**1. Introduction**

Pesticides play an important role in modern agriculture. Synthetic pesticides are recognized as a cost-effective method of controlling pests, improving productivity and food quality. However, while pesticides may have a beneficial effect on agricultural productivity, their indiscriminate use causes many serious problems to the environment and human health, since these compounds are toxic to non-target species (Diez, 2010; Coutinho *et al.*, 2005).

The fate of pesticides in the environment is influenced by many processes (biological, chemical and physical) that determine their persistence and mobility (Gravilescu, 2005). Millions of tons of pesticides are applied annually, but it is believed that only a small fraction of these products effectively reaches the target organisms, and the remainder are deposited on the soil, contam‐ inating non-target organisms and moving into the atmosphere and water (Eerd *et al.*, 2003). Since many pesticide types are recalcitrant, they remain for a long time in soils and sediments, where they can enter the food chain directly or percolate into the groundwater (Rissato *et al.*, 2004; Gravilescu, 2005).

Detoxification of pesticides *in situ* has been achieved by treatment of the contaminated soil with certain microorganisms or plants, a technology known as bioremediation or more specifically, phytoremediation in the case of plants (Sutherland *et al.*, 2002). These microor‐ ganisms are the main biological agents capable of removing and degrading waste materials, to enable their recycling in the environment (Chowdhury *et al.*, 2008). Since the conventional treatment options for the pesticide residues clean-up in the environment include removal of the contaminated material to be incinerated or disposed in landfills, *in situ* biological reme‐

© 2013 da Silva et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. diation is seen as a safer, less disruptive and more cost-effective alternative treatment (Sutherland *et al.*, 2004).

Effective techniques for soil bioremediation are bioaugmentation, biostimulation, phytoreme‐ diation and enzymatic bioremediation. However, the three first techniques are limited by their dependence upon the growth rate of the remediating plants and microbes, which will vary with nutrients, aeration, pH and other factors relating to the contaminated soil (Scott *et al.*, 2008; Sutherland *et al.*, 2004). A successful bioremediation technique requires efficient organ‐ isms that can degrade pollutant to a minimum level. In the case of pesticides, an adequate rate of biodegradation is required to attain the acceptable level of a pesticide residue or its metab‐ olites at the contaminated site in a limited period of time (Singh, 2008).

Organophosphate pesticides (OPs) are used worldwide in agriculture, municipal hygiene, disease vector control and against household pests; they were also a group of compounds used historically as chemical warfare agents (Yang *et al.*, 2008; Zheng *et al.*, 2007; Edwards and Tchounwou, 2005). OPs are phosphorus-containing pesticides whose insecticidal qualities were first observed in Germany during World War II (Edwards and Tchounwou, 2005). The principal types are phosphotriesters, thiophosphotriesters, and phosphorothiolesters. Phos‐ photriesters contain a phosphate center with three *O*-linked groups, thiophosphotriesters have the phosphoryl oxygens replaced by sulfur and in phosphorothiolesters, one or more of the ester oxygen are replaced by sulfur (Figure 1) (Bigley and Raushel, 2013 ).

This chemical class of pesticides has been used to replace the organochlorine pesticides, banned in the United States since the 1970s (Jauregui *et al.*, 2003). However, the OPs are also highly toxic pesticides, since they are potent irreversible acetylcholinesterase (AChE) inhibitors that have a profound effect on the nervous system of exposed organisms, including human beings (Edwards and Tchounwou, 2005).

The hydrolysis mechanism normally catalyzed by AChE depends on the attack of a serine residue at the active site on the carbonyl group in ACh, but in the presence of organophos‐ phates, this residue is readily phosphorylated, as follows: a histidine residue at the active site captures a proton from the serine residue, increasing its nucleophilic character, so that it readily attacks the electrophilic phosphorus atom, releasing the leaving group (X) (Figure 2). Unlike the acetylated enzyme, the phosphorylated enzyme reacts slowly with water, allowing the dealkylation of the alkoxy substituent (R2) attached to the phosphorus atom. The organophos‐ phate compounds thus inactivate acetylcholinesterase by phosphorylation of the serine at the enzyme active site. The result is the formation of a strong hydrogen bond between a protonated histidine residue of the catalytic site and the negatively charged oxygen atom of the inhibitor. Therefore, the protonated histidine cannot function as a general base catalyst for the hydrolysis of the phosphorylated enzyme, which is a necessary step for the reactivation of AChE (Figure 2) (Mileson *et al.*, 1998; Santos *et al.*, 2007)

