Biodegradation of Xenobiotic Compounds Xenobiotics

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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
  microorganismsEx. 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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Why compounds are recalcitrant?

• Failure of the compound to induce the synthesis
  of degrading enzyme.
• Failure of the compound to enter the m.o. cell
  for lack of suitable permease.
• Unavailability of the compound due to
  insolubility or adsorption.

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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.

monooxygenase
OH
dioxygenase
OH
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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 Organis
ms 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 Hydrocar
bon E. coli, P. putida, P. Aeruginosa,
Candida Surfactants Alcaligenes,
Achromobacter, Bacillus, Flavobacterium, P
seudomonas, 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-Dichlorophe
noxy 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
COOH
3H2
H2O
Metabolic pool
CH3
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
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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
O
O
H2O
NADPH
OH
C HOOC CH2 HOOC CHOH
CH2
Intermediary metabolism
OH
OH
OH
OH
OH
OH
5) Methanogenic fermentation
OH
3H2
H
OH
OH
O
H2
OH
OH
H2O
OH
H2
H
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Mechanisms of Dehalogenation
1) reductive dehalogenation two-electron
transfer reaction which involves the release of
the halogen as a halogenide ion and its
replacement by hydrogen
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.
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
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6) dehydrodehalogenation HX is eliminated from
the molecule, leading to the formation of a
double bond
Cl
Cl
Cl
Cl
Cl
HCl
Cl
Cl
Cl
Cl
Cl
Cl
7) hydratation a hydratase-catalyzed addition
of water molecule to an unsaturated bond can
yield dehalogenation of vinylic compounds
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Chlorophenols
Metabolism of monochlorophenols by Pseudomonas
sp. B13
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Proposed degradation pathway for
2,4,5-trichlorophenol in P. cepacia
Proposed degradation pathway for
pentachlorophenol in Flavobacterium sp. and
coryneform-like strain KC-3
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Organism 1
Organism 2
reductive
decarboxylation
reductive
dechlorination
dechlorination
phenol
2,4-dichlorophenol
4-chlorophenol
4-hydroxybenzoate
carboxylation
in p -position
Organism 3
ring fission
Organism 4
methanogenesis
Organism 5 6
Sequential degradation of 2,4-dichlorophenol
under anaerobic (methanogenic) conditions in a
lake sediment
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Polycyclic Aromatic Hydrocarbon (PAHs)
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Polycyclic Aromatic Hydrocarbon (PAHs)

• 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

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• 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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BTEX

• benzene degrading bacteria
• e.g. P. putida 39 /D, Moraxella
• spp. , Arthrobacter spp.and
• Aerobacter aerogenes

• toluene degrading bacteria
• e.g. P. aeruginosa , P. putida
• Pseudomonas spp.,
• Achromobacter spp. and
• Nocardia corallina

Biodegradation of benzene (Experiments with
Pseudomonas putida by Gibson and his colleagues
showed two routes to catechol from benzene)
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Biodegradation of toluene
Anaerobic pathway
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)

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Theoretical 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
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Conclusion
MMO

• TCE 1. TCE - epoxide (major
  product)
• 2. Chloral (minor product)

Toluene dioxygenase

• TCE 1. D - formate (major product)
• 2. Glyoxylate (minor product)

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e.g. heme, vitamin B12, coenzyme F430
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Bacterial transition-metal coenzyme
Vitamine B12
Coenzyme F430
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Polychlorinated biphenyls (PCBs)
Cln
Cln
General structure (there are 210 theoretically
possible PCB molecules)
Microorganism-specific nature of PCB degradation
(Unterman et al., 1988)
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Major steps in the conversion of PCBs into
chlorobenzoates (Sylvestre and Sandossi,1994)
Proposed metabolic pathway of 2,20,5-trichlorobiph
enyl via 2,3 attack (Komancova et al., 2003)
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Anaerobic degradation
- reductive dechlorination of Arochlor PCBs by
Hudson River sediment - each peak in the gas
chromatographic tracing represents a specific
PCB congener
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Potential pathway for anaerobic dechlorination of
a highly chlorinated congener (Fish and Principe,
1994)

• Both anaerobic and aerobic metabolism modes
  transform PCBs.
• Different microorganisms show preferential
  attack on PCBs
• resulting in different patterns of degradation.
  
• The degree of chlorination of the congener is a
  major factor,
• which influences degradation potential of the
  compound.

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Pesticides

• 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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Proposed pathways for the microbial degradation
of Carbaryl (carbamates) e.g. Pseudomonas
spp., Rhodococcus sp., Bacillus sp.,
Micrococcus sp.
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Proposed pathways for the microbial degradation
of parathions (organophosphates) e.g.
Pseudomonas sp., Flavobacterium sp., Bacillus
strains., Arthrobacter strains
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Lindane (gamma-hexachlorocyclohexane, g-HCH)
Microorganisms
Sphingomonas paucimobilis UT 26
Anabeana sp. PCC7120
Nostoc ellipsosporum
Enzymes
g-HCH dehydrochlorinase (Lin A)
1,4-TCDN halidohydrolase (Lin B)
2,5-DDOL dehydrogenase (Lin C)
2,5-DCHQ reductive dehalogenase (Lin D)
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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
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The assay systems for biodegradability test

• Measurement of oxygen consumption by manometric
  and electrolytic system
• 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)