an overview of biodegradation of organic pollutants

International Journal of Scientific and Innovative Research 2016; 4(1) P-ISSN 2347-2189, E- ISSN 2347-4971

AN OVERVIEW OF BIODEGRADATION OF ORGANIC POLLUTANTS Arpna Ratnakar, Shiv Shankar and *Shikha Department of Environmental Science, Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar, Raebareli Road, Lucknow, Uttar Pradesh, India *

Address for correspondence: Dr. Shikha, Assistant Professor, Department of Environmental Science, Babasaheb Bhimrao Ambedkar University (A Central University) ,Vidya Vihar, Raebareli Road, Lucknow, Uttar Pradesh, India Email: [email protected] ABSTRACT An increase in organic pollutant is a major concern in the contemporary world. Organic pollutants may be treated through physical and chemical processes, but these processes are toxic and not environmental friendly because their final product may still remains toxic till the very end. Hence, the biological approaches may be a suitable alternative towards bioremediation practices being not only cost effective but eco-friendly as well. Moreover, the final product happens to be less toxic as compared to other approaches. The microorganisms and plants (bioremediation) are used to remediate the polluted environments widely and is emerging as a promising and appealing area of environmental biotechnology. Apart from using the whole cell microorganisms, the use of their extracellular and/or cell-free enzymes has been advocated as an innovative technique to abate pollution. Employment of extracellular enzyme for the removal of pollutants has several advantages over using whole microbial cell. The present review attempts a brief survey of many aspects dealing with the characteristics and potential abilities of both cell-present and cell free extra cellular enzymes in bioremediation of various organic pollutants. Keywords: Bioremediation; Organic Pollutant; Microbial Enzymes; Extracellular enzymes;

INTRODUCTION 1. Organic pollutants: Origin & Occurrence The late 1800s and early 1900, has witnessed a dramatic increase in the range of chemically synthesized products which include pesticides, plastics, hydrocarbon fuels, soaps, detergents and other useful substance. The effects of these chemical substances on the environment are a consequence of a sequence of processes that depend on the properties of individual chemical. Halogenated organic pollutants (HOPs), such as polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT), polybrominated diphenyl ethers (PBDEs), dechlorane plus (DP), and decabromodiphenyl ethane (DBDPE), have been of great concern due to their persistence,

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bioaccumulation and potential toxicity to wildlife and human. PCBs were used primarily as dielectric and coolant uids in capacitors, transformers and electric uids [1]. PBDEs (including Penta-, Octaand Deca-BDE commercial formulations), DP and DBDPE are some widely used ame retardants in electronics, textiles, thermoplastics, polyurethane foams and building materials [2,3,4]. Among them, Penta- and Octa-BDE technical mixtures have been added to the list of emerging POPs by the Stockholm Convention in 2009 and Deca-BDE technical mixtures have been phasedout in Europe and America, while Deca-BDE, DP [5] and DBDPE are still widely used in China .

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Fig.1 Major Sources of pollution [6]. A wide spectrum of hazardous pollutants with diverse structures originates from anthropogenic sources and pollute environment continuously. Three major sources of organic pollutants can be identied viz; industrial activities, military waste and agricultural practices (Fig.1). Important organic contaminants include petroleum product, polycyclic aromatic hydrocarbons (PAHs), dioxins, chlorophenoxy acetic acids (2, 4-D and 2, 4, 5-T), organophosphates, triazines, carbamates, plastics and related compounds and [7] organometallic compounds . Modern agriculture depends on the four main factors viz: water, fertilizers, seed and pesticides. Pesticides are the integral part of modern agriculture. About 35-45 % crop production is lost due to insects, weeds and diseases, while 35% crop produces are lost during storage. Indian Agrochemical Industry size is estimated to be US$ 3.8 billion in year 2012. Over the 12th plan period, the segment is expected to grow at 12-13% per annum to reach 7.0 billion. The Indian domestic demand is growing at the rate of 8-9% and export demand at 15-16% [8]. Motor vehicles are major sources of petroleum hydrocarbons, polycyclic aromatic hydrocarbons and dioxins which are often discharged to the atmosphere in particulate 74