According the Brazillian Food, Drug and Sanitary Surveillance Agency (ANVISA), analysis of pesticide waste in food showed that OPs are those with the greatest number of occurrences in unsatisfactory samples. Among then, chlorpyrifos, methamidophos and acephate are the main active ingredients responsible for food contamination. Profenofos appeared to be the 12th

6

APPENDIX

NO2

Cl (A) Phosphotriesters

Cl

chlorpyrifos methyl parathion

P S O O O

Figure 1. Chemical structures of main class of OPs. (A) Phosphotriesters; (B) Thiophosphotriesters; (C) Phosphorothiolesters

**Figure 1.** Chemical structures of main class of OPs. (A) Phosphotriesters; (B) Thiophosphotriesters; (C) Phosphorothio‐

Ser O

Figure 5. Marine fungi growing on solid culture medium containing various concentrations of profenofos pesticide (10 days

oxydemeton methyl

S

O-

O

H H

Glu

O-

Glu

H N N

H N N

R2

+


His

O P O

<sup>R</sup> <sup>O</sup> <sup>1</sup>

+

His

O P R2O R1

+ -

H

O X

Ser

Ser

O

P OO S O

dichlorvos

P OO O O

NO2

Cl Cl

Biodegradation of the Organophosphate Pesticide Profenofos by Marine Fungi

P OO S O N

P OO O O

Cl

tetrachlorvinphos

Cl

http://dx.doi.org/10.5772/56372

149

diazinon

N O

O

phosmet

P S O O O

N

Cl

P OO S O

P S O S O

:

P X

R2O <sup>O</sup>

R1

His

inactive enzyme

H N N

+

H O ¨

O <sup>P</sup> O-

<sup>R</sup> <sup>O</sup> <sup>1</sup>

Ser

**Figure 2.** Mechanism of inhibition of acetylcholinesterase by organophosphate pesticides (Santos *et al.*, 2007)

His

malathion

P OO O O

(B) Thiophosphotriesters

(C) Phosphorothiolesters

O

O

Glu

O-

Glu

<sup>O</sup>- <sup>H</sup> <sup>N</sup> N

at 35ºC)

lesters

N Cl

paraoxon

O O

O O

Cl

Biodegradation of the Organophosphate Pesticide Profenofos by Marine Fungi http://dx.doi.org/10.5772/56372 149

diation is seen as a safer, less disruptive and more cost-effective alternative treatment

Effective techniques for soil bioremediation are bioaugmentation, biostimulation, phytoreme‐ diation and enzymatic bioremediation. However, the three first techniques are limited by their dependence upon the growth rate of the remediating plants and microbes, which will vary with nutrients, aeration, pH and other factors relating to the contaminated soil (Scott *et al.*, 2008; Sutherland *et al.*, 2004). A successful bioremediation technique requires efficient organ‐ isms that can degrade pollutant to a minimum level. In the case of pesticides, an adequate rate of biodegradation is required to attain the acceptable level of a pesticide residue or its metab‐

Organophosphate pesticides (OPs) are used worldwide in agriculture, municipal hygiene, disease vector control and against household pests; they were also a group of compounds used historically as chemical warfare agents (Yang *et al.*, 2008; Zheng *et al.*, 2007; Edwards and Tchounwou, 2005). OPs are phosphorus-containing pesticides whose insecticidal qualities were first observed in Germany during World War II (Edwards and Tchounwou, 2005). The principal types are phosphotriesters, thiophosphotriesters, and phosphorothiolesters. Phos‐ photriesters contain a phosphate center with three *O*-linked groups, thiophosphotriesters have the phosphoryl oxygens replaced by sulfur and in phosphorothiolesters, one or more of the

This chemical class of pesticides has been used to replace the organochlorine pesticides, banned in the United States since the 1970s (Jauregui *et al.*, 2003). However, the OPs are also highly toxic pesticides, since they are potent irreversible acetylcholinesterase (AChE) inhibitors that have a profound effect on the nervous system of exposed organisms, including human beings

