BIODEGRADATION OF

XENOBIOTICS

HYDROCARBONS, PLASTICS &

PESTICIDES

DONE BY

SUSHMITA PRADHAN

II SEM M.Sc Microbiology

XENOBIOTICS

• It is derived from a greek word “XENOS” meaning ‘foreign or strange’.

• Xenobiotics are those chemicals which are man-made and do not

occur naturally in nature.

• They are usually synthesized for industrial or agricultural purposes e.g.

aromatics, pesticides, hydrocarbons, plastics , lignin etc.

• They are also called RECALCITRANTS as they can resist degradation

to maximum level.

BIODEGRADATION

• According to the definition by the International Union of Pure

and Applied Chemistry, the term biodegradation is “Breakdown

of a substance catalyzed by enzymes in vitro or in vivo.

• In other words, defined as the ability of microorganisms to

convert toxic chemicals (xenobiotics) to simpler non-toxic

compounds by synthesis of certain enzymes

• Biodegradation of xenobiotics can be affected by substrate

specificity, nutrition source, temperature, pH etc.

SOURCES OF XENOBIOTICS

1. Petrochemical industry :

-oil/gas industry, refineries.

- produces 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

3. Pesticide industry :

- most commonly found.

-structures are benzene and benzene derivatives,

4. Paint industry :

- major ingredient are solvents,

- xylene, toluene, methyl ethyl ketone, methyl

5. Others :

- Electronic industry, Textile industry, Pulp and Paper industry,

Cosmetics and Pharmaceutical industry, Wood preservation

BIODEGRADATION OF PESTICIDES

• Pesticides are substances meant for destroying or mitigating any pest.

• They are a class of biocide.

• The most common use of pesticides is as plant protection products

(also known as crop protection products).

• It includes: herbicide, insecticide, nematicide, termiticide,

molluscicide, piscicide, avicide, rodenticide, insect repellent, animal

repellent, antimicrobial, fungicide, disinfectant, and sanitizer.

DIFFERENT METHODS - 4

a) Detoxification:

Conversion of the pesticide molecule to a non-toxic compound.

A single moiety in the side chain of a complex molecule is

disturbed(removed), rendering the chemical non-toxic.

b) Degradation:

Breakdown or transformation of a complex substrate into

simpler products leading to mineralization.

E.g. Thirum (fungicide) is degraded by a strain of

Pseudomonas and the degradation products are dimethylamine,

proteins, sulpholipids, etc (Raghu et al., 1975).

c) Conjugation (complex formation or addition reaction):

An organism makes the substrate more complex or combines the pesticide

with cell metabolites.

Conjugation or the formation of addition product is accomplished by those

organisms catalyzing the reaction of addition of an amino acid, organic

acid or methyl crown to the substrate thereby inactivating the pestcides

d) Changing the spectrum of toxicity:

Some pesticides are designed to control one particular group of pests, but

are metabolized to yield products inhibitory to entirely dissimilar groups of

organisms, for e.g. the fungicide PCNB is converted in soil to chlorinated

benzoic acids that kill plants.

There are many mechanisms involved on the biodegradation of pesticides and

other contaminants. These may be summarised as follows:

Dehalogenation- nitrofen, DDT, cyanazine, propachlor.

Deamination- fluchloralin

Decarboxylation- DDTc, biofenox, dichlorop-methyl

Methyl oxidation- bromacil

Hydroxylation- benthiocarb, bux insecticide

BIODEGRADTION OF PLASTICS

• Plastic is a broad name given to different polymers with high molecular

weight, which can be degraded by various processes.

• The biodegradation of plastics by microorganisms and enzymes seems to

be the most effective process.

• It consist of two steps- fragmentation and mineralization. But at the core,

reaction occurring at molecular level are oxidation and hydrolysis.

• The decomposition of major condensation polymers (e.g. polyesters and

polyamides) takes place through hydrolysis, while decomposition of

polymers in which the main chain contains only carbon atoms (e.g.

polyvinyl alcohol, lignin) includes oxidation which can be followed by

hydrolysis of the products of oxidation.

METHOD

HYDROLYSIS-

The process of breaking these chains and dissolving the polymers into smaller

fragments is called hydrolysis. E.g. Pseudomonas sps

Polymeric Chains is broken down into constituent parts for the energy potential

by microorganisms. Monomers are readily available to other bacteria and is used.

Acetate and hydrogen produced is used directly by methanogens. Other molecules,

such as volatile fatty acids (VFAs) with a chain length greater than that of acetate

is first catabolized into compounds that can be directly used by methanogens.

ACIDOGENESIS-

This results in further breakdown of the remaining components by acidogenic

(fermentative) bacteria into ammonia, ethanol, carbon dioxide, and hydrogen

sulfide. E.g Streptococcus acidophilus.

ACETOGENESIS-

Simple molecules created through the acidogenesis phase are further

digested by Acetogens to produce largely acetic acid, as well as carbon

dioxide and hydrogen.

METHANOGENESIS-

Here, methanogens use the intermediate products of the preceding stages

and convert them into methane, carbon dioxide, and water.