form. Urban and industrial wastes are often, disposed directly into pits dug into the ground which leads to contamination of the soil and in some cases the adjacent ground water. Outside urban areas agricultural activities are the major sources of pollutants [7]. 1.1 Organic pollutants: Types and properties Organic pollutants are chemical compounds that contain carbon and have a demonstrably negative effect on one or more components of the environment. Organic pollutant can be placed into three general classes: (i) hydrocarbons, (ii) oxygen, nitrogen and phosphorus compounds and (iii) organometallic compounds. The major category of organic pollutants includes the hydrocarbons and related compounds, which contains such compounds as Dichloro Diphenyl Trichloroethane (DDT), the dioxins and the polycyclic a r o m a t i c h y d r o c a r b o n s ( PA H s ) . T h e s e compounds contain the elements of carbon and hydrogen, with some containing chlorine and oxygen as well. There are a limited number of types of chemical bonds present, which are principally C-H, C-C, C-Cl, C=C and C=C (aromatic). All of these bonds are relatively stable and have limited polarity and this property www.ijsir.co.in


is then conferred onto the related compounds [7]. (1) Owing to low polarity, hydrocarbons, in general are lipophilic, poorly soluble in water and persistent in the environment. This class includes the most toxic organic compound, abiotic in origin:-2, 3, 7, 8-tetrachlorodibenzo (1, 4) dioxin, also known as 2, 3, 7, 8-TCDD or TCDD [7]. (2) The group containing oxygen, nitrogen and phosphorus compounds is extremely diverse but as a general rule may contain compounds with relatively high solubility in water, low fat solubility and relatively low persistence in the environment. This might be due to the presence of bonds with relatively high levels of polarity due to carbon and other atoms being attached to oxygen, nitrogen or phosphorus conferring a high level of polarity onto the related compounds [7] . (3) The organometallic group is considered the least important in view of environmental perspective and includes compounds which may be combinations of metal, such as lead and tin, with organic components based on carbon [7]. 1.1.1. Persistent Organic Pollutants (POPs) A group of chemicals have posed particular environmental problems owing to their fat solubility, bioaccumulation potential and environmental persistence, along with usage patterns. These are referred to as Persistent

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Organic Pollutants (POPs) and these substances have often been found distributed over long distances up to a global scale. They have been observed to persist for long periods of time in the environment and can accumulate and pass from one species to the next through the food chain [9, 10]. Persistent organic pollutants include two types of important compounds: (i) Polycyclic aromatic hydrocarbons and (ii) halogenated hydrocarbons. The latter group includes the organochlorines viz., DDT (Dichloro Diphenyl Trichloroethane), PCBs (Per Chloro Biphenyls). The dioxins produced at large scale are released to the environment. They are not easily degraded by microbes. Highly chlorinated biphenyls tend to concentrate up to greater extent than less chlorinated PCBs [11]. Less chlorinated PCBs are removed easily from the body in comparison to high chlorinated biphenyls. In general, it is known that the more highly chlorinated biphenyls tend to accumulate to a greater extent than the less chlorinated PCBs; similarly, metabolism and excretion are also more rapid for the less chlorinated PCBs than for the highly chlorinated biphenyls. Under the treaty, known as the Stockholm Convention, countries agreed to reduce or eliminate the production, use, and/or release of 12 key POPs (Table 1) and specied under the Convention a scientic review process that has led to the addition of other POPs chemicals of global concern. [12]

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Table 1: List of POPs Given By UNEP at Stockholm Convention S.N.

Agency

[13]

:

Name of Compounds 1995

2001

1.

Aldrin

Chlordecone

2.

Chlordane

α- Hexachlorocyclohexane and β-Hexachlorocyclohexane

3.

Dieldrin

heptabromodiphenyl ether.

UNEP at 4. 5. 6.

Stockholm Convention

Hexabromodiphenyl ether and

Endrin

Lindane

Heptachlor

Pentachlorobenzene

Hexachlorobenzene

Tetrabromodiphenyl ether and Pentabromodiphenyl ether.

7.

Mirex,

Peruorooctane sulfonic acid

8.

Toxaphene

Endosulfans

9.

Polychlorinated biphenyls

Hexabromocyclododecane

(PCBs) 10

Dichlorodiphenyltrichloro

.

ethane (DDT)

11

Dioxins

Tributyltin

. 12

Polychlorinated

.

