Comparative analysis of fungal and bacterial enzymes in biodegradation of xenobiotic compounds

CHAPTER 10

Comparative analysis of fungal and bacterial enzymes in biodegradation of xenobiotic compounds Saurabh Gangola1, Samiksha Joshi2, Saurabh Kumar3 and Satish Chandra Pandey4 1

Department of Microbiology, Crop Production Division, ICAR-VPKAS, Almora, India Department of Environment Science, Graphic Era University, Dehradun, India Translational Health Science and Technology Institute, Faridabad, India 4 Cell and Molecular Biology Laboratory, Department of Zoology, Kumaun University, SSJ Campus, Almora, India 2 3

10.1 Introduction The quality of life on Earth is linked inextricably to the overall quality of the environment. Unfortunately, progress in science, technology, and industry has resulted in a large amount of waste ranging from raw sewage to nuclear waste which is let out or dumped into the ecosystem, thereby posing a serious problem for survival of humankind itself on Earth (Karigar & Rao, 2011). Manmade compounds or chemical compounds which are foreign to the biosphere are known as xenobiotics. Xenobiotic substances are becoming an increasingly large problem since they are relatively new substances and are very difficult to remove from the environment. Elements and compounds undergo a number of processes in the environment, namely condensation, sedimentation, coagulation, reaction, diffusion, and scavenging. During these processes chemicals react and transform into other compounds, persist longer in the environment, and do not degrade easily, and are known as recalcitrant compounds. Diffusion is the movement of particles from an area where their concentration is high to an area that has a lower concentration. Diffusion includes not only diffusion of particles, but all transport phenomena occurring within thermodynamic systems under the influence of thermal fluctuations. Polyaromatic, nitrogen- and halogen-containing organic compounds are recalcitrant compounds which are difficult to degrade by Smart Bioremediation Technologies DOI: https://doi.org/10.1016/B978-0-12-818307-6.00010-X

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microorganisms. Such compounds have higher bioaccumulation and biomagnification potency when they enter into biotic entities. In bioaccumulation, the organism absorbs a substance at a rate greater than that at which the substance is lost. Compounds accumulate in living things any time they are taken up and stored faster than they are broken down (metabolized) or excreted. Biomagnification describes a process that results in the accumulation of a chemical in an organism at higher levels than are found in its food. This occurs when a chemical becomes more and more concentrated as it moves up through a food chain with the dietary linkages between single-celled plants and increasingly larger animal species. Understanding the dynamic process of bioaccumulation and biomagnification is very important in protecting humans and other organisms from the adverse effects of chemical exposure, and it has become a critical consideration in the regulation of chemicals (Karigar & Rao, 2011).

10.2 Points of xenobiotic release Major sources of xenobiotic compounds entering into the environment are (1) chemical and pharmaceutical industry xenobiotics and synthetic polymers; (2) pulp and paper bleaching, which are the main sources of natural and manmade chlorinated organic compounds in the environment; (3) mining, which releases heavy metals into biogeochemical cycles; (4) fossil fuels (coal and petroleum), which may be accidentally released in large amounts into the ecosystem (oil spills); and (5) intensive agriculture, which releases massive amounts of fertilizers, pesticides, and herbicides. These are some of the examples through which xenobiotic compounds enter into the environment. Due to their potential toxicity to both wildlife and humans, several persistent organic pollutants (POPs) have now been totally banned from production and use in many countries around the world. Halogenated organic substances constitute one of the most important groups of environmental pollutants as a result of their widespread use as herbicides, insecticides, fungicides, solvents, hydraulic and heat transfer fluids, plasticizers, and intermediates for chemical syntheses. Because of their toxicity, bioconcentration, persistence, and ubiquity, halogenated compounds have raised concern over their possible effects on the quality of life (Fetzner & Lingens, 1994). Many xenobiotics (pesticides) produce a variety of biological effects, which are used when they are characterized using bioassay. Before they can be registered for sale in most countries,

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xenobiotic pesticides must undergo extensive evaluation for risk factors, such as toxicity to humans, ecotoxicity, or persistence in the environment.