The hydrolysis mechanism normally catalyzed by AChE depends on the attack of a serine residue at the active site on the carbonyl group in ACh, but in the presence of organophos‐ phates, this residue is readily phosphorylated, as follows: a histidine residue at the active site captures a proton from the serine residue, increasing its nucleophilic character, so that it readily attacks the electrophilic phosphorus atom, releasing the leaving group (X) (Figure 2). Unlike the acetylated enzyme, the phosphorylated enzyme reacts slowly with water, allowing the dealkylation of the alkoxy substituent (R2) attached to the phosphorus atom. The organophos‐ phate compounds thus inactivate acetylcholinesterase by phosphorylation of the serine at the enzyme active site. The result is the formation of a strong hydrogen bond between a protonated histidine residue of the catalytic site and the negatively charged oxygen atom of the inhibitor. Therefore, the protonated histidine cannot function as a general base catalyst for the hydrolysis of the phosphorylated enzyme, which is a necessary step for the reactivation of AChE (Figure

According the Brazillian Food, Drug and Sanitary Surveillance Agency (ANVISA), analysis of pesticide waste in food showed that OPs are those with the greatest number of occurrences in unsatisfactory samples. Among then, chlorpyrifos, methamidophos and acephate are the main active ingredients responsible for food contamination. Profenofos appeared to be the 12th

olites at the contaminated site in a limited period of time (Singh, 2008).

ester oxygen are replaced by sulfur (Figure 1) (Bigley and Raushel, 2013 ).

(Sutherland *et al.*, 2004).

148 Applied Bioremediation - Active and Passive Approaches

(Edwards and Tchounwou, 2005).

2) (Mileson *et al.*, 1998; Santos *et al.*, 2007)

Figure 1. Chemical structures of main class of OPs. (A) Phosphotriesters; (B) Thiophosphotriesters; (C) Phosphorothiolesters **Figure 1.** Chemical structures of main class of OPs. (A) Phosphotriesters; (B) Thiophosphotriesters; (C) Phosphorothio‐ lesters

**Figure 2.** Mechanism of inhibition of acetylcholinesterase by organophosphate pesticides (Santos *et al.*, 2007)

at 35ºC)

Figure 5. Marine fungi growing on solid culture medium containing various concentrations of profenofos pesticide (10 days

6

 commonest active ingredient in irregular samples of food, being found in samples of orange, strawberry and pepper (ANVISA, 2012).

Profenofos, *O*-(4-bromo-2-chlorophenyl) *O*-ethyl *S*-propyl phosphorothioate, is a broad spectrum, non-systemic foliar insecticide and acaricide. It is effective against a wide range of chewing and sucking insects and mites on various crops (Reddy and Rao, 2008). In the United States, profenofos is a "restricted use" pesticide sprayed only on cotton crops (McDaniel and Moser, 2004; EPA, 2012). However, in Brazil, this pesticide can also be used for foliar applica‐ tion on cotton, peanuts, potatoes, coffee, onions, peas, beans, green beans, watermelon, corn, cucumber, cabbage, soybean, tomato and wheat (ANVISA, 2011).

Classified as a moderately hazardous (Toxicity class II) pesticide by the World Health Organization (WHO) (Abass *et al.*, 2007; Malghani *et al.*, 2009), profenofos has a moderate order of acute toxicity following oral and dermal administration (McDaniel and Moser, 2004; Abass *et al.*, 2007). According to US Environmental Protection Agency (EPA), profenofos was first registered in the United States in 1982 and about 775,000 pounds (lbs.) of active ingredient are applied to cotton each year (EPA, 2012).

Chemical decontamination of organophosphates relies on bleach treatment, alkaline hydrol‐ ysis or incineration, but these conditions are harsh and the byproducts can be toxic (Ghanem and Raushel, 2005). Specific bioremediation of OPs requires highly specialized enzymes, so genetic engineering has been used to improve the properties of enzymes from various sources to enhance catalytic rates, stability and substrate range (Sutherland *et al.*, 2004).

A number of enzymes capable of detoxifying OPs have been discovered and the majority of them belong to the class of phosphotriesterases (PTE). Various PTEs have been identified: organophosphate hydrolase (OPH), methyl parathion hydrolase (MPH), organophosphorus acid anhydrolase (OPAA), diisopropylfluorophosphatase (DFP), and paraoxonase 1 (PON1) (Bigley and Raushel, 2013). All of these enzymes are found to promote the hydrolysis of organophosphate compounds. The most frequently cited enzyme in the literature, OPH, isolated from the bacteria *Pseudomonas diminuta* or *Flavobacterium* ATCC 27551, catalyzes the hydrolysis of a wide range of OP pesticides (Rogers, 1999; Chen and Mulchandani, 1988). Another enzyme reported involving the hydrolysis of OPs are carboxylesterases (CbEs), although the hydrolysis of OPs by PTEs is more efficient in the detoxification than the CbEs (Sogorb and Vilanova, 2002).

