PCBs and Bioremediation

1. PCBs and Bioremediation Pamela Windy Ravi

2. Overview • What are PCBs? • Why are they a problem? • What can we do with them? • How do the microbial methods work?

3. PCBs • Synthesized chemicals from petro-chemical industry used as lubricants and insulators in heavy industry • Used because • Low reactivity • Non-flammable • High electrical resistance • Stable when exposed to heat and pressure

4. Hydraulic fluid Casting wax Carbonless carbon paper Compressors Heat transfer systems Plasticizers Pigments Adhesives Liquid cooled electric motors Fluorescent light ballasts Uses

5. Manufacturing • First manufactured in 1929 by Montesano • Manufactured around the world • Production ended in 1977 in the US • Manufacture and unauthorized use banned in 1978 by USEPA • Made in countries world wide – Europe (France), Japan, former USSR

6. Where found • They are ubiquitous: • Water: rain and groundwater • Soil: through direct disposal and leaching from disposal sites • Animals: bioaccumulation • Food: bioaccumulation and production methods

7. Basic Aromatic Carbon structure

8. Nomenclature • Ortho: Cl on carbons 2 or 2' and/or 6 or 6‘ • Meta: Cl on carbons 3 or 3' and/or 5 or 5‘ • Para: Cl on carbons 4 or 4‘ • Named for company or given numbers • First two digits = number carbons • Second two indicate percent by weight of chlorine (ie 1242)

9. Interesting Facts • Between 1929 and 1970, 4*10^5 tons produced in US • Around 4,000 tons per year get into waterways through dumping and leakage • As of 1975, 4.5 million kg lost to environment through vaporization, leaks, spills, and landfills

10. PCBs are toxic Soluble in fats, oils, solvents Some more than others: more Cl = more toxic Position of Cl affects toxicity Ortho position less toxic than meta or para Para + meta = “dioxin like”; flat plane molecule, particularly toxic Risks

11. Food Chain Effects • Bioaccumulation • Primary producers and lower trophic level organisms take up PCBs, accumulates in the food chain • Higher organisms eating primary producers get more concentrated amounts of toxin • Often people consuming higher organisms are exposed to more toxic forms than factory workers

12. carcinogenous: Liver cancer melanoma immune system studies done on rhesus monkeys which have similar systems effects noted in people exposed to PCB contaminated rice oil suppressed swollen thymus gland in infants reproductive system humans and animals reduced birth weight reduced conception weight decrease in gestational ages still births and abortions Human Health

13. nervous system infant neurological functions Recognition short-term memory Learning effects seen at levels present in breast milk endocrine system thyroid health other health effects Human Health Cont.

14. Inhibits plankton growth and photosynthesis affecting the food chain reduce trophic pathways Reduce plankton size Reduce size of higher feeders Divert carbon flow to non-harvestable species less plankton = less bigger food fish Toxic to crustaceans, mollusks, and fish at concentrations of only a few ppb Human health concerns apply to animals as well Marine and Animal Health

15. Mitigation • Extraction or separation of contaminants from environmental media • Immobilization of contaminants • Destruction or alteration of contaminants • Chemical • Thermal • Biological

16. Bioremediation • The process by which organisms use toxic chemicals as a food source in an environment which is favorable to that process • Generally enhanced by addition of nutrients, oxygen, moisture or adjusting pH levels

17. Pathways of PCB Degradation • Anaerobic/Aerobic removal • Photochemical Degradation • Thermal Degradation • Fungal Degradation

18. Methods for PCB removal • Natural Attenuation: Microbes already in the soil are allowed to degrade as they can naturally and the site is closely monitored. • Biostimulation: Microbes present in the soil are stimulated with nutrients such as oxygen, carbon sources like fertilizer to increase degradation. • Bioaugmentation: Microbes that can naturally degrade PCB’s are transplanted to the site and fed nutrients if necessary

19. Microbial Methods • Microbes either: • Use PCBs as a carbon source • Microbes initiate reductive de-chlorination • Problems • Generally slow • Use other carbon sources in natural systems first • Microbes prefer lower chlorinated biphenyl • Prefer para and meta positions

