Biodegradation of Xenobiotic compound, PAH, PCB, Nitrobenzene & DDT

Gunjan Mehta,

VSC,

Rajkot

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Biodegradation of Xenobiotic Compounds

• Xenobiotics : compound have been

produced artificially by chemical synthesis for

industrial or agricultural purposes e.g.

halogenated H.C., aromatics, pesticides, PCB,

PAH, lignin, humic substances

• Recalcitrant : compound totally resistant to

biodegradation e.g. unusual substitute (Cl- or

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H-), unusual bond sequences (3΄ & 4΄), highly

condensed aromatic rings, and excessive

molecular size (polyethylene)

• Co-metabolism : an organic compound is

converted to metabolic products but does

not serve as a source of energy or nutrients

to microorganisms

Ex. insecticides, aliphatic & aromatic H.C.

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Source of xenobiotic compounds

1. Petrochemical industry : oil/gas industry, refineries,

and the production of basic chemicals e.g. vinyl

chloride and benzene

2. Plastic industry :

- closely related to the petrochemical industry

- uses a number of complex organic compounds

such as anti-oxidants, plasticizers, cross-linking

agents

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3. Pesticide industry : most commonly found central

structures are benzene and benzene derivatives,

often chlorinated and often heterocyclic

4. Paint industry : major ingredient are solvents,

xylene, toluene, methyl ethyl ketone, methyl

isobutyl ketone and preservatives

5. Others : Electronic industry, Textile industry, Pulp

and Paper industry, Cosmetics and Pharmaceutical

industry, Wood preservation

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1. Failure of the compound to induce the

synthesis of degrading enzyme.

2. Failure of the compound to enter the m.o. cell

for lack of suitable permease.

3. Unavailability of the compound due to

insolubility or adsorption.

Why compounds are recalcitrant?

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4. Excessive toxicity of the parent compound

or its metabolic products.

5. Unavailability of the proper electron acceptor.

6. Unfavorable environmental factors e.g. temp.,

light, pH, O2 , moisture.

7. Unavailability of the other nutrients (N, P)

and growth factors.

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Factors effect the xenobiotic biodegradation by m.o.

1. Substrate specificity: such as for the type of

aromatic cpd., for the ring position (o-, m-, or p- ),

and for the atom or group removed. Specificities

could reside at the level of enzymes, organisms, or

broad physiological groups

2. Electron acceptors : oxygen, nitrate, and sulfate

most often inhibit dehalogenation by anaerobic

communities

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3. Other nutrients : addition of various nutrients as

electron donors, C-source, N-source, P-source, or

micronutrients can stimulate the reaction or support

the growth of the microorganisms

4. Temperature : affected both the acclimation period

and the rate of biodegradation activity

5. Substrate availability : the hydrophobicity of many

xenobiotic cpd. affects their biodegradation through

its effect on their availability to microorganisms

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Biodegradation of Petroleum compounds

Petroleum compounds are categorized into 2 groups Aliphatic hydrocarbon e.g. alkane, alcohol,

aldehyde Aromatic hydrocarbon e.g. benzene, phenol,

toluene, catechol

H.C. (substrate) + O2 H.C.-OH + H2O

H.C. (substrate) + O2 H.C.OH

OH

monooxygenase

dioxygenase

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Straight chain

aliphatic H.C.

compounds

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Cyclic aliphatic

H.C. compounds

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Aerobic degradation

of aromatic compounds

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- are metabolized by a variety of bacteria, with ring fission

accomplished by mono- and dioxygenases

- catechol and protocatechuate are the intermediates

mostly found in aromatic cpd. degradation pathway

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Some m.o. involved in the biodegradation of xenobiotics

Organic Pollutants Organisms

Phenolic - Achromobacter, Alcaligenes,

compound Acinetobacter, Arthrobacter,

Azotobacter, Flavobacterium,

Pseudomonas putida

- Candida tropicalis

Trichosporon cutaneoum

- Aspergillus, Penicillium

Benzoate & related Arthrobacter, Bacillus spp.,

compound Micrococcus, P. putida

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Organic Pollutants Organisms

