Grantee Research Project Results
2002 Progress Report: Interaction of Various Plant Species with Microbial PCB-Degraders in Contaminated Soils
EPA Grant Number: R828770C008Subproject: this is subproject number 008 , established and managed by the Center Director under grant R828770
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Health Effects Institute (2005 — 2010)
Center Director: Greenbaum, Daniel S.
Title: Interaction of Various Plant Species with Microbial PCB-Degraders in Contaminated Soils
Investigators: Rugh, Clayton , Dutta, Sisir
Institution: Michigan State University , Howard University
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2001 through September 30, 2003
Project Period Covered by this Report: October 1, 2001 through September 30, 2002
Project Amount: Refer to main center abstract for funding details.
RFA: Hazardous Substance Research Centers - HSRC (2001) Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management
Objective:
The main objective of this research project is to investigate the interactive roles that plants and microbes play in the rhizosphere for the biological degradation of polychlorinated biphenyl (PCB) contaminants. It has been suggested that plants possess benefits for microbial PCB degradation through a potential combination of augmented microbial densities and/or stimulation of biodegradative activities. This research project will utilize both field and laboratory analyses to describe interactions of plant species with microbial degraders.
Progress Summary:
The combination of field and laboratory studies allows for a better understanding of the biological processes that are involved with PCB cycling in the environment and permits the development of habitat management strategies for in situ containment and decontamination. Isolation of specific plant-microbe treatments under controlled laboratory conditions permits the determination of the rate of PCB degradation with regard to plant and microbial species, plant growth rate, and microbial activity. Furthermore, it allows for the identification of plants that negatively effect biodegradative processes by inhibition of growth or suppression of microbial activity. This approach contributes to our overall understanding of PCB biogeochemical cycling and for the advancement of rhizosphere-based remediation strategies.
Dutta Laboratory, Howard University (HU). We obtained 12 PCB-contaminated soil samples from the U.S. Army Waterways Research Station in Vicksburg, MS. This study provides data on known and unknown microorganisms indigenous to the soils contaminated with varying PCBs. We used colony forming unit (CFU) counts to quantitate growth of microorganisms and to estimate the effect of PCB toxicity. PCB dechlorination was estimated by chlorine (Cl-) release assay (at 460 nm) and by high performance liquid chromatography (HPLC). We extracted unidentified microorganisms with the ability to degrade spiked 2', 3, 4-PCB congener from highly to moderately contaminated soils. The higher the concentration of 2', 3, 4-PCB in the soil, the greater the ability of the soil-derived microbial consortia to deplete the pollutant. Indigenous unidentified cultures were at least equal in degradation assays than known PCB degraders such as Comamonas testosteroni and Rhodoccocus sp. Mixed microbial communities were consistently superior in degradation assays than pure cultures.
The presence of plants significantly increased the rhizosphere CFU counts relative to unplanted treatments (see Table 1). We initiated the identification of selected, indigenous PCB-degrading microbes using 16S rDNA sequence analysis. We electroporated the plasmid pPE43, which contains the ohb DNA region and relevant genes for PCB degradation, into Sinorhizobium meliloti. The PCB depletion and growth of S. meliloti electrotransformants in the presence of the alfalfa plant were significantly enhanced. Genetically modified nitrogen-fixing S. meliloti U.S. Department of Agriculture (USDA) 1936 and its symbiont, the alfalfa plant, would be particularly suited for treatment of PCB-contaminated soils with concurrent enhancement of soil fertility as green manure.
Table 1. Preliminary Results of CFUs at 10-6 Serial Dilution From Isolated Cultures. PCB concentrations in ppm were estimated using 2', 3, 4-congener as standard by Gas Chromatography/Mass Spectrometry (GC/MS). N = 3, standard deviation from the mean was less than 4 percent in general. CFU enhancement shows significant phytostimulation by alfalfa plants (Dutta, et al., 2002).
Soil Sample | PCB (ppm) | CFUs Without Plants | CFUs With Plants |
C3 | <0.05 | 15 x 106 | 166 x 106 |
PIII-1 | 48 | 20 x 106 | 175 x 106 |
PIII-2 | 57 | 46 x 106 | 189 x 106 |
PIII-3 | 77 | 38 x 106 | 200 x 106 |
Control | None | None | None |
The growth of shoots and roots of 2', 3, 4-PCB-grown (approximately 100 ppm) alfalfa plants with various microbial treatments is shown in Figures 1 and 2. Both the unidentified consortia (see Figure 1: Panel C) and the transformed S. meliloti (see Figure 1: Panel B) containing PCB-degrading ohb gene cluster showed robust growth and development in alfalfa shoots. In contrast, the alfalfa plant (see Figure 1:Panel A) treated with transformed Escherichia coli containing ohb, displayed stunted shoot growth and leaf development.