Zheng *et al.* showed that when OPH was coexpressed with CbE, the mixed enzymes degraded a variety of P-O bond containing OPs (chlorpyrifos, methyl parathion, dichlorvos and phoxim), whereas OPH had a very low catalytic activity for P-S bond containing OPs (malathion) (Figure 3). Thus, the hydrolase activities usually vary among structurally different OPs, ranging from the nearly diffusion-controlled limit for paraoxon to several orders of magnitude lower for phosphothiolesters, such as malathion (Zheng *et al.*, 2007).

that occur by subsequent biotransformations, yielding novel polar metabolites, such as glycosylated and sulfated derivatives. According to the Food and Agriculture Organization of the United Nations (FAO), in aerobic soil conditions, profenofos degraded rapidly, with mineralization and formation of unextracted residues. In sterilized soil, cleavage of the phenolphosphorus ester bond in profenofos proceeded via chemical hydrolysis, with accumulation of 4-bromo-2-chlorophenol and formation of unextracted residues. The metabolic biotrans‐ formations of profenofos in plants and animals are similar and occur via hydrolysis to 4-

P OO

N

chlorpyrifos

Cl

Cl

Cl

methyl parathion

Cl

N

Cl

OPH + CbE

OPH + CbE

OPH + CbE

OPH + CbE

OPH + CbE

NO2

HO

HO

N

Cl

Biodegradation of the Organophosphate Pesticide Profenofos by Marine Fungi

Cl

Cl

N

HO Cl

N

HO

O O

O O

HS

Cl

+

+

+

+

+

NO2

P OH <sup>O</sup>

P OH <sup>O</sup>

P OH <sup>O</sup>

S

<sup>P</sup> OH <sup>O</sup>

P SH <sup>O</sup>

O

S

O

O

O

S

O

http://dx.doi.org/10.5772/56372

S

151

S

P OO

P OO

P OO

<sup>O</sup> <sup>N</sup>

S

P S O

O

S

O O

phoxim

dichlorvos

O O

**Figure 3.** Hydrolysis of different OPs pesticides by OPH + CbE enzymes

malathion

O

O

O

S

O

Some degradation pathways are described in the literature for profenofos. The metabolic pathway of profenofos in cotton plants involves the cleavage of the phosphorothioate ester bond to yield 4-bromo-2-chlorophenol, followed by conjugation with glucose (Capps *et al.*, 1996). In the literature there are some cases of (bio)degradation of organophosphate pesticides Biodegradation of the Organophosphate Pesticide Profenofos by Marine Fungi http://dx.doi.org/10.5772/56372 151

**Figure 3.** Hydrolysis of different OPs pesticides by OPH + CbE enzymes

commonest active ingredient in irregular samples of food, being found in samples of orange,

Profenofos, *O*-(4-bromo-2-chlorophenyl) *O*-ethyl *S*-propyl phosphorothioate, is a broad spectrum, non-systemic foliar insecticide and acaricide. It is effective against a wide range of chewing and sucking insects and mites on various crops (Reddy and Rao, 2008). In the United States, profenofos is a "restricted use" pesticide sprayed only on cotton crops (McDaniel and Moser, 2004; EPA, 2012). However, in Brazil, this pesticide can also be used for foliar applica‐ tion on cotton, peanuts, potatoes, coffee, onions, peas, beans, green beans, watermelon, corn,

Classified as a moderately hazardous (Toxicity class II) pesticide by the World Health Organization (WHO) (Abass *et al.*, 2007; Malghani *et al.*, 2009), profenofos has a moderate order of acute toxicity following oral and dermal administration (McDaniel and Moser, 2004; Abass *et al.*, 2007). According to US Environmental Protection Agency (EPA), profenofos was first registered in the United States in 1982 and about 775,000 pounds (lbs.) of active ingredient are

Chemical decontamination of organophosphates relies on bleach treatment, alkaline hydrol‐ ysis or incineration, but these conditions are harsh and the byproducts can be toxic (Ghanem and Raushel, 2005). Specific bioremediation of OPs requires highly specialized enzymes, so genetic engineering has been used to improve the properties of enzymes from various sources

