2003 Progress Report: Interaction of Various Plant Species with Microbial PCB-Degraders in Contaminated Soils

EPA Grant Number: R828770C008
Subproject: 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: HSRC (2001) - Midwest Hazardous Substance Research Center
Center Director: Banks, M. Katherine
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: Lasat, Mitch
Project Period: October 1, 2001 through September 30, 2003
Project Period Covered by this Report: October 1, 2002 through September 30, 2003
Project Amount: Refer to main center abstract for funding details.
RFA: Hazardous Substance Research Centers - HSRC (2001) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management


The overall objective of this research project is to investigate the interactive roles of plants and microbes for the biological degradation of polychlorinated biphenyl (PCB) contaminants in the rhizosphere. 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. Soil samples will be obtained from PCB-contaminated field sites and analyzed for PCB congener profile and microbial community structure. Microbial communities from distinct vegetated habitats will be classified using molecular genetic methods (e.g., Terminal-Restriction Fragment Length Polymorphism [T-RFLP]). Individual species of plants and microbes will be used in treatment combinations in PCB-contaminated soils under controlled conditions.

The combination of field and laboratory studies will allow better understanding of the biological processes involved with PCB cycling in the environment and permit the development of habitat management strategies for in situ containment and decontamination. Isolation of specific plant-microbe treatments under controlled laboratory conditions will permit determination of the rate of PCB degradation with regard to plant and microbial species, plant growth rate, and microbial activity. Furthermore, it will allow identification of plants that negatively affect biodegradative processes by suppression of microbial growth or activity. This approach will contribute to our overall understanding of PCB biogeochemical cycling and for advancement of rhizosphere-based remediation strategies.

The specific objectives of this research project are to: (1) elucidate specific PCB biodegradative enzymatic pathways; (2) analyze plant effects on PCB soil degradation rates; (3) characterize plant-microbe interactions in PCB-contaminated rhizosphere soils; and (4) evaluate soil amendments and PCB contaminant properties on biodegradation rates.

Progress Summary:

Reductive Dechlorination of PCBs Using Microbes and Plants

PCB degradation requires two basic steps: reductive dechlorination, usually mediated by anaerobes, followed by enzymatic ring cleavage by aerobic microorganisms. In the present investigation, we hypothesize that aerobic microbes, fungi, or plants may perform reductive dechlorination of PCB without assistance of anaerobes. Nitrate reductase (NR) is a ubiquitous and inducible enzyme involved mainly in conversion of nitrate to ammonia. The mechanism of NR-mediated PCB dechlorination will be investigated in various bacterial, fungal, and plant systems. Experiments are being designed to characterize the effects of pure PCBs (e.g., Tetra- [PCB 77]; Hexa- [PCB 153]), mixed PCB congeners (e.g., Aroclor 1242), and KNO3 on NR gene expression and enzyme activity in these organisms.

The inducible NR enzyme was shown to mediate PCB dechlorination in experiments using white-rot-fungi (Phanerochaete chrysosporium) and confirmed using pure NR enzyme (Sigma-Aldrich, USA). NR requires molybdenum (Mo) and iron as cofactors to promote reductive dechlorination, though tungsten (W) acts as a competitive inhibitor to retard the dechlorination process. Results are shown in Figures 1-3.

Figure 1. Effects of Different Metal Ions on PCB Biodegradation. PCBs were incubated in the presence of P. chrysosporium with or without the NR-associated metal ions, Mo, and W. Bars represent the average of replicate assays (N = 3; mean ± SE).

Figure 2. Chlorine Release Assay of Treated PCBs. Two separate metal ions were coincubated with the fungus P. chrysoporium in a PCB biodegradation assay. The addition of Mo increased PCB dechlorination, whereas tungsten (W) reduced chlorine release (measured at A460). These findings suggest that Mo may be an inducer and W an inhibitor of the specific PCB-degrading enzymes (N = 3; mean ± SE). Figure 2A represents in vivo studies and Figure 2B represents in vitro studies.

Figure 3. PCB Chlorine Release Assay Using Pure NR Enzyme. This chart shows chlorine release from 1,000 ppm Aroclor 1242 by pure NR enzyme with or without added Mo. The bars represent free Cl- measured at A460 (N = 3; mean ± SE).

PCB Phytostimulation Greenhouse Study

PCB-contaminated soils were obtained from an impacted settling basin in southeast Indiana for use in greenhouse and laboratory phytoremediation studies. The PCB level of the homogenized soil mixture was determined to be 1.10 +/- 0.17 ppm of approximately equal parts Aroclor 1254 and Aroclor 1260. The site had been impacted by manufacturing runoff less than 30 years prior to collection. Our intent was to perform a pilot-scale phytoremediation greenhouse trial using a modest number of plants reported or observed previously to accelerate biodegradation of PCBs or other persistent organic contaminants.