Figure 1. Alfalfa (Medicago sativa) Plants Grown for 8 Weeks in Leonard Jar Setups in Humidity-Controlled Growth Chambers at 30°C and 8/16 Hours Day/Night Cycles. Each jar contained sterile soil:vermiculite (50:50) spiked with 2', 3, 4- PCB (approximately 100 ppm) and inoculated: Jar (A) transformed E. coli containing PCB-degrading plasmids/genes "ohb" gene clusters; Jar (B) - transformed S. meliloti (an N2-fixing bacteria) containing similar PCB-degrading plasmids/genes; and Jar (C) uncharacterized microbial consortia isolated from the PCB-contaminated soil (Dutta, et al., 2002; unpublished).
Figure 2. Growth of Alfalfa Roots in Approximately 100 ppm PCB in the Presence of Different Microbial Inocula. (A) Transformed E. coli containing PCB-degrading plasmids/genes; (B) transformed S. meliloti containing PCB-degrading plasmids/genes; and (C) uncharacterized microbial consortia isolated from PCB-contaminated soil (Dutta, et al., unpublished 2002).
Distribution of PCBs in Different Plant Tissues. After the application of approximately 100 ppm PCBs to the reactor media, translocation and/or adsorption of PCB to various parts of the plants, the soil, water, and the walls of the Leonard bioreactor jar are determined by HPLC analysis. We harvested alfalfa plants after 4 weeks of growth in the spiked soils. We extracted PCBs from 1.0 g of leafs, roots, and stems by acetone:hexane mixture (50:50 v/v) and currently are analyzing them.
Rugh Laboratory, MSU
Soil Sample Acquisition. We received permission to obtain PCB-contaminated soils in October and November 2002, from sites in Dearborn, MI (Ford Motor Company) and Clarksville, IN (U.S. Army Corps of Engineers [USACE], Indiana Army Ammunition Plant [INAAP]). Ford Motor Company currently is finalizing a consent order for remedial action with the Michigan Department of Environmental Quality and the U.S. Environmental Protection Agency (EPA), allowing us to acquire sufficient amounts of contaminated soil for our research program. In October 2002, we worked alongside the site remediation contractor to characterize the excavated soils for PCB content and congener using field immunoassays (Strategic Diagnostics, Inc.) for the identification and collection of PCB-impacted soils. Preliminary analyses of these soils showed Aroclor 1248 (approximately 100 ppm) and Aroclor 1260 (approximately 50 ppm) with lesser amounts of Aroclors 1242 and 1254. We worked with the Primary Remedial Contractor, URS (Omaha, NE), to collect impacted soils and sediments from the INAAP in November 2002. Preliminary analyses revealed generally low levels of PCBs throughout the 3-acre site, though "hotspots" of 0.5-1.2 ppm of Aroclors 1242 and 1254 have been identified. Michigan State University (MSU) will be working with the URS Analytical Laboratory to develop methods for reliable detection and analysis of these low-level PCBs. Both sites represent historic depositions with significant contaminant-soil aging (<20-30 years).
For experimental comparison to the PCB-contaminated field soils, we also have obtained analytical grade stocks for Aroclors 1242, 1248, 1254, and 1260 to be used to spike prepared soils. These spiked soils will be used in concurrent parallel experiments with the field soils to determine the effects of aging upon rhizoremediative capabilities for each of the Aroclor mixtures under different biological treatment regimes. In general, aged soils have been shown to be recalcitrant to bioremediative treatments, relative to freshly spiked soils. It is expected that we will observe a similar phenomenon, though it is important to determine if this effect is consistent for all congener types under varied biological treatment regimes.
Plant Selection. In a different research study, we conducted laboratory, greenhouse, and field trials of a wide variety of Michigan native plant species for their ability to enhance indigenous polycyclic aromatic hydrocarbon (PAH) biodegradation in industrially impacted soils. From more than 50 tested species in laboratory and greenhouse experiments, we identified approximately 20 species as superior PAH phytoremediator species. We hypothesize that plants capable of phytostimulation of PAH microbes will possess similar capabilities for PCBs, because both microbial processes utilize dioxygenase enzymatic pathways that are inducible by phenolic compounds in root exudates.
