Xinobiotics: Threatening Pollutants and Their Biodegradation

amit.pratush@sjtu.edu.cn/ amit.pratush@gmail.
com
10/14/16
What is Waste?
What is degradation?
What is Biodegradation?
Breakdown of a substance catalyzed by enzymes in vitro or in vivo
OR
Ability of microorganisms to convert toxic chemicals (xenobiotics) to simpler
non-toxic compounds by synthesis of certain enzymes
What is the need of Biodegradation?

amit.pratush@sjtu.edu.cn/ amit.pratush@gmail.com
10/14/16
Biodegradation is the initial process that results to bioremediation .

in general it is bio mediated decomposition of paper, paint, textiles, hydrocarbons and other pollutants.
Superior technique over using chemicals why?
1.
Microorganisms easy to handle.
2.
Plants easy to grow.
Enzymatic processes in bioremediation

Major types of reactions
Oxidation, Reduction, demethylation, deamination, conjugation;
Decarboxylation in which the CO2 is replaced with an H atom or OH group.
Hydrolysis which involves the addition of H2O to a molecule accompanied by cleavage of the molecule
into two species.
Substitution in which one group of atom is replaced by another (such as OH for Cl - ).
Elimination whereby atoms or group of atoms are removed from adjacent carbon atoms, which remained
joined by a double bond.
condensation, in which two smaller molecules are joined to produce a larger one
Isomer formation conversion of one isomer of a compound to another with a same molecular formula but
different structure
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Biodegradation has at least 3 outcomes: Example of 2,6-Dichlorobenzonitrile can explain all steps
1.
A minor change in an organic

molecule leaving the main
structure intact.
2.
Fragmentation of a complex
organic structure in such a way
that the fragments could be
reassembled to yield the original
structure.
3.
Complete mineralization, which in

the transformation of organic
molecules to mineral forms.
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Use of various types of microorganisms for biodegradation

Aerobic bacteria:
Examples include: Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and Mycobacterium.
Shown to degrade pesticides and hydrocarbons; alkanes and polyaromatics.
May be able to use the contaminant as sole source of carbon and energy.
Methanotrophs:
Aerobic bacteria that utilize methane for carbon and energy.
The enzyme Methane monooxygenase has a broad substrate range and it is active against a wide
range of compounds (e.g. chlorinated aliphatic such as trichloroethylene and 1,2-dichloroethane)
Anaerobic bacteria:
Not used as frequently as aerobic bacteria.
Can often be applied to bioremediation of polychlorinated biphenyls (PCBs) in river sediments,
trichloroethylene (TCE) and chloroform.
Fungi:
Able to degrade a diverse range of persistent or toxic environmental pollutants.
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How Microbes Use the Contaminant

Primary substrate
Enough available to be the sole energy source.
Secondary substrate
Provides energy,
concentration.
Co -metabolic substrate
Utilization of a compound by a microbe relying on some

other primary substrate.
pH conditions for microorganisms
not
available
in
high
enough
Temp. conditions for microorganisms
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What are the XENOBIOTICS?

derived from a Greek word XENOS meaning foreign or strange.
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.
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SOURCES OF XENOBIOTICS
S.
No.
Types of Industries
Major waste
A.
Petrochemical industry
oil/gas industry, refineries.

- Produces basic chemicals e.g. vinyl chloride and benzene.
B.
Plastic industry
closely related to the petrochemical industry

- uses a number of complex organic compounds: such as anti-oxidants,
plasticizers, cross-linking agents etc.
C.
Paint industry
major ingredient are solvents,

- xylene, toluene, methyl ethyl ketone,
D.
Pesticide industry
most commonly found.

