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USE OF ENZYMES IN BIOREMEDIATION

BACHELOR OF TECHNOLOGY (BIOTECHNOLOGY)

BY  

SNEHAL.S.MENON

 DEPARTMENT OF BIOTECHNOLOGY,

STES’S SINHGAD COLLEGE OF ENGINEERING, VADGAON (BK), OFF SINHGAD ROAD,

PUNE- 411041

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OUTLINE Introduction Bioremediation Enymatic bioremediation Enzymological background Extracellular enzymes used in bioremediation Advantages and disadvantages of extracellular enzymes Soluble and immobilized enzymes Plants and their associated enzymes in bioremediation Major enzymes used in bioremediation Case studies Scope of enzymatic bioremediation and future prospects Conclusion References

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INTRODUCTIONENVIRONMENTAL POLLUTION

Environment

Atmosphere

Earth

Water

Space

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TYPES OF POLLUTION Atmospheric pollution: pollution of air. Water pollution: pollution of hydrosphere or water. Industrial effluents pollution: pollution due to disposal of waste water. Domestic effluent pollution: pollution due to indiscriminate dispersal of domestic sewage. Soil pollution: pollution of lithosphere or land.

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OTHER TYPES OF POLLUTION Noise pollution

Vibration

Noxious odours

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BIOREMEDIATION: According to E.K. Nyer, the term “bioremediation” refers to all biochemical

reactions of natural attenuation, which includes all biotic and abiotic processes used to reduce contaminant levels.

Biodegradation is the primary mechanism to reduce biodegradable contaminants by employing organisms like bacteria, fungi, algae or plants.

Figure 1: The process of waste bioremediation

.

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TYPES OF BIOREMEDIATION Microbial bioremediation: Bioremediation can occur either naturally, or by the use of bioaugmentation (whole cell introduction) or Biostimulation approaches (use of nutrients or conditions to stimulate the native

microbial community). Enzymatic bioremediation: Isolated enzymes may also be used to transform the contaminant into less-toxic or non-

toxic compounds. Extracellular enzymes: Extracellular enzymes are either secreted from organisms such as white rot fungi or are

produced during a fermentation process. Phytoremediation: It is the in situ use of plants, their enzymatic system, their roots and associated

microorganisms to degrade harmless pollutants present in different environmental systems (soil, sediments, groundwater and air).

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ENZYMATIC BIOREMEDIATION: Enzymological background: Enzymes are biological catalysts that facilitate the conversion of substrates

into products by providing favourable conditions that lower the activation energy of the reaction.

The regions of the enzyme that are directly involved in the catalytic process are called the active sites.

Figure 2: Mechanism of enzymes

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EXTRACELLULAR ENZYMES IN BIOREMEDIATION: Extracellular enzymes refer to those enzymes that are either secreted by the

microbes, such as white rot fungi or those that enter the aqueous phase during an aerobic submerged fermentation process.

Such enzymes are naturally produced by the microbes and then harvested. Enzymes from white rot fungi have been shown to be

effective degraders of TNT, phenols, PCBs, PAHs and dyes.

Figure 3: Some xenobiotics amenable to enzymatic bioremediation

Chrysene(PAHs) Lindane(pesticide) Polychlorinated biphenyls(PCBs)

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ADVANTAGES AND DISADVANTAGES OF EXTRACELLULAR ENZYMES

ADVANTAGES

• Can work in multiple environments.

• Can be recovered and recycled.

• Can be used with different substrates.

• They are biodegradable.

DISADVANTAGES

• Difficult to maintain enzyme concentration.

• Difficult to optimize.• Limits overall success.• High cost.

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SOLUBLE AND IMMOBILIZED ENZYMES Soluble enzymes Mobile enzymes can be added at a single point and then spread due to

diffusion, dispersion. and the flows of groundwater and surface water . Immobilized enzymes Enzymes can he immobilized onto a carrier, which can be granular, fibrous,

a tube or a membrane.