Bacterial Strain Characterization. We obtained several well-characterized biphenyl-utilizing bacterial strains, including: Burkholderia (Pseudomonas sp.) LB400, Rhodococcus erythreus NY05, Rhodococcus sp. RHA1, and C. testosteroni VP44. For Burkholderia LB400, R. erythreus NY05, and Rhodococcus sp. RHA1, we performed biphenyl sole-carbon source assays, morphological and growth rate analyses, and DNA extractions for confirmation of strain ID using 16S rDNA sequence analysis and terminal restriction fragmented length polymorphism (T-RFLP) fingerprinting. We currently are conducting comparisons of soil DNA extraction protocols for microbial identification and community analysis. We refined the T-RFLP analysis for microbial community analysis of rhizosphere soils using FAM-labeled polymerase chain reaction (PCR) primers, rather than the recommended HEX-labeled primers. We experienced considerable reduction in reaction quality with the HEX-tag, relative to unlabeled primers. This problem was corrected by the use of FAM-tag and several other minor reaction modifications to produce consistent results.
Future Activities:
The previously described preliminary studies allowed for the development of basic experimental tools, including bioreactor systems, selected plant species, microbial resources, and analytical procedures for PCB rhizoremediation research. We are engaged in technology transfer between our two research laboratories of the independently developed protocols. We scheduled reciprocal visits by key members of the research teams at 2 to 3-month intervals to maintain strategic interactions, protocol refinement, and research material exchange. The Dutta Laboratory at HU will focus on smaller-scale treatment systems (e.g., Leonard bioreactors), individual congener exposure, and fate studies. The Rugh Laboratory at MSU will emphasize greenhouse-scale treatments using larger soil volumes with complex PCB mixtures (e.g., Aroclors 1242, 1248, 1254, and 1260) in both spiked and industrially supplied soils to evaluate contaminant weathering in addition to other environmental variables.
We will perform the phytostimulation of PCB biodegradation with a variety of proven hydrophobic pollutant remediating plant species, including: Joe-Pye Weed (Eupatorium purpureum), Little Bluestem (Andropogon scoparius), Leadplant (Amorpha canadensis), and New England Aster (Aster novae-anglicae). We also will perform phytostimulation with the demonstrated PCB phyto-species, White Mulberry (Morus alba) and Alfalfa (Medicago sativa), and suspected PCB phyto-species, including Spicebush (Lindera benzoin) and Sassafras (Sassafras albidum).
It is expected that each of the tested species will display distinct PCB degradation rates, treated soil congener composition, tissue accumulation patterns, and rhizosphere microbial communities. These parameters will be examined using the methods described here and in greater detail in the research proposal drafts. Early results indicate that biological treatments can accelerate PCB degradation, although this process is influenced by both abiotic and biotic factors, including microbial community structure, plant species, initial contaminant concentration, and profile.
We will obtain plant and soil samples from PCB-contaminated field sites and analyze them for PCB congener profile and content, plant species ID, and microbial isolation and identification. Microbial populations will be classified using molecular genetic methods (e.g., T-RFLP) from distinct vegetated habitats. Individual species of plants and microbes will be used in treatment combinations in PCB-contaminated soils under controlled conditions. Laboratory assessment of specific plant-microbe combinations under a variety of growth conditions will enhance our understanding of biogeochemical cycling and biodegradative processes for PCBs. This knowledge can be used to design biological approaches for management of PCB impacted sites by enhancing natural degradation of these toxicants.
Journal Articles:
No journal articles submitted with this report: View all 8 publications for this subprojectSupplemental Keywords:
bioremediation, hydrophobic, organic pollutant, phytoremediation., RFA, Scientific Discipline, Waste, Contaminated Sediments, Environmental Chemistry, Microbiology, Environmental Microbiology, Hazardous Waste, Bioremediation, Hazardous, degradation, plant species, microbial degradation, industrial waste, bioavailability, biodegradation, contaminated sediment, contaminated soil, microbes, PCB contaminated soil, contaminants in soil, bioremediation of soils, PCB, biochemistry, phytoremediationRelevant Websites:
http://bridge.ecn.purdue.edu/~mhsrc/ Exit
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R828770 Health Effects Institute (2005 — 2010) Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R828770C001 Technical Outreach Services for Communities
R828770C002 Technical Outreach Services for Native American Communities
R828770C003 Sustainable Remediation
R828770C004 Incorporating Natural Attenuation Into Design and Management
Strategies For Contaminated Sites
R828770C005 Metals Removal by Constructed Wetlands
R828770C006 Adaptation of Subsurface Microbial Biofilm Communities in Response to Chemical Stressors
R828770C007 Dewatering, Remediation, and Evaluation of Dredged Sediments
R828770C008 Interaction of Various Plant Species with Microbial PCB-Degraders
in Contaminated Soils
R828770C009 Microbial Indicators of Bioremediation Potential and Success
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: R828770
108 publications for this center
14 journal articles for this center