10.3 Alternative degradation approach Degradation of such compounds by physical and/or chemical processes is costly and often produces undesirable products and intermediates which are toxic. The main degraders in nature are microorganisms, mostly bacteria and some fungi. These organisms, due to their rapid rates of multiplication and great metabolic potential, are able to adapt to new substrates. Selection of degradative potent microorganisms and their successive adaptation to a naturally persistent compound might be a powerful means for environmental detoxification. Better understanding of metabolic pathways for the biodegradation of specific organic compounds as well as a more thorough knowledge of degrading microorganisms will make purposeful application of biodegradation possible. Biological methods, being ecofriendly and cheap techniques, were proposed for xenobiotic degradation purposes in order to overcome these problems. Successful bioremediation requires potential microbial strains which can degrade hazardous pollutants in a short time without producing toxic intermediates. Knowledge of microbial interactions in the soil environment is advantageous to expedite the process in minimal time. Microorganisms participate in biological recycling of various natural and manmade chemicals. They have evolved extensive range of enzymes, pathways, and biochemical control mechanisms in order to degrade and utilize pollutants as a source of carbon and energy. Mechanisms of bioremediation include both genetically engineering microorganisms and isolating the naturally occurring xenobiotic-degrading microbes. Xenobiotics may be limited in the environment and difficult to access in areas such as the subsurface environment. Degradative organisms can be engineered to increase mobility in order to access these compounds, including enhanced chemotaxis (Díaz, 2004). One limitation of the bioremediation process is that optimal conditions are required for proper metabolic functioning of certain microorganisms, which may be difficult to meet in an environmental setting (Singh, 2004). In some cases a single microorganism may not be capable of performing all metabolic processes required for degradation of a xenobiotic compound and so “syntrophic bacterial consortia” may be employed (Díaz, 2004). In this case, a

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group of bacteria work in conjunction, resulting in dead-end products from one organism being further degraded by another organism (Singh, 2004). In other cases, the products of one microorganisms may inhibit the activity of another, and thus a balance may be maintained (Díaz, 2004).

10.4 Limitations of bioremediation The process of bioremediation is a very slow process. Only certain species of bacteria and fungi have proven their ability as potent pollutant degraders. Many strains are known to be effective as bioremediation agents but only under laboratory conditions. Seasonal variation of microbial activity results in direct exposure to prevailing environmental factors, and lack of control of these factors. Although microorganisms can exist in extreme environments, most prefer optimal conditions—a situation that is difficult to achieve outside the laboratory (Dana & Bauder, 2011; Vidali, 2001). There can be problematic utilization of treatment additives such as nutrients, surfactants, and oxygen. Microorganisms act well only when the waste materials help them to generate more cells. When native microorganisms lack biodegradation capacity, genetically engineered microorganisms may be added to the site during in situ bioremediation. Most bioremediation systems operate under aerobic conditions, but anaerobic environments may also permit microbial degradation of recalcitrant molecules. Both bacteria and fungi rely on the participation of different intracellular and extracellular enzymes, respectively, for the remediation of recalcitrant and organopollutants (Vidali, 2001). Bacterial species like Pseudomonas, Escherichia, Sphingobium, Pandoraea, Rhodococcus, Gordonia, Bacillus, Moraxella, Micrococcus (aerobic bacteria), Pelatomaculum, Desulfotomaculum, Syntrophobacter, Syntrophus, Desulphovibrio, Methanospirillum, Methanosaeta (anaerobic bacteria) and fungal species like Phanerochaete chrysosporium, Aspergillus niger, Aspergillus ochraceus, Penicillium chrysogenum, Fusarium sp., Cladosporium sp., Candida tropicalis SK21 (aerobic), Neocallimastix, Piromyces, Ontomyces, Buwchfawromyces, Anaeromyces, Orpinomyces, Cyllamyces, Caecomyces (Anaerobic), etc. have been isolated from soil and characterized for their biodegradation potential of xenobiotic compounds (DDT, lindane, PCBs, TNT, and crystal violet) (Ellouze & Sayadi, 2016; Leis et al., 2014; Liggenstoffer, Youssef, Couger, & Elshahed, 2010). Communities of fungi and bacteria have vital functions in the environment, as they individually or in combination degrade and mineralize organic compounds (Frey-Klett et al., 2011). It is therefore also

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very plausible that the consortia consisting of fungi and bacteria in some cases may be more suitable in a bioremediation than single strains. Different parameters are considered to be taken into account to construct efficient consortia for bioremediation. Interactions between the two groups are important to understand the mechanisms within the consortia. Therefore, the goal of bioremediation is to reduce pollutant levels to acceptable levels, that is, within the limits set by regulatory agencies (Pointing, 2001). However, from an environmental point of view, total mineralization of toxic chemicals is desirable as it represents complete detoxification (Gan & Koskinen, 1998).

