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Author’s Accepted Manuscript Biotransformation and biodegradation of methyl parathion by Brazilian bacterial strains isolated from mangrove peat Natália Alvarenga, Willian G. Birolli, Eloá B. Meira, Simone C.O. Lucas, Iara L. de Matos, Marcia Nitschke, Luciane P.C. Romão, André L.M. Porto

PII: DOI: Reference:

S1878-8181(16)30396-6 BCAB681

To appear in: Biocatalysis and Agricultural Biotechnology Received date: 27 October 2016 Revised date: 17 December 2017 Accepted date: 31 December 2017 Cite this article as: Natália Alvarenga, Willian G. Birolli, Eloá B. Meira, Simone C.O. Lucas, Iara L. de Matos, Marcia Nitschke, Luciane P.C. Romão and André L.M. Porto, Biotransformation and biodegradation of methyl parathion by Brazilian bacterial strains isolated from mangrove peat, Biocatalysis and Agricultural Biotechnology, This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Biotransformation and biodegradation of methyl parathion by Brazilian bacterial strains isolated from mangrove peat

Natália Alvarenga a, Willian G. Birolli a, Eloá B. Meira a, Simone C. O. Lucas b, Iara L. de Matos a, Marcia Nitschke a, Luciane P. C. Romão b, André L. M. Porto a,*


Instituto de Química de São Carlos, Universidade de São Paulo, Av. João Dagnone, 1100, Química

Ambiental, J. Santa Angelina, 13563-120, São Carlos, São Paulo, Brazil b

Universidade Federal de Sergipe, Av. Marechal Rondon, s/n, J. Rosa Elze, 49100-000, São Cristóvão,

Sergipe, Brazil

* Corresponding author. Tel. +55 16 33738103 E-mail address: [email protected] (André L. M. Porto)

Highlights 

Bacterial strains catalyzed the biotransformation of methyl parathion after 24 h.

Biodegradation occurred through the hydrolysis of ester bond by phase I reaction.

Biotransformation occurred by reduction of nitro group followed by acetylation of the amino group.

Selected strains were able to reduce p-nitrophenol levels by phase II reaction.


Abstract Four bacterial strains (Bacillus sp. CBMAI 1833, Bacillus cereus P5CNB, Kosakonia sp.

CBMAI 1836 and Kosakonia sp. CBMAI 1835), isolated from a Brazilian

mangrove peat were evaluated by the biodegradation of methyl parathion. Strains Bacillus sp. CBMAI 1833 and B. cereus P5CNB showed better methyl parathion degradation at 36 h than the Kosakonia strains. By HPLC-UV analysis, in the presence of both strains, it was observed that all methyl parathion was biotransformed and biodegraded with 24 h of incubation. HPLC-ToF and GC-MS analysis were employed for identification of metabolites from the methyl parathion reactions. The first biodegradation pathway showed the direct hydrolysis of the pesticide to yield pnitrophenol by phase I reaction. The biotransformation of methyl parathion occurs via the nitro group reduction, with the formation of an amine group in the phenolic moiety, followed by the amine acetylation to yield an acetamide derivative by phase II reactions. Further biodegradation proceeded with the hydrolysis of the acetamide product, forming N-(4-hydroxyphenyl) acetamide. Bacillus sp. CBMAI 1833 and B. cereus P5CNB were also able to promote the reduction of p-nitrophenol levels in 12 days.

Graphical abstract


Keywords: Organophosphate pesticides; Enzymatic hydrolysis; p-Nitrophenol; Bioconjugation reactions.

1. Introduction

Organophosphate pesticides (OP) are a pesticides class widely spread for the protection of a variety of agricultural crops (Bigley and Raushel, 2013). These pesticides represent a risk for nontarget organisms since they act inhibiting the acetylcholinesterase, the enzyme responsible for the hydrolysis of the neurotransmitter acetylcholine. OP degradation pathways have received considerable attention due to the health concern caused by their deliberated use (Pakala et al., 2007; Reddy and Rao, 2008). The most extensively used OP includes methyl parathion, chlorpyrifos and malathion (Ojha et al., 2011). Methyl parathion (MP) (O,O-dimethyl-O-(4-nitrophenyl) phosphorothioate) is an important broad-spectrum non-systemic pesticide and it was classified as a highly toxic insecticide (Edwards and Tchounwou, 2005; Pino and Peñuela, 2011). In Brazil, according to the Brazilian National Health Surveillance Agency (ANVISA), MP presented a foliar application in cultures as cotton and soybeans, but its use was recently prohibited (Anvisa, 2015). In USA, its use was restricted by the U.S. Environmental Protection Agency on several crops and banned for indoor purposes. Despite of the restrictions against MP, its illegal use has led to the concern about health problems to the exposed organisms (Karanth et al., 2004; Edwards and Tchounwou, 2005).