dibenzofurans

DEGRADATION OF ORGANIC POLLUTANTS

Organic chemicals that are introduced into the environment are subjected to various physical, chemical, and biological processes which act in an interconnected way in environmental systems to determine the overall fate of the compound. The neutralization when done through chemical means, a huge amount of acid is used, which is neither economical, nor safe and poses serious health hazards [14]. There are many processes for the degradation of organic pollutants. Some processes for degradation of organic pollutant are listed below: 1. Physical processes Physical processes have been used for the degradation of organic pollutant from many decades, which may include various processes like 76

photocatalytic degradation by using Ag-modied Zn 2 GeO 4 nanorods, TiO 2 /graphene oxide nanocomposite hydrogels, Bio-silica coated with amorphous manganese oxide etc. Decomposition of these organic pollutants via catalytic/ photocatalytic oxidation is considered to be the most efcient green method for organic waste management [15, 16, 17, 18]. Visible-light response semiconductors have attracted interest of many researchers as the efcient photo catalysts [19]. There are many catalysts used for photo catalytic degradation of organic pollutants. TiO2 used as a photo catalyst because of its low cost, chemical stability, non-toxicity [20]. TiO2 is preferred because it is a promising photo-oxidation catalyst and has strong oxidizing ability of photo-induced holes. Many researchers coupled TiO2 with narrow band gap semiconductors which enhanced the www.ijsir.co.in


separation of photo induced charges by formation of heterogenous junctions. Researchers modied the surface of Ag3PO4 using TiO2 by sol gel process [21] . Researches in their study deposited Ag3PO4 [22] nanoparticles onto TiO2 to form heterostructure . R e s e a r c h e r s p r e p a r e d A g / A g 3 P O 4 / Ti O 2 heterostructure photo electrodes using a sequential chemical deposition and UV-reduction method [23]. One of the effective methods was using TiO2 nanoparticles as a photocatalyst for the degradation of organic compounds due to their non-toxicity, low cost, physical and chemical stability, and high [24, 25, 26] reactivity . 2. Chemical Processes The chemical methods for bioremediation include electrochemical dehalogenation of chlorinated benzenes [27, 28, 29, 30, 31] , in this the chlorine is eliminated step by step from the highly chlorinated benzenes to yield less-chlorinated benzenes and [27] nally transform to benzene. It was analyzed that chlorobenzenes and the cathodic reaction pathway for hexachlorobenzenes as follows: hexachlorobenzene pentachlorobenzene 1, 2, 3, 5-tetrachlorobenzene 1, 2, 4-trichlorobenzene 1, 4-dichlorobenzene monochlorobenzene benzene. The catalytic degradation of organic molecules through MnO 2 nanostructures are concerned, several groups reported the mineralization of various organic compounds/ dyes, such as Rhodamine B (RhB), MB, Benzyl alcohol (BA) in the presence of strong oxidizing agents at elevated temperature [17,18]. 3. Biological processes Bioremediation of organic pollutant contaminated soil offers a cost-competitive treatment for many sites that are currently facing costly incineration or the extended liability of land disposal. In the eld, under conditions of full-scale site remediation, this [32, technology has been shown to be cost effective 33] . Different types of biological processes include bio-attenuation, bio-stimulation and bioaugmentation. 3.1. Bio-attenuation (Natural Attenuation) In bio-attenuation, the pollutants are transformed to less harmful forms or immobilized forms. Such transformation and immobilization processes are largely due to biodegradation by microorganisms [34] and to some extent by the reactions with www.ijsir.co.in

naturally-occurring chemicals and sorption on the geologic media. 3.2. Bio-stimulation Bio-stimulation is a process of decontamination of polluted soil in which growth of microbes is facilitated by modifying environment.The pace of microbial transformation of chemical pollutants heavily rely on supply and availability of nutrients like Carbon, Nitrogen, Potassium, available oxygen, optimum pH, redox potential and also on the type and concentration of organic pollutants [35]. To stimulate microbial degradation, nutrients in the form of fertilizers, slow release and oleophilic are [36] added . 3.3. Bio-augmentation Bio-augmentation is the addition of bacterial cultures to speed up the rate of degradation of contaminants. The contaminated soil sediments contain microora which are very well adapted to high concentration of organic pollutants. Microorganism isolated from contaminated soil sediments can be utilized for remediating soils freshly contaminated with hydrocarbons. Priming with 2% bio-remediated soil has been found to facilitate biodegradation of PAH components of [37] soil treated with fuel oil . ADVANTAGE OF BIOLOGICAL PROCESSES Biological processes have more advantages in comparison to physico-chemical processes. Harmful intermediates are not generated. Complete destruction of the xenobiotic to C1 is feasible. In-situ bioremediation of nitro-aromatics or aromatic compounds is feasible. Bioprocesses are eco-friendly and economically sustainable and enjoys wide public acceptance [38,39]. Bioremediation can often be carried out on site, often without causing a major disruption of normal activities. This also eliminates the need to transport quantities of waste off site and the potential threats to human health and the [40] environment that can arise during transportation . TYPES OF BIOPROCESS FOR ORGANIC POLLUTANT REMEDIATION Bioremediation is the process where, in controlled conditions, microorganisms have been used for the removal of pollutants from 77