-are benzene and benzene derivatives
E.
Other Industries
Electronic industry, Textile industry, Pulp and Paper industry, Cosmetics

and Pharmaceutical industry, Wood preservation etc.
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A. Biodegradation of Petrochemical industry waste

Major waste is oil: which is mixture of Hydrocarbons &
Organic compounds.
9
Effect
Effect on
on water
water bodies
bodies after11
after11 million
million gallon
gallon oil
oil
spill
spill from
from the
the supertanker
supertanker EXXON
EXXON VALDEZVALDEZALASKA
ALASKA in
in March-1989
March-1989
Main principle of aerobic degradation of

Hydrocarbon
hydrocarbons by microorganisms
degradation rates in different environm
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10
Aromatic hydrocarbon Biodegradation

1.
2.
Polycyclic aromatic hydrocarbon (PAH)

Polychlorinated biphenyl's (PCB)
Adverse effects of PCB & PAH pollution on human health

Causes reproductive disabilities in animals, human, birds.
Carcinogenic
Nervous system damage
Endocrine gland malfunction
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Microorganism involved in biodegradation of

hydrocarbons
Organic
Pollutants
Organisms
Phenolic compound
Achromobacter,Alcaligenes,Acinetobacter,
Arthrobacter, Azotobacter, Aspergillus, Flavobacterium,
Pseudomonas, Penicillium putida,Candida tropicalis,
Trichosporon cutaneoum
Benzoate related
compound
Factors affecting Hydrocarbon

Biodegradation
Arthrobacter, Bacillus sp.,
Hydrocarbon
E. coli, P. putida, P. Aeruginosa, Candida
Surfactants
Alcaligenes, Achromobacter, Bacillus, Flavobacterium,

Pseudomonas, Candida
Biostimulation
PCB
Bioremediations
Natural
Attenuation
Bioaugmentation
amit.pratush@sjtu.edu.cn/
Attenuation amit.pratush@gmail.com
10/14/16
12
12
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13
B. Biodegradation of Plastic industry waste
What is Plastic:
Plastic is a broad name given to different polymers

with high molecular weight, which can be degraded
by various processes
OR
Plasticis a material consisting of any of a wide
range ofsyntheticor semi-syntheticorganic
compoundsthat
aremalleableand
can
bemoldedinto solid objects.
Plastics are typicallyorganic polymersof
highmolecular mass, but they often contain other
substances. They are usually synthetic, most
commonly derived from petrochemicals, but many
are partially natural.
Plastic in common mans Life: 7 types of
plastic as shown in table
n
Ethylene monomer
Life without Plastic:????
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14
Plastic as a waste and its

danger to society Low degradability.
Not immediate impact on our

environment, but its continuous
dumping
eventually
create
problems for future generations
Additives of plastic can

cause permanent harm
to our metabolism.
Chemicals
such
as
phthalates and BPA are
interfere
with
our
natural hormone levels
which
can
cause
serious problems in
humans
Need log time (400 to 1000 years)

for decomposing.
waste plastic will continue to clog
our waterways, oceans, forests,
amit.pratush@sjtu.edu.cn/ amit.pratush@gmail.com and other natural habitats
Chemical dangers are also high,
because
both
creation
and
Children toys are

manufactured with
plastic. Small parts
may be unknowingly
engulfed.
Plastic
bags
(grocery or trash
bags)who
can
sometimes end up
wrapped
around
children
faces,
disrupting
their
breathing.
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degradation of Plastic a Process Over view:
Anaerobic digestion
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16
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Alternative of Plastic OR Biodegradable

plastic/Bio
Plastic
Bioplasticsareplasticsderived
from
renewablebiomasssources, such asvegetable fats
and oils,corn starch, ormicrobiota.Bioplastic can be
made from agriculturalby-productsand also from
usedplastic bottlesand other containers using
microorganisms.
Environmentally
friendly
plastics fall into three types:
1. Bioplasticsmade from natural materials such as
corn starch
2. Biodegradable plasticsmade from traditional
petrochemicals, which are engineered to break down
more quickly.
3. Eco/recycled plastics, which are simply plastics
made
from recycled plastic materials rather than raw
petrochemicals.
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C. Biodegradation of Paint industry waste
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20
Paint industry waste

treatment
Need all Physical,
Chemical as well as
biological treatment.
1:
Physical
Treatment
for
removal
of
hydrocarbons,
esters,
alcohols,
volatile,
semivolatile and nonvolatile chlorinated
organic pollutants.
10/14/16
21
D. Biodegradation of Pesticide industry waste

