Fenpropathrin Biodegradation Pathway in Bacillus sp. DG-02 and Its


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Fenpropathrin Biodegradation Pathway in Bacillus sp. DG-02 and Its Potential for Bioremediation of Pyrethroid-Contaminated Soils Shaohua Chen,†,§,‡ Changqing Chang,†,§ Yinyue Deng,†,§ Shuwen An,§ Yi Hu Dong,§ Jianuan Zhou,†,‡ Meiying Hu,† Guohua Zhong,*,† and Lian-Hui Zhang*,†,§,‡ †

College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, People’s Republic of China § Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673, Republic of Singapore ‡ Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, People’s Republic of China S Supporting Information *

ABSTRACT: The widely used insecticide fenpropathrin in agriculture has become a public concern because of its heavy environmental contamination and toxic effects on mammals, yet little is known about the kinetic and metabolic behaviors of this pesticide. This study reports the degradation kinetics and metabolic pathway of fenpropathrin in Bacillus sp. DG-02, previously isolated from the pyrethroid-manufacturing wastewater treatment system. Up to 93.3% of 50 mg L−1 fenpropathrin was degraded by Bacillus sp. DG-02 within 72 h, and the degradation rate parameters qmax, Ks, and Ki were determined to be 0.05 h−1, 9.0 mg L−1, and 694.8 mg L−1, respectively. Analysis of the degradation products by gas chromatography−mass spectrometry led to identification of seven metabolites of fenpropathrin, which suggest that fenpropathrin could be degraded first by cleavage of its carboxylester linkage and diaryl bond, followed by degradation of the aromatic ring and subsequent metabolism. In addition to degradation of fenpropathrin, this strain was also found to be capable of degrading a wide range of synthetic pyrethroids including deltamethrin, λ-cyhalothrin, β-cypermethrin, β-cyfluthrin, bifenthrin, and permethrin, which are also widely used insecticides with environmental contamination problems with the degradation process following the first-order kinetic model. Bioaugmentation of fenpropathrin-contaminated soils with strain DG-02 significantly enhanced the disappearance rate of fenpropathrin, and its half-life was sharply reduced in the soils. Taken together, these results depict the biodegradation mechanisms of fenpropathrin and also highlight the promising potentials of Bacillus sp. DG-02 in bioremediation of pyrethroidcontaminated soils. KEYWORDS: Bacillus sp. DG-02, fenpropathrin, bioremediation, metabolites, biodegradation pathway, kinetics



INTRODUCTION Synthetic pyrethroids (SPs) are the chemical analogues of pyrethrins, which are natural chemicals derived from the flowers of Chrysanthemum cinerariaefolium.1 The usage of SPs has been increasing steadily over the past two decades, and these insectcides have now become one of the most widely used classes of pesticides.2 Especially with the ban or restricted use of organophosphates in more and more countries, SPs have generally been regarded as the suitable replacement, and their commercial production and usage are anticipated to be further increased.3 Currently, they are the dominant insecticides,4 contributing to >25% of worldwide insecticide sales.5 Most pyrethroids contain cyclopropanecarboxylic acid moieties (or an equivalent group) linked to aromatic alcohols through a central ester (or ether) bond (Figure 1). Fenpropathrin [α-cyano-3-phenoxybenzyl 2,2,3,3-tetramethylcyclopropanecarboxylate] is one of the most popular SPs, used for the control of a broad spectrum of insect pests.6 However, a consequence of the increased use of this insecticide is widespread environmental pollution problems, leading to serious damages to nontarget organisms.7,8 Recent studies showed that fenpropathrin was detected in nearly all tested agricultural and residential runoff samples.3,4,9−11 Such a © 2014 American Chemical Society

widespread environmental contamination situation also increases the potential of human exposure to the insecticide.12−16 Although SPs are considered safer than organophosphorous insecticides, evidence is accumulating that developmental exposure to fenpropathrin may cause severe toxic effects on human beings, including reproductive damages, neurotoxicity, immunotoxicity, endocrine disruption, and carcinogenesis.17−20 Furthermore, fenpropathrin is also highly toxic to fish and invertebrates.21 Thus, effective measures for reducing this contamination problem are critically needed to ensure that human health and environmental sustainability will not be compromised by the continued use of fenpropathrin. Microorganism-based bioremediation is the most promising and efficient strategy for removal of environmental pollutants compared to the conventional physicochemical methods such as photodecomposition, fenton degradation, ozonation, and adsorption, which are known for high operating cost and low efficiency.22−24 There is a growing body of evidence that Received: Revised: Accepted: Published: 2147