The usual chemical methods for detoxification of contaminated sites involve the hydrolysis of the OP with strong alkalines solutions, producing metabolites and potentially toxic wastes (Munnecke and Hsieh, 1974). A milder and more complete degradation process is usually achieved with microbial degradation. Microorganisms are able to chemically and physically interact with pesticides, promoting structural changes (biotransformation) or complete degradation of the molecule in a simple, inexpensive and more environmentally friend fashion when compared to non-biological processes (Gavrilescu, 2005; Diez, 2010). The process involving pesticides metabolism by microorganisms may be described in three phases: in the initial phase (Phase I) the pesticide might be transformed by oxidation, reduction or hydrolysis reactions, usually introducing polar groups into the molecule. Subsequently, in the Phase II, the biotransformed molecule is conjugated to sugars, amino acids or glutathione, making the compound less toxic, more soluble in water and more easily excreted; the Phase III involves the conversion of conjugated metabolites into secondary conjugates, which are less toxic (Derelanko and Hollinger, 2001; Van Eerd et al., 2003). Many enzymes are involved in the metabolism of organophosphate compounds, including esterases, which is an important class of enzymes that hydrolyzes these synthetic pesticides. The best characterized OP-degrading esterases are the phosphotriesterases (PTE). The natural substrate of these enzymes is still unknown and its ability of OP hydrolysis is believed to be a promiscuous activity from PTE (Ghanem and Raushel, 2005; Bigley and Raushel, 2013; Bigley et al., 2015; Mabanglo et al. 2016). Studies in OP biodegradation by fungi have been reported by us. The pesticide profenofos and its main hydrolysis product, 4-bromo-2-chlorophenol, was degraded by


marine-derived fungi deposited on Brazilian Collection of Environmental and Industrial Microorganisms – CBMAI, Penicillium raistrickii CBMAI 931 and Aspergillus sydowii CBMAI 935 (Silva et al., 2013). MP was completely degraded by A. sydowii CBMAI 935 in 20 d of reaction, whereas Penicillium decaturense CBMAI 1234 degraded all the pesticide in 30 d with the bioactivation of the pesticide to its toxic

oxon form

(Alvarenga et al., 2014). The biodegradation through hydrolytic process may reduce the toxicity of MP by almost 120-fold, leading to the formation of the dialkylthiophosphate and p-nitrophenol (PNP) (Shimazu et al., 2001). PNP is less toxic than MP but it is also considered by the U.S. Environmental Protection Agency as a priority pollutant because of its large use in drugs and pesticides manufacture, leather treatment and for military purposes (Yi et al., 2003). This way, it is required microorganisms capable of not only biodegrade MP but also reduce the PNP levels in environment. The bacterial strains employed in this work were Kosakonia sp., B. cereus and Bacillus sp. isolated from a Brazilian mangrove peat, in Aracaju, Sergipe state. According to Franchi et al. (2003) the peat is “a fossil, organic and mineral substance originated from the decomposition of vegetal wastes, found in wetlands as in rivers floodplains, coastal plains and lake regions” (Franchi et al., 2003). Microorganisms isolated from organic matter decomposition areas may afford innumerous enzymatic activities, being promising sources of enzymes for a variety of biodegradation reactions. The potential of these bacterial strains has been studied by our research group for the biodegradation of xenobiotic compounds and promising results has been obtained for the degradation of pesticides, such as esfenvalerate (Birolli et al., 2016). Bacteria from Bacillus genus has been demonstrated as a good source of organophosphate degrading enzymes. Bacillus cereus isolated from malathion


contaminated field degraded 87% of malaoxon and 49% of malathion after 7 d (Singh et al., 2012). High concentrations (300 mg L-1) of chlorpyrifos was degraded by Bacillus pumilus C2A1 (Anwar et al., 2009) and Bacillus cereus degraded 84% chlorpyrifos after 20 d (Lakshmi et al., 2009). Since the taxonomic reclassification of the genus Enterobacter into the genus Cronobacter and three novel genera (Lelliottia, Pluralibacter and Kosakonia), to the best of our knowledge, no report about the OP biotransformation with Kosakonia sp. has been reported. The aim of the present study was to assess the biotransformation and/ or biodegradation of MP by bacterial strains isolated from the Brazilian peat through the quantification of the residual pesticide and identification of the degradation metabolites and biotransformation products. It was also studied the ability of the bacterial strains to reduce the levels of PNP, which is also an important environmental pollutant.

