Rotation I Presentation


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  • Stress need for terminal electron acceptor; specific Rdase enzymes, N and P process generally preferred.
  • 36 in others
  • Anoxic reagents, anaerobic chamber, crimp and purge vials, limit oxygen We know that Dehalococcoides dechlorinates mixtures of PCBs, but don’t know which congeners.
  • Retention times show how long specific congeners remain on GC column. Shows where product is seen and substrate
  • MV provides ample supply of H, no need for hydrogenase activity by microbes.
  • Both 234 and 2345 are doubly flanked congeners, significant product with removal of meta chlorine.
  • 234 and 2345 are doubly flanked chlorines that get removed, don’t really know why.
  • Rotation I Presentation

    1. 1. Dechlorination of Polychlorinated Biphenyl (PCB) Congeners by Dehalococcoides By: David VanHoute First Year Graduate Student Rensselaer Polytechnic Institute 11/12/2010
    2. 2. PCBs <ul><li>Persistent pollutant of aquatic habitats and watersheds </li></ul><ul><li>Used for manufacture of dielectric fluids, transformers, capacitors, and coolers </li></ul><ul><li>Linked to toxicity within food webs and as a possible human carcinogen </li></ul><ul><li>Exist in a variety of forms (209) from original Aroclor mixtures: </li></ul><ul><li>Ex: 234 CB, 235 CB, 236 CB </li></ul>
    3. 3. Genus Dehalococcoides <ul><li>Strict anaerobe and slow growing </li></ul><ul><li>Sediment dwelling </li></ul><ul><li>Only means of respiration is through using a halogenated compound as terminal electron acceptor. </li></ul><ul><li>Laboratory studies show that Dehalococcoides can effectively dechlorinate PCB mixtures </li></ul><ul><li>Reductive Dehalogenating Enzymes (RDases) </li></ul><ul><li>Generally shown to have specific dechlorination activity*: </li></ul><ul><ul><ul><ul><li>Meta and Para (process N and P respectively) </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Ortho and unflanked Para (process LP) </li></ul></ul></ul></ul><ul><li>*Bedard, Donna. A Case Study for Microbial Biodegradation: Anaerobic Bacterial Reductive Dechlorination of Polychlorinated Biphenyls-From Sediment to Defined Medium. Annu. Rev. Microbiol. 2008. 62:253-70. </li></ul>
    4. 4. Reductive Dehalogenating Enzymes (RDases) <ul><li>Studies have shown a wide range of dechlorinating activity: </li></ul><ul><ul><ul><ul><ul><li>Vinyl Chlorides </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Di-, Tri-, Tetrachlorinated Ethenes </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Chlorobenzenes </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>PCB Congeners </li></ul></ul></ul></ul></ul><ul><li>Upwards of 32 possible RDase genes found in strain CBDB1. </li></ul><ul><li>Specific RDases for specific PCB substrates? </li></ul>
    5. 5. Rotation <ul><li>1. Anaerobic techniques </li></ul><ul><li>2. Data Collection </li></ul><ul><li>3. Goals for rotation: </li></ul><ul><ul><ul><ul><li>Generate PCB calibration standards </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Assess dechlorinating activity of individual PCB congeners </li></ul></ul></ul></ul><ul><li>4. Purpose: To test and determine dehalogenation of 234 CB, 235 CB, 236 CB, 245 CB, 2345 CB congeners by pure cultures of Dehalococcoides cultured using 236-236 PCB as a terminal electron acceptor. </li></ul>
    6. 6. PCB Calibration Standards <ul><li>Generated calibration standards of the PCB congeners for the GC at conc. 50, 100, 200, 500, 1000 ppb (=ng/ml). </li></ul><ul><li>Generation of standards allows for quantification of product formation. </li></ul><ul><li>Figure out retention times on GC of individual congeners. </li></ul>