20. Ortho, Meta and Para Chlorination of Biphenyl

21. Pathways of Aerobic Degradation Oxygenaseattack dihydroxybiphenyl Bugs attack benezoate

22. Pathways of Aerobic Degradation cont. metaclevage Most bugs degrade this M

23. Pathways of Aerobic Degradation cont. Harder to degrade than cholorbenzoate

24. Degradation of PCB Products Gives rise to chlorobenzoate Dioxygenase sttack on 2,3 catechol

25. Degradation of PCB Products cont.

26. Degradation of Chloroacetophenones

27. Take Home Message of Aerobic Degradation • Works at 10% oxygen content or at 4ppm minimum. • Anaerobic degradation needs to occur first if more that 4 chlorines exist per ring. • Degradation means chlorine removal from the ortho, meta or para positions. • Degradation happens most often through 2,3-dioxygenase on the 2,3 carbons with a metacleavage, or a metacleavage of unchlorinated 2,3-carbons.

28. Photochemical Degradation • Photochemical degradation is environmental degradation. This is also known as reductive dechlorination • Dechlorinates PCB’s by using mercury lamps as UV sources (If not degraded on site) with radiowave lengths of 254nm • The higher the chlorine content the faster they are photodegraded.

29. Photochemical Degradation cont. • Main pathway represented is reductive dechlorination by C-Cl bond clevage. • Environmental Degradation by Photochemical processes can increase presence of organic compounds that sensitize the reaction.

30. Thermal Degradation • Basically incineration of PCB’s • Must be at no lower that 700 degrees Celsius to decompose completely. At lower temperatures toxic compounds are produced (PCDF’s). • Method can be adopted on an industrial scale as a recommended waste disposal technique with 50 to 500ppm.

31. Thermal Degradation cont. • Chemical procedures of complete PCB dechlorination using catalysts such as 5% platinum, palladium, nickel boride in alcohol with excess sodium borohydride and LiAlH4 (Lithium Aluminum Hydride).

32. Fungal Degradation • Aspergillus niger: fillamentous with cytochrome p450 that attacks lower chlorinated PCB’s • Phanerochaete chrysosporium: White rot fungi can attack even highly chlorinated PCB’s at low conc. (less than 500ppb) while aerobic degradation is occuring at a level of 10ppm.

33. Anaerobic Reductive Dechlorination of PCBs • Not as well-characterized a process as aerobic degradation • Anaerobic bacteria responsible were not identified until more recently • Anaerobic PCB degradation first observed in Hudson river sediments (a site of historic contamination) • Since then, it has been noted in many other places

34. For the uncontaminated (PCB-free) sites, this was determined by introducing PCBs to sediments from these areas in the lab • Indicates that dechlorinating activity may be due to a common, widespread reductive pathway (Abramowicz,1995)

35. Anaerobic dechlorination is complementary to aerobic degradation • The less chlorinated products of anaerobic pathways are better substrates for aerobic pathways than more chlorinated congeners • A combination of the two could result in complete PCB breakdown

36. A reduction pathway, with Cl as the terminal electron acceptor • At least one species (o-17) likely uses acetate as the electron donor

37. Anaerobic congener selectivity • Most (but not all) observed microbial degradation of PCBs removes Cl only in the meta or para positions (primarily ortho products) • Even highly chlorinated congeners can be mostly dechlorinated

38. Aerobic PCB Degraders • Numerous soil bacteria break down PCBs via dioxygenase pathways • Most identified seem to be Pseudomonas species • Others: Achromobacter, Acinetobacter, Alcaligenes, Arthrobacter, Corynebacterium, Rhodococcus, Burkholderia (fairly diverse) • In general, the more highly chlorinated the PCB is, the fewer species that are able to degrade it aerobically.

39. (Abramowicz, 1990)

40. Some aerobic bacteria are capable of degrading a broader range of PCB congeners, notably: • Burkholderia xenovorans LB400 (Gram -) • Widest range of congener substrates • ~9.5 Mb genome - one of the largest sequenced • Rhodococcus erythropolis RHA (Gram +) • These possess different enzymatic pathways, and the genes for them(“ohb” and “rod/cat+” respectively) are often used in the genetic construction of PCB degraders

41. Anaerobic PCB degraders • Although PCB dechlorination in anaerobic sediments was noted fairly early on, first responsible bacteria was not identified until 2001 • “o-17”, from Baltimore Harbor sediments • Was discovered by monitoring 16s rRNA of an enriched ortho-PCB degrading culture. • Growth of o-17 was dependant on the growth and dechlorination of 2,3,5,6-tetrachlorobiphenyl