contaminated sites. Bioremediation of toxic wastes can be categorized as in situ and ex situ bioremediation. The main objective is to degrade organic chemicals to concentration below the permissible limits established by higher authorities. An inoculation of potential strain enhances the destruction of pollutants in the soil. Initially, such studies have been carried out with 10–30 g of soil at rhizospheric depths. In contaminated soil (e.g.) 5.0 g parathion/kg or more than 90% of parathion was destroyed within 3 weeks because of direct inoculation of Pseudomonads Pseudomonas stutzeri and Pseudomonas aeruginosa [41]. 1. In-situ Bioremediation In situ , process is applied on contaminated site without removing contaminated material from its original position. Bioremediation which employs microorganisms to degrade toxic compounds, is an [42] attractive technology . In-situ processes include (i) Bio-attenuation (ii) Bio-stimulation (iii) Bioventing etc. Complete bioremediation of TNT (Tri Nitro Toluene) at laboratory-scale suggested that an initial anaerobic treatment was essential before the aerobic phase [43]. Rhizoremediation of TNT by colonizing Pseudomonas in plant rhizosphere has [44, 45] been reported by the researchers . Transgenic plants (tobacco) that express the onr gene from Enterobacter cloacae have also been designed for bioremediation of TNT [46]. 2. Ex-situ Bioremediation In this type of bioremediation techniques, removal of contaminated material from the source

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(soil/water) and processing it in bioreactors under controlled operating parameters (temperature, pH, and aeration) is carried out. Ex-situ processes include (i) land farming (ii) soil bio-piles (iii) composting (iv) phytoremediation (v) biorestoration and (vi) bio-stimulation. It has been reported that nitro-aromatic explosives from contaminated sediments were effectively degraded [43] at eld scale using composting . In comparison to in-situ approach this process is more expensive. In Ex-situ bioremediation process, heavily contaminated soil in a soil: water ratio of 1:1 (w/w) is stirred in a reactor to form slurry and treated under aerobic and anaerobic conditions [47]. The main drawbacks of these techniques include long incubation period within composting and lack of effective control. In land-farming, contaminated soil is mixed with nutrients and moisture and periodically aerated. Ex- situ treatment uses free and immobilized cell systems. In free cell systems, live bacteria/fungi or their consortia has been used as an inoculum to degrade the organics. It has been investigated the performance of P. putida on a sintered-glass lter plate for the bioremediation of wastewater containing 4-nitrophenol [ 4 8 ] . Researchers have reported immobilized enzymes on porous glass or silica beads which successfully decont-aminated organophosphate pesticides from soils [49]. It has been analyzed that enzymes (hydrolases) adsorbed on to soil, hydrolyzed more than 90% parathion to non-toxic products in 4 h [43]. Similarly, laccase has also been suggested to degrade nitro-phenol. In-situ and ex-situ technique of bioremediation has been summarized in Fig. 2, with reference to immobilization of biocatalysts.

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Fig. 2: In-situ and ex-situ techniques [50] degradation of these pollutants, they have evolved ADVANTAGE OF MICROORGANISMS novel pathway(s) for their metabolism. Microbes FOR ORGANIC POLLUTANT are the only entities present in the biosphere which BIOREMEDIATION possess an exceptional ability to exploit various During the last few decades, extensive research organic/inorganic compounds for their growth. has resulted in isolation of novel microbial strains Microbes are empowered to inhabit various having potential of degrading a vast array of ecological niches and pursue unusual metabolic organic compounds. Though several organic and physiological activities [38]. pollutants are relatively new for the microbes, for

Fig. 3: Bioremediation of pollutants utilizing biodegradation abilities of microorganisms [51] www.ijsir.co.in

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The bioremediation and biotransformation methods that endeavor to harness the astonishing, naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds including hydrocarbons (e.g. oil), polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), radionuclides and metals is [52] summarized in Figure 3.

PROCESS/ MECHANISM OF BIODEGRADATION OF ORGANIC POLLUTANTS In the soil, enzymes are present in complex, three dimensional assemblages of mineral and organic particles which restrict their mobility and affect their activity (Fig. 4) [53,54,55,56].