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Figure 1. Molecular structures of SPs. concentration of 10 g L−1, and stored in dark bottles at 4 °C prior to use. The stock solutions were sterilized by membrane filtration and rationed into medium to get the desired concentrations. At high concentrations, polysorbate 80 was used as emulsifier to disperse the possible precipitate in the liquid medium. All other chemicals and solvents used in this study were of analytical grade. Bacterium Used in This Study. The mineral salt medium (MSM) containing (g L−1) (NH4)2SO4, 2; MgSO4·7H2O, 0.2; CaCl2·2H2O, 0.01; FeSO4·7H2O, 0.001, Na2HPO4·12H2O, 1.5; and KH2PO4, 1.5, was used for the isolation of potential degrading strains. The final pH was adjusted to 7.5. The bacterial strain DG-02, which was deposited in the China Center for Type Culture Collection (collection no. CCTCC M 20111382), was isolated from a pyrethroid-manufacturing wastewater treatment system using an enrichment culture technique.34 In this study, the bacterium was further identified by API 50 CHB systems (bioMérieux Inc., France) according to the instructions of the manufacturer. Inoculum Preparation. The bacterial strain was stored in 15% glycerol at −80 °C. Before each experiment the strain was thawed and grown in 250 mL Erlenmeyer flasks containing 50 mL of sterile Luria− Bertani (LB) medium (containing tryptone, 10 g L−1; yeast extract, 5 g L−1; NaCl, 10 g L−1). At 30 °C, the flasks were placed on a platform shaker at 180g. The bacterial cells in the late-exponential growth phase were harvested by centrifugation (5 min, 4000g) at 4 °C and washed twice in 0.9% normal saline (N-saline) before inoculation. Unless otherwise stated, the densities of strain DG-02 were adjusted with sterile N-saline to approximately 1.0 × 108 colony-forming units (CFU) mL−1. One percent of this suspension was used as the inocula for the subsequent studies. Growth and Degradation Experiments. For the growth and degradation experiments, triplicate cultures were grown in MSM supplemented with 50 mg L−1 of fenpropathrin as the sole carbon source at 30 °C and 180g on a rotary shaker. A noninoculated culture served as a control. Samples were collected periodically from the cultures. The bacterial growth was monitored by counting the CFU per liter of serial dilutions, and the amount of residual fenpropathrin was determined by high-performance liquid chromatography (HPLC) as described below.

xenobiotic compounds can be successfully eliminated by diverse microorganisms belonging to different taxonomic groups.25−29 Recently, Cycoń et al.24 reported that Bacillus sp. TDS-2 isolated from sandy soil successfully removed thiophanatemethyl (100 mg kg−1) from soils within 24 days. Chen et al.26 reported that about 80% of the initial dose of β-cypermethrin (50 mg kg−1) was removed from the sterilized soils by Streptomyces aureus HP-S-01 within 10 days. A few bacterial species, including Sphingobium sp. JZ-2,30 Sphingobium sp. JQL4-5,31 Clostridium sp. ZP3,32 and Ochrobactrum trtici pyd1,33 have been reported for the degradation of fenpropathrin. However, these isolates commonly degraded only a couple of pyrethroid insecticides, which may limit their use in bioremediation as various regions are usually contaminated by multiple pyrethroid compounds.3,4,9−11 Furthermore, little is known about the biodegradation pathway and catalytic mechanisms of fenpropathrin. In our previous study, Bacillus sp. DG-02 was isolated from the pyrethroid-manufacturing wastewater treatment system.34 In the present study, we demonstrated that strain DG-02 was capable of degrading not only fenpropathrin but also other related SPs. The aims of this study were to investigate the biodegradation kinetics and to determine the bacterial metabolic products of fenpropathrin. Finally, our findings highlight the promising potentials and advantages of Bacillus sp. DG-02 for bioremediation of pyrethroid-contaminated environments.