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.
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Degradation of pesticides is the breaking down of toxic chemicals into nontoxic compounds
and, in some cases, back into their original elements. The degradation or breakdown of
pesticides can occur in plants, animals, and in the soil and water; or it can take place upon
exposure to ultra-violet (UV) radiation.
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Microorganism involved in Pesticide biodegradation

Name of Pesticide
Microorganism
DDT
P. aeruginosa
(dichlorodiphenyltrichloroeth
ane)
Linurin
B. sphaerica
Arthrobacter, P.
2,4-D (2,4Dichlorophenoxyacetic acid) cepacia
2,4,5-T
(2,4,5Trichlorophenoxyacetic
acid) , Parathion
P. cepacia
Factors affecting Pesticide degradation

1.Temperature
2. pH
3.Moisture
4. Organic matter
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E. Biodegradation of other industry waste such as Chemical,

Pharmaceutical, textile & cosmetic
The waste composition of any
pharmaceutical company depends on
the nature of drugs manufactured in
that industry. All waste must be
properly treated (Physical, Chemical
or Biological) before final disposal, so
that their will be least harm to the
environment
place. produce large
The chemicaltook
companies
number of nitrile containing chemicals like
Acetonitrile,
Propionitrile,
Isobutyronitrile,
Adiponitrile
etc.
which are used an various
chemical
processes as during the formation of
several usable products.
Most of the pharmaceutical industries

produce drugs having nitrogen &
sulfur as a base, so the waste contain
mostly nitrogen in the from of nitrile
or sulfur in the form of sulphite.
There are other several kind of waste
material found in the Pharma
company effluent.
Presently I am focusing here only
10/14/16
the Nitrile Biodegradation
25
Nitrile Biodegradation
The biological degradation of nitriles are carried out a couple of microbial systems.
These microbes produce enzyme (Intracellular /extracellular), which further hydrolyze the nitrile to its
corresponding by products either carboxylic acid or amides, respectively.
The enzymatic degradation of various substrates is known as Green Chemistry.
The
major key enzymes of the microbial metabolism of

nitriles are:
1. Nitrilase (EC 3.5.5.1). 2. Nitrile hydratase (EC 4.2.1.84). 3.
TheAmidase
microbial
of nitriles proceeds through two enzymatic pathways. One is
(ECdegradation
3.5.1.4).
the nitrilase pathway, in which nitrilase hydrolyzes nitriles directly to the corresponding
carboxylic acids and ammonia (Equation 1).
RCN
2H2O
Nitrilase
RCOOH
NH3
The other pathway is a combination of nitrile hydratase (NHase) and amidase. Firstly, nitriles are hydrated into
the corresponding amides by NHase (Equation 2). Secondly, amides are hydrolyzed into the corresponding
carboxylic acids and ammonia by amidase (Equation 3)
RCN + H2O
RCONH2 + H2O
NHase
Amidase
RCONH2
RCOOH + NH3
3
26
Nitrile degradation by Nitrilase enzyme

Nitrilase in Biological Systems
The nitrile pathway consists of aldoxime nitrile amide

acid acyl-CoA and the enzymes involved in this pathway are
aldoxime dehydratase, nitrilase/(amidase and nitrile
hydratase) and acyl-CoA synthetase
Bacteria
Yeast
Cyanobac Molds
treia
Plants
Pseudomonas
Candida
Synechococcus
Aspergillus
Arabidopsis
Nocardia
Cryptococcus
Phormidium
Penicillium
Zea
Bacillus
Kluyveromyc
es
Lyngbya
Fusarium
Brassica
Rhodococcus
Rhodotorrula
Nostocc
Trichoderma
Hordium
Alcaligenes
Myrothecium
Lupinus
Arthrobacter
Nicotiana
10/14/16
27
Use of Nitrilases in Green