Hydrocarbon E. coli, P. putida, P. Aeruginosa, Candida

Surfactants Alcaligenes, Achromobacter,

Bacillus, Flavobacterium,

Pseudomonas, Candida

Pesticides P. Aeruginosa DDT

B. sphaericus Linurin

2,4-D Arthrobacter, P. cepacia

P. cepacia 2,4,5-T

Parathion Pseudomonas spp., E. coli,

P. aeruginosa

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Genetic Regulation of Xenobiotic Degradation

plasmid-borne

mostly in the genus Pseudomonas

PLASMID SUBSTRATE

TOL Toluene, m-xylene, p-xylene

CAM Camphor

OCT Octane, hexane, decane

NAH Napthalene

pJP1 2,4-Dichlorophenoxy acetic acid

pAC25 3-Chlorobenzoate

SAL Salicylate

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Anaerobic degradation

1) Photometabolism : in bacteria this light-induced

“bound oxygen” (OH•) was used to oxidized substrates

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2) under nitrate-reducing condition : Nitrate-reducing bacteria couple the oxidation of org. cpd. with water to the exergonic reduction of nitrate via nitrite to N2

OH OH O

Metabolicpool

3H2 H2O

H2

3) dissimilation through sulfate respiration: Sulfate- reducing bacteria couple the oxidation of org. cpd. with water to the exergonic reduction of sulfate via

sulfite to sulfide

CH3

COOH

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4) The anaerobic fermentation of many polyphenolic substances : m.o. derive their energy from substrate-level phosphorylation while org. cpd. serve as e-donors and acceptors.OH O

Intermediarymetabolism

NADPH H2O

5) Methanogenic fermentation :

OH OH

O

OH OH OH OH

OH C

HOOC CH2

HOOC CHOH

CH2

O

OH

OH

H

H

OH 3H2

OHOH

H2

O

OH O

H2

H2

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1) reductive dehalogenation : two-electron transfer reaction which involves the release of the halogen as a halogenide ion and its replacement by hydrogen

Cl Cl H Cl

C C + XH- C C + Cl - + X

Cl Cl Cl Cl

Mechanisms of Dehalogenation

COOHCl

+ O2 + NADPH + H+

OHOH

+ NAD+ + CO2 + HCl

2) oxygenolytic dehalogenation : catalyzed by mono- or dioxygenases, which incorporate atom of molecular oxygen into the substrate

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3) hydrolytic dehalogenation : catalyzed by halido-

hydrolases, the halogen is replaced by a OH group

which is derived from water.

H2O Cl-

COOH Cl COOH OH

4) thiolytic dehalogenation : in dichloromethane-utilizing

bacteria, a dehalogenating glutathione S-transferase

catalyzes the formation of a S-chloromethyl glutathione

conjugate, with a concomitant declorination taking place.

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5) intramolecular substitution: involved in the

dehalogenation of vicinal haloalcohols

CH2OH-CHOH-CH2Cl CH2OH-CH-CH2 + HCl

O

CH2Cl2 + GSH [GS-CH2Cl] + HCl

[GS-CH2Cl] + H2O GS-CH2OH + HCl

GS-CH2OH CH2O + GSH

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7) hydratation : a hydratase-catalyzed addition of water

molecule to an unsaturated bond can yield dehalogenation

of vinylic compounds

HOOC-CH=CHCl + H2O [ HOOC-CH2-CHOHCl]

HOOC-CH2-CHO + HCl

Cl

Cl

Cl

Cl

ClCl

Cl

Cl

Cl

Cl

Cl

+ HCl

6) dehydrodehalogenation : HX is eliminated from the

molecule, leading to the formation of a double bond

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Metabolism of monochlorophenols by Pseudomonas sp. B13

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Cl

Cl

Cl

OH

Cl

Cl

Cl

Cl

OH

OH

Cl

Cl

Cl

OH

OH

Cl

Cl

Cl

OH

OH

Cl

HCl HCl HCl

Proposed degradation pathway for pentachlorophenol in Flavobacterium sp. and coryneform-like strain KC-3