A number of enzymes capable of detoxifying OPs have been discovered and the majority of them belong to the class of phosphotriesterases (PTE). Various PTEs have been identified: organophosphate hydrolase (OPH), methyl parathion hydrolase (MPH), organophosphorus acid anhydrolase (OPAA), diisopropylfluorophosphatase (DFP), and paraoxonase 1 (PON1) (Bigley and Raushel, 2013). All of these enzymes are found to promote the hydrolysis of organophosphate compounds. The most frequently cited enzyme in the literature, OPH, isolated from the bacteria *Pseudomonas diminuta* or *Flavobacterium* ATCC 27551, catalyzes the hydrolysis of a wide range of OP pesticides (Rogers, 1999; Chen and Mulchandani, 1988). Another enzyme reported involving the hydrolysis of OPs are carboxylesterases (CbEs), although the hydrolysis of OPs by PTEs is more efficient in the detoxification than the CbEs

Zheng *et al.* showed that when OPH was coexpressed with CbE, the mixed enzymes degraded a variety of P-O bond containing OPs (chlorpyrifos, methyl parathion, dichlorvos and phoxim), whereas OPH had a very low catalytic activity for P-S bond containing OPs (malathion) (Figure 3). Thus, the hydrolase activities usually vary among structurally different OPs, ranging from the nearly diffusion-controlled limit for paraoxon to several orders of magnitude lower for

Some degradation pathways are described in the literature for profenofos. The metabolic pathway of profenofos in cotton plants involves the cleavage of the phosphorothioate ester bond to yield 4-bromo-2-chlorophenol, followed by conjugation with glucose (Capps *et al.*, 1996). In the literature there are some cases of (bio)degradation of organophosphate pesticides

to enhance catalytic rates, stability and substrate range (Sutherland *et al.*, 2004).

strawberry and pepper (ANVISA, 2012).

150 Applied Bioremediation - Active and Passive Approaches

applied to cotton each year (EPA, 2012).

(Sogorb and Vilanova, 2002).

phosphothiolesters, such as malathion (Zheng *et al.*, 2007).

cucumber, cabbage, soybean, tomato and wheat (ANVISA, 2011).

that occur by subsequent biotransformations, yielding novel polar metabolites, such as glycosylated and sulfated derivatives. According to the Food and Agriculture Organization of the United Nations (FAO), in aerobic soil conditions, profenofos degraded rapidly, with mineralization and formation of unextracted residues. In sterilized soil, cleavage of the phenolphosphorus ester bond in profenofos proceeded via chemical hydrolysis, with accumulation of 4-bromo-2-chlorophenol and formation of unextracted residues. The metabolic biotrans‐ formations of profenofos in plants and animals are similar and occur via hydrolysis to 4bromo-2-chlorophenol which is then conjugated by several enzymatic reactions (Figure 4) (FAO, 2012).

(Malghani *et al.*, 2009). Filamentous fungi of the genus *Aspergillus* have been used in the biodegradation of OPs. For instance, *Aspergillus niger* showed high biodegradation of mala‐ thion pesticide (Ramadevi *et al.*, 2012), *Aspergillus flavus* and *Aspergillus sydowii* were capable of degrading pirimiphos-methyl, pyrazophos and malathion, even at high concentrations (1,000 ppm), utilizing these compounds as sole phosphorus and carbon sources, releasing the phosphorus moiety from these pesticides by means of their phosphatases (Hasan, 1999).

Biodegradation of the Organophosphate Pesticide Profenofos by Marine Fungi

http://dx.doi.org/10.5772/56372

153

Marine enzymes have a great potential for use in biocatalytic reactions, as in biodegradation of pesticides, due to the peculiar characteristics of the marine environment. As the sea covers more than three quarters of the Earth's surface and provide abundant resources for biotech‐ nological research and development (Rush *et al.*, 2007), marine organisms offer a dramatically different environment for the biosynthesis of molecules than terrestrial organisms, and are a vast untapped source of enzymes (Venter *et al.*, 2004; Venter *et al.*, 2010). In recent years, a variety of new enzymes with specific activities have been isolated from bacteria, fungi and other marine organisms; moreover, some can produce a considerable number of molecules with potential to be transformed into commercial drugs (Ghosh *et al.*, 2005; Haefner, 2003). In fact, the marine environment is a very rich source of extremely potent compounds exhibiting significant activities in anti-tumor, anti-inflammatory, analgesic, immunomodulatory, allergic

