Xinobiotic Biodegradation

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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?


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 reactionsOxidation, 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 oneIsomer formation conversion of one isomer of a compound to another with a same molecular formula but different structure


Biodegradation has at least 3 outcomes: Example of 2,6-Dichlorobenzonitrile can explain all steps1. 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.


Use of various types of microorganisms for biodegradationAerobic 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.


How Microbes Use the ContaminantPrimary substrate Enough available to be the sole energy source.

Secondary substrate Provides energy, not available in high enough concentration.

Co -metabolic substrate Utilization of a compound by a microbe relying on some other primary substrate.

pH conditions for microorganisms Temp. conditions for microorganisms


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.

SOURCES OF XENOBIOTICS

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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.

Effect on water bodies after11 million gallon oil spill from the supertanker EXXON VALDEZ-ALASKA in March-1989


Hydrocarbon degradation rates in different environmentsMain principle of aerobic degradation of hydrocarbons by microorganisms


Adverse effects of PCB & PAH pollution on human healthCauses reproductive disabilities in animals, human, birds.CarcinogenicNervous system damageEndocrine gland malfunction

Aromatic hydrocarbon Biodegradation1. Polycyclic aromatic hydrocarbon (PAH)2. Polychlorinated biphenyl's (PCB)


Factors affecting Hydrocarbon Biodegradation

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Microorganism involved in biodegradation of hydrocarbonsOrganic Pollutants

Organisms

Phenolic compound Achromobacter,Alcaligenes,Acinetobacter, Arthrobacter, Azotobacter, Aspergillus, Flavobacterium, Pseudomonas, Penicillium putida,Candida tropicalis, Trichosporon cutaneoum

Benzoate related compound

Arthrobacter, Bacillus sp.,

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

Surfactants Alcaligenes, Achromobacter, Bacillus, Flavobacterium, Pseudomonas, Candida

PCB Bioremediations

Biostimulation

BioaugmentationNatural Attenuation



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 ORPlastic is a material consisting of any of a wide range of synthetic or semi-synthetic organic compounds that are malleable and can be molded into solid objects. Plastics are typically organic polymers of high molecular mass, but they often contain other substances. They are usually synthetic, most commonly derived from petrochemicals, but many are partially natural.Plastic in common man’s Life: 7 types of plastic as shown in table

nEthylene monomer

Life without Plastic:????

https://en.wikipedia.org/wiki/Ethylene

https://en.wikipedia.org/wiki/Monomer


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

Low degradability. Not immediate impact on our environment, but its continuous dumping eventually create problems for future generations

Need log time (400 to 1000 years) for decomposing.waste plastic will continue to clog our waterways, oceans, forests, and other natural habitatsChemical dangers are also high, because both creation and recycling of plastic produce toxic materials of many kinds.

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.

Plastic as a waste and its danger to society


Biodegradation of Plastic a Process Over view:

Anaerobic digestion



Alternative of Plastic OR Biodegradable plastic/Bio PlasticBioplastics are plastics derived from renewable biomass sources, such as vegetable fats and oils, corn starch, or microbiota. Bioplastic can be made from agricultural by-products and also from used plastic bottles and other containers using microorganisms.

Environmentally friendly plastics fall into three types:1. Bioplastics made from natural materials such as

corn starch

2. Biodegradable plastics made 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.



C. Biodegradation of Paint industry waste


Need all Physical, Chemical as well as biological treatment. 1: Physical Treatment for removal of hydrocarbons, esters, alcohols, volatile, semi-volatile and non-volatile chlorinated organic pollutants.

Paint industry waste treatment


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.

D. Biodegradation of Pesticide industry waste


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.


Microorganism involved in Pesticide biodegradation

Name of Pesticide MicroorganismDDT (dichlorodiphenyltrichloroethane)

P. aeruginosa

Linurin B. sphaerica

2,4-D (2,4-Dichlorophenoxyacetic acid )

Arthrobacter, P. cepacia

2,4,5-T (2,4,5-Trichlorophenoxyacetic acid) , Parathion

P. cepacia

Factors affecting Pesticide degradation1.Temperature2. pH3.Moisture4. Organic matter


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 took place.

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 the Nitrile Biodegradation

The chemical companies produce large 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.

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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. Amidase (EC 3.5.1.4).

RCN + 2H2O RCOOH + NH3Nitrilase 1

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 NHase RCONH2 2

RCONH2+ H2O RCOOH + NH3Amidase

3

The microbial degradation of nitriles proceeds through two enzymatic pathways. One is the nitrilase pathway, in which nitrilase hydrolyzes nitriles directly to the corresponding carboxylic acids and ammonia (Equation 1).


Nitrilase in Biological Systems

Bacteria Yeast Cyanobactreia

Molds Plants

Pseudomonas Candida Synechococcus Aspergillus Arabidopsis

Nocardia Cryptococcus Phormidium Penicillium Zea

Bacillus Kluyveromyces

Lyngbya Fusarium Brassica

Rhodococcus Rhodotorrula Nostocc Trichoderma Hordium

Alcaligenes - - Myrothecium Lupinus

Arthrobacter - - - Nicotiana

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

a. Nitrile degradation by Nitrilase enzyme


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


b.Nitrile degradation by Nitrile hydratase (NHase) enzyme

Nitriles are hydrated into the corresponding amides by NHase

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.


N N

CONH2

Nicotinamide3- Cyanopyridine

H2O

CN

Nitrile Hydratase

The NHase mediated conversion of 3-cyanopyridine to nicotinamide is shown below:

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.

NHase has been successfully adopted in chemical industry for production of acrylamide, nicotinamide and 5-cyanovaleramide by mean of Green chemistry.


Both eukaryotic as well as prokaryotic microorganisms produce the NHase enzyme. NHase producing Eukaryotic microorganisms Microorganism Substrates Enzyme activity

Candida guilliermondii CCT 7207

(cyclo) alkylnitriles, arylnitriles, heterocyclicnitriles

NHase

Candida famata Alkylnitriles NHase

Cryptococcus sp. UFMG-Y28 Acetonitrile, propionitrile

NHase

Cryptococcus flavus UFMG-Y61

Isobutyronitrile NHase

Rhodotorula glutinis UFMG-Y5

Methacrylonitrile NHase

Monosiga brevicollis Acrylamide NHase

NHase producing Prokaryotic microorganisms Microorganism 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

NHase

Rhodococcus sp. R312 (CBS 717.73)

alkylnitriles, benzonitrile NHase

Rhodococcus sp. YH3-3 alkylnitriles, arylnitriles, heterocyclic nitriles, phenylacetonitrile

NHase

R. rhodochrous J1 (FERM BP-1478) Alkylnitriles, Arylnitriles, hetero-cyclic nitriles

NHase

R. rhodochrous PA34 cyanopyridine NHase

R. rhodochrous PA34 mutant 4D 3 cyanopyridine 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.


Biochemistry of nitrile hydratase



Recombinant DNA Technology (RDT) in

XENOBIOTICS degradation1.Increase in secondary metabolite production of

microorganisms2.Construction of Broad-host-range plasmid cloning

vectors3.Detection of Containment of engineered

organisms (e.g. streptavidin gene expression.)

4. Increasing the substrate range of biodegrading enzymes

5. Tracking recombinant microorganisms

Streptavidin gene

Presence of growth

substrate

Gene expressedGene repressed

Absence of growth

substrate

very less production of Streptavidin + binds with D

biotin

Kill the host organism


Pros & Cons of Biodegradation

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.

Pros (Advantages)


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.


Thanks for your attention

Presentations & Public Speaking

Xinobiotic Biodegradation