Soil PCB Analytical Protocol Development

We surveyed a variety of soil PCB extraction procedures for analysis with gas chromatograph-electron capture detector (GC-ECD) (Agilent 6890) via U.S. Environmental Protection Agency (EPA) Method 8082. The soil PCB extraction protocols examined included Soxhlet Extraction (EPA Method 3540), Sonication Extraction (EPA Method 3550), and Accelerated Solvent Extraction. All soil extracts were further concentrated on a boiling water bath and a nitrogen evaporation bath. Quantitation of PCB extracts was based on 10 unique peaks identified in mixed standards of Aroclors 1254 and 1260, (50:50) with 5 of the peaks unique to each Aroclor. For method development, we used soils spiked with Aroclors, surrogate compounds (tetrachloroxylene and decachlorobiphenyl), and a PCB-soil sample previously analyzed by a certified laboratory. Extraction efficiency was determined by both spike recovery and yield in successive extraction cycles.

The Soxhlet method was found to be very laborious (~10 samples/week) and displayed poor extraction consistency, displaying approximately 40-120 percent soil PCB recovery for split samples. The Sonication method was more easily performed (~20 samples/week), but gave consistently low yields (~13-30 percent soil PCB recovery). Both methods were abandoned in favor of Accelerated Soil Extraction using Acetone-Hexane (50:50) with a Dionex ASE Instrument. The ASE protocol gave approximately 95-102 percent PCB spike recovery and allowed higher rates of sample processing (qreater than or equal to 60 samples/week). In addition, the ASE procedure required less soil per extraction, less solvent, less extract processing, and less laboratory use/contamination. Subsequently, the ASE protocol was used for soil PCB analyses for this study.

PCB Phytoremediation Greenhouse Study Design and Results

Unamended PCB-contaminated soil was placed in a 6" plastic pot and planted with a single plant for each planted treatment, including: alfalfa (Medicago sativa), big bluestem (Andropogon gerardii), green bulrush (Scirpus atrovirens), monkeyflower (Mimulus ringens), switchgrass (Panicum virgatum), and white mulberry (Morus alba). Five pots of each planted and unplanted control treatments were sacrificed at 10 and 16 weeks after planting. Over the course of the study, pots were watered as needed, fertilized with standard nutrients weekly, and kept weed-free.

Figure 4. Soils Were Analyzed by Accelerated Solvent Extraction (HEX:ACE 50:50) and GC-ECD. Shown are soil PCB levels for the treated soils at 10 and 16 weeks after planting (time-zero soil PCB = 1.10 +/- 0.17 ppm).

Statistically significant reduction in soil PCB levels was not observed for any of the planted treatments over the course of the 16-week study relative to the starting concentration or unplanted treatment. Some plants may have achieved minor reductions in PCB content, though substantial analyte variation rendered differences among the treatments insignificant. Given the well-documented recalcitrance of Aroclors 1254 and 1260 to aerobic biodegradation, it is possible that longer treatment periods or the addition of soil amendments are required to achieve effective reduction in these high molecular weight PCBs. Other possible limits to observable bioremediation may result from low-soil PCB levels (~1.0 ppm), resulting in low bioavailability or substantial contaminant heterogeneity and causing high analytical variation.

Microbial Community Analyses using 16S rDNA T-RFLP

T-RFLP analyses are used to produce diagnostic DNA "fingerprints" of target biological communities. We are working to develop this protocol to identify microbial responses in planted and/or amended soil treatments for PCB biodegradation. T-RFLP uses PCR amplification of the variable regions of the microbial 16S rDNA sequence followed by digestion with diagnostic sets of three to four restriction enzymes. The initial PCR amplification reaction uses one or both primers labeled with a fluorescent dye(s), allowing analysis of the restriction fragments on automated sequencing instruments.

To advance molecular genetic analysis of PCB soil microbial communities, we are sharing laboratory resources, reagents, and travel between Michigan State University (MSU) and Howard University (HU) facilities for research updates and experimental troubleshooting. The HU research team came to MSU in February 2003 for a "T-RFLP Workshop" to review progress and introduce the protocols to newer members of the MSU-HU research team. Additional reciprocal visits are planned for the coming academic year.

In summary, the T-RFLP procedure is performed on DNA extracted from treated soils with a commercially available preparation (MoBio, Solana Beach, CA). Soil DNA is PCR-amplified using 16S rDNA primers; the 5' primer of the pair labeled with the fluorescent conjugant FAM. The PCR product was run on a 1 percent agarose gel, and the band corresponding to approximately 1,100 bp was excised and purified (QIAGEN, Valencia, CA). We are utilizing four different restriction enzymes for DNA digestion: HhaI, HaeIII, MspI, and RsaI. Digested DNA samples are desalted and analyzed by the MSU Genomic Center for Capillary Electrophoresis.

