: November 1, 2001 through October 31, 2004 (Extended to December 14, 2005)
Project Period Covered by this Report: November 1, 2001 through October 31, 2002
Project Amount: $401,241
RFA: Phytoremediation (2001) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management
Objective:
This research project is designed to investigate the role of root exudates in the plant uptake of persistent organic pollutants (POPs) from soil. Preliminary data have shown that two weathered organic pollutants (p,p'-DDE and chlordane) readily are translocated from soil to the roots of a very narrow range of plant species. These findings contradict a significant body of scientific evidence indicating time-dependent reductions in contaminant availability in soil (i.e., sequestration). The fundamental objective of this research project is to determine the mechanism by which the uptake occurs. The following hypotheses are being tested:
· The root exudates of certain plant species facilitate the mobility and subsequent availability of weathered organic pollutants. Plant root exudates are isolated and characterized from plants shown to have differential pollutant uptake/remediation abilities in the field. A series of abiotic assays comparing contaminant release in the presence and absence of these exudates are testing the central hypothesis. Biosensors are constructed for visual detection of the spatial and temporal release of critical exudate constituents from roots.
· Contaminant solubilization by exudates occurs by direct or indirect mechanisms. In direct enhancement, the exudate molecules directly induce contaminant release from the soil. Possible mechanisms include the formation of exudate/contaminant complexes or the partial solubilization/reformation of soil structure organic fractions through the chelation of polyvalent metals (iron and aluminum). A second hypothesis considers indirect enhancement, where root exudates stimulate a microbial community that promotes contaminant availability to the plant. The microbial communities of plants with differential uptake/remediation potentials are characterized by 16s rRNA-targeted terminal restriction fragment length polymorphism (T-RFLP) profiling, and critical microbial exudates are investigated for their role in promoting pollutant availability.
Progress Summary:
CAES: White, Gent, Mattina
We completed a series of field and greenhouse studies this year.
Field Studies. A series of field experiments addressed the uptake and translocation of highly weathered DDE by crops in two genera: Cucumis and Cucurbita. Data from our group and from the literature indicate that certain species in the Cucurbita genus extract significantly higher than expected quantities of weathered, POPs; six varieties were planted, including a zucchini (summer) squash and two winter squash (both Cucurbita pepo), and three pumpkin varieties (one C. pepo and two Cucurbita maxima). The data in the literature and from our laboratory are contradictory regarding the Cucumis genera, with some indications of high contaminant uptake by melon species (Cucurbita melo), and poor uptake by cucumber (Cucurbita sativas). Consequently, we grew three different cultivar varieties of each species (cucumber and melon) in DDE-contaminated soil.
Somewhat expectedly, there was significant variability in the phytoextraction of weathered DDE between the different genera. Root to soil bioconcentration factors (BCFs), defined as the contaminant concentration in the roots (dry weight) divided by that in the soil (dry weight), were up to 1.8 for the Cucumis sp., but ranged up to 16 for the Cucurbita sp. Somewhat unexpectedly, large variability in uptake existed within the Cucurbita genera (BCFs of 0.47-16) and even within a single species (C. pepo zucchini BCF was 16; C. pepo winter squash was 2.0). The concentration of contaminant was highest in the roots of all species tested, but when accounting for biomass in the calculation of absolute contaminant removal, at least 86 percent of the extracted DDE was in aboveground tissue. In terms of phytoremediation, the percent of contaminant removed from soil by the "non-uptaker" Cucumis sp. was low (0.03 percent), but C. pepo removed up to 2.4 percent of the highly weathered contaminant in a single growing season. These values approach those observed in the commercial phytoremediation of heavy metals.
Greenhouse Studies. We conducted a series of experiments to assess the effect of organic acids on the bioavailability of weathered DDE. We tested a series of six organic acids (citric, tartaric, malonic, malic, oxalic, and succinic) known to be root exudates, and ethylenediamine tetra-acetic acid (EDTA) under abiotic conditions for their ability to facilitate the desorption of weathered DDE through soil matrix destruction mediated by the chelation of inorganic soil constituents. Each acid was present at a concentration range of 1-100 mM. At concentrations of 50 mM, all organic acids increased the desorption of weathered DDE by 19-80 percent, with EDTA, citric, and oxalic acids being most effective. The organic acids also increased the aqueous concentration of eight inorganic elements extracted from the soil matrix, including six-fold increases in the concentrations of Al, Fe, Mn, and P at 1 mM.