10.5 Strategies for biodegradation Among others, the composition and size of soil microbial populations, as well as the status of metabolic activity, are the determining factors as to whether or not biodegradation is feasible as a remediation option. Biodegradation refers to the metabolic ability of microorganisms to transform organic contaminants into less harmful compounds (Margesin, Walder, & Schinner, 2000). According to McFarland, Salladay, Ash, and Baiden (1996) bioremediation techniques aim to accelerate the naturally occurring biodegradation process by optimizing the conditions under which it occurs. In many contaminated areas, even though suitable microbial populations may be available for biodegradation of a given contaminant, environmental conditions may limit or even inhibit this process (Margesin et al., 2000). In such cases, biostimulation of the degrading potential of native microbial populations and/or the addition of selected degrading microorganisms to contaminated soil (bioaugmentation) have been effective at enhancing pesticide metabolism (Ghani, Wardle, Rahman, & Lauren, 1996; Han, Choi, & Song, 2004). Biostimulation typically involves the addition of limited nutrients (e.g., carbon and nitrogen sources, O2), acid or bases for pH optimization, or water or specific substrates to stimulate specific enzymes. It is an effective bioremediation strategy (Margesin et al., 2000; McFarland et al., 1996), although it may have poor reproducibility and be dependent on the characteristics of microbial populations (Gadd, 2001). Alternatively, bioaugmentation is an attractive option. Indeed, the major advantage offered by bioaugmentation is the ability to choose the introduced species based on the goals of the process and the conditions of the matrix. In this case, the success of bioremediation is mainly dependent on the

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competition/proliferation capability of the introduced species and the bioavailability of the xenobiotic compounds (Khadrani, Seigle-Murandi, Steiman, & Vroumsia, 1999). Bioavailability here refers to the acquisition and subsequent transformation/degradation of a compound (Gadd, 2001) and is closely related to its chemical properties, as well as to a wide range of soil physical and chemical parameters. Complete biodegradation ultimately results in the mineralization of xenobiotic compounds to CO2 and water (Han et al., 2004). In addition to mineralization, which implies the use of xenobiotics as a carbon source, microorganisms can also cometabolize xenobiotics, for example, transforming them into metabolites while growing and obtaining energy from other substrates found in soil (Gadd, 2001; Khadrani et al., 1999). Many of the reactions involved in cometabolism of pesticides, including oxidation reduction, dehalogenation, ring-cleavage, and hydrolysis, occur simultaneously (Gadd, 2001). Transformation can lead to complete detoxification, breakdown of products, which may be further attacked by other microbial groups. This technology offers the potential to treat contaminated soil and groundwater on site without the need for excavation (Balba, Al-Awadhi, & Al-Daher, 1998; Khadrani et al., 1999), requires relatively little energy input, and preserves the soil structure (Houot, Barriuso, & Bergheaud, 1998). Perhaps the most attractive feature of bioremediation is its reduced impact on natural ecosystems, which should be well received by the public (Zhang & Qiao, 2002). For fungal systems, bioremediation requires the soil to be aerobic with the provision of enough oxygen to enable effective colonization to occur.

10.6 Aerobic and anaerobic degradation As reflected by the vast knowledge about aerobic degradation mechanisms in comparison to anaerobic ones (e.g., Neilson & Allard, 2008), aerobic conditions are generally reported to be more effective for the removal of most organic contaminants.

10.6.1 The essential characteristics of aerobic microorganisms degrading organic pollutants 1. Metabolic processes for optimizing the contact between the microbial cells and the organic pollutants. The chemicals must be accessible to the organisms having biodegrading activities. For example,