Fig. 4: Soil Bound Enzymes [6] Enzyme molecules can be adsorbed, immobilized, or entrapped in such matrices giving rise to the so[55] called naturally immobilized enzymes . Aromatic hydrocarbons are common environmental pollutants with toxic, genotoxic, mutagenic [57] and carcinogenic properties . The microorganisms have been found to be very useful in bioremediation of petroleum hydrocarbons because microbes have been found to use PAH compound as a carbon source for their growth and development. Researchers have isolated different microbial strains of Flavobacterium sp. and Pseudomonas sp from petroleum hydrocarbon contaminated soil sample; these isolates were found to degrade hydrocarbon [58] . Bacterial degradation of multiple petroleum hydrocarbons requires more than single strain to degrade it. Microbial populations that consist of strains that belong to various genera have been detected in [59] petroleum-contaminated soil or water . Microbial strains were selected based on the criteria that showed good growth in crude oil, individual [60] hydrocarbon compounds or both . Strain ability to degrade hydrocarbon contamination in the environment was investigated using soil samples that were contaminated with diesel, crude oil or 80

[60]

engine oil . Textile efuent treatment using biological methods was more suitable than [61] chemical and physical methods . Biological methods for decolorization mainly involve the use of bacteria, fungi and plants [62, 63]. Decolorization of textile dyes by using number of microorganisms has been reported [61]. ENZYMATIC DEGRADATION OF ORGANIC POLLUTANT Enzymes are biological catalysts rendering favorable conditions to enhance the conversion of substrates into products by providing favorable conditions. Enzymes tend to lower the activation energy of the reaction. Enzymes possess catalytic property which can be effective and used in bioremediation. Bioremediation as spontaneous or a managed strategy is the application of biological processes for the cleanup of hazardous chemicals present in the environment. For efcient biodegradation of pollutants it must interact with microbial enzyme system of the microbes employed. Soluble pollutants are easily taken by microbial cell while insoluble pollutants are transformed into simpler units by enzymatic action of microbes. Extra cellular enzymes include a wide www.ijsir.co.in


spectrum of oxidoreductases and hydrolases. Such enzymes may explicate a degradative function and convert polymeric substances into partially degraded or oxidized products which can be easily taken by the cells (Fig. 5). These latter in turn bring

about their complete mineralization. The partial oxidation of recalcitrant pollutants such as PAHs by extra cellular oxidative enzymes give rise to products of increased polarity and water solubility [64] and thus with a higher biodegradability .

Fig . 5: Roles of extra cellular enzymes in cell metabolism [6] It has been demonstrated that several oxidoreductases and hydrolases were extractable in a free form from soil, being the two classes of enzymes involved in the transformation of both [65] xenobiotic molecules and natural products . Enumerable bacteria and fungi have explicit enzymatic machinery to metabolize nitriles of geologic and anthropogenic origin. Oxidase activities in comparison to hydrolytic activities are more dynamic. This partly reects their varied functions such as detoxication of phenolics and reactive metals, pigment production and [66, 67, 68, 69] antimicrobial defense as well as the degradation of lignin and humics to obtain C, N and P [70]. MICROBIAL ENZYMES IN BIOREMEDIATION Microbial enzymes are catalysts of biological origin known to enhance the conversion of substrates into products by rendering favourable conditions by lowering the activation energy of the reaction. An enzyme may be a protein or a www.ijsir.co.in

glycoprotein and consists of at least one polypeptide moiety. 1. Microbial Hydrolytic Enzymes (Hydrolases) Bacteria have been very well documented to play signicant role in degradation of organic pollutants. Extracellular enzyme activity is a signicant step towards the degradation and utilization of organic polymers of high molecular weight, since pollutants with molecular mass lower than 600 daltons have the ability to pass through cell pores. Hydrolytic enzymes generally are involved in degradation of pollutants and reduction of their toxicity since they break down chemical linkages present in the toxic molecules. In the degradation of oil spill, organophosphate and carbamate insecticides, the use of hydrolytic enzymes has been found very effective. DDT, an organochlorine insecticide and heptachlor are although stable in well-aerated soil, but can be degraded readily in anaerobic environments [71, 72]. Hemicellulase, cellulose and glycosidase are few important examples of such enzymes. 81