MATERIALS AND METHODS

Chemicals. Deltamethrin (98%), fenpropathrin (93%), λ-cyhalothrin (95%), β-cypermethrin (94.8%), β-cyfluthrin (95%), bifenthrin (98%), and permethrin (95%) were obtained from Zhongshan Aestar Fine Chemical Inc., Ltd., China. The chromatographic grade acetonitrile were purchased from Sigma-Aldrich, USA. The chemicals were dissolved in dimethyl sulfoxide (DMSO) or acetone at a stock 2148

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Figure 2. Degradation and utilization of fenpropathrin (50 mg L−1) during growth of strain DG-02: (□) fenpropathrin control; (■) fenpropathrin degradation by strain DG-02; (▲) cell growth. Error bars indicate the standard deviation of three replicates. The mineral salt media containing different final concentrations of fenpropathrin (25, 50, 100, 200, 400, 600, 800, 1000, and 1200 mg L−1) were inoculated respectively with strain DG-02 and incubated at 30 °C and 180g on a rotary shaker. Each treatment was performed in triplicate, and fenpropathrin residues were measured at an interval of 12 h. Identification of Metabolites during Fenpropathrin Degradation. The metabolic products of fenpropathrin in cell-free filtrates of strain DG-02 cultures containing different concentrations of fenpropathrin (50, 100, and 200 mg L−1) were extracted and identified by gas chromatopraphy−mass spectrometry (GC-MS) (Agilent 6890N/5975, USA). The cell-free filtrates were taken at an interval of 12 h. The same bacterial culture supernatants lacking fenpropathrin were used as a negative control, and noninoculated control containing the same amount of fenpropathrin was included as well. The metabolites were extracted with ethyl acetate from the cell-free filtrates after acidification to pH 2 with 2 M HCl. The organic layer was dehydrated, dried, and redissolved in methanol.30,35 After filtration with a 0.45 μm membrane (Millipore, USA), the samples were subjected to GC-MS as described below. The metabolites identified by mass spectrometry analysis were matched with authentic standard compounds from the National Institute of Standards and Technology (NIST, USA) library database. Degradation Kinetics of Various SPs. To determine its ability to degrade various SPs, strain DG-02 was inoculated into fresh MSM supplemented with 50 mg L−1 deltamethrin, λ-cyhalothrin, βcypermethrin, β-cyfluthrin, bifenthrin, and permethrin, respectively. Triplicate cultures were grown in MSM at 30 °C. Degradation measurements were carried out periodically as described above. Biodegradation of Fenpropathrin in Soils. The soil samples were collected from the top 0−20 cm from a farm in South China Agricultural University, Guangzhou, southern China, that had never been treated with fenpropathrin or organic and inorganic fertilizers. The detailed physicochemical properties of the soils were (g kg−1 of dry weight): organic matter, 10.5; total N, 0.5; total P, 0.4; total K, 18.2; and pH, 6.9. The soils have a sandy loam texture (sand, 65.0%; silt, 28.0%; clay, 7.0%).34 In the laboratory, the soils were gently airdried to the point of soil moisture suitable for sieving. After sieving to a maximum particle size of <2 mm, the soils were used in the bioremediation experiments. The bioremediation experiment with strain DG-02 was performed in sterile and nonsterile soils. In the first case, soils were sterilized by autoclaving for 1 h at 121 °C. The stock solution of fenpropathrin was sprayed on the surface of 200 g of sterile and nonsterile soils.24 The applied dose of insecticide corresponded to a soil concentration of 50 mg kg−1. After thorough mixing, the bacterial suspension was