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hydrocarbons are water-insoluble and their degradation requires the production of biosurfactants (Dana & Bauder, 2011). 2. The initial intracellular attack of organic pollutants is an oxidative process, the activation and incorporation of oxygen is the enzymatic key reaction catalyzed by oxygenases and peroxidases. 3. Peripheral degradation pathways convert organic pollutants step by step into intermediates of the central intermediary metabolism, for example, the tricarboxylic acid cycle. 4. Biosynthesis of cell biomass from the central precursor metabolites, for example, acetyl-CoA, succinate, pyruvate. Sugars required for various biosyntheses and growth must be synthesized by gluconeogenesis (Fritsche & Hofrichter, 2000). Enzymatic key reactions of aerobic biodegradation are oxidations catalyzed by oxygenases and peroxidases. Oxygenases are oxidoreductases that use O2 to incorporate oxygen into the substrate. Degradative organisms need oxygen at two metabolic sites, at the initial attack of the substrate and at the end of the respiratory chain. Distinct higher fungi have developed a unique oxidative system for the degradation of lignin based on extracellular ligninolytic peroxidases and laccases. This enzymatic system possesses increasing significance for the cometabolic degradation of persistent organopollutants. In most cases the domain of basidiomycetous fungi is reported, which requires a deeper insight and extensive consideration.

10.6.2 Anaerobic microorganisms degrading organic pollutants In the absence of oxygen (anaerobic condition), aromatic compounds (xenobiotic) act as an electron-donating substrate and the microorganisms’ growth occurs by oxidation of these compounds in the presence of a terminal electron acceptor. A basic pathway for anaerobic biodegradation of polycyclic aromatic hydrocarbons is demonstrated in Fig. 10.1. Due to lack of aeration and transformation of organic compounds to methane, which acts as an energy source, the anaerobic process requires less energy and has the ability to control temperature. Hydrolytic enzymes adsorb to the degradable surface areas and their adsorption capabilities determine the hydrolysis rate of particulate organic compounds. The bioenergy produced from the different breakdown phases of complex organic matter can be used as a biofuel (Gupta & Verma, 2015). Besides the advantages of the anaerobic process, there is one downside which is the requirement

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Figure 10.1 Bioremediation of xenobiotics via an anaerobic mechanism.

for a longer start-up time for improving required biomass and sensitivity to temperature (Sayara, Pognani, Sarrà, & Sánchez, 2010). Several pollutants, such as highly halogenated aromatic compounds, however, have been shown to be more easily degraded under strictly anaerobic conditions (Vogel, Criddle, & McCarty, 1987). However, little is known about the organisms and enzymatic processes involved in anaerobic degradation of these compounds. Anaerobic conditions frequently occur in environmental matrices, such as soils, sediments, and aquifers (Bethke, Sanford, Kirk, Jin, & Flynn, 2011). Nevertheless, the types of microorganisms and metabolisms adapted toward these oxygen-free habitats strongly depend on the availability of electron acceptors and their respective energy yield. After oxygen depletion by aerobic respiration, nitrate can be used as an alternative electron acceptor by nitrate-reducing or denitrifying bacteria. As denitrifying bacteria are commonly facultative anaerobes which easily switch between aerobic and anoxic respiration, it is difficult to distinguish between an aerobic and anoxic reaction under denitrifying conditions, if the exclusion of oxygen cannot be guaranteed (Meiberg, Bruinenberg, & Harder, 1980; Wilson & Bouwer, 1997). Azo groups are generally amenable to both, aerobic and anaerobic cleavage (Pandey, Singh, & Iyengar, 2007; Saratale, Saratale, Chang, &

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Govindwar, 2011; Singh, Singh, & Singh, 2015), most azo dyes are not cleaved under aerobic conditions in conventional wastewater treatment, while strictly anaerobic conditions facilitate the reductive cleavage of the azo-bond to amino moieties (Dos Santos, Cervantes, & Van Lier, 2007; Solís, Solís, Pérez, Manjarrez, & Flores, 2012). This has been extensively studied for the treatment of wastewater from dyeing and dyemanufacturing industries, in anaerobic enrichment and pure cultures as well as in cell-free extracts. Due to the high molecular weight and commonly added sulfonate groups on azo-dyes, the degradation is thought to primarily proceed via extracellular cleavage of the azo-bond by the transfer of two times two electrons. The electrons are transferred by extracellular redox mediators which are reduced by a membrane-bound azo reductase, which in return depends on the supply of intracellular reducing equivalents, such as NAD(P)H or FADH2 (Solís et al., 2012).