1.1 Microbial Lipases Enzyme lipase is known to degrade lipids present in biomass of wide spectrum of microbes, animals and plants. Lipases catalyse the hydrolysis of triacylglycerols to glycerol and free-fatty acids. Lipases have been shown to be responsible for the drastic reduction in total hydrocarbon from contaminated soil [73, 74]. Lipases have been extracted from variety of organisms including bacteria, plant, actinomycetes and animal cell. Microbial lipases have been deemed as more diverse due to their enumerable applications in industries. Reactions such as hydrolysis, inter- esterication, esterication, alcoholysis and aminolysis are generally catalyzed by microbial lipases [75]. Lipase assay has been proposed as the most signicant parameter for interpreting the extent of degradation [73, 74] of hydrocarbon in soil . Lipases are used in industrial production of specic pharmaceutical products, diagnostic, bioremediation of pollutants, food, chemical, detergent manufacturing, cosmetic and paper making industries, but high cost of the production of lipases limits their industrial [76, 77] applications . 1.2 Microbial Cellulases Enzyme cellulases catalyze the conversion of waste cellulosic material into foods to cater the growing food demand of population. Cellulases have been the subject of intense research [78]. Some organisms produce cell bound, cell envelope associated or extra cellular cellulases. Cellulases are a mixture of several enzymes and three major groups of cellulases have been found to indulge in the hydrolysis of (1) endoglucanase (EG, endo1,4-D-glucanohydrolase) known to attack area of less crystallinity in the cellulose ber, creating free chain ends; (2) exoglucanase or cellobiohydrolase (CBH, 1,4-b-D-glucan cellobiohydrolase) with a potential to degrade the cellulose molecule further by eliminating cellobiose units from the free chain ends; (3) β-glucosidase which catalyzes further hydrolysis of cellobiose to glucose units. Besides of above mentioned enzymes, some ancillary enzymes are also present in microbial cell. Cellulose after enzymatic hydrolysis by cellulases is converted to reducing sugars that can be fermented by yeasts or bacteria for the production of ethanol [79]. 82

1.3 Microbial Proteases Microbial proteases catalyze the hydrolysis of proteinaceous substrate entering environment due to shedding and death of animals, moulting of appendages and also as byproduct of some industries like poultry, shery and leather. Proteases hail to the group of enzymes known to hydrolyze peptide bonds in aqueous environment and synthesize them in non-aqueous environment. In food, leather, detergent and pharmaceutical industries, proteases have diverse applications [80, 81]. Proteases as ubiquitous enzymes have gained signicant applications in biotechnology sector. Among the different proteases known, alkaline proteases have profound applications in industries like pharmaceutical, laundry detergents, leather processing proteinaceous waste bioremediation [82] and food industry . Extracellular thermoalkaline bacterial proteases are exclusively signicant for the hydrolysis of waste proteins and enable the bacteria to absorb and utilize hydrolytic products by surviving easily under extreme [83, 84] environments . It has been reported that bacterial alkaline proteases are important proteases employed in leather processing and laundry detergents [85].The alkaline protease production by Bacillus subtilis and its possible application as a depilating agent have been reported [ 8 6 ] . Researchers have reported the production of a salt tolerant protease from Pseudomonas aeruginosa BC1 and its application in tannery saline wastewater treatment [87]. However, these enzymes are mostly not signicantly active at broad temperature and pH range. 2. Microbial Oxidoreductases Oxidoreductases are known to catalyze the degradation of toxic organic compounds by enumerable bacteria and fungi and higher plants via [88] oxidative coupling . Microorganisms may harness energy through energy-yielding biochemical reactions dominated by these enzymes to break chemical linkages and to consolidate the electron transfer from a reduced organic substrate (donor) to another chemical compound (acceptor). The oxidoreductases catalyze the process of humication of variety of compounds which are of phenolic in nature produced during degradation of lignin in soil sediments. Similarly, oxidoreductases have the potential to degrade toxic xenobiotics, www.ijsir.co.in