Marine organisms in general (fungi, bacteria, algae, sponges, fish, prawns and other crusta‐ ceans) can be rich sources of novel enzymes, but most of the current bioprospecting activity focuses on microbial ones. A marine enzyme is a protein molecule with unique properties as it is derived from an organism whose natural habitat is saline or brackish water (Trincone, 2010; Sarkar *et al.*, 2010). These enzymes can be biocatalysts with properties such as high salt tolerance, hyperthermostability, barophilicity and cold adaptability. Microorganisms isolated from ocean sediment and seawater are the most widely studied sources of marine enzymes,

Enzymatic reactions catalyzed by marine fungi can be used when the fungi are cultured in media based on artificial seawater. The filamentous marine fungi *Aspergillus sydowii* CBMAI 933, *Penicillium raistrickii* CBMAI 931, *Penicillium miczynskii* CBMAI 930 and *Trichoderma* sp. CBMAI 932, grown in artificial seawater were able to catalyze the hydrolysis of benzyl glycidyl ether (Martins *et al.*, 2011). Similar results were observed in a study of ligninolytic enzyme production by the marine fungi *Aspergillus sclerotiorum* CBMAI 849, *Cladosporium cladospor‐ ioides* CBMAI 857 and *Mucor racemosus* CBMAI 847 (Bonugli-Santos *et al.*, 2010). Other studies have shown that marine bacteria and fungi cultured in the laboratory have specific require‐ ments for salts, especially sodium, potassium, magnesium and chloride ions (Martins *et al.*,

In this chapter, the first results obtained in the biodegradation of profenofos by whole cells of marine fungi are presented. Marine fungi were selected by us, with high potential to biode‐ grade profenofos and its main metabolite. The results presented in this chapter explore the potential of marine fungi in biotransformation and biodegradation of a xenobiotic (pesticide profenofos). The fungal biodegradation of OPs is still underexplored by researches, especially with regard to the biodegradation of profenofos, making this work extremely relevant. The

and anti-viral assays (Newman and Cragg, 2004; San-Martín *et al.*, 2008).

especially proteases, carbohydrases and peroxidases (Ghosh *et al.*, 2005).

2011; MacLeod, 1965; Kogure, 1998; Rocha *et al.*, 2009).

**Figure 4.** Proposed metabolic pathway of profenofos in soil, plant and enzymatic reactions

Fungi degrade a wide variety of compounds, a process known as mycodegradation. This process involves degradation to smaller molecules which may be toxic or non-toxic, as well as the removal of the pesticide molecule through a simple absorption or adsorption mechanism (Ramadevi *et al*., 2012).

The ability of bacterial species to degrade organophosphates is well established and researches have even proposed possible degradation mechanisms for the OPs (Van Eerd *et al.*, 2003). However, the mechanisms of fungal degradation of these compounds are less established than those used by bacteria, since there are few studies on fungal degradation of OPs.

There are few studies on the biodegradation of profenofos by microorganisms. Malghani *et al.* reportedthe successful biodegradationofthis compoundby bacteria, andatthe time of writing, theauthor statedthatnostudiesonbacterialdegradationofprofenofoshadbeenreportedearlier (Malghani *et al.*, 2009). Filamentous fungi of the genus *Aspergillus* have been used in the biodegradation of OPs. For instance, *Aspergillus niger* showed high biodegradation of mala‐ thion pesticide (Ramadevi *et al.*, 2012), *Aspergillus flavus* and *Aspergillus sydowii* were capable of degrading pirimiphos-methyl, pyrazophos and malathion, even at high concentrations (1,000 ppm), utilizing these compounds as sole phosphorus and carbon sources, releasing the phosphorus moiety from these pesticides by means of their phosphatases (Hasan, 1999).

bromo-2-chlorophenol which is then conjugated by several enzymatic reactions (Figure 4)

O

4-bromo-2-chlorophenol

profenofos 4-bromo-2-

Fungi degrade a wide variety of compounds, a process known as mycodegradation. This process involves degradation to smaller molecules which may be toxic or non-toxic, as well as the removal of the pesticide molecule through a simple absorption or adsorption mechanism

The ability of bacterial species to degrade organophosphates is well established and researches have even proposed possible degradation mechanisms for the OPs (Van Eerd *et al.*, 2003). However, the mechanisms of fungal degradation of these compounds are less established than

There are few studies on the biodegradation of profenofos by microorganisms. Malghani *et al.* reportedthe successful biodegradationofthis compoundby bacteria, andatthe time of writing, theauthor statedthatnostudiesonbacterialdegradationofprofenofoshadbeenreportedearlier

those used by bacteria, since there are few studies on fungal degradation of OPs.