introduced in triplicate into the soils by drip irrigation to give a final bacterial count of approximately 1.0 × 108 CFU g−1 of soil. The triplicate samples of sterile and nonsterile soils without strain DG-02 were kept as controls, respectively. The same amount of pesticide was applied to these soils. The water contents were adjusted to approximately 40% of water-holding capacity by the addition of sterile deionized water.24 All of the soil samples were incubated in a darkened thermostatic chamber maintained at 30 °C for 15 days. Soil treatments (20 g) were removed aseptically at 0, 3, 6, 9, 12, and 15 days for the determination of fenpropathrin concentrations. Chemical Analyses. Pyrethroid quantification was performed on an Agilent 1100 HPLC equipped with a ternary gradient pump, programmable variable-wavelength UV detector, column oven, electric sample valve, and C18 reversed-phase column (Hypersil ODS2 5 μm × 4.6 mm × 250 mm) with array detection from 190 to 400 nm (total scan) based on retention time and peak area of the pure standard. The samples were performed using a mobile phase of 90:10 acetonitrile/ water. Sample injection volume was 10 μL, and mobile phase was programmed at a flow rate of 1 mL min−1.36,37 The metabolites of fenpropathrin were confirmed on an Agilent 6890N/5975 GC-MS system equipped with an autosampler and oncolumn, split/splitless capillary injection system, with a HP-5MS capillary column (30.0 m × 250 μm × 0.25 μm) with array detection from 30 to 500 nm (total scan). The operating conditions were as follows: the column was held initially at a temperature of 90 °C for 2 min, then raised at 6 °C min−1 to 150 °C for 1 min, at 10 °C min−1 to 180 °C for 4 min, and finally at 20 °C min−1 to 260 °C for 10 min. The temperatures corresponding to the transfer line and the ion trap were 280 and 230 °C, respectively, and the ionization energy was 70 eV. The injection volume was 1.0 μL with splitless sampling at 250 °C. Helium was used as a carrier gas at a flow rate of 1.5 mL min−1.38 Kinetics Analyses. The substrate inhibition model (eq 1) was used to fit the specific degradation rate (q) at different initial concentrations of fenpropathrin.

q=

qmax S S + K s + (S2/K i)

(1)

qmax is the maximum specific fenpropathrin degradation rate (h−1), Ki is the substrate inhibition constant (mg L−1), Ks is the half-saturation constant (mg L−1), and S is the inhibitor concentration (mg L−1). The disappearance of fenpropathrin and other pyrethroids in MSM or soils was fitted to the first-order kinetic model (eq 2).

Ct = C0 × e−kt 2149

(2)

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Figure 3. Relationship between initial fenpropathrin concentration and specific degradation rate by strain DG-02. C0 is the amount of substrate at time zero, Ct is the amount of substrate at time t, and k and t are the degradation rate constant (h−1) and degradation period in hours, respectively. The algorithm as expressed in eq 3 was used to determine the theoretical half-life (t1/2) values of fenpropathrin and other pyrethroids.

t1/2 =

ln(2) k

Biodegradation of Fenpropathrin with Different Initial Concentrations. To test the effect of fenpropathrin concentration on its degradation, strain DG-02 was inoculated to the media containing various initial fenpropathrin concentrations ranging from 25 to 1200 mg L−1 (Figure S1 in the Supporting Information). Strain DG-02 rapidly degraded and utilized the added fenpropathrin up to the concentration of 1200 mg L−1, and a lag period was observed at higher fenpropathrin concentrations. At a concentration of 25 mg L−1, the residual level of fenpropathrin could not be detected in just over 72 h. Further loading of the organic compound in subsequent experiments demonstrated that strain DG-02 was capable of degrading about 93.3 and 90.4% of fenpropathrin at a dose of 50 and 100 mg L−1 in 72 h, respectively, and the strain completely degraded the added doses in approximately 120 h. When the concentration was increased to 200, 400, and 600 mg L−1, respectively, about 87.6, 84.7, and 80.5% degradations of this compound were observed in 72 h, respectively, and complete degradation occurred after a prolonged culture. Over this time period the cells could utilize only 75.8, 67.2, and 61.2% of fenpropathrin when it was given 800, 1000, and 1200 mg L−1, respectively. These findings suggest that increased concentration of fenpropathrin has a slight effect on biodegradation performance of strain DG-02 with a modest increase in the duration of lag phase, but did not lead to complete inhibition or cell death. Previous studies showed that the pyrethroid-degrading strains usually transform the added pesticides with a concentration lower than 200 mg L−1.39,40 At higher concentrations, however, the metabolic activity of the reported pyrethroid-degrading strains was subject to complete catabolite repression.41 Noticeably, our results suggest this particular isolate could tolerate and degrade fenpropathrin up to a concentration as high as 1200 mg L−1. This feature gives the pesticide degrader a competitive advantage in variable environments, as they could survive and utilize the pollutants even exposed to a high concentration.42 The decrease in fenpropathrin degradation rate following an increase in the initial fenpropathrin concentration implies that fenpropathrin may act as a partial inhibitor to strain DG-02. To address this possibility, the substrate inhibition model (eq 1) adapted from Luong was used to fit the specific degradation rate (q) at different initial concentrations.43 The relationship between q and S is shown in Figure 3. The kinetic parameters of strain DG-02 determined from nonlinear regression using

(3) −1

ln 2 is the natural logarithm of 2, and k is the rate constant (h ).