such as phenolics or anilinic compounds, via polymerization, co-polymerization with other [89] substrates, or binding to humic substances . Microbial enzymes have also been employed in the [90, 40, 91] decolorization and degradation of azo dyes . Chlorinated phenolic compounds, produced while partial degradation of lignin during the pulp bleaching process, happen to be the most abundant recalcitrant wastes present in the waste water generated from the pulp and paper industry. Several fungal species in general and white rot fungi in particular are deemed as appropriate for removal of chlorinated phenolic compounds from the contaminated environments. The principal mechanism involved in the degradation of pollutants is the secretion of cluster of ligninolytic enzymes like laccase, lignin peroxidae and manganese peroxidase in soil by fungal mycelium. Filamentous fungi have large surface area to access [92] soil pollutants more effectively than bacteria . 2.1. Microbial Oxygenases Oxygenases belong to oxidoreductase class of enzymes and take part in oxidation of reduced substrates by transferring oxygen from molecular oxygen (O2) utilizing FAD/NADH/NADPH as a co-substrate. On the basis of number of oxygen atoms used for oxygenation, oxygenases can be categorized into; (i) monooxygenases and (ii) dioxygenases [93, 94, 95]. These enzymes either increase reactivity or water solubility of aromatic compounds or through cleavage of the aromatic ring hence, play important role in the metabolism of organic compounds. Oxygenases have a very broad substrate range and can act on wide range of compounds including the chlorinated aliphatics. Generally, the incorporation of O2 atoms into the organic molecule by oxygenase leads in cleavage of the aromatic rings. The most studied enzymes in bioremediation happen to be bacterial mono- or [93, 94, 95] dioxygenases . Haloginated compounds represent the largest group of environmental pollutants bearing widespread application as insecticides, herbicides, hydraulic, fungicides, heat transfer uids, intermediates for chemical synthesis and plasticizers. The degradation of such pollutants may be achieved by specic oxygenases. Oxygenases are also known to catalyze dehalogenation reactions of halogenated methanes, ethanes, and ethylenes in association with www.ijsir.co.in

[94]

multifunctional enzymes . 2.2 Monooxygenases Enzyme Monooxygenases act by incorporating one atom of the oxygen molecule into the substrate. Monooxygenases consists of a versatile super family of enzymes catalyzing oxidative reactions of substrates ranging from alkanes to complex endogenous molecules such as steroids and fatty acids [96, 97]. Mono-oxygenases have been very well documented to act as biocatalysts in bioremediation process and synthetic chemistry owing to their highly region-selectivity and stereo selectivity on diverse substrates. Monooxygenases catalyze a wide spectrum of reactions such as dehalogenation, desulfurization, ammonication, denitrication, biotransformation, hydroxylation and biodegradation of various aromatic and aliphatic compounds. Such properties of oxygenases have been researched properly and have been employed in recent years for several important applications in the form of biodegradation and biotransformation of aromatic [97] compounds . Methane mono-oxygenase has been characterized very well among all the monooxygenases studies up till now. Oxygenases have been reported to play important role in degradation of hydrocarbon such as substituted methanes, alkanes, cycloalkanes, alkenes, haloalkenes, ethers, and aromatic and heterocyclic hydrocarbons [98,99]. 2.3. Microbial Dioxygenases Dioxygenases are multicomponent enzyme systems reported to incorporate molecular oxygen into substrates. These dioxygenases catalyze the oxygenation of diverse substrates. In the beginning, Dioxygenases were known to catalyze the oxidation of aromatic compounds and, hence, have applications in environmental remediation. The crystal structure of naphthalene dioxygenase has conrmed the presence of a Rieske (2Fe–2S) cluster and mononuclear iron in each alpha subunit [100] . 2.4. Microbial Laccases Laccases (p-diphenol:dioxygen oxidoreductase) are a family of multicopper oxidases secreted by some plants, fungi, insects and bacteria, that has the potential to catalyze the oxidation of a wide range of reduced phenolic and aromatic substrates with 83


concomitant reduction of molecular oxygen to [88, 101] water . Several microbes have been reported to produce intra and extracellular laccases capable of catalyzing the oxidation of ortho and paradiphenols, aminophenols, polyphenols, polyamines, lignins, and aryl diamines as well as some inorganic [101,102,103] ions . Laccases besides of catalyzing the oxidation of phenolic and methoxy-phenolic acids, also perform decarboxylation and demethylation. These enzymes have been reported to be actively involved in depolymerization of lignin. Moreover, these compounds are employed as nutrients by microorganisms or repolymerized to humic materials by laccase [104]. In case of laccase, the substrate specicity and afnity has been reported to differ with change in pH. Laccase may be inhibited by variety of reagents such as halides (excluding iodide), azide, cyanide and hydroxide [104] . It has been reported that different laccases have differential tolerance toward inhibition by halides, indicating differential halide accessibility. Laccase production has been found sensitive to the nitrogen concentration in fungi. High nitrogen levels are usually essential to obtain greater amounts of [88] laccase . 2.5. Microbial Peroxidases Peroxidases (donor: hydrogen peroxide oxidoreductases) are ubiquitous enzymes bearing boundless potential to catalyze oxidation of lignin and other phenolic compounds using hydrogen peroxide in the presence of a mediator. Heme and non- heme proteins may be present in peroxidases. Peroxidases are generally involved in biological processes such as immune system or hormone regulation in mammals, However in plants, they play important role in metabolism of auxin, synthesis of lignin and suberin, defense against pathogens, cell wall component cross-linking, or [105,106] cell elongation . 2.5.1. Classification of Peroxidase Enzymes Peroxidases have been classied into different types based on its source and activity (http://peroxibase.toulouse.inra.fr/). Among peroxidases, lignin peroxidase (LiP), manganesedependant peroxidase (MnP) and versatile peroxidase (VP) have been studied extensively due to their high potential to degrade toxic substances in nature. 84

