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PCBs and Bioremediation. Pamela Windy Ravi. Overview. What are PCBs? Why are they a problem? What can we do with them? How do the microbial methods work?. PCBs. Synthesized chemicals from petro-chemical industry used as lubricants and insulators in heavy industry Used because

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  • What are PCBs?
  • Why are they a problem?
  • What can we do with them?
  • How do the microbial methods work?
  • 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
Hydraulic fluid

Casting wax

Carbonless carbon paper


Heat transfer systems




Liquid cooled electric motors

Fluorescent light ballasts

  • 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
where found
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
  • 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)
interesting facts
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
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

food chain effects
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
human health

Liver cancer


immune system

studies done on rhesus monkeys which have similar systems

effects noted in people exposed to PCB contaminated rice oil


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
human health cont
nervous system

infant neurological functions


short-term memory


effects seen at levels present in breast milk

endocrine system

thyroid health

other health effects

Human Health Cont.
marine and animal health
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
  • Extraction or separation of contaminants from environmental media
  • Immobilization of contaminants
  • Destruction or alteration of contaminants
    • Chemical
    • Thermal
    • Biological
  • 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
pathways of pcb degradation
Pathways of PCB Degradation
  • Anaerobic/Aerobic removal
  • Photochemical Degradation
  • Thermal Degradation
  • Fungal Degradation
methods for pcb removal
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
microbial methods
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
pathways of aerobic degradation
Pathways of Aerobic Degradation



Bugs attack benezoate

pathways of aerobic degradation cont
Pathways of Aerobic Degradation cont.


Most bugs degrade this


pathways of aerobic degradation cont23
Pathways of Aerobic Degradation cont.

Harder to degrade than cholorbenzoate

degradation of pcb products
Degradation of PCB Products

Gives rise to chlorobenzoate

Dioxygenase sttack on 2,3 catechol

take home message of aerobic degradation
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.
photochemical degradation
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.
photochemical degradation cont
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.
thermal degradation
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.
thermal degradation cont
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).
fungal degradation
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.
anaerobic reductive dechlorination of pcbs
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

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


anaerobic dechlorination is complementary to aerobic degradation
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
A reduction pathway, with Cl as the terminal electron acceptor
  • At least one species (o-17) likely uses acetate as the electron donor
anaerobic congener selectivity
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
aerobic pcb degraders
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.
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
anaerobic pcb degraders
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
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 +)
genetic construction of pcb degrading bacteria
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
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
    • 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)
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

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),


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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

references cont
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

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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

references cont cont
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

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