Proposed degradation pathway for 2,4,5-trichlorophenol in P. cepacia

Cl

Cl

Cl

OH

Cl

Cl

OH

OH

OH

Cl

OH

OH

HCl HCl

hydroquinone

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2,4-dichlorophenol

reductive

dechlorination

4-chlorophenolphenol

reductive

dechlorination

carboxylation in p -position4-hydroxybenzoate

Cl

Cl

OH

Cl

OH OH

COOH

benzoate

3 Acetate + CO2 + 3 H2

CH4 + CO2

COOH

OH

ring fission

methanogenesis

decarboxylation

Sequential degradation of 2,4-dichlorophenol under anaerobic (methanogenic)conditions in a lake sediment

Organism 1 Organism 2

Organism 3

Organism 4

Organism 5 & 6

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• Bacteria, fungi, yeasts, and algae have the ability to metabolize both lower and higher M.W. PAHs found in the natural environment most bacteria have been found to oxygenate the PAH initially to form dihydrodiol with a cis-configuration, which can be further oxidized to catechols.

• Most fungi oxidize PAHs via a cytochrome P450 catalyzed mono- oxygenase reaction to form reactive arene oxides that can isomerize to phenols

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• White-rot fungi oxidize PAHs via ligninases (lignin peroxidases and laccase) to form highly reactive quinones.

• little is known about the potential of PAHs for anaerobic metabolism

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Compound Organisms Metabolite

Naphthalene Acinetobacter calcoaceticus , Alcaligenes denitrificans,Mycobacterium sp. , Pseudomonas sp., Pseudomonas putida , Pseudomonas fluorescens , Pseudomonas paucimobilis , Pseudomonas vesicularis , Pseudomonas cepacia , Pseudomonas testosteroni , Rhodococcus sp. , Corynebacterium renale , Moraxella sp., Bacillus cereus , Streptomyces sp.

Naphthalene cis -1,2 – dihydrodiol,1,2 – dihydroxynaphthalene, 2 - hydroxychromene - 2 – carboxylic acid, trans – o – hydroxybenzylidene pyruvic acid, salicylaldehyde, salicylic acid, catechol, gentisic acid, naphthalene trans – 1,2 – dihydrodiol .

Acenaphthene Beijerinckia sp., Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas cepacia

1- Acenaphthenol, 1- acenaphthenone, acenaphthene – cis – 1,2 – dihydrodiol,1,2 – acenaphthenedione,1,2 – dihydroxyacenaphthylene,7,8 – diketonaphthyl – l – acetic acid,1,8 – naphthalenedicarboxylic acid, 3 – hydroxyphthalic acid .

Bacterial strain degrading PAHs

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Compound Organisms Metabolite

Anthracene Beijerinckia sp., Mycobacterium sp., Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas paucimobilis, Pseudomonas cepacia, Rhodococcus sp., Flavobacterium sp, Arthrobacter sp.

Anthracene cis – 1,2 – dihydrodiol,1,2 – dihydroxyanthracene, cis – 4 – ( 2 – hydroxynaphth – 3 – yl ) – 2 – oxobut – 3 – enoic acid, 2 – hydroxy – 3 – naphthaldehyde, 2 - hydroxy – 3 – naphthoic acid, catechol, 2,3 –dihydroxynaphthalene, salicylic acid

Phenanthrene Aeromonas sp., Alcaligenes denitrificans , Arthrobacter polychromogenes , Beijerinckia sp. , Mycobacterium sp. , Micrococcus sp., Vibrio sp., Pseudomonas putida, Pseudomonas paucimobilis, Rhodococcus sp., Nocardia sp., Flavobacterium sp., Streptomyces griseus, Acinetobacter sp.

3,4 – dihydroxyphenanthrene,cis – 4 – ( 1- hydroxynaphth – 2 – yl ) – 2- oxobut – 3 – enoic acid,1 – hydroxy – 2 – naphthaldehyde, 1 – hydroxy – 2 – naphthoic acid,1,2 – dihydroxynaphthalene, o – phthalic acid , 2 – carboxybenz- aldehyde, protocatechuic acid .