42. (Cutter et. al. 2001)

43. o-17 most closely related to Dehalococcoides sp. (one of the green non-sulfur bacteria) • Since then, other species have been identified using similar techniques. • Relatives of: • Desulfovibrio caledonienssi (a  -proteobacteria) • Aminobacterium columbiense, (Gram +)

44. Genetic Construction of PCB Degrading Bacteria • Most of the work thus far done with aerobic species, especially. Burkholderia, Rhodococcus, Pseudomonas • Some success with aerobically degrading contaminated river sediments in the laboratory

45. Potential advantages: • Easier to control the growth of a single strain than a consortium of bacteria, making it desirable to put many complementary catabolic pathways into one strain • Can expand the metabolic pathway of a bacteria “horizontally” to increase the number of substrates (i.e., the number of different PCB congeners) it can act on • Can expand pathway “vertically” by adding genes coding for additional enzymes to break down a compound further • Potential to accelerate degradation/bioremediation (use of strong promoters on introduced genes, etc) • Create strains capable of utilizing PCBs as preferential sole carbon source. • Possibility to increase the degrading potential or natural fitness of existing species

46. Challenges/Disadvantages • When combining pathways, often have to alter the regulation, activity and/or specificity of critical enzymes • Intermediates from one PCB-degrading pathway can inhibit the enzymes of another pathway • Unproductive pathways must be inactivated • Often these bacteria have reduced fitness in natural environments (aerobic PCB breakdown is a co-metabolic pathway) • Ethical concerns, gene swapping with indigenous species, unexpected effects, etc. • Still, Cl not completely mineralized (persists in organic products)

47. Introduction of pathways on plasmids • Several dioxygenase pathway-coding plasmids have been identified • Ex. Rhodococcus RH1 and RH2 • Different plasmids have different host ranges • Need to combine with strong promoters • Can effect large genetic changes in a single step • Splice genes directly into genome • Standard mating procedures : conjugation, transformation, spontaneous mutation • Gene exchange between complementary species • Use of chemostat • Slow, largely uncontrolled • Enzyme manipulation

48. References Abramowicz, D. 1990. Aerobic and Anaerobic Biodegradation of PCBs: A Review. Biotechnology 10(3):241-251 Abramowicz, D. 1990. Aerobic and Anaerobic Biodegradation of PCBs: A Review. Biocatalysis, Abramowicz, D.A., Eds., New York, N.Y.: Van Nostrand Reinhold Abramowicz, D. 1995. Aerobic and Anaerobic PCB Biodegradation in the Environment. Environmental Health Perspectives Supplements 103(S5), (http://ehp.niehs.nih.gov/members/1995/Suppl-5/abra-full.html) Barbalace, R. The hemistry of Polychlorinated Biphenyls, PCBs the Manmade Chemicals that Won’t Go Away. Environmentalchemistry.com <http://environmentalchemistry.com/yogi/chemistry/pcb.html> Bedard, D., J. Quensen. Microbial Reductive Dechlorination of Polychlorinated Biphenyls. Microbial Transformation and Degradation of Toxic Organic Chemicals. L. Young and C.E. Cernigalia. New York, Wiley-Liss:127-216  Brenner, V, J. Arensdorf, D. Focht. 1994. Genetic Construction of PCB Degraders. Biodegradation 5:359-377 Cutter, L., J. Watts, K. Sowers, H. May. 2001. Identification of a microorganism that links its growth to the reductive dechlorination of 2,3,5,6-chlorobiphenyl. Environmental Microbiology 3(11):699-709 ESRM 490: Lecture on Aerobic Biodegradation of Polychlorinated Biphenyls Spring 2005 Federal Remediation Technologies Roundtable. Remediation Technologies Screening Matrix and Reference Guide, Version 4.0, Section 3 Treatment Perspectives. Website. <http://www.frtr.gov/matrix2/section3/sec3_int.html#t31> Hammond, A. 1972. Chemical Pollution: Polychlorinated Biphenyls. Science New Series No 4018. 175:1155-156. http://links.jstor.org/sici?sici=0036-8075%2819720114%293%3A175%3A4018%3C155%3ACPPB%3E2.0.CO%3B2-A