10.7 Enzymatic bioremediation For enzymatic bioremediation, first an enzyme is selected for a bioremediation application; it needs to have the capacity to degrade the target contaminant into less-toxic products. It is also important to search for enzymes that do not depend on cofactors, which would increase process costs at the commercial level. After screening, the next step is to identify the gene encoding the selected enzyme and, if necessary, improve enzymatic production. Commercial companies produce their enzymes via large-scale industrial fermentation; unlysed cells are removed during downstream processing. The shelf-life and environmental stability of enzymes must be evaluated to ensure effectiveness of the enzyme against the contaminant (Fig. 10.2). Enzymatic bioremediation by PAH detoxification can be achieved by the use of laccases (Alcalde et al., 2005) (enzymes capable of catalyzing the oxidation of phenols, polyphenols, and anilines, coupled to the four-electron reduction of molecular oxygen to water) (Smith, Thurston, & Wood, 1997). A great advantage of the enzymatic bioremediation of xenobiotics that are either hydrophobic or poorly soluble in aqueous solutions, such as PAHs, is that enzymatic oxidation can occur in the presence of organic solvents (Alcalde et al., 2005). A disadvantage is that the relevant enzymes can be unstable, inhibited, or denatured in organic solvents. In the work of Bulter and colleagues (Bulter et al., 2003) laccase was expressed from Myceliophthora thermophila (MtL) in Saccharomyces cerevisiae, using directed evolution, and extensively improved laccase expression.

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Figure 10.2 Schematic approach toward enzymatic bioremediation of xenobiotic compounds.

10.8 Microbial enzymes in bioremediation 10.8.1 Microbial oxidoreductases The detoxification of toxic organic compounds by various bacteria and fungi (Gianfreda, Xu, & Bollag, 1999) through oxidative coupling is mediated with oxidoreductases. Microbes extract energy via energyyielding biochemical reactions mediated by these enzymes to cleave chemical bonds and to assist the transfer of electrons from a reduced organic substrate (donor) to another chemical compound (acceptor). During such oxidation reduction reactions, the contaminants are finally oxidized to harmless compounds. The oxidoreductases participate in the humification of various phenolic substances that are produced from the decomposition of lignin in a soil environment. In the same way, oxidoreductases can also detoxify toxic xenobiotics, such as phenolic or anilinic compounds, through polymerization, copolymerization with other substrates, or binding to humic substances (Park, Park, & Kim, 2006). Microbial enzymes have been exploited in the decolorization and degradation of azo dyes (Husain, 2006). Many fungal species are considered to be suitable for the removal of chlorinated phenolic compounds from contaminated environments. The activity of fungi is mainly due to the action of extracellular

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oxidoreductase enzymes, like laccase, manganese peroxidase, and lignin peroxidase, which are released from fungal mycelium into their nearby environment. Being filamentous, fungi can reach soil pollutants more effectively than bacteria (Rubilar, Diez, & Gianfreda, 2008).

10.8.2 Microbial oxygenases Oxygenases belong to the oxidoreductase group of enzymes. They participate in oxidation of reduced substrates by transferring oxygen from molecular oxygen (O2) utilizing FAD/NADH/NADPH as a cosubstrate. Oxygenases are grouped into two categories, the monooxygenases and dioxygenases, on the basis of the number of oxygen atoms used for oxygenation. They play a key role in the metabolism of organic compounds by increasing their reactivity or water solubility or bringing about cleavage of the aromatic ring. Oxygenases have a broad substrate range and are active against a wide range of compounds, including the chlorinated aliphatics. Generally the introduction of O2 atoms into the organic molecule by oxygenase results in cleavage of the aromatic rings. Historically, the most studied enzymes in bioremediation are bacterial mono- or dioxygenases (Karigar & Rao, 2011). Halogenated organic compounds comprise the largest groups of environmental pollutants as a result of their widespread use as herbicides, insecticides, fungicides, hydraulic and heat transfer fluids, plasticizers, and intermediates for chemical synthesis. The degradation of these pollutants is achieved by specific oxygenases. Oxygenases also mediate dehalogenation reactions of halogenated methanes, ethanes, and ethylenes in association with multifunctional enzymes (Fetzner & Lingens, 1994).