Chemistry
Substrate
Product
Acrylonitrile
Acrylic acid
Acetonitrile
Acetic acid
Phenylpropanenitrile
Indole-3-acetic acid
2-Hydoxypropionitrile
Lactic acid
3-hydroxyglutaronitrile
(R)-3-hydroxy-4-cyanobutanoic acid
Benzonitrile
Benzohydroxamic acid
Alpha hydroxycarbxalic acid
Poly carboxylic acid
10/14/16
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b.Nitrile degradation by Nitrile

hydratase (NHase) enzyme
Nitrile hydratase (NHase, EC 4.2.1.84) is one of the key enzymes of nitrile metabolism in a
large number of microbes that catalyses the hydration of nitriles to corresponding
amides.
Nitriles are hydrated into the corresponding amides by NHase

10/14/16
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NHase has been successfully adopted in chemical industry for production of acrylamide,
nicotinamide and 5-cyanovaleramide by mean of Green chemistry.
The NHase mediated conversion of 3-cyanopyridine to nicotinamide is shown below:
CONH2
CN
Nitrile Hydratase
H2O
N
3- Cyanopyridine
Nicotinamide
Nicotinamide (Vit. B3), which is an important part of vitamin B complex group. Vitamin B3 is mainly used in pellagra
and niacin deficiency. It also shows the antioxidant and cryoprotective effects.
10/14/16
30
eukaryotic as well as prokaryotic microorganisms produce the NHase e

NHase producing Eukaryotic
microorganisms
NHase producing Prokaryotic

microorganisms
Microorganism
Substrates
Enzyme activity
Microorganism
Candida guilliermondii CCT

7207
(cyclo) alkylnitriles,
arylnitriles,
heterocyclicnitriles
NHase
Candida famata
Alkylnitriles
NHase
Cryptococcus sp. UFMG-Y28
Acetonitrile,
propionitrile
NHase
Cryptococcus flavus UFMGY61
Isobutyronitrile
NHase
Rhodotorula glutinis UFMGY5
Methacrylonitrile
NHase
Monosiga brevicollis
Acrylamide
NHase
NHase production exist both inducible as

well as constitutive form but most of them
(80%) are inducible a few only (20%) of the
organism produce constitutive in nature.
Substrates
Enzyme activity
A. tumefaciens d3 (DSM 9674)
arylnitriles, arylalkylnitriles
NHase
A. tumefaciens B-261
Indole-3-acetonitrile
NHase
B. cereus
Acrylonitrile
NHase
Bacillus sp. BR449
Acrylonitrile
NHase
Bacillus sp. RAPc8
(cyclo) alkylnitriles
NHase
B. pallidus Dac521
Alkylnitriles
NHase
Rhodococcus sp. C3II (DSM 9685) alkylnitriles, arylnitriles,

arylalkylnitriles
Rhodococcus sp. R312 (CBS
alkylnitriles, benzonitrile
717.73)
Rhodococcus sp. YH3-3
alkylnitriles, arylnitriles,
heterocyclic nitriles,
phenylacetonitrile
R. rhodochrous J1 (FERM BP-1478) Alkylnitriles, Arylnitriles,
hetero-cyclic nitriles
R. rhodochrous PA34
cyanopyridine
R. rhodochrous PA34 mutant 4D
3 cyanopyridine
NHase
NHase
NHase
NHase
NHase
10/14/16
NHase
31
Biochemistry of nitrile hydratase
10/14/16
32
10/14/16
33
Recombinant DNA Technology (RDT)