RESULTS AND DISCUSSION Identification of Strain DG-02 Using API Systems. In our previous studies, stain DG-02 was tentatively identified as Bacillus sp. based on the morphology and 16S rDNA gene analysis.34 In this study, the bacterium was further classified as Bacillus cereus by API 50 CHB systems (bioMérieux Inc., France) according to the instructions of the manufacturer, with good identification (95.3%). It was positive in tests such as glycerol, maltose, ribose, trehalose, adonitol, cellobiose, gluconate, and N-acetylglucosamine. It was negative in tests such as erythritol, sannose, sorbose, rhamnose, melibiose, 5keto-D-gluconate, and methyl α-D-mannoside. Detailed biochemical properties of strain DG-02 are presented in Table S1 in the Supporting Information. Utilization of Fenpropathrin as a Growth Substrate by Strain DG-02. The growth of strain DG-02 on fenpropathrin and the ability of this bacterium to degrade fenpropathrin are shown in Figure 2. Strain DG-02 degraded fenpropathrin rapidly with about 82% of the initial dose being eliminated within 48 h. During this time, the number of strain DG-02 cells was increased to its maximum level. After incubation for 72 h, approximately 93.3% of the added fenpropathrin was degraded by strain DG-02 when fenpropathrin was used as the growth substrate. It is worth mentioning that partial bacterial strain DG-02 perhaps utilize their dead cells for growth. Finally, the residual amount of the pesticide was not detectable by HPLC at 120 h post incubation (data not shown). In contrast with previous studies, the current study showed that the strain DG02 could efficiently degrade fenpropathrin, suggesting this isolate may be an ideal candidate for bioremediation of a fenpropathrin-contaminated environment. In the control test, no significant change in fenpropathrin concentration was observed in the uninoculated medium. 2150

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Table 1. Chromatographic Properties of Metabolites of Fenpropathrin during Degradation by Strain DG-02

with formation of seven new compounds (Figure 4b−h). The changes in fenpropathrin peak area over time are shown in Figure S2 in the Supporting Information. The seven compounds were characterized as 2,2,3,3-tetramethylcyclopropanecarboxylic acid phenyl ester, 3,4-dihydroxybenzoic acid, 3phenoxybenzoate, 3,4-dimethoxyphenol, 3-phenoxybenzaldehyde, α-hydroxy-3-phenoxybenzeneacetonitrile, and phenol, respectively, on the basis of the similarity of their fragment retention times and molecular ions to those of corresponding authentic compounds in the NIST library database (Figure S3 in the Supporting Information). Among these products, 3phenoxybenzaldehyde, 3-phenoxybenzoate, and 3,4-dimethoxybenzoic acid were also reported during the degradation of fenpropathrin in previous studies,30,33 but the other four metabolic products of fenpropathrin were detected for the first time in this study. These metabolite concentrations were very low. We also noted that these metabolites were transient, suggesting that fenpropathrin was completely degraded by the strain DG-02 without any noncleavable metabolites at the end of the experiment. In the negative control lacking fenpropathrin, none of these metabolites was detected. In the noninoculated control containing the same amount of fenpropathrin, only fenpropathrin was detected (Figure S4 in the Supporting Information).

matrix laboratory (MATLAB) software package (version 7.8) were qmax = 0.05 h−1 and Ks = 9.0 mg L−1. The inhibitory effect of fenpropathrin was considered to occur in a linear manner at Ki = 694.8 mg L−1. The critical inhibitor concentration (Sm) was established to be 78.9 mg L−1 by calculating the square root of Ki × Ks. The coefficient of determination (R2) was 0.9549, which indicates that the experimental data were well correlated with the model. As shown in Figure 3, when the initial concentration of fenpropathrin was <78.9 mg L−1, the q value was gradually increased. At higher concentrations, inhibition by fenpropathrin became substantial and the q value was proportionally decreased in a dosage-dependent manner. This reveals that the fenpropathrin degradation activity of strain DG02 could be partially inhibited at a high concentration of fenpropathrin but may not lead to a complete repression. Degradation Products and Degradation Pathway of Fenpropathrin. The predicted chemical structures, retention times (RT), and characteristic ions of the mass spectra (m/z) are summarized in Table 1. Compound A, with a RT of 17.293 min, showed a prominent protonated molecular ion at m/z 349, which is similar to the characteristic parental ion of fenpropathrin (Figure 4a). Additionally, compound A also has the same RT as the authentic fenpropathrin. Therefore, compound A was identified as fenpropathrin. Along with the degradation process, compound A disappeared concomitantly 2151