2. Material and methods 2.1 Chemicals Methyl parathion (99.7%) and p-nitrophenol (99.9 %) analytical grade were purchased from Sigma-Aldrich (São Paulo, Brazil) and the commercial formulation of methyl parathion named ‘‘Folisuper 600 BR’’ was obtained from Agripec Química e Farmacêutica S/A (Ceará, Brazil). Solvents were obtained from Sigma–Aldrich and Synth (São Paulo, Brazil). Nutrient broth (3 g of beef extract and 5 of enzymatic digest of gelatin per liter) and enzymatic digest of soy 2 (peptone S2) were purchased from Acumedia (São Paulo, Brazil) and Agar powder from Himedia (Paraná, Brazil). Salts for saline medium preparation were obtained from Synth (São Paulo, Brazil). 2.2 Bacterial strains 6

The Brazilian bacterial strains Kosakonia sp. CBMAI 1835, Kosakonia sp. CBMAI 1836, B. cereus P5CNB and Bacillus sp. CBMAI 1833 were isolated from a mangrove peat collected in Santo Amaro das Brotas, Sergipe state, Brazil (N 07º 20.729´/ W 880º 35.113´), by Simone C. O. Lucas (Federal University of Sergipe – UFS). The bacteria were purified in the Environmental Analytical Chemistry Laboratory supervised by Prof. Dra. Luciane P. C. Romão. They were identified by both conventional and molecular methods at the Chemical, Biological and Agricultural Multidisciplinary Research Center (CPQBA/UNICAMP, Brazil) and the strains coded were deposited in the CBMAI collection (

2.3 Composition of culture media Saline medium solution (1 L): CaCl2.2H2O (1.36 g), MgCl2.6H2O (9.68 g), KCl (0.61 g), NaCl (30.0 g), Na2HPO4 (0.014 mg), Na2SO4 (3.47 g), NaHCO3 (0.17 g), KBr (0.1 g), SrCl2.6H2O (0.040 g), H3BO3 (0.030 g). Solid culture medium: The bacterial strains were grown in solid medium composed of enzymatic digest of soy 2 (5 g L-1), nutrient broth (5 g L-1) and Agar (20 g L-1) in saline medium (1 L). The pH of culture media was adjusted to 7 with a 0.7 M KOH solution. Solid culture medium supplemented with MP: The bacterial strains were grown in solid medium composed of enzymatic digest of soy 2 (5 g L-1), nutrient broth (5 g L1

), Agar (20 g L-1) and commercial MP (50 mg L-1) in saline medium solution (1 L). The

pH of culture media was adjusted to 7 with a 0.7 M KOH solution.


Liquid culture medium: Reactions were conducted in liquid medium composed of enzymatic digest of soy 2 (5 g L-1) and nutrient broth (5 g L-1) in saline medium (1 L). The pH of culture media was adjusted to 7 with a 0.7 M KOH solution. The culture media were sterilized in autoclave (AV-50, Phoenix, Brazil) at 121 ºC for 20 min. The manipulations involving the bacterial strains were carried out in a laminar flow cabinet (Veco) under sterile conditions. 2.4 Bacterial inoculum Bacteria were inoculated in Petri dishes containing the solid medium culture and incubated for 24 h (32 ºC). The plates containing the grown bacterial strain were washed with 5 mL of sterilized sodium chloride solution (0.86% NaCl in distilled water) and diluted in a test-tube to set the optical density (OD) to 0.1 in 610 nm. The measurement of colony forming units (CFU mL-1) of bacterial solution was quantified by the dilution plate count technique (Table SM-1). For the biodegradation experiments, 1 mL of bacterial suspension (OD610 nm 0.1) was inoculated in a 250 mL Erlenmeyer flask containing 100 mL of liquid medium and incubated in orbital shaker for 24 h (130 rpm, 32 ºC). 2.5 Evaluation of growth conditions and bacterial strain selection Biotransformation assays were evaluated in two different conditions: bacterial inoculums with strains grown in solid medium in absence and presence of commercial MP (50 mg L-1). The inoculation of bacterial suspension a 250 mL Erlenmeyer flask containing the liquid medium was performed as described in section 2.4. After the incubation of the bacteria for 24 h, the medium was supplemented with commercial MP (50 mg L-1) and incubated in orbital shaker for 36 h (130 rpm, 32 ºC). After the reaction


