The document summarizes research on the dechlorination of polychlorinated biphenyl (PCB) congeners by the anaerobic bacterium Dehalococcoides. Key findings include: 1) Dehalococcoides uses PCBs as a terminal electron acceptor and can effectively dechlorinate various PCB mixtures. 2) Experiments showed some PCB congeners (234 CB and 2345 CB) underwent more dechlorination than others (245 CB and 235 CB). 3) Further research is needed to understand congener specificity and identify reductive dehalogenase enzymes responsible for dechlorinating different PCBs.

1. Dechlorination of Polychlorinated Biphenyl (PCB) Congeners by Dehalococcoides By: David VanHoute First Year Graduate Student Rensselaer Polytechnic Institute 11/12/2010

2. PCBs Persistent pollutant of aquatic habitats and watersheds Used for manufacture of dielectric fluids, transformers, capacitors, and coolers Linked to toxicity within food webs and as a possible human carcinogen Exist in a variety of forms (209) from original Aroclor mixtures: Ex: 234 CB, 235 CB, 236 CB

3. Genus Dehalococcoides Strict anaerobe and slow growing Sediment dwelling Only means of respiration is through using a halogenated compound as terminal electron acceptor. Laboratory studies show that Dehalococcoides can effectively dechlorinate PCB mixtures Reductive Dehalogenating Enzymes (RDases) Generally shown to have specific dechlorination activity*: Meta and Para (process N and P respectively) Ortho and unflanked Para (process LP) *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.

4. Reductive Dehalogenating Enzymes (RDases) Studies have shown a wide range of dechlorinating activity: Vinyl Chlorides Di-, Tri-, Tetrachlorinated Ethenes Chlorobenzenes PCB Congeners Upwards of 32 possible RDase genes found in strain CBDB1. Specific RDases for specific PCB substrates?

5. Rotation 1. Anaerobic techniques 2. Data Collection 3. Goals for rotation: Generate PCB calibration standards Assess dechlorinating activity of individual PCB congeners 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.

6. PCB Calibration Standards Generated calibration standards of the PCB congeners for the GC at conc. 50, 100, 200, 500, 1000 ppb (=ng/ml). Generation of standards allows for quantification of product formation. Figure out retention times on GC of individual congeners.

7. Methyl Viologen Assays (MV) All reagents MUST be anoxic. MV as electron donor PCB as electron acceptor Titanium(III) Citrate as a reductant. Assess dechlorination of congener triplicates at three different time points: 0, 2, 24 hrs. Reaction terminated with Hydrogen Peroxide.

8. Collecting Data Extract product and substrate samples in 2mL Hexane Gas Chromatography (GC) with a 63 Ni electron capture detector

9. Assessing Dechlorination Early MV assays (4th shown) showed small amounts of dechlorination Data for MV assays 1-3 not shown Final MV (5th) assay shows more dechlorination of 234 CB, 2345 CB

10. Significant Dechlorination of Congener 234 CB Congener 234 CB had the most dechlorination activity of all three congeners Doubly flanked chlorine at meta position. Raw data from GC: Blue= 234 CB Black=24 CB

11. Significant Dechlorination of Congener 2345 CB Congener 2345 CB also had significant dechlorination. 245 CB preferred dechlorination pathway over 235 CB? Para position more difficult to dechlorinate than meta. Doubly flanked chlorine

12. Positive Control of 236 CB What Happened? 236 CB (positive control) Known to show extensive dechlorinating activity Two of the three triplicates oxidized prior to 24hr.

13. Conclusions Dechlorination by this strain appears to be sensitive to certain PCB congeners 234 CB and 2345 CB were congeners most likely to be dechlorinated; positive control should have shown more dechlorination Congeners 245 CB and 235 CB showed no dechlorination Is this determined by stereochemistry or action by specific RDases? Are transcription levels for particular RDase genes higher? Anaerobic research VERY difficult

14. Future? Continue research into congener specificity Identification of novel RDases and characterize their substrate specificity Hopeful purification and sequencing Finding new models to implement more efficient means of bioremediation of PCB pollutants.

15. Acknowledgements Special thanks to Dr. Donna Bedard Special thanks to Sarah LaRoe Biology Department Literature: 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. 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. 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 4 – Adrian, Lorenz et al. (2000). Bacterial Dehalorespiration with Chlorinated Benzenes. Nature, 408:580-583. 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 .