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Compound Organisms Metabolite

Fluoranthene Alcaligenes denitrificans , Mycobacterium sp. , Pseudomonas putida , Pseudomonas paucimobilis, Pseudomonas cepacia , Rhodococcus sp.

7- Acenaphthenone, 1- acenaphthenone, 7- hydroxyacenaphthylene, benzoic acid, phenylacetic acid, adipic acid, 3- hydroxymethyl – 4,5- benzocoumarin,9- fluorenone – 1 – carboxylic acid,8- hydroxy – 7- methoxyfluoranthene,9- hydroxyfluorene , 9- fluorenone,phthalic acid, 2- carboxybenzaldehyde

Pyrene Alcaligenes denitrificans , Mycobacterium sp. , Rhodococcus sp.

Pyrene cis - and trans - 4,5 – dihydrodiol,4 – hydroxyperinaphthenone, phthalic acid, 4- phenanthroic acid, 1,2 - and 4,5 – dihydroxypyrene, cinnamic acid, cis – 2 –hydroxy – 3 – ( perinaphthenone -9-yl ) propenic acid

Chrysene Rhodococcus sp. None determined

Benz [a] anthracene

Alcaligenes denitrificans , Beijerinckia sp. , Pseudomonas putida

Benz [a] anthracene cis – 1,2, cis- 8,9-, and cis – 10,11- dihydrodiols, 1- hydroxy – 2 – anthranoic acid, 2- hydroxy – 3 – phenanthroic acid, 3- hydroxy – 2 – phenanthroic acid .

Benz [a] pyrene

Beijerinckia sp., Mycobacterium sp.

Benz [a] pyrene cis -7,8 - and cis -9,10 – dihydrodiols .

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Biodegradation of toluene

Anaerobicpathway

Aerobic pathway

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p-Xylene

The metabolic enzymes in this pathway have been shown to have similar specificty for toluene, para-xylene, and meta-xylene

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Biodegradation of ethyl benzene

• Under aerobic conditions

ethylbenzene degradation involves

oxygenase reactions that can

proceed in either of two primary

pathways

• A Pseudomonas sp. (strain NCIB

10643) has been shown to utilize a

wide range of n-alkylbenzenes

(C2-C7), of which ethylbenzene is

a single example.

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Bacterial co-metabolism of halogenated organic compounds

• TCE (trichloroethylene), widely used industrial solvent,

is unreactive halocarbon compound. • Anaerobic bacteria can reductively dehalogenate

TCE to form vinylchloride (strongly carcinogenic).• No one has succeeded in obtaining a TCE-degrading

bacteria able to use TCE as sole carbon and energy

source, that driven the studies on the co-metabolism

of TCE

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• An oxygenase

• monooxygenase might yield TCE- epoxide

• dioxygenase yield 1,2 – dihydroxy TCE

e.g. Toluene dioxygenase from

Pseudomonas putida F1

• Methane monooxygenase (MMO) from

Methanotrophs (Methylococcus, Methylosinus)

40Theoretical pathways of TCE oxidation by monooxygenases and dioxygenases

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TCE oxidation by soluble methane monooxygenase (MMO) oxidizes methane to methanol in the first step of C1 oxidative metabolism by methanotrophs

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Oxygenases and organisms implicated in TCE oxidation Organism enzyme

Methylosinus trichosporium OB 3 b

Pseudomonas cepacia G 4

P. mendocina

P. putida

Nitrosomonas europaea

Methylocystis parvus OBBP

Mycobacterium sp.

Alcaligenes eutrophus JMP 134

Rhodococcus erythropolis

soluble methane monooxygenase

Toluene 2- monooxygenase

Toluene 4- monooxygenase

Toluene dioxygenase

Ammonia monooxygenase

Particulate methane monooxygenase

Propane monooxygenasePhenol hydroxylase

Isoprene oxygenase

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• TCE 1. TCE - epoxide (major product)

2. Chloral (minor product)

• TCE 1. D - formate (major product)

2. Glyoxylate (minor product)

MMO

Conclusion

Toluene dioxygenase

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e.g. heme, vitamin B12, coenzyme F430

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Bacterial transition-metal coenzyme

Coenzyme F430

Vitamine B12

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Cl

n

Cln

General structure(there are 210 theoretically possible PCB molecules)

Microorganism-specific nature of PCB degradation (Unterman et al., 1988)

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 Low reactivity Non-flammable High electrical resistance Stable when exposed to heat and pressure

Uses

Hydraulic fluid Casting wax Carbonless carbon

paper Compressors Heat transfer systems

Plasticizers Pigments Adhesives Liquid cooled electric

motors Fluorescent light

ballasts

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

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

Basic Aromatic Carbon structure

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

Risks

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 EffectsBioaccumulation

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 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 Cont.