period, samples were extracted and analyzed by HPLC. The best growth condition in solid medium and the best strains were selected for further biodegradation studies.

2.6 Extraction of methyl parathion and metabolites After the experiment period, 70 mL of ethyl acetate was added to each 250 mL Erlenmeyer flask containing the reaction and submitted for magnetic stirring for 30 min. The reaction was transferred to a 500 mL centrifugation flask and centrifuged (20 min, 10,000 rpm) to obtain the cell free extract. The pH of the medium was adjusted to 7 and the sample was extracted with ethyl acetate (2 x 25 mL). The aqueous phase was discarded and the organic phase was dried with anhydrous Na2SO4, filtrated, evaporated under vacuum. The residue was suspended in a 10 mL volumetric flask with methanol HPLC grade. MP and PNP were quantified by HPLC and the identification of the metabolites was perfomed by GC-MS and LC-MS. The experiments were carried out in triplicates. 2.7 Biodegradation reactions with Bacillus sp. CBMAI 1833 and B. cereus P5CNB MP biodegradation: After the incubation of Bacillus sp. CBMAI 1833 and B. cereus P5CNB separately in orbital shaker (24 h, 130 rpm, 32 ºC) the liquid medium was supplemented with commercial MP (50 mg L-1) and incubated for 6, 12, 24 and 36 h. The reactions were performed in triplicates and the extraction procedures were the same as described in section 2.6. PNP biodegradation: After the incubation of Bacillus sp. CBMAI 1833 and B. cereus P5CNB separately in orbital shaker (24 h, 130 rpm, 32 ºC) the liquid medium was supplemented with PNP (50 mg L-1) and incubated for 3, 6, 9 and 12 d. The


reactions were performed in triplicates and the extraction procedures were the same as described in section 2.6.

2.8 Methyl parathion and p-nitrophenol method recovery The method recovery of MP and PNP were evaluated with B. cereus P5CNB cultures inoculated as described in section 2.4 in 250 mL Erlenmeyer flask containing 100 mL of liquid medium, incubated in orbital shaker (24 h, 130 rpm, 32⁰C) and sterilized in autoclave in order to stop all the enzymatic activity, which may interfere in the recovery process. After cooled in room temperature, the xenobiotic compound (MP and PNP) was added (50 mg L-1) and stirred in orbital shaker for 30 min (130 rpm, 32⁰C). The samples were extracted as described in section 2.6 and then quantified by HPLC (through analytical curves of standard solutions). The analyses were performed in quintuplicates. 2.9 Analytical procedures High-performance







biodegradation reactions were performed in a Shimadzu Prominence series equipped with a photodiode array detector (SPD-M20A). The HPLC (equipped with a 0.46 x 25 cm Shimadzu CLC-ODS (M) C18 column) conditions were: mobile phase acetonitrile and water (50:50), detection wavelength of 278 nm, 10 µL injection volume, flow rate of 0.8 mL min-1, oven at 40 °C and run time of 25 min. The retention times for PNP and MP were 6.1 and 17.6 min, respectively. MP and PNP concentrations were quantified using a calibration curve of standard solutions (Alvarenga et al., 2014) (Figs. SM-1 and SM-2). 10