10.8.3 Monooxygenases Monooxygenases incorporate one atom of the oxygen molecule into the substrate. Monooxygenases are classified into two subclasses based on the presence cofactor: flavin-dependent monooxygenases and P450 monooxygenases. Flavin-dependent monooxygenases contain flavin as a prosthetic group and require NADP or NADPH as coenzyme. P450 monooxygenases are heme-containing oxygenases that exist in both eukaryotic and prokaryrotic organisms. The monooxygenases comprise a versatile superfamily of enzymes that catalyzes oxidative reactions of substrates ranging from alkanes to complex endogenous molecules such as steroids and fatty acids. Monooxygenases act as biocatalysts in the bioremediation process

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and synthetic chemistry due to their highly region selectivity and stereoselectivity on a wide range of substrates. The majority of monooxygenases studied previously have a cofactor, but there are certain monooxygenases which function independently of a cofactor. These enzymes require only molecular oxygen for their activities and utilize the substrate as a reducing agent (Arora, Srivastava, & Singh, 2010; Cirino & Arnold, 2002). The desulfurization, dehalogenation, denitrification, ammonification, hydroxylation, biotransformation, and biodegradation of various aromatic and aliphatic compounds are catalyzed by monooxygenases. Methane monooxygenase enzyme is the best characterized one among the monooxygenases. This enzyme is involved in the degradation of hydrocarbons such as substituted methanes, alkanes, cycloalkanes, alkenes, haloalkenes, ethers, and aromatic and heterocyclic hydrocarbons (Grosse et al., 1999). Under oxygen-rich conditions, monooxygenase catalyzes oxidative dehalogenation reactions, whereas under low oxygen levels, reductive dechlorination takes place.

10.8.4 Microbial dioxygenases Dioxygenases are multicomponent enzyme systems that introduce molecular oxygen into their substrate. Aromatic hydrocarbon dioxygenases belong to a large family of Rieske nonheme iron oxygenases. These dioxygenases catalyze enantiospecifically the oxygenation of a wide range of substrates. Dioxygenases primarily oxidize aromatic compounds and, therefore, have applications in environmental remediation. All members of this family have one or two electron transport proteins preceding their oxygenase components. The crystal structure of naphthalene dioxygenase has confirmed the presence of a Rieske (2Fe 2S) cluster and mononuclear iron in each alpha subunit (Dua, Singh, Sethunathan, & Johri, 2002). The catechol dioxygenases serve as part of nature’s strategy for degrading aromatic molecules in the environment. They are found in soil bacteria and are involved in the transformation of aromatic precursors into aliphatic products. The intradiol cleaving enzymes utilize Fe(III), while the extradiol cleaving enzymes utilize Fe(II) and Mn(II) in a few cases (Que & Ho, 1996).

10.8.5 Microbial laccases Laccases (p-diphenol:dioxygen oxidoreductase) constitute a family of multicopper oxidases produced by certain plants, fungi, insects, and bacteria,

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that catalyze the oxidation of a wide range of reduced phenolic and aromatic substrates with a concomitant reduction of molecular oxygen to water (Mai, Schormann, Milstein, & Hüttermann, 2000). Laccases are known to occur in multiple isoenzyme forms, each of which is encoded by a separate gene, and, in some cases, the genes have been expressed differently depending upon the nature of the inducer (Rezende, Barbosa, Vasconcelos, Haddad, & Dekker, 2005). Many microorganisms 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 ions (Couto & Herrera, 2006). Laccases not only oxidize phenolic and methoxyphenolic acids, but also decarboxylate them and attack their methoxy groups (demethylation). These enzymes are involved in the depolymerization of lignin, which results in a variety of phenols. In addition, these compounds are utilized as nutrients for microorganisms or repolymerized to humic materials by laccase (Kim, Park, Lee, & Kim, 2002). Among the biological agents, laccases represent an interesting group of ubiquitous, oxidoreductase enzymes that show promise of offering great potential for biotechnological and bioremediation applications (Gianfreda et al., 1999).

10.8.6 Microbial peroxidases Peroxidases (donor: hydrogen peroxide oxidoreductases) are ubiquitous enzymes that catalyze the oxidation of lignin and other phenolic compounds at the expense of hydrogen peroxide (H2O2) in the presence of a mediator.

10.8.7 Microbial lignin peroxidases Lignin peroxidases are heme proteins secreted mainly by the white rot fungus during secondary metabolism. In the presence of cosubstrate H2O2 and mediator-like veratryl alcohol LiP degrade lignin and other phenolic compounds. During the reaction, H2O2 is reduced to H2O with the gaining of an electron from LiP (which itself gets oxidized). The LiP (oxidized), by gaining an electron from veratryl alcohol, returns to its native reduced state, and veratryl aldehyde is formed. Veratryl aldehyde then is again reduced back to veratryl alcohol by gaining an electron from the substrate. This results in the oxidation of halogenated phenolic compounds, polycyclic aromatic compounds, and other aromatic compounds,

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followed by a series of nonenzymatic reactions (Ten Have & Teunissen, 2001; Yoshida, Chatani, Honda, Watanabe, & Kuwahara, 1998).