Figure 4: Types of immobilization of enzymes In general, immobilization makes the enzyme more resistant to

temperature, pH and substrate concentration swings, giving it a longer lifetime and higher productivity per active unit.

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PLANTS AND THEIR ASSOCIATED ENZYMES IN BIOREMEDIATION The involvement of plants in the bioremediation of pollutants is called as

phytoremediation. The process of phytoremediation is an emerging green technology that

facilitates the removal or degradation of the toxic chemicals in soils, sediments, groundwater, surface water and air.

Figure 5: Enzymatic and microbial activities responsible for enhanced remediation in rhizospheric zone

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MAJOR ENZYMES USED IN BIOREMEDIATION Enzymes from white rot fungi have been found to be very capable of

degrading a large number of different contaminants.

White rot fungi are unique among eukaryotes because they are able to cleave the carbon-carbon bonds in contaminants such as PAHs.

During the secondary metabolism of plant life, white rot fungi produce and secrete LiP, manganese peroxidase (MnP) and laccase.

Each of the enzymes can catalyze the one-electron oxidation of phenols and non-phenolitic substrates.

This results in the production of cation-radical intermediates, which can be used to futher oxidize non-phenolitic substrates.

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Lignin peroxidase(LiP) LiPs are hemoproteins which catalyze reactions in the presence of hydrogen

peroxide. LiP is very effective in the bioremediation of PAHs. LiP from Phanerochete chrysosporium. for example. is able to degrade

PAHs. LiP is also capable of degrading benzo[a]pyrene into 52% 1,6-quinone,

25% 3,6-quinone. and 23% 6,12-quinone. These product ratios are very similar to those found from the degradation of

benzo[a]pyrene using chemical and electrochemical means.

Benzo[a]pyrene(PAHs)

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Manganese peroxidase (MnP): MnP is also a hydrogen peroxide dependent enzyme,but it can only oxidize

organics when in the presence of Mn(Il). MnP oxidizes Mn(II) to Mn(III), which acts as an obligatory oxidation

intermediate for the oxidation of various compounds. The Mn(IlI) ions migrate away from the enzyme and start the oxidation of the

lignin and other compounds.

Figure 6: Mechanism of MnP

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Laccase: Laccases are multi-copper oxidases that catalyze the one electron oxidation

of substituted phenols, anilines, and aromatic thiols to the corresponding radicals with the concomitant reduction of molecular oxygen to water.

These radicals produce polymeric products by self-coupling or cross-coupling with other molecules, and dechlorination, demethoxylation and decarboxylation during coupling and polymerization of differently substituted substrates may also occur.

These enzymes appear suitable and versatile catalysts, very useful for the application in several biotechnological processes.

Figure 7: Designer laccases

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OTHER EXTRACELLULAR ENZYMES USED IN BIOREMEDIATION

HRP is a peroxidase that is secreted by the root hairs of the horseradish plant and can catalyze the oxidation of compounds such as phenols, biphenols, anilines and benzidines over a large range of pHs and temperatures .

Extracellular enzymes such as proteases, amylases and lipases are produced during the aerobic fermentation of organic matter by yeast or other microbes.

Environments such as waste-water require a different type of enzyme and these enzymes, rather than catalyzing the oxidation of recalcitrant compounds, catalyze the degradation of organic matter .

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CASE STUDIESBIOREMEDIATION OF TEXTILE EFFLUENT USING ENZYMES

Enzymes can act on specific recalcitrant pollutants to remove them by precipitation or transformation to other products.

White-rot fungi were able to degrade dyes using lignin peroxidase (LiP) and manganese dependent peroxidase (MnP)

The manganese peroxidase produced by Phanerochaete sordida showed higher range of 90% decolorization of azo and anthraquinone dye.

The comparison of the fungal isolates and enzymatic treatment in the degradation of reactive blue 5 dyes was carried out and the degrading capacity of the enzyme manganese peroxidase was 1.5 times greater than the fungal isolates.