Liquid chromatography-mass spectrometry (LC-MS) analyses were performed in a Shimadzu Prominence coupled to a mass espectrometer Bruker (Microtof-Q model) equipped with a hybrid quadrupole/Tof detector in electrospray ionization mode. The HPLC-ToF (equipped with a 0.46 x 25 cm Shimadzu CLC-ODS (M) C18 column) conditions were negative and positive ionization mode, mobile phase acetonitrile and water (gradient conditions in Table SM-2), detection wavelength of 280 nm, flow rate of 1.0 mL min-1, spliter of 0.2 mL min-1, oven at 40 C and run time of 35 min. The retention times in negative ionization mode for N-(4-hydroxyphenyl)acetamide and PNP were 4.0 and 10.9 min, respectively. The retention times in positive ionization mode for O-(4-aminophenyl)-O,O-dimethyl phosphorothioate and O-(4-acetamidophenyl)-O,Odimethyl phosphorothioate were 7.6 and 15.0 min, respectively (Alvarenga et al., 2014). Gas chromatography-mass spectrometry (GC-MS) analyses were performed in a Shimadzu GC2010plus coupled to a mass selective detector (Shimadzu MS2010plus) in electron ionization (EI, 70 eV) mode. The GC-MS (equipped with a 30 m x 0.25 mm x 0.25 µm J&W Scientific DB5 column) conditions were: oven temperature started at 90 C and kept for 2 min, increased to 280 C at 6 C min-1 and held for 6.3 min; injector and detector temperature maintained at 250 C and 200 C, respectively; injector mode was splitless; column flow of 0.75 mL min-1; helium used as the carrier gas at a pressure of 60 kPa and run time of 40 min. The retention times for PNP and MP were 13.3 and 17.4 min, respectively.

3. Results and discussion 3.1 MP and PNP recovery method in biodegradation reactions


The recovery of MP and PNP (50 mg L-1 initial concentrations) were determined following the same extraction procedures as for the biodegradation experiments. The average MP recovery was 91% and the standard deviation between quintuplicates was 4.4%. PNP recovery was 95% and the standard deviation was 1.8% between quintuplicates. The recovery method for both compounds presented reliable values for biodegradation studies. 3.2 Evaluation of growth conditions and bacterial strain selection The biodegradation was evaluated with the inoculum of the bacterial strains grown in both presence and absence of MP (50 mg L-1) on solid culture medium to evaluate the possibility of the pesticide act as an inductor for the production of the MPdegrading enzymes. After bacterial growth for 24 h in liquid medium, commercial MP (50 mg L-1) was added and the reactions were incubated during 36 h. Figure 1 shows the residual MP for each strains with inoculums of bacteria grown in absence of MP (grey bars) and in presence of MP (black bars). Strain







biodegradation/biotransformation when the inoculum was performed with bacteria grown in absence of MP (16.4 mg L-1 of residual MP) than in reactions with bacteria previously grown in presence of MP (22.6 mg L-1 of residual MP). Kosakonia sp. CBMAI 1836, presented no significant differences for inoculum in absence and presence of MP (21.7 and 23.9 mg L-1, respectively). B. cereus P5CNB and Bacillus sp. CBMAI 1833 showed a complete degradation in 36 h with both strains growth condition. Once the biodegradation proceeded better when the bacteria were grown in absence of MP, this was set as the better growth condition to further biodegradation studies. Probably the growth of bacteria in presence of the pesticide on solid culture medium produced some toxic metabolite that inhibited bacterial growth and reduced the 12

bacterial population and, therefore, the biodegradation. Bacterial strains of B. cereus P5CNB and Bacillus sp. CBMAI 1833 were selected for further biodegradations studies to evaluate the biodegradation kinetic, as well as the metabolites formed during the reaction course.

Figure 1 here

3.3 Biodegradation reactions with Bacillus sp. CBMAI 1833 and B. cereus P5CNB The kinetic of MP biodegradation/biotransformation by bacterial strain Bacillus sp. CBMAI 1833 and B. cereus P5CNB was performed in 6, 12, 24 and 36 h. The abiotic control (MP in absence of bacterial strains) evaluated the spontaneous degradation of MP in the liquid medium at the same reaction conditions in which were developed the biodegradation assays. These controls showed that, in absence of any microorganism, 11% of MP was degraded in 36 h. The abiotic degradation occurs trough chemical and physical transformations by oxidation/reduction, hydrolysis, rearrangements and photolysis (Van Eerd et al., 2003). In the specific case of MP, the most probably pathway of abiotic degradation is the hydrolysis reaction. Bacillus sp. CBMAI 1833 strain presented a high rate of degradation in 6 h (> 99%), and thus, the biodegradation value was estimated by extrapolation of the analytical curve. The formation of PNP remained constant during the reaction course (about 2 mg L-1) in the experiments with Bacillus sp. CBMAI 1833 (Fig SM-03), this metabolite probably is continuous produced by the MP biodegradation but it is also consumed by biodegradation. In the presence of B. cereus P5CNB the degradation was slower than for Bacillus sp. CBMAI 1833. In 6 h, 83% of MP was biodegraded/biotransformed, reaching a high level of degradation in 12 h (Fig SM-04). 13