10.8.8 Microbial manganese peroxidases MnP is an extracellular heme enzyme from the lignin-degrading Basidiomycetes fungus, that oxidizes Mn21 to the oxidant Mn31 in a multistep reaction. Mn21 stimulates MnP production and functions as a substrate for MnP. The Mn31 generated by MnP acts as a mediator for the oxidation of various phenolic compounds. The resulting Mn31 chelate oxalate is small enough to diffuse into areas 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 (Ten Have & Teunissen, 2001).

10.8.9 Microbial versatile peroxidases VP enzymes are able to directly oxidize Mn21, methoxybenzenes, phenolic aromatic substrates like that of MnP, LiP, and horseradish peroxidase. VP has extraordinary broad substrate specificity and a tendency to oxidize the substrates in the absence of manganese when compared to other peroxidases. It has also been demonstrated that VP is able to oxidize both phenolic and nonphenolic lignin model dimers (Ruiz-Duenas et al., 2007). Therefore, a highly efficient VP overproduction system is desired for biotechnological applications in industrial processes and bioremediation of recalcitrant pollutants (Tsukihara, Honda, Sakai, Watanabe, & Watanabe, 2006). Bacterial activity is the major process involved in the hydrolysis of organic pollutants. Extracellular enzyme activity is a key step in the degradation and utilization of organic polymers, since only compounds with a molecular mass lower than 600 Da can pass through cell pores (VasilevaTonkova & Galabova, 2003). Hydrolytic enzymes disrupt major chemical bonds in the toxic molecules which results in the reduction of their toxicity. This mechanism is effective for the biodegradation of oil spills and organophosphate and carbamate insecticides. Organochlorine insecticides such as DDT and heptachlor are stable in well-aerated soil but readily degrade in anaerobic environments (Vasileva-Tonkova & Galabova, 2003). Hydrolases also catalyze several related reactions including condensations and alcoholysis. The main advantages of this enzyme class are ready

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availability, lack of cofactor stereoselectivity, and tolerance of the addition of water-miscible solvents (http://www.wiley-vch.de/publish/dt/).

10.8.10 Microbial lipases Lipase degrades lipids derived from a large variety of microorganisms, animals, and plants. Recent works have shown that lipase is closely related to the organic pollutants present in soil. Lipase activity was responsible for the drastic reduction in total hydrocarbon from contaminated soil. Research undertaken in this area is likely to progress the knowledge in the bioremediation of oils spills (Riffaldi, Levi-Minzi, Cardelli, Palumbo, & Saviozzi, 2006). Lipases have been extracted from bacteria, plants, actinomycetes, and animal cells. Among these, microbial lipases are more versatile because of their potent application in industries. These enzymes can catalyze various reactions such as hydrolysis, interesterification, esterification, alcoholysis, and aminolysis (Prasad & Manjunath, 2011).

10.8.11 Esterase Esterase belongs to the hydrolase group and is capable of hydrolyzing a large number of ester-containing compounds, such as carbamates, organophosphates, and pyrethroids. Some pyrethroid-degrading enzymes have been purified and characterized. To date only a few pyrethroid-degrading genes, such as estP, pytH, pye3, pytH, and pytZ, have been reported by Ruan et al. (2013). These genes responsible for the degradation of cypermethrin may also be involved in the degradation of the sulfosulfuron and fipronil. More than 300 genes involved in the catabolism of aromatic compounds have been cloned and identified from culturable bacteria (Goyal & Zylstra, 1996). In general there is a positive correlation between the relative abundance of the genes involved in bioremediation and the potential for contaminant degradation (Rogers, Ong, Kjartanson, Golchin, & Stenback, 2002). Therefore, there has been an increased emphasis on quantifying the levels of mRNA to characterize bioremediation genes. Often an increased mRNA concentration can be, at least qualitatively, associated with higher rates of contaminant degradation. Analysis of the mRNA level for expression of genes other than those directly involved in bioremediation might yield an additional insight into the mechanisms that control the rate and extent of bioremediation (Lovley, 2003).

