Dechlorination of Polychlorinated Biphenyl (PCB)

Congeners by Dehalococcoides

By: David VanHouteFirst Year Graduate Student

Rensselaer Polytechnic Institute11/12/2010

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

Genus DehalococcoidesStrict anaerobe and slow growing Sediment dwellingOnly 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, D onna. A Case Study for M icrobial Biodegradation: Anaerobic Bacterial Reductive D echlorination of Polychlorinated Biphenyls-From Sed im ent to D efined

M ed ium . Annu. Rev. M icrobiol. 2008. 62:253-70.

Reductive Dehalogenating Enzymes (RDases)

Studies have shown a wide range of dechlorinating activity:

Vinyl ChloridesDi-, Tri-, Tetrachlorinated EthenesChlorobenzenesPCB Congeners

Upwards of 32 possible RDase genes found in strain CBDB1.

Specific RDases for specific PCB substrates?

Rotation1. Anaerobic techniques2. Data Collection3. 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.

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.

PCB Congener

mix

Congener Peak Retention time of

congener

Mix 2 2,6 5 10.699

Mix 1 2,5 6 11.69

Mix 2 2,4 6 11.841

Mix 2 2,3 8 12.497

Mix 4 3,5 9 13.33

Mix 8 2,4,6 11 14

Mix 5 3,4 13 14.1

Mix 2 2,3,6 16 14.993

Mix 9 2,3,5 19 16.003

Mix 3 2,4,5 20 16.437

Mix 6 2,3,4 25 17.636

Mix 6 3,4,5 33 19.783

Mix 6 2,3,5,6 35 20.01

Mix 9 2,3,4,6 35 20.23

Mix 6 2,3,4,5 46B 23.057

PCB Retention Times

10

12

14

16

18

20

22

24

2,6 2,5 2,4 2,3 3,52,4,6

3,42,3,6 2,3,5 2,4,5 2,3,4 3,4,5

2,3,5,62,3,4,62,3,4,5

Congener

Retention Time (min)

Methyl Viologen Assays (MV)All reagents MUST be

anoxic.MV as electron donorPCB as electron acceptorTitanium(III) Citrate as a

reductant. Assess dechlorination of

congener triplicates at three different time points: 0, 2, 24 hrs.

Reaction terminated with Hydrogen Peroxide.

QuickTime and aªTIFF (Uncompressed) decompressor

are needed to see this picture.

Collecting Data

Time (min)

26

0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

236

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

1 1 0

0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

T24-235

Time (min)

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

QuickTime and aªTIFF (Uncompressed) decompressor

are needed to see this picture.

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

Dechlorination of PCB Congeners of MV4 Assay: 234,235,236,245,2345

0 0

151

0 0 00 0

151

0 0 00

5473

0

50

100

150

200

250

300

350

400

0 2 24

Time (hrs.)

pmol/ml Product

Released

234

235

236

245

2345

Dechlorination of PCB Congeners in MV5: 234, 235, 236, 245, 2345

0 0

367

0 0 00 0

135

0 0 00

100

289

0

50

100

150

200

250

300

350

400

0 2 24

Time (hrs.)

pmol/ml of Product

Released

234

235

236

245

2345

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

234

0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

24

Ti me (m in)

Dechlorination of Congener 234

536

430

210

0 0

367

01002003004005006007008009001000

0 2 24

Time (hrs.)

pmol/ml of 24 Released

234

24

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 f lanked chlori ne

Dechlorination of PCB Congener 2345 to 245 and 235

0

100

289

0 0

78

635

564

282

0

100

200

300

400

500

600

700

0 2 24

Time (hrs.)

pmol/ml 245 or 235

Released

245

235

2345C lC l

C l

C l

C l

C l

C l

C lC l

C l

2 4 5 - C B

2 3 5 - C B

2 3 4 5 - C B

Positive Control of 236 CBWhat Happened?

Dechlorination of Congener 236

1407

992 1000

0 0

135

0

200

400

600

800

1000

1200

1400

1600

0 2 24

Time (hrs)

pmol/ml of 26 Product Released

236

26

Dechlorination of Congener 236 (3rd triplicate only)

1325

1100 1080

0 0

323

0

200

400

600

800

1000

1200

1400

0 2 24

Time (hrs)

pmol/ml of 26 Product Released

236

26

• 236 CB (positive control)• Known to show extensive dechlorinating activity• Two of the three triplicates oxidized prior to 24hr.

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

C lC l

C l

C l

C l

2 4 - C B2 3 4 - C B

PCB Congener Dechlorination 234 Yes 235 No 236 Yes 245 No 2345 Yes

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.

Acknowledgements Special thanks to Dr. Donna Bedard Special thanks to Sarah LaRoe Biology Department

Literature:1 – Adrian, Lorenz et al. dehalococcoides 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.