The PNP remained in low concentration with B. cereus P5CNB, reaching an accumulation of 2.5 ppm in 36 h (Table 1). Comparing to data of abiotic control, both strains were very effective in promote the biotransformation of MP. These










biotransformation/biodegradation, since the degradation values were very higher than in the abiotic conditions or in the method recovery. Bacterial degradation of MP presented in this work was extremely faster than degradation promoted by marine-derived fungi strains previously studied by our research group, in which completed biodegradation of MP was reached on 20-30 days of reaction (Alvarenga et al., 2012; Rodrigues et al., 2016). Various studies have also reported bacterial strains as effective catalysts for the biodegradation of MP in only a few hours (Shimazu et al., 2001; Yang et al., 2008; Fernández-López et al. 2017).

Table 1 here

Since the presumable pathway of MP biodegradation is the pesticide hydrolysis, generating dialkylthiophosphate and PNP (Shimazu et al., 2001), bacterial strains were also evaluated by their ability of degrading these main MP hydrolysis product, i.e., PNP. Literature reported the PNP biodegradation through the formation of the 4nitrocatechol, followed by the removal of the nitro group yielding the 1,2,4-benzenetriol and, through a ring cleavage dioxygenase, yielding the maleyalcetate, which is converted to 3-ketoadipate (Jain et al., 1994; Kadiyala and Spain, 1998). The PNP biodegradation via formation of 4-benzoquinone, instead the 4-nitrocatechol was also described (Spain and Gibson, 1991). However, the PNP is often toxic to many


microorganisms because of the presence of the nitro group, which makes the microbiological treatment more challenging (Torres et al., 2003; Li et al., 2005). The biodegradation of PNP was monitored in 3, 6, 9 and 12 d. The abiotic control (PNP with no bacterial inoculum) presented a spontaneous degradation of 7% in 12 d. However, considering that 5% of PNP is lost by the extraction methods, this compound showed to be very stable in the reaction medium (Table 2). Comparing to the abiotic control, both strains showed a biodegradation potential for this xenobiotic compound, with Bacillus sp. CBMAI 1833 and B. cereus P5CNB reaching 23% and 18% of PNP degradation in 12 d, respectively (Table 2). Although the nitro group is toxic for many microorganisms, Bacillus sp. CBMAI 1833 and B. cereus P5CNB were capable of reducing the PNP levels, however, no degradation metabolites were found in GC-MS and HPLC-ToF analyses. The importance of microorganisms capable of the biodegradation of methyl parathion and the reduction of PNP levels has been reported before by us and other research groups, with the rate of degradation of both compounds varying according to the type of microorganism and reaction conditions (Shimazu et al., 2001; Yang et al., 2008; Alvarenga et al., 2012, Rodrigues et al., 2016; FernándezLópez et al. 2017).

Table 2 here

3.4 Metabolites identification In the HPLC-ToF analysis of the MP biotransformation and degradation with bacteria Bacillus sp. CBMAI 1833 in positive mode, there were identified peaks from the reduction of the nitro group of MP, forming the protonated ion of O-(4aminophenyl)-O,O-dimethyl phosphorothioate with m/z 234 (tr = 7.6 min) (Figure 2a).


The acetylated product of the arylamine was identified as O-(4-acetamidophenyl)- O,Odimethyl phosphorothioate with m/z 276 from the protonated ion (tr = 15.0 min) (Figure 2a). In the reaction with B. cereus P5CNB only the acetylated product was identified (Figure 2b). The biodegradation chromatograms (12 and 24 h) for each strain were compared to the bacteria control chromatogram to prove that the metabolites peaks were product of MP degradation and not from bacterial metabolites.

Figure 2 here

The HPLC-ToF analysis in negative mode showed the formation of two hydrolysis products, p-nitrophenol and N-(4-hydroxyphenyl) acetamide by Bacillus sp. CBMAI 1833 and B. cereus P5CNB (Figure 3). p-Nitrophenol is a direct hydrolysis product of MP by the action of PTE enzymes and presents m/z 138 from the deprotonated ion (tr = 11.0 min). The N-(4-hydroxyphenyl) acetamide compound is the hydrolysis product of the acetylated compound, O-(4-acetamidophenyl)-O,O-dimethyl phosphorothioate with m/z 150 from the deprotonated ion (tr = 4.0 min).