(1) Microbial Lignin Peroxidases White rot fungus during secondary metabolism secrete lignin peroxidases which are heme proteins. In the presence of substrate like veratryl alcohol and co-substrate H2O2, LiP has been found to degrade lignin and other phenolic compounds. Lignin peroxidases have been shown to oxidize halogenated phenolic compounds, polycyclic aromatic compounds and other aromatic compounds followed by a series of non-enzymatic reactions[107,108]. (2) Microbial Manganese Peroxidases MnP is an heme containing extracellular enzyme produced by the lignin-degrading basidiomycetes 2+ fungus, which catalyzes oxidation of Mn to the 3 2+ oxidant Mn + in a series of reactions. Mn not only stimulates the production of MnP but also acts as a 3+ substrate for MnP. The Mn , produced by MnP has been found to act as a mediator for the oxidation of various phenolic compounds. The resulting Mn³+ chelate oxalate is small enough to diffuse into areas otherwise inaccessible even to the enzyme, as in the case of lignin or analogous structures such as xenobiotic pollutants buried deep within the soil, which are not necessarily available to the enzymes for action[108]. (3) Microbial Versatile Peroxidases Versatile Peroxidase (VP) enzymes have capacity to catalyze the oxidation of Mn 2+, methoxy benzenes, phenolic aromatic substrates like that of MnP, LiP, and horseradish peroxidase. Versatile peroxidase has peculiar broad substrate specicity and has the potential to oxidize the substrates in the absence of manganese as compared to other peroxidases. It has been well established that versatile peroxidases can oxidize both phenolic and nonphenolic lignin model [109] dimmers . Therefore, a signicantly efcient VP overproduction system is needed for biotechnological applications in industrial processes and [110] bioremediation of recalcitrant pollutants . ADVANTAGES OVER NON- ENZYMATIC METHODS The use of microbes for bioremediation deals with many rate-limiting factors. Costly and timewww.ijsir.co.in


consuming methods may be necessary to produce microbial cultures. Many other factors that could restrict the use of microbes include limited mobility of the cells within the soil, alternate carbon sources and weakness of the inoculated microorganisms in competition with the [111] indigenous population . Biotransformation involves a series of enzyme-catalyzed reactions. Biodegradation is mediated by microbial enzymes [33] . Enzymes are complex proteins functioning as bio-oxidation catalysts that cause a host of reactions. Enzymes exhibit specicity and are characterized by showing an optimum temperature and pH for their actions. Enzymes act as catalysts and accelerate the chemical reaction rate by lowering the activation energy for a particular reaction. The activation energy for the enzymecatalyzed reaction is much smaller than that for the non-enzyme-catalyzed reaction. This results in a [111] much faster reaction rate . Enzymatic catalysis involves the formation of an intermediate complex on binding sites on the enzyme. The use of enzymes instead of chemicals or microorganisms [112] undoubtedly presents some advantages . Biotransformation does not generate toxic side products as is often the case with chemical and some microbiological processes and the enzymes are digested, in situ, by the indigenous microorganisms after the treatment. CONCLUSION Organic pollutant contaminated sites treatment through bioremediation is best because the approach is not only sustainable but eco-friendly. Moreover, there is no collateral loss of habitat q u a l i t y. T h e i m m e n s e b i o d i v e r s i t y o f microorganism in our environment offers greater potential for transformation of toxic compounds to less toxic by-products. Microbial enzymes play a major and crucial role in biodegradation of soil contaminated with organic pollutants such as diesel, petrol or PAHs compound etc. In future, these enzymes open further exploration by researchers which is likely to open a new era of microbiology aiding various environment clean up technologies. FUTURE PERSPECTIVES A great deal of work is still a waiting to establish a complete understanding of the molecular basis for catabolic sequences. Microbial bioremediation www.ijsir.co.in

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