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Lipase activity was found to be the most useful indicator parameter for testing hydrocarbon degradation in soil (Prasad & Manjunath, 2011; Riffaldi et al., 2006). Lipase is of much interest in the production of regiospecific compounds which are employed in the pharmaceutical industry. Along with its diagnostic usage in bioremediation, lipase has many potential applications in food, chemical, detergent manufacturing, cosmetic, and paper-making industries, but its production cost has restricted its industrial use (Sharma, Sharma, & Shukla, 2011).

10.9 Molecular biology, metabolic engineering, and future prospects Despite all the advantages related to enzymatic bioremediation, high production costs, low yields, and enzymatic inhibition are some of the problems that must be overcome before its more widespread application. Many production improvements are necessary to avoid nonprohibitive processes. Therefore, molecular tools are being widely explored to provide competitive enzymatic bioremediation products. Molecular tools allow us to detect genes related to degrading enzymes in environmental samples or isolates, thus serving as powerful tools for bioprospection. Furthermore, DNA engineering can considerably improve enzyme yield with lower costs (Alcalde, Ferrer, Plou, & Ballesteros, 2006). Many PCR primers that target genes related to petroleum-degrading enzymes, both in aerobic and anaerobic conditions, have already been described. The utilization of these already-characterized primers may facilitate environmental screening of degrading abilities and may help to evaluate the potentials of microbial isolates. More primers can be described for specific pathways or to improve the comprehensiveness of known primers using available databases (Peixoto, Vermelho, & Rosado, 2011). The benefits provided by molecular tools can open unlimited windows of opportunity, as it is possible to detect genes from cultivable or noncultivable organisms (using metagenomics) and to express these genes in cultivable organisms, using enzymes that are not yet described. For instance, the use of fosmid and cosmid shotgun metagenomic libraries offers a great improvement to the bioprospection of new enzymes. The possibility of identifying and using genes from yet-undescribed microorganisms increases possible enzyme targets from about 0.1% to 1% of microbial cells (consisting of cultivable microbial cells in environmental samples), including all available DNA in that sample (Zeyaullah et al., 2009).

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Molecular tools also allow us to increase expression levels, manipulating not only physiochemical conditions (optimal conditions), but also the organisms at a genetic level, to improve enzyme production in many different conditions, for instance, improving the efficiency and speed of petroleum degradation, decreasing the time of the remediation process. Genetic manipulation would be also useful to allow or improve petroleum degradation in extreme environments, such as cold or hypersaline sites. The use of free extremozymes would be advantageous in these environments, since it avoids some of the limitations of bioremediation using whole cells in extreme conditions, such as microbial competitiveness.

10.10 Conclusion Considering that bioremediation remains a field with much work to be done, with few extremely effective field applications due to the extremely diverse conditions found in different ecosystems, the development of alternative or complementary strategies is continually encouraged. Despite the fact that in many cases the costs are still prohibitive, enzymatic bioremediation can provide real benefits to the environment, avoiding the conditions that are required for whole-cell applications, especially in extreme environments. Furthermore, enzymatic effectiveness can be improved in vitro also using molecular tools, such as DNA engineering, to generate super bioremediators, which can present advantages in the field. Enzymatic bioremediation also influences other biological areas, such as medicine, since Rittmann and Schloendorn (2007) proposed the idea of “medical bioremediation,” based on and inspired by environmental bioremediation principles. Medical bioremediation studies have proposed the utilization of one or several microbial enzymes to degrade intracellular accumulators that impair cellular function and viability and cause diseases such as atherosclerosis, macular degeneration, and neurodegenerative diseases. Medical bioremediation could be effective enough to eliminate intracellular accumulators from affected cells. Our experience with whole cells indicates that bacterial consortia are a better alternative for the degradation of diverse and complex petroleum compounds. Likewise, the use of an enzyme mixture is probably a more suitable tool for use against petroleum contamination in the environment because specific enzymes for recalcitrant and toxic compounds can be applied together.

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If we see the overall growth rate and the adaptation to the environment, bacteria are more dominant over fungi because bacteria multiply more rapidly and adapt to low acidic pH to higher alkaline pH in the environment. Meanwhile most of the fungi usually grow in acidic pH and the growth rate of fungi is also slower than in bacteria. In contrast the metabolic rate of fungi is much faster than bacteria. Therefore to solve the adaptation and growth rate problem, bacterial and fungal enzymes are utilized for the bioremediation or a fungal degradative gene could be cloned into the bacterium genome.

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