These components make up the majority of the biogas emitted.

Methanogenesis is sensitive to both high and low pHs and occurs between

pH 6.5 and pH 8. The remaining, indigestible material the microbes cannot

use and any dead bacterial remains constitute the digestate.

Some of the microorganism that can degrade plastics are:-

Aliphatic Polyesters

PolyEthylene Adipate (PEA)- lipases from R. arrizus, R. delemar, Achromobacter sp.

and Candida cylindracea

Poly (β-Propiolactone) PPL - estereases from Acidovorax sp., Variovorax

paradoxus, Sphingomonas paucimobilis.

Aromatic Polyesters

Poly-3-Hydroxybutyrate (PHB) – estereases from Pseudomonas

lemoigne, Comamonas sp. Acidovorax faecalis, Aspergillus fumigatus

Poly Lactic Acid (PLA) - proteinase K from Tritirachium album, Amycolatopsis sp

Strains of Actinimycetes has been reported to degrade polyamide (nylon),

polystyrene, polyethylene.

BIODEGRADATION OF

HYDROCARBONS

• A hydrocarbon is an organic compound consisting entirely of hydrogen

and carbon.

• The majority of hydrocarbons found on earth naturally occur in crude

oil.

• Aromatic hydrocarbons (arenes), alkanes,

alkenes, cycloalkanes and alkyne-based compounds are different types

of hydrocarbons.

BIODEGRADATION OF PETROLEUM

Petroleum compounds are categorized into 2 groups

1. Aliphatic hydrocarbon e.g. alkane, alcohol, aldehyde

2. Aromatic hydrocarbon e.g. benzene, phenol, toluene, catechol

Aromatic hydrocarbons are degraded aerobically and anaerobically.

AEROBIC DEGRADATION

• 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 compound degradation pathway.

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OTHER MECHANISMS

1) Photometabolism : in bacteria, this light-induced

“bound oxygen” (OH•

) is used to oxidize substrates

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2) under nitrate-reducing condition : Nitrate-reducing bacteria

couple the oxidation of organic compound with water to the

exergonic reduction of nitrate via nitrite to N2..

3) dissimilation through sulfate respiration: Sulfate- reducing

bacteria couple the oxidation of organic compound with

water to the exergonic reduction of sulfate via sulfite to

sulfide.

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Some microorganisms involved in the biodegradation of

hydrocarbons

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. sphaericu Linurin

Arthrobacter, P. cepacia 2,4-D

P. cepacia 2,4,5-T , Parathion

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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

POLYCYCLIC AROMATIC HYDROCARBONS

(PAH)• Bacteria, fungi, yeasts, and algae have the ability to metabolize both lower

and higher molecular weight 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.

• White-rot fungi oxidize PAHs via ligninases (lignin peroxidases and

laccase) to form highly reactive quinones.

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Compound Organisms Metabolite

Naphthalene Acinetobacter calcoaceticus ,

Alcaligenes denitrificans,

Mycobacterium sp. , Pseudomonas sp.,

Pseudomonas putida ,

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

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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 .

POLYCHLORINATED BIPHENYLS (PCBs)

• Synthesized chemicals from petro-chemical industry used as lubricants

and insulators in heavy industry.

• First manufactured in 1929 by Monsanto.

• Manufacture and unauthorized use banned in 1978 by USEPA

• Used because-

• Low reactivity

• Non-flammable

• High electrical resistance

• Stable when exposed to heat and pressure

• Used as Hydraulic fluid, Casting wax, Carbonless carbon paper,

Compressors, Heat transfer systems, Plasticizers, Pigments, Adhesives,

Liquid cooled electric motors, Fluorescent light.

RISKS-

Causes reproductive disabilities in animals, human, birds.

Carcinogenic

Bioaccumulation

Soluble in almost all the solvents, fats, oils

Nervous system damage

Endocrine gland malfunction

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.

PATHWAYS FOR PCB REMOVAL

FUNGAL DEGRADATION –

• Aspergillus niger: fillamentous with cytochrome p450 that attacks

lower chlorinated PCB’s

• Phanerochaete chrysosporium: White rot fungi can attack lignin (PCB)

at low concentration with the help og ligninases.

BACTERIAL DEGRADATION-

• Soil bacteria breaks down PCBs via dioxygenase pathways.

• Most identified seem to be Pseudomonas species, Achromobacter,

Acinetobacter, Alcaligenes, Arthrobacter, Corynebacterium,

Rhodococcus, Burkholderia .

REFERENCE

BOOKS-

Sullia S.B and Shantharam S.; General microbiology, Second

edition

Page No. 348-350

Dubey R. C and Maheshwari D.K; A textbook of microbiology,

second revised edition 2009; Page No. 832-836

ARTICLES

Biodegradation of polymers- Dr Rolf Joachim Miller

Biodegradation of pesticides- Andre Luiz Porto, Marcia Nitcshke

and Gleiseda Melgar

Microbial Degradation of Petroleum Hydrocarbon -Nilanjana Das

and Preethy Chandran