Cl

Br

4-bromo-2-chloro-1 methoxybenzene

Br

Cl

O

chlorophenol

Cl

3-chloro-4-methoxy phenol

HO

Br

S

3-(5-hydroxy-2-methoxyphenylthio) propanoic acid

Cl O

OH

HO OH O

O

H H OH O

glucoside

<sup>O</sup> <sup>S</sup> PTE

H HO

OH H

OH

Cl O

Br

S O O OH

O

SH O

Cl O

3-chloro-4 methoxyphenol

HO

4-bromophenol

Br

HO

4-bromo-2-chloro-1 ethoxybenzene

Cl

4-bromo-2-chlorophenyl hydrogen sulfate

3-mercapto-4-methoxyphenol

OH

Br

(FAO, 2012).

Cl O

P OH O S

Br

Cl O

152 Applied Bioremediation - Active and Passive Approaches

P SH O O *O*-4-bromo-2-chlorophenyl *O*-ethyl *S*-hydrogen phosphorothioate

*O*-4-bromo-2-chlorophenyl *S*-propyl *O*-hydrogen phosphorothioate

Cl O

Br

4-bromo-2-chlorophenyl dihydrogen phosphate

(Ramadevi *et al*., 2012).

P OH O OH

> Cl O

Br

4-bromo-2-chlorophenyl ethyl hydrogen phosphate

P OH O O

**Figure 4.** Proposed metabolic pathway of profenofos in soil, plant and enzymatic reactions

Cl O

P O

Br

Br

Marine enzymes have a great potential for use in biocatalytic reactions, as in biodegradation of pesticides, due to the peculiar characteristics of the marine environment. As the sea covers more than three quarters of the Earth's surface and provide abundant resources for biotech‐ nological research and development (Rush *et al.*, 2007), marine organisms offer a dramatically different environment for the biosynthesis of molecules than terrestrial organisms, and are a vast untapped source of enzymes (Venter *et al.*, 2004; Venter *et al.*, 2010). In recent years, a variety of new enzymes with specific activities have been isolated from bacteria, fungi and other marine organisms; moreover, some can produce a considerable number of molecules with potential to be transformed into commercial drugs (Ghosh *et al.*, 2005; Haefner, 2003). In fact, the marine environment is a very rich source of extremely potent compounds exhibiting significant activities in anti-tumor, anti-inflammatory, analgesic, immunomodulatory, allergic and anti-viral assays (Newman and Cragg, 2004; San-Martín *et al.*, 2008).

Marine organisms in general (fungi, bacteria, algae, sponges, fish, prawns and other crusta‐ ceans) can be rich sources of novel enzymes, but most of the current bioprospecting activity focuses on microbial ones. A marine enzyme is a protein molecule with unique properties as it is derived from an organism whose natural habitat is saline or brackish water (Trincone, 2010; Sarkar *et al.*, 2010). These enzymes can be biocatalysts with properties such as high salt tolerance, hyperthermostability, barophilicity and cold adaptability. Microorganisms isolated from ocean sediment and seawater are the most widely studied sources of marine enzymes, especially proteases, carbohydrases and peroxidases (Ghosh *et al.*, 2005).

Enzymatic reactions catalyzed by marine fungi can be used when the fungi are cultured in media based on artificial seawater. The filamentous marine fungi *Aspergillus sydowii* CBMAI 933, *Penicillium raistrickii* CBMAI 931, *Penicillium miczynskii* CBMAI 930 and *Trichoderma* sp. CBMAI 932, grown in artificial seawater were able to catalyze the hydrolysis of benzyl glycidyl ether (Martins *et al.*, 2011). Similar results were observed in a study of ligninolytic enzyme production by the marine fungi *Aspergillus sclerotiorum* CBMAI 849, *Cladosporium cladospor‐ ioides* CBMAI 857 and *Mucor racemosus* CBMAI 847 (Bonugli-Santos *et al.*, 2010). Other studies have shown that marine bacteria and fungi cultured in the laboratory have specific require‐ ments for salts, especially sodium, potassium, magnesium and chloride ions (Martins *et al.*, 2011; MacLeod, 1965; Kogure, 1998; Rocha *et al.*, 2009).