We are conducting T-RFLP analyses of PCB soil planted with alfalfa. Bacterial communities are being examined from the following treatments: (1) PCB contaminated soils, unplanted (#P3-2, U.S. Army); (2) #P3-2 soils planted with alfalfa; and (3) #P3-2 soils spiked with additional 100 ppm 2¢,3,4-PCB planted with alfalfa.

We currently are working to overcome inconsistent product yield and reproducibility problems with the T-RFLP products. In general, the primary step of DNA extraction works well for these soils, though the PCR amplification procedure is especially sensitive to coextractants and contamination (e.g., failed negative controls). Therefore, we frequently obtain insufficient amplification products for T-RFLP analyses. Our T-RFLP optimization strategies include evaluation of alternative DNA purification techniques, primer designs, fluorescent conjugate effects, and PCR amplification conditions.

Future Activities:

Reductive Dechlorination of PCBs Using Aerobic Microbes: Role of NR, Effect of Cofactors and Inhibitors

We have conducted detailed experiments using white-rot-fungi (P. chrysosporium) and purified NR from the fungus, Aspergillus, indicating that PCBs can be dechlorinated both in vitro and in vivo under aerobic conditions (manuscript submitted to Biochemical and Biophysical Research Communications, 2003). We will continue testing a wide range of bacterial, fungal, and plant systems for the presence of NR enzymes and their effectiveness for PCB dechlorination.

Mechansistic Understanding of NR-Mediated PCB Reductive Dechlorination

NR is an inducible enzyme requiring KNO3 as a substrate, several cofactors, such as Mo and iron, and an electron donor such as nicotinamide adenine dinucleotide/nicotinamide adenosine dinucleotide phosphate. We will continue analyzing the biochemical parameters for the NR enzyme complex within the various biological systems, including fungi, bacteria, and plants.

Standardization of Microbial Community Structure Analyses Using T-RFLP

As previously described, the T-RFLP protocol has not worked consistently in our hands. We will continue efforts to develop this procedure in our laboratory to provide a means to characterize the microbial community structure and response under various treatment conditions.

Influence of Soil Amendments and Contaminant Chemistry on PCB Biodegradation

Various soil modifications (e.g., biosurfactants, bioaugmentation) and the influence of PCB-contaminant properties (e.g., weathered, spiked, individual congeners, different Aroclors) will be surveyed for their influence on PCB phytoremediation effectiveness. We currently are using 2',3,4-PCB and hexachloro PCBs (PCB 153) for spiked bioremediation experiments, though these studies are being extended using tetrachloro- (PCB 77) and Aroclors (1242, 1248, 1254, 1260). Comparative studies using more highly chlorinated PCBs will be applicable to conditions in impacted field sites and more responsive to regulatory concerns.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 8 publications 2 publications in selected types All 2 journal articles
Other center views: All 108 publications 22 publications in selected types All 14 journal articles
Type Citation Sub Project Document Sources
Journal Article Dutta SK, Adam A, Toure O, Williams AL, Chen YQ. Indigenous mixed soil bacteria in presence of compatible plants are more efficient in PCB degradation. Fresenius Environmental Bulletin 2003;12(3):314-319. R828770 (Final)
R828770C008 (2003)
  • Full-text: Howard University PDF
  • Abstract: PSP-PARLAR Abstract
  • Journal Article Toure O, Chen YQ, Dutta SK. Sinorhizobium meliloti electrotransporant containing ortho-dechlorination gene shows enhanced PCB dechlorination. Fresenius Environmental Bulletin 2003;12(3 Spec Iss):320-322. R828770 (2003)
    R828770 (Final)
    R828770C008 (2003)
  • Abstract: Parlar Scientific Publications-Abstract
  • Supplemental Keywords:

    bioremediation, PCBs, polychlorinated biphenyls, microbial degraders, waste, analytical chemistry, contaminated sediments, environmental microbiology, hazardous, hazardous waste, microbiology, molecular biology/genetics, PCB-contaminated soil, bioavailability, biochemistry, biodegradation, bioremediation of soils, contaminants in soil, contaminated soils, degradation, industrial waste, microbes, microbial degradation, phytoremediation, hydrophobic, organic pollutant., 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, phytoremediation

    Relevant Websites:

    http://www.mhsrc.org Exit

    Progress and Final Reports:

    Original Abstract
  • 2002 Progress Report
  • Final

  • Main Center Abstract and Reports:

    R828770    HSRC (2001) - Midwest Hazardous Substance Research Center

    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