49. References Cont. Harding, L. J. Phillips Jr. 1978. Polychlorinated Biphenyls: Transfer from Microparticulates to Marine Phytoplankton and the Effects on Photosynthesis. Science, New Series (4373)202:1189-1192. http://links.jstor.org/sici?sici=0036-8075%2819781215%293%3A202%3A4373%3C1189%3APBFMT%3E2.0.CO3B2-Q Mitchell, S.,R. Scadden, R. Weston. 2001. PCB Decontamination Methods fro Achieving TSCA Compliance During Facility Decommissioning Projects. Technical Paper #0102. National Defense Industrial Association 27th Environmental Symposium and Exhibition. Austin:1-12. Jacobson, S., G. Fein, J. Jacobson, P. Schwarz, J. Dowler. 1985. The Effect of Intrauterine PCB Exposure on Visual Recognition Memory. Child Development (4)56:853-860. Mosser, J., N. Fisher, T. Teng, C. Wurster. 1972. Polychlorinated Biphenyls: Toxicity to Certain Phytoplankers. Science, New Series (4018)175:191-192.http://links.jstor.org/sici?sici=0036-8075%2819720114%293%3A175%3A4018%3C191%3APBTTCP%3E2.0.CO%3B2-Y   O’Connors, Harold., C. Wurster, C. Powers, D. Biggs, R. Rowland. 1978. Polychlorinated Biphenyls May Alter Marine Trophic Pathways by Reducing Phytoplankton Size and Production. Science, New Series (4357)201:737-739.<http://links.jstor.org/sici?sici=0036-8075%2819780825%293%3A201%3A4357%3C737%3APBMT%3E2.0.CO%3B2-4> Ohtsubo, Y., M. Shimura, M. Delawary, K. Kimbara, M. Takagi, Kudo, A. Ohta, Y. Nagata. 2003. Novel Approach to the Improvement of Biphenyl and Polychlorinated Biphenyl Degradation Activity: Promoter Implantation by Homologous Recombination” Applied and Environmental Microbiology 69(1):146-153 Polychlorinated Biphenyls (PCB’s) in the Environment. University of Waterloo, Canada. http://wvlc.uwaterloo.ca./biology/molecules/intro/pcb_pjpa.htm. Qingzhong, W., J. Watts, K. Sowers, H. May. 2002. Identification of a Bacterium That Specifically Catalyzes the Reductive Chlorination of Polychlorinated biphenyls with Doubly Flanked Chlorines” Applied and Environmental Microbiology 68(2):807-812

50. References Cont. Cont. Shimizu, S., H. Kobayashi, E. Masai, M. Fukuda. Characterization of the 450kbLinear Plasmid in a Polychlorinated Biphenyl Degrader, Rhodococcus sp. Strain RHA1. Applied and Environmental Microbiology 67(5):2021-2028 Suenaga,H., T. Watanabe, M. Sato, Ngadiman, K. Furukawa. 2002. Alteration of Regiospecificity in Biphenyl Dioxygenase by Active-Site Engineering. Journal of Bacteriology 184(13):3682–3688, Sanggoo, K. and F. Picardal. 2001. Microbial Growth on Dichlorobiphenyls Chlorinated on Both Rings as a Sole Carbon and Energy Source. Applied and Environmental Microbiology 67 Tiedje, J. 2002. Principles and Prospects for Bioremediation of PCBs in Soils and Sediments. Seminar Presentation. <http://www.clu-in.org/conf/tio/pcb_100902/download.cfm?name=pcb_aprt1100402pdf.pdf&one=1> *Chemical structures drawn using the open-source Unix “XDrawChem” software* http US EPA. Polychlorinated Bipheyls (PCBs), Health effects of PCBs. Website. <http://www.epa.gov/opptintr/pcb/effects.html>://xdrawchem.sourceforge.net/ Van Briesen, J., M. Blough, W. Brown, and E. Minkley Jr. 2004. Critical Oxygen Concentrations for Biodegradation of PCB’s, Symposia Papers Presented Before the Division of Environmental Chemistry American Chemical Society, Philadelphia, PA, 1-5. Wikipedia, the World’s Open Source Encyclopedia. Polychlorinated Biphenyl. Website. <http://en.wikipedia.org/wiki/Polychlorinated_biphenyl> Wunderlich, M., R. Scrudato, and R. Chiarenzelli. 1999. Containment Facilities to Promote Containment Separation and Degradation, Proceedings of the 1999 Conference on Hazardous Waste Research. 318-321.