Figure 3 here

The nitro compounds are toxic and mutagenic to microorganisms and its removal from the aromatic ring have been reported by several bacterial genera (MarvinSikkema and Bont, 1994; Donlon et al., 1995). The mechanism of mineralization of nitro compounds occur by four main pathways: oxygenation reaction yielding nitrite, initial reduction yielding aromatic amines, complete elimination of the nitro group and partial reduction to produce a hydroxylamine group (Marvin-Sikkema and Bont, 1994).


Nitroreductases are the enzymes responsible by the initial reduction of the nitro group to an amine (Marvin-Sikkema and Bont, 1994). The subsequent acetylation of the amino group may be catalyzed by aryalmine N-transferases (NATs), a family of enzymes found in different organisms, both prokaryotes and eukaryotes (Sandy et al., 2005). NATs are responsible for the transfer of an acetyl group from the acetyl coenzyme A (acetyl CoA) to the terminal nitrogen and oxygen atom of arylamines, arylhydroxylamines and arylhydrazines (Sandy et al., 2005; Suzuki et al., 2007; Sim et al., 2008). Bacterial NATs have been found in more than 25 bacterial species and, the enzymes derived from these microorganisms showed the ability to acetylate of important drugs (Suzuki et al., 2007). In some cases, the enzyme activity is supposed to make the aromatic amines less toxic, since the acetylation led to the convertion of the ionizable group (amine) to a nonionizable one (the acetyl group attached to the substrate) (Manahan, 2003). Further hydrolysis of the acetylated group was promoted in a smaller extention than the direct hydrolysis of MP, as can be seen by intensity of the p-nitrophenol and N(4-hydroxyphenyl) acetamide peaks in Figure 3 for Bacillus sp. CBMAI 1833 and B. cereus P5CNB.

The hydrolysis of the O-(4-acetamidophenyl)-O,O-dimethyl

phosphorothioate may be promoted by a PTE or another class of esterase enzymes. The metabolites were also identified by GC-MS analysis and their formations were monitored in 6, 12, 24 and 36 h. The metabolites N-(4-hydroxyphenyl) acetamide and p-nitrophenol were also assigned by comparison with standard compounds (Figure 4, Figs. SM-5, SM-6 and SM-7).

Figure 4 here


Based on the HPLC-ToF and GC-MS analysis it was proposed a pathway for MP metabolization (Figure 5). Although the B. cereus P5CNB did not show the peak corresponding to the arylamine derivative, it was supposed that acetylation may only occur after the reduction of the nitro group. Therefore, both strains promoted the MP degradation by the same pathway: MP was hydrolyzed through the action of PTE enzymes yielding p-nitrophenol; simultaneously MP was biotransformed to O-(4aminophenyl)-O,O-dimethyl phosphorothioate with the reduction of the nitro group by nitroreductases and, subsequent acetylation of the amino group by NATs to yield O-(4acetamidophenyl)-O,O-dimethyl phosphorothioate; the hydrolysis of the acetylated group, forming N-(4-hydroxyphenyl) acetamide, proved the bacterial strains is capable of further degradation of the biotransformed compound.

Figure 5 here

4. Conclusion Bacillus sp. CBMAI 1833 and B. cereus P5CNB isolated from a Brazilian peat showed different pathways for commercial MP metabolization. Both strains were capable of not only degrade MP by direct hydrolysis yielding PNP, but also to biotransformation of pesticide to the acetamide derivative, to further promote its hydrolysis in a subsequent step of degradation process. Bacillus sp. CBMAI 1833 and B. cereus P5CNB were also capable of the biodegrade PNP.



The authors acknowledge to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant no 558062/2009-1) and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, grant no 2012/19934-0) for the financial support provided to this research. NAS and WGB are indebted to CNPq (141844/2013-2 and 141656/2014-0, respectively) and ILM is indebt to CAPES (1313652) for the provision of their scholarships. The authors also acknowledge Prof. M. R. V. Lanza (Instituto de Química de São Carlos - USP) for donating commercial pesticide MP and the Chromatography Group (Instituto de Química de São Carlos –USP), including Dr. Guilherme M. Titato for the HPLC-ToF analysis (FAPESP, grant nº 2004/09498-2). References Alvarenga, N., Birolli, W.G., Seleghim, M.H.R., Porto, A.L.M., 2014. Biodegradation of methyl parathion by whole cells of marine-derived fungi Aspergillus sydowii and Penicillium decaturense. Chemosphere 117, 47-52.