Lignin peroxidase obtained from Phanerochaete chrysosporium is effective against methylene blue and azure B dyes

VP (Versatile peroxidase) has been recently described as new family of ligninolytic peroxidases,together with lignin peroxidase and manganese peroxidase obtained from Phanerochaete chrysosporium .

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These enzymes exhibited both lignolytic peroxidase and manganese peroxidase activity and therefore these enzymes were called as hybrid manganese peroxidase-lignin peroxidase or versatile peroxidase.

Recalcitrant dyes could be successfully decolorized by peroxidases in the presence of some suitable redox mediators.

Treatment of recalcitrant pollutants by using enzyme-redox mediator system will be significantly useful procedure for targeting number of dyes with diversified structures.

Laccase produced by Pycnoporus snnguineus in liquid cultures can completely decolor bromophenol blue and malachite green (both triphenylmethane dyes) and partially decolor orange G and amaranth (both azo dyes).

lmmobilization of the enzyme on alumina increased its thermal stability and made it less affected by inhibitors, such as halides and dye additives.

lmmobilized laccase was also able to decrease the toxicity of the dyes by up to 80%

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The enzymatic decolorization of industrial dyes is a big challenge due to large diversity of chemical structures.

Enzymes offered several advantages such as greater specificity, better standardization, easy handle and store and no dependence on bacterial growth rates.

A major obstacle that will have to he overcome is the long contact time required for decolorization to occur.

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SCOPE OF ENZYMATIC BIOREMEDIATION AND FUTURE PROSPECTS

The scope of bioremediation is to decrease the concentration of organic pollutants at undetectable levels or, if measurable, lower than the limits established as safe or tolerable by regulatory agencies.

There are several fields in which enzymes can be applied:

Figure 8: Overview of enzymology of biological remediation Enzymatic bioremediation improved with molecular tools can be

particularly suitable for situations where rapid remediation is required.

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CONCLUSION Enzymes present environmental advantages against chemicals and

microorganisms. They are:- the biotransformation does not generate toxic side products as is often the

case with chemical and some microbiological processes; the enzymes are digested, in situ, by the indigenous microorganisms after

the treatment; the requirement to enhance bio-availability by the introduction of organic

co-solvents or surfactants is much more feasible from an enzymatic point of view than using whole cells;

the production of enzymes at a higher scale, with enhanced stability and/or activity and at a lower cost is feasible by using recombinant-DNA technology .

However, a more extensive effort is required to overcome several bottlenecks: high enzyme cost, low activity and/or stability under given conditions, low reaction yields and the low biodiversity in organisms screened so far.

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REFERENCES Khopkar.S.M, Environmental Pollution Monitoring and Control, Newage

publishers,2007. Miguel Alcalde, Manuel Ferrer, Francisco J. Plou and Antonio Ballesteros; 2006;

Environmental biocatalysis: frommremediation with enzymes to novel green processes; TRENDS in Biotechnology; Vol.24; No.6; 1-7.

Timothy P. Ruggaber and Jeffrey W. Talley; 2006, Enhancing Bioremediation with Enzymatic Processes; 1-13.

M.A. Rao, R. Scelza, R. Scotti and L. Gianfreda; 2010; Role of enzymes in the remediation of polluted environments; Soil science. Plant nutrition, 10(3): 333- 353

R. S. Peixoto, A. B. Vermelho, and A. S. Rosado; 2011; Petroleum-Degrading Enzymes: Bioremediation and New Prospects; Volume 2011; 1-7.

Chandrakant S. Karigar and Shwetha S. Rao, 2011, Role of Microbial Enzymes in the Bioremediation of Pollutants,Enzyme Research, Volume 2011,1-11.

Palanivelan Ramachandran,Rajakumar Sundharam,Jayanthi Palaniyappan and Ayyasamy Pudukkadu Munusamy, 2013, Potential process implicated in bioremediation of textile effluents, Advances in Applied Science Research, 4(1),131-145.