In a parallel set of experiments, we grew zucchini seedlings under dense conditions in pots of DDE-contaminated soil. The seedlings were divided into three treatments, and over a 28-day growing period, two treatments received periodic amendments of 10 mM citric or oxalic acid to the soil. Plants receiving water alone extract 1.7 percent of the contaminant, but in soils receiving citric and oxalic acid amendments, the plants removed 2.1 and 1.9 percent of the DDE, respectively (significantly different from water alone at p <0.05). In addition, the aboveground biomass of plants grown with organic acid amendments was greater, resulting in up to 66 percent more DDE in the aerial tissue as compared to plants grown in soil receiving water alone. In a second cropping of zucchini on the same soils, plants grown in the absence of organic acids removed 2.5 percent of the DDE (significantly greater than crop 1), but organic acid amendments had no effect on uptake.
UC: Smets
Methods Obtaining Rhizobacteria. To date, we have grown four types of
plants (lettuce, wheat, zucchini, and pumpkin) in polycyclic aromatic hydrocarbon
(PAH)-contaminated soil. The amount of contaminated soil was varied as follows:
0 percent, 0.3 percent, 3 percent, and 30 percent contaminated soil in the potting
media. After 3 months of growth, we harvested the plants and removed the roots
from the shoot section of the plant. Root-associated bacteria were dislodged
from the roots by sonication. We separated the bacteria from the soil particles
through a series of centrifugation and filtration.
Methods for PAH-Degradation Selection. We isolated PAH-degrading bacteria
by spreading the rhizobacteria on minimal media plates amended with a layer
of PAH on the surface of the plate. We used four PAHs (phenanthrene, pyrene,
anthracene, and chrysene), but anthracene and chrysene provided the best discernable
selection. We chose colonies capable of degradation based on clearing zones
of the PAH. Figure 1 shows an example of a PAH-degrader on a selection plate.
Figure 1. Picture of Colony With Cleared Zone of Surrounding PAH
We have isolated more than 300 anthracene or chrysene-degrading rhizobacteria from the four host plants' rhizospheres and the bulk-contaminated soil. We took 20 isolates from each selection plate and we incubated a selection plate for anthracene and chrysene for each cell sample. Presently, we are differentiating among these isolates using the BOX-PCR method.
The gel in Figure 2 indicates how BOX-PCR provides a band pattern that can be used as a fingerprint to differentiate among isolated bacteria.
Figure 2. Gel of BOX-PCR Using DNA From Chrysene-Degrading Isolates
Methods for Investigating Iron Importance. We have considered how the availability of iron can impact PAH degradation. We have used microplate assay using the tetrazolium salt dye WST-1 to indicate growth of incubating cells. We used a range of iron to anthracene ratios to observe when iron was limiting, and when it was in excess for a specific anthracene-degrader based on the growth indicated by adding the WST-1 stain. Figure 3 shows that for this specific anthracene-degrader (isolated from the Winsted bulk soil), the best growth was exhibited when the iron concentration to anthracene concentration ratio was 0.6 x 10-3.
Figure 3. Influence of Iron Concetration on Athracene Catabolism
T-RFLP Analysis of Rhizosphere Microbial Community Subjected to Different Levels of Environmental Stress. We are performing microbial community analysis on all of the rhizosphere samples collected from the four different plant types (lettuce, wheat, zucchini, and pumpkin) grown in the different degrees of PAH-contaminated soils. We employed a 16 S-rDNA targeted T-RFLP method. We loaded samples on native gel and ran them on ABI 377 sequencer in GeneScan mode. We examined the profiles in GeneScan software (ABI) and analyzed them in BioNumerics software (Applied-Maths, Belgium) via principal component analysis.
UC: Gage
We constructed a biosensor for detecting plant-released dicarboxylic acids by fusing the dctA promoter from Sinorhizobium meliloti to the gene for green fluorescent protein (GFP).