    7. 7. Methyl Viologen Assays (MV) <ul><li>All reagents MUST be anoxic. </li></ul><ul><li>MV as electron donor </li></ul><ul><li>PCB as electron acceptor </li></ul><ul><li>Titanium(III) Citrate as a reductant. </li></ul><ul><li>Assess dechlorination of congener triplicates at three different time points: 0, 2, 24 hrs. </li></ul><ul><li>Reaction terminated with Hydrogen Peroxide. </li></ul>
    8. 8. Collecting Data <ul><li>Extract product and substrate samples in 2mL Hexane </li></ul><ul><li>Gas Chromatography (GC) with a 63 Ni electron capture detector </li></ul>
    9. 9. Assessing Dechlorination <ul><li>Early MV assays (4th shown) showed small amounts of dechlorination </li></ul><ul><li>Data for MV assays 1-3 not shown </li></ul><ul><li>Final MV (5th) assay shows more dechlorination of 234 CB, 2345 CB </li></ul>
    10. 10. Significant Dechlorination of Congener 234 CB <ul><li>Congener 234 CB had the most dechlorination activity of all three congeners </li></ul><ul><li>Doubly flanked chlorine at meta position. </li></ul><ul><li>Raw data from GC: </li></ul><ul><ul><ul><ul><li>Blue= 234 CB </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Black=24 CB </li></ul></ul></ul></ul>
    11. 11. Significant Dechlorination of Congener 2345 CB <ul><li>Congener 2345 CB also had significant dechlorination. </li></ul><ul><li>245 CB preferred dechlorination pathway over 235 CB? Para position more difficult to dechlorinate than meta. </li></ul><ul><li>Doubly flanked chlorine </li></ul>
    12. 12. Positive Control of 236 CB What Happened? <ul><li>236 CB (positive control) </li></ul><ul><li>Known to show extensive dechlorinating activity </li></ul><ul><li>Two of the three triplicates oxidized prior to 24hr. </li></ul>
    13. 13. Conclusions <ul><li>Dechlorination by this strain appears to be sensitive to certain PCB congeners </li></ul><ul><li>234 CB and 2345 CB were congeners most likely to be dechlorinated; positive control should have shown more dechlorination </li></ul><ul><li>Congeners 245 CB and 235 CB showed no dechlorination </li></ul><ul><li>Is this determined by stereochemistry or action by specific RDases? </li></ul><ul><li>Are transcription levels for particular RDase genes higher? </li></ul><ul><li>Anaerobic research VERY difficult </li></ul>
    14. 14. Future? <ul><li>Continue research into congener specificity </li></ul><ul><li>Identification of novel RDases and characterize their substrate specificity </li></ul><ul><li>Hopeful purification and sequencing </li></ul><ul><li>Finding new models to implement more efficient means of bioremediation of PCB pollutants. </li></ul>
    15. 15. Acknowledgements <ul><li>Special thanks to Dr. Donna Bedard </li></ul><ul><li>Special thanks to Sarah LaRoe </li></ul><ul><li>Biology Department </li></ul><ul><li>Literature: </li></ul><ul><li>1 – Adrian, Lorenz et al. de halococcoides sp. Strain CBDB1 Extensively Dechlorinates the Commercial Polychlorinated Biphenyl Mixture Aroclor 1260. Applied and Environmental Microbiology, 75: 4516-4524. </li></ul><ul><li>2 – Bedard, Donna. (2008) A Case Study for Microbial Biodegradation: Anaerobic Bacterial Reductive Dechlorination of Polychlorinated Biphenyls-From Sediment to Defined Medium. Annual Review of Microbiology, 18: 35. </li></ul><ul><li>3 – Hoelscher, Tina. Et al. (2003). Reductive Dehalogenation of Chlorobenzene Congeners in Cell Extracts of Dehalococcoides sp. Strain CBDB1. Applied and Environmental Microbiology, 69: 2999-3001 </li></ul><ul><li>4 – Adrian, Lorenz et al. (2000). Bacterial Dehalorespiration with Chlorinated Benzenes. Nature, 408:580-583. </li></ul><ul><li>5 - Fung, Jennifer M. et al. (2007). Expression of Reductive Dehalogenase Genes in Dehalococcoides ethenogenes Strain 195 Growing on Tetrachloroethene, Trichloroethene, or 2,3-Dichlorophenol. Applied and Environmental Microbiology. 73: 4439-4445 . </li></ul>