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Figure 4. continued

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Figure 4. GC-MS analysis of the metabolites produced from fenpropathrin degradation by strain DG-02. (a−h) Characteristic ions of compounds A−H in GC-MS. The retention times of the compounds were 17.293, 15.919, 11.034, 9.744, 9.576, 8.025, 7.907, and 4.392 min, respectively, which were identified as fenpropathrin, 2,2,3,3-tetramethylcyclopropanecarboxylic acid phenyl ester, 3,4-dihydroxybenzoic acid, 3-phenoxybenzoate, 3,4dimethoxyphenol, 3-phenoxybenzaldehyde, α-hydroxy-3-phenoxybenzeneacetonitrile, and phenol, respectively.

DG-02 (Figure 5). According to this pathway, fenpropathrin was initially hydrolyzed by cleavage of the carboxylester linkage to produce α-hydroxy-3-phenoxybenzeneacetonitrile and

On the basis of the chemical structures of fenpropathrin and the metabolic products formed during fermentation, we propose a degradation pathway of fenpropathrin in strain 2153

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Figure 5. Proposed pathway for degradation of fenpropathrin in strain DG-02.

Figure 6. Degradation kinetics of various SPs by strain DG-02: (◆) deltamethrin; (◇) fenpropathrin; (●) β-cypermethrin; (△) β-cyfluthrin; (▲) λ-cyhalothrin; (□) permethrin; (■) bifenthrin. Error bars indicate the standard deviation of three replicates.

2,2,3,3-tetramethylcyclopropanecarboxylic acid phenyl ester. The intermediate product α-hydroxy-3-phenoxybenzeneacetonitrile was unstable in the environment, which was spontaneously converted to 3-phenoxybenzaldehyde. Subsequent oxidization of 3-phenoxybenzaldehyde yielded 3-phenoxyben-

zoate, which was then subject to diaryl cleavage, resulting in the formation of 3,4-dihydroxybenzoic acid, 3,4-dimethoxyphenol, and phenol. However, these compounds were transient. Eventually, no persistent accumulative product was detected by GC-MS analysis, indicating that these compounds under2154

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neously degrade methyl parathion and fenpropathrin in MSM.31 Clostridium sp. ZP3 was reported to be capable of degrading fenpropathrin in liquid medium with the presence of an extra carbon source (glucose).32 One recently isolated fenpropathrin-degrading strain, O. trtici pyd-1, could also utilize fenpropathrin as a growth substrate and degrade approximately 90% of 100 mg L−1 of the pesticide in 72 h in liquid culture.33 However, these strains either could degrade only a limited number of pyrethroids or showed a low degradation efficiency, which limits their use in bioremediation of environments contaminated with multiple pyrethroids. It is noteworthy that strain DG-02 could degrade not only fenpropathrin but also other pyrethroids, which is rarely seen in other microorganisms. Previous papers suggest that the bacterial isolates from Bacillus genus could be the most metabolically active microorganisms, as they are capable of degrading a wide variety of xenobiotic aromatic compounds, including thiophanate-methyl,24 chlorpyrifos,29 and 4-chloro-2-nitrophenol.46 The results from this study further expand the metabolic list of the Bacillus species. In our previous studies, strain DG-02 was found to be highly efficient in degrading 3-phenoxybenzoic acid.34 3-Phenoxybenzoic acid is a major product common to most of the pyrethroids.47 Degradation of this compound by the same strain that degrades pyrethroids is very important because 3-phenoxybenzoic acid is generally antimicrobial, and it would be accumulated if not degradable, which prevents further degradation and may cause growth arrest.37 Importantly, the ability of strain DG-02 in degrading a wide range of pyrethroids suggests that this particular isolate has promising potentials and advantages as a bioremediation organism in removing pyrethroid residues from various environments as most contaminated regions were affected by multiple pyrethroid compounds.3,4,9−11 To predict the ability of strain DG-02 in biodegradation of various substrates in environment, the degradation process was fitted to the first-order kinetic model (eq 2). The kinetic parameters for all runs calculated from the equations are presented in Table 2. In these studies, the degradation rate and