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

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

Mitigation     Extraction or separation of contaminants from

environmental media

Immobilization of contaminants

Destruction or alteration of contaminants Chemical Thermal Biological

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

Pathways of PCB Degradation

Anaerobic/Aerobic removal Photochemical Degradation Thermal Degradation Fungal Degradation

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

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

Ortho, Meta and Para Chlorination of Biphenyl

Pathways of Aerobic Degradation

dihydroxybiphenyl

Oxygenase attack

Bugs attack benezoate

Pathways of Aerobic Degradation cont.

metaclevage

Most bugs degrade this

M

Degradation of PCB Products

Dioxygenase attack on

2,3 catechol

Degradation of PCB Products cont.

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.

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

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

(Abramowicz,1995)

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

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

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.

(Abramowicz, 1990)

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

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

(Cutter et. al. 2001)

Biodegradation of Nitrobenzene

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Nitrobenzene degradation pathways.

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• in about 1868 a chemical compound used as a commercial pesticide was Paris green [Copper acetate

meta-arsenate, Cu(CH3COO)2 • 3Cu(AsO2)2]• DDT (dichloro-diphenyl-trichloroethane) is the first of a number of chlorinated H.C. to be developed as pesticides in 1939• Pesticides can be classified in a number of different ways for example; by their chemical nature ( natural organic cpd., inorganic cpd., chlorinated hydrocarbon, organophosphates, carbamates, and others)

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Proposed pathways for the microbial degradation of DDT

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Summary of Biodegradation of Pesticides 

There are many mechanisms involved on the biodegradation of pesticides and other contaminants. These may be summarised as follows: CATEGORY/REACTION EXAMPLE

Dehalogenation

RCH2C1 Õ RCH2OH Propachlor (C,S)

ArC1 Õ ArCH Nitrofen (S)

ArF Õ ArCHFlam-prop methyl

(S,C)

ARC1 Õ ArH Pentachlorophenol

(S,C)

Ar2CHCH2C1 Õ Ar2C=CH2 DDT (C)

ArCHCHC12 Õ Ar2C=CHC1 DDT (C,S,W)

Ar2CHCC13 Õ Ar2CHCHC12 DDT (C,S)

Ar2CHCC13 Õ Ar2C=CC12 DDT (S,C)

RCC13 Õ RCOOH N-Serve (S), DDT (C)

HetC1 Õ HetOH Cyanazine (S)

CATEGORY/REACTION EXAMPLE

Deamination

ArNH2 Õ ArOH  Fluchloralin (S)

Decarboxylation

ArCOOH Õ Ar4 Biofenox (S)

ArCHCOOH Õ Ar2CH DDT (C)

RCH(CH3)COOH Õ RCH2CH2  Dichlorfop-methyl (S)

ArN(R)COOH Õ ArN(R)H DDD(S)

Methyl oxidation

RCH3 Õ RCH2OH Õ RCHO Õ RCOOH

Bromacil (S)

Hydroxylation and ketone formation

ArH Õ ArOH  Benthiocarb (S)

R(R’)CHR" Õ, (R’)CHOH(R") Bux insecticide (S)

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1. Measurement of oxygen consumption by manometric and electrolytic system

2. Measurement of CO2 evolution by infrared or

chemical methods

3. Use of radio-labeled substrates

4. Measurement of the disappearance of the chemicals

by GC

5. Determination of the reduction of DOC

6. Chemical biodegradability under anaerobic conditions

(measuring gas production, CH4+ CO2)

The assay systems for biodegradability test