in
XENOBIOTICS degradation
1.Increase in secondary metabolite production of
microorganisms
2.Construction of Broad-host-range plasmid cloning
vectors
3.Detection of Containment of engineered
organisms (e.g. streptavidin gene expression.)
Absence of
growth
substrate
Presence of
growth
substrate
Streptavidin
gene
Gene repressed
Gene expressed
very less
production of
Streptavidin +
binds with D
biotin
Kill the host

organism
10/14/16
4. Increasing the substrate range of biodegrading

enzymes
34
Pros & Cons of Biodegradation

Pros (Advantages)
Bioremediation is a natural process and is therefore perceived by the public.
Bioremediation is useful for the complete destruction of a wide variety of contaminants.
Instead of transferring contaminants from one environmental medium to another, for example,
from land to water or air, the complete destruction of target pollutants is possible.
Bioremediation can often be carried out on site, often without causing a major disruption of
normal activities.
Bioremediation can prove less expensive than other technologies that are used for cleanup of
hazardous waste.
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35
Cons (Disadvantages)
Bioremediation is limited to those compounds that are biodegradable. Not all compounds
are susceptible to rapid and complete degradation.
There are some concerns that the products of biodegradation may be more persistent or
toxic than the parent compound.
Biological processes are often highly specific. microbial populations, suitable
environmental growth conditions, and appropriate levels of nutrients and contaminants.
It is difficult to extrapolate (deduce) from bench and pilot-scale studies to full scale field
operations.
Bioremediation often takes longer than other treatment options.
10/14/16
36
Thanks for your attent

10/14/16
37

Xinobiotics: Threatening Pollutants and Their Biodegradation

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amit.pratush@sjtu.edu.cn/ amit.pratush@gmail.

What is the need of Biodegradation?

Biodegradation is the initial process that results to bioremediation .

Microorganisms easy to handle.

Plants easy to grow.

Enzymatic processes in bioremediation

A minor change in an organic

Complete mineralization, which in

Use of various types of microorganisms for biodegradation

How Microbes Use the Contaminant

Enough available to be the sole energy source.

Utilization of a compound by a microbe relying on some

pH conditions for microorganisms

Temp. conditions for microorganisms

What are the XENOBIOTICS?

those chemicals which are man-made and do not occur naturally in

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

oil/gas industry, refineries.

closely related to the petrochemical industry

major ingredient are solvents,

most commonly found.

Electronic industry, Textile industry, Pulp and Paper industry, Cosmetics

A. Biodegradation of Petrochemical industry waste

Main principle of aerobic degradation of

degradation rates in different environm

Aromatic hydrocarbon Biodegradation

Polycyclic aromatic hydrocarbon (PAH)

Adverse effects of PCB & PAH pollution on human health

Microorganism involved in biodegradation of

Factors affecting Hydrocarbon

Arthrobacter, Bacillus sp.,

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

Alcaligenes, Achromobacter, Bacillus, Flavobacterium,

B. Biodegradation of Plastic industry waste

Plastic is a broad name given to different polymers

Life without Plastic:????

Plastic as a waste and its

Not immediate impact on our

Additives of plastic can

Need log time (400 to 1000 years)

Children toys are

degradation of Plastic a Process Over view:

Alternative of Plastic OR Biodegradable

C. Biodegradation of Paint industry waste

Paint industry waste

D. Biodegradation of Pesticide industry waste

Microorganism involved in Pesticide biodegradation

Factors affecting Pesticide degradation

E. Biodegradation of other industry waste such as Chemical,

Most of the pharmaceutical industries

major key enzymes of the microbial metabolism of

Nitrile degradation by Nitrilase enzyme

The nitrile pathway consists of aldoxime nitrile amide

Use of Nitrilases in Green

Alpha hydroxycarbxalic acid

Poly carboxylic acid

b.Nitrile degradation by Nitrile

Nitriles are hydrated into the corresponding amides by NHase

eukaryotic as well as prokaryotic microorganisms produce the NHase e

NHase producing Prokaryotic

Candida guilliermondii CCT

Cryptococcus sp. UFMG-Y28

Cryptococcus flavus UFMGY61