In this chapter, the first results obtained in the biodegradation of profenofos by whole cells of marine fungi are presented. Marine fungi were selected by us, with high potential to biode‐ grade profenofos and its main metabolite. The results presented in this chapter explore the potential of marine fungi in biotransformation and biodegradation of a xenobiotic (pesticide profenofos). The fungal biodegradation of OPs is still underexplored by researches, especially with regard to the biodegradation of profenofos, making this work extremely relevant. The main objective of this study was the screening of Brazilian marine fungi with the enzymes required for detoxification of organophosphate pesticides (phosphotriesterases-PTEs and /or carboxylesterases-CBEs). The biodegradation of profenofos in the presence of these selected fungi was evaluated, assessing the degradation of the pesticide, as well as the formation of the metabolite, 4-bromo-2-chlorophenol. This results are environmentally important, because the pesticides applied to crops can be leached into rivers, lakes and seas under these different conditions, where they may suffer different biodegradation processes.

**2.4. Composition of marine fungi growth media**

(1L) and adjusted to pH 5 by addition of 3M KOH or 1M HCl.

(1L), adjusted to pH 7 by addition of 3M KOH or 1M HCl.

of the plates, relative to the control culture.

chlorpyrifos (used as internal standard) were prepared.

**2.6. Analytical curve**

mL).

(10.0 mL).

medium (25 mL), in Petri dishes, maintained at 4°C in the refrigerator.

**2.5. Cultivation of marine fungi on solid medium in the presence of profenofos**

SrCl2.6H2O (0.040g), H3BO3 (0.030g).

to pH 8 by addition of 3M KOH.

1M HCl.

*Composition of Artificial Sea Water* (ASW) (1L): CaCl2.2H2O (1.36g), MgCl2.6H2O (9.68g), KCl (0.61g), NaCl (30.0g), Na2HPO4 (0.014 mg), Na2SO4 (3.47g), NaHCO3 (0.17g), KBr (0.1g),

Biodegradation of the Organophosphate Pesticide Profenofos by Marine Fungi

http://dx.doi.org/10.5772/56372

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*Solid medium for stock cultures:* agar (20 g. L-1) and malt extract (20 g.L-1) in ASW (1L) and adjusted

*Solid medium for fungal screening:* agar (20 g. L-1) and malt extract (20 g.L-1) dissolved in ASW

*Liquid medium:* malt extract (20 g.L-1) in ASW (1L), adjusted to pH 7 by addition of 3M KOH or

*Liquid mineral medium supplemented with KNO3(12.5 ppm)*: KNO3 (12.5 mg.L-1) dissolved in ASW

The culture media were sterilized in autoclave for 20 minutes (at 121 °C, 1.5 kPa). All manip‐ ulations involving marine fungi were carried out under sterile conditions in a Veco laminar flow cabinet. The stock cultures of the marine microorganisms were stored on solid culture

Marine fungi were screened by culturing on Petri dishes containing 25 mL of solid culture medium (2.0 g of malt extract, 2.0 g of agar and 100 mL of ASW) with the addition of profenofos and without (control culture). After the medium sterilization in the autoclave, the agar was cooled to 40-45°C and the profenofos was added at three different concentrations: 5.0, 10.0 and 15.0 μL per plate, solubilized in 100.0, 200.0 and 300.0 μL of dimethyl sulfoxide (DMSO), respectively. At room temperature, fungal mycelia from recent cultures were transferred to the surfaces of the agar plates with an inoculating loop. The fungi were incubated for 10 days at 35°C. Tolerance of profenofos was estimated by the size of the colony formed on the surface

Stock solutions of 500.0 ppm of profenofos, 4-bromo-2-chlorophenol (main metabolite) and

*Profenofos 500.0 ppm:* 3.4 μL (1.3 mmol) of profenofos analytical standard and ethyl acetate (10.0

*4-bromo-2-chlorophenol 500.0 ppm*: 5.0 mg (2.4 mol) of 4-bromo-2-chlorophenol and ethyl acetate

All standard solutions were prepared in a volumetric flask and made up to the containing 10.0 mL mark with ethyl acetate (HPLC grade). From these stock solutions were prepared the

*Chlorpyrifos 500.0 ppm*: 5.0 mg (1.4 mol) of chlorpyrifos and ethyl acetate (10.0 mL).