went aromatic ring cleavage and further metabolism. Very interestingly, in our previous studies we demonstrated that strain DG-02 also degraded 3-phenoxybenzoic acid to give 3,4dihydroxybenzoic acid, 3,4-dimethoxyphenol, and phenol.34 Clearly, the bacterial strain appears to harbor a complete metabolic pathway for degradation and metabolism of fenpropathrin, indicating that the isolate could be an ideal candidate for bioremediation of the environments contaminated by fenpropathrin and the related pyrethroid insecticides. It was evident from the results that strain DG-02 degrades fenpropathrin by first cleavage of the carboxylester linkage and diaryl bond of fenpropathrin. Similar findings were also observed in biodegradation of other pyrethroid insecticides such as cyfluthrin and cypermethrin.38,42 However, our results were very different from the previous findings of Zhang et al.,32 who reported that Clostridium sp. ZP3 degraded fenpropathrin with an oxidization process to yield 3,5-dimethylamphetamine, benzenemethanol, and benzyl alcohol. It was generally regarded that ester hydrolysis via carboxylesterases was the primary degradation pathway of various pyrethroids in a multitude of species, from mammals to insects to microorganisms.30,40,44,45 The fenpropathrin degradation pathway in strain DG-02 appears to be similar to the initial steps of most environmental isolates in pyrethroid biodegradation processes, in which pyrethroids were converted to 2,2,3,3-tetramethylcyclopropanecarboxylic acid and 3-phenoxybenzoic acid through hydrolysis of the carboxylester linkage.30,35,39 Unfortunately, however, it is not clear whether these microorganisms are capable of further metabolism of pyrethroids. In this study, we showed that strain DG-02 could further metabolize the intermediate 3phenoxybenzoate by cleavage of the diaryl bond (Figure 5), leading to complete degradation of the pesticide, which is rarely seen in other pyrethroid-degrading strains. Degradation Kinetics of Various SPs. The abilities of strain DG-02 to degrade various pyrethroids are shown in Figure 6. The strain was capable of utilizing all of the pyrethroids tested. As shown in Figure 6, degradation of all tested compounds started rapidly at the beginning of incubation with no apparent lag period. Deltamethrin and fenpropathrin were the most preferred substrates, with degradation rates of 94.1 and 93.3% within 72 h of incubation, respectively. β-Cypermethrin, β-cyfluthrin, and λ-cyhalothrin were degraded relatively more slowly than deltamethrin and fenpropathrin, with degradation rates of 89.2, 85.6, and 82.7%, respectively. Bifenthrin and permethrin were the most persistent; only 65.1 and 63.6% of the added substrates were degraded under the same experimental conditions, suggesting that absence of the cyano group may cause a substantial reduction in the hydrolysis rate. These results contrast with the previous findings that degradation reduction in pyrethroid compounds with a cyano group is due to its toxic effect.33 Our results provide a strong endorsement to the previous view that different capacities of various microorganisms to utilize pyrethroid compounds could be attributed to a number of factors, including the differences in substrate transportation, enzyme specificity, and molecular structures of these compounds.34 Several papers on the microbial degradation of fenpropathrin have been published. Sphingobium sp. JZ-2 could use fenpropathrin as the sole carbon source in MSM and could degrade >80% of the added pesticide at a concentration of 50 mg L−1 after 4 days of incubation.30 A genetically engineered microorganism named JQL4-5-mpd was also able to simulta-

Table 2. Kinetic Parameters of Degradation of Various SPs by Strain DG-02 treatment deltamethrin fenpropathrin β-cypermethrin β-cyfluthrin λ-cyhalothrin bifenthrin permethrin

regression eq Ct Ct Ct Ct Ct Ct Ct

= = = = = = =

−0.0355t

52.0e 52.3e−0.0315t 54.8e−0.0286t 53.2e−0.0277t 51.9e−0.0234t 52.0e−0.0155t 50.7e−0.0160t

k (h−1)

t1/2 (h)