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Residual MP (mg L-1)

25 20 15 10 5 0

Figure 1. Residual concentration of MP in reactions containing inoculums of bacteria previously grown in absence (grey bars) and presence (black bars) of the pesticide (36


h, 130 rpm, 32 ºC). The data are mean of ± standard deviation for three independent experiments.


Bacterial control (pesticide absence) Bacillus sp. CBMAI 1833 + MP (12 h) Bacillus sp. CBMAI 1833 + MP (24 h)

b) Bacterial control (pesticide absence) Bacillus cereus P5CNB + MP (12 h) Bacillus cereus P5CNB + MP (24 h )

Figure 2. Extracted Ion Chromatogram in positive ionization mode at m/z 234.00±0.05 and m/z 276±0.05 for a) MP biotransformation by Bacillus sp. CBMAI 1833; b) MP biotransformation by B. cereus P5CNB.



Bacterial control (pesticide absence) Bacillus sp. CBMAI 1833 + MP (12 h) Bacillus sp. CBMAI 1833 + MP (24 h)


Bacterial control (pesticide absence) Bacillus cereus P5CNB + MP (12 h) Bacillus cereus P5CNB + MP (24 h)

Figure 3. Extracted Ion Chromatogram in negative ionization mode at m/z 138.00±0.05 and m/z 150±0.05 for a) MP biotransformation by Bacillus sp. CBMAI 1833; b) MP biotransformation by B. cereus P5CNB.



(x10,000,000) 2.50


















































Figure 4. GC-MS chromatogram of methyl parathion biotransformation by a) Bacillus sp. CBMAI 1833 and b) B. cereus P5CNB. Bacterial control (black line) and reaction between bacterial strain and methyl parathion in 6 h (red line), 12 h (blue line), 24 h (brown line), 36 h (green line).


Figure 5. Proposed biodegradation and biotransformation pathway of methyl parathion by bacterial strains Bacillus sp. CBMAI 1833 and B. cereus P5CNB.

Table 1. Quantitative biodegradation of methyl parathion (50 mg L-1) by Bacillus sp. CBMAI 1833 and B. cereus P5CNB until 36 h of reaction in a liquid mediuma. Time (h) cb PNP (mg L-1) cb MP (mg L-1) Abiotic control 36 2.3 ± 0.1 44.7 ± 1.6 Bacillus sp. CBMAI 1833 (50 mg L-1 of methyl parathion) 6 2.1 ± 0.2 0.23 ± 0.81 12 2.4 ± 0.1 0.05 ± 0.11 24 1.9 ± 0.2 nd 36 2.1 ± 0.1 nd -1 Bacillus cereus P5CNB (50 mg L of methyl parathion) 6 2.0 ± 1.3 8.6 ± 3.5 12 2.2 ± 0.5 0.5 ± 1.4 24 2.4 ± 2.9 0.01 ± 0.2 36 2.5 ± 1.0 nd

% of MP degraded 11 >99 >99 100 100 83 99 >99 100


MP method recovery: 91%. cb: concentration determined by HPLC-UV. nd: not detected.

Table 2. Quantitative biodegradation of p-nitrophenol (50 mg L-1) by Bacillus sp. CBMAI 1833 and B. cereus P5CNB until 36 h of reaction in a liquid mediuma. Time (d) ca PNP (mg L-1) Abiotic control 12 46.3 ± 0.07 Bacillus sp. CBMAI 1833 (50 mg L-1 of PNP) 3 45.3 ± 1.18 6 43.7 ± 2.12 9 42.6 ± 1.41 12 39.1 ± 2.98 -1 Bacillus cereus P5CNB (50 mg L of PNP) 3 46.1 ± 0.09 6 43.4 ± 0.07 9 41.9 ± 1.35 12 41.0 ± 3.78

% of PNP degraded 7 9 13 14 23 8 13 16 18


PNP method recovery: 95%. cb: concentration determined by HPLC-UV.


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