The dctA gene is a member of the dicarboxylate transport system in S. meliloti. This transport system is responsible for the facilitated movement of the C4-dicarboxylate acids, succinate, fumarate, malate, and aspartate across the cell membrane. The dctA gene specifically encodes a gene for the permease that allows the movement of the carbon into the cell. The dctA promoter region was amplified by polymerase chain reaction (PCR) using lab-designed primers to create the 320bp promoter fragment for the fusion. We cloned this in front of a gene from a bright variant of gfp. The final biosensor plasmid also contained a trp terminator and stop codons in all three reading frames in front of the promoter and gfp gene, respectively, to prevent any transcriptional and translational read-through from the plasmid vector. We conducted the plasmid construction in an Escherichia coli host, and then transferred it to our wild-type S. meliloti strain Rm1021 for expression. The final plasmid, pPG12, was tested on a variety of carbon sources and showed the highest green fluorescent protein (GFP) with fumaric acid, followed by maleic and succinic acids. We innoculated alfalfa (Medicago sativa) roots with the biosensor, and fluorescence was apparent along the root during plant growth, indicating that the organic acids were released from roots, and that biosensor was functional in an applied setting.
Future Activities:
CAES. The most pressing future activities involve the characterization of the root exudates of both non-uptaker and uptaker plant species, determining the major difference that will explain the disparity between the two groups. These studies will focus first on the exudation of low molecular weight organic acids as a consequence of nutrient status (plus and minus phosphorus being the starting point). The experiments will involve hydroponic, modified hydroponic (sand or soil), and real soil conditions.
We also will seek to address some of the uptake variability that has been observed within the C. pepo group, where both uptakers and non-uptakers have been identified. We will begin greenhouse studies on the potential phytoextraction of highly weathered PAHs by C. pepo and some of the abiotic batch studies utilizing isolated root exudates.
UC: Smets. We will identify or associate unique chrysene- and anthracene-degrading species and strains through 16s rDNA sequencing. From this analysis, we can discern whether host-specific/rhizosphere-located PAH degraders exist. We will conduct a series of experiments to examine the ability and range of PAH degradation for these identified bacteria.
We will conduct further experiments to assess the importance of iron availability on PAH-degradation. These experiments will incorporate various isolates, several PAHs, as well as more common carbon sources to determine if the amount of iron needed is greater for growth on PAH. Isolates also will be used for siderophore production assays. Siderophores are small extracellular proteins used by bacteria and plants to sequester iron when it is limiting.
We will subject the community T-RFLP profiles to continued statistical analysis to evaluate the extent to which the plant host-type or the soil-type (degree of contamination) contributes to the microbial community structure.
UC: Gage. We will attempt to construct biosensor or reporter bacteria that will fluoresce in the presence of other organic acids, specifically citric and oxalic acids. These reporters will then be inoculated into the rhizospheres of uptaker and non-uptaker plants at various growth stages, and under different nutrient limitations to gain an impression of the spatial and temporal release of low molecular weight organic acids.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 39 publications | 15 publications in selected types | All 15 journal articles |
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Type | Citation | ||
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White JC. Differential bioavailability of field-weathered p,p’-DDE to plants of the Cucurbita and Cucumis genera. Chemosphere 2002;49(2):143-152. |
R829405 (2002) R829405 (Final) |
not available |
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White JC, Mattina MJI, Lee W-Y, Eitzer BD, Iannucci-Berger W. Role of organic acids in enhancing the desorption and uptake of weathered p,p'-DDE by Cucurbita pepo. Environmental Pollution 2003;124(1):71-80. |
R829405 (2002) R829405 (Final) |
not available |
Supplemental Keywords:
phytoremediation, plant-based remediation, bioremediation, clean-up, risk assessment, bioavailability, environmental microbiology, remediation, chlordane, bioaccumulation, biological treatment, contaminant removal, in situ bioremediation, in situ remediation, organic contaminants, organic pollutants, sequestration, soil contaminants, soil microbes, soil pollution., RFA, Scientific Discipline, Waste, Ecosystem Protection/Environmental Exposure & Risk, Bioavailability, Contaminated Sediments, Remediation, Environmental Chemistry, Chemistry, Environmental Microbiology, Bioremediation, Ecology and Ecosystems, risk assessment, sequestration, plant-based remediation, organic pollutants, rhizospheric, in situ remediation, biological treatment, contaminated sediment, plant uptake studies, contaminated soil, soils, toxicity, contaminants in soil, in situ bioremediation, bioremediation of soils, persistent organic pollutants, phytoremediation, bioacummulation, soil microbes, organic contaminants, soil reclamationProgress 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.