R2

0.0355 0.0315 0.0286 0.0277 0.0234 0.0155 0.0160

19.5 22.0 24.2 25.0 29.6 44.7 43.3

0.9826 0.9735 0.9657 0.9688 0.9564 0.9574 0.9718

the substrate concentration were in direct proportion, and the degradation process corresponded to the first-order kinetic model, with an R2 ranging from 0.9564 to 0.9826, indicating that the experimental data were well-correlated with the model. The degradation process was characterized by a k ranging from 0.0160 to 0.0355 h−1 and a t1/2 varying from 19.5 to 43.3 h (Table 3). Given that the reported t1/2 values for various pyrethroids in water or soil are usually between 17 and 600 days,1 addition of strain DG-02 significantly shortened the t1/2 for pyrethroids, suggesting that this strain is suitable for the efficient and rapid degradation of pyrethroid residues and cleanup of contaminated environments. 2155

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Table 3. Kinetic Parameters of Degradation of Fenpropathrin in Sterile and Nonsterile Soils Inoculated with Strain DG-02 treatment sterile soils + fenpropathrin nonsterile soils + fenpropathrin sterile soils + fenpropathrin + DG-02 nonsterile soils + fenpropathrin + DG-02

regression eq Ct = 49.8e Ct = 49.9e

−0.0098t −0.00187t

Ct = 51.6e−0.0981t

k (day−1)

t1/2 (days)

R2

0.0098

70.7

0.9791

0.0187

37.1

0.9913

0.0981

7.1

0.9849

AUTHOR INFORMATION

Corresponding Authors

*(G.Z.) E-mail: [email protected] *(L.-H.Z.) Phone: +86-20-85288229. Fax: +86-20-85280292. E-mail: [email protected] Funding

Financial support for some of this research was received from the Guangdong Hopson-Pearl River Education Development Foundation (HPEDF). Notes

Ct = 51.0e

−0.1281t

0.1281

5.4

The authors declare no competing financial interest.



0.9916

REFERENCES

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Biodegradation of Fenpropathrin in Soils. The abilities of strain DG-02 to degrade fenpropathrin in sterile and nonsterile soils were investigated, respectively. The kinetic parameters for all treatments are presented in Table 3. Kinetic data showed that the degradation process corresponded to the first-order model and was characterized by k ranging from 0.0098 to 0.1281 day−1. The coefficient of determination R2 was determined to be 0.9791−0.9916, showing that the experimental data are well-correlated with the model. Without strain DG-02, the t 1/2 values were 70.7 and 37.1 days for fenpropathrin in sterile and nonsterile soils, respectively. In contrast, the t1/2 values for fenpropathrin in sterile and nonsterile soils inoculated with strain DG-02 were shortened, giving t1/2 values of 7.1 and 5.4 days, respectively. Our results confirmed previous observation made by Khan et al.48 indicating that biodegradation is the main mechanism of SP dissipation in soils, whereas its abiotic breakdown is less important. Studies on other SPs have shown that microorganisms which can degrade them in culture conditions also perform their degradation in soils.26,49 In our bioremediation experiment, strain DG-02 efficiently degraded fenpropathrin in soils, suggesting that strain DG-02 may be ideal for bioremediation of soils contaminated with fenpropathrin. In conclusion, Bacillus sp. DG-02 was found to be highly efficient in degrading fenpropathrin in different contaminated soils and water resources. This strain was able to utilize fenpropathrin as the growth substrate and rapidly degraded the pesticide in a concentration as high as 1200 mg L−1. This is an important feature of a microorganism to be employed for bioremediation of variable environments. Moreover, we presented evidence that the bacterium harbors the metabolic pathway for complete degradation and metabolism of fenpropathrin. The strain could degrade both the carboxylester linkage and the diaryl bond of fenpropathrin, which plays a key role in the fenpropathrin biogeocycle. Another important feature which is worth mentioning is that this particular strain was capable of degrading a wide range of synthetic pyrethroids, suggesting that the isolate has promising potentials and advantages as a bioremediation organism in removing pyrethroid residues from water, soils, or crops.



Article

ASSOCIATED CONTENT

S Supporting Information *

Table S1 and Figures S1−S4. This material is available free of charge via the Internet at http://pubs.acs.org. 2156

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