Simultaneous Degradation of Cypermethrin and Its Metabolite, 3

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Simultaneous Degradation of Cypermethrin and Its Metabolite, 3‑Phenoxybenzoic Acid, by the Cooperation of Bacillus licheniformis B‑1 and Sphingomonas sp. SC‑1 Fangfang Liu, Yuanlong Chi, Shuang Wu, Dongying Jia, and Kai Yao* College of Light Industry and Food Engineering, Sichuan University, 610065 Chengdu, Sichuan, People’s Republic of China ABSTRACT: Cypermethrin (CY) and its metabolite, 3-phenoxybenzoic acid (3-PBA), generally coexist in agricultural soil and cause a toxic effect on the human body. In this study, CY and its metabolite 3-PBA were simultaneously degraded by the cooperation of Bacillus licheniformis B-1 and Sphingomonas sp. SC-1. The effects of the inoculation proportion and inoculation method of these two strains, cultivation time, and initial CY content on the degradation of CY and 3-PBA were investigated. Furthermore, the degradation of CY and 3-PBA in soil environment by using the cooperation of these two strains was also determined. When the inoculation proportion of the biomass of strain B-1/strain SC-1 was 3.3:6.7, strain B-1 was inoculated first, and strain SC-1 was inoculated after 24 h of cultivation, 75.60% CY (100 mg L−1) was degraded at 72 h and the 3-PBA content was 10.31 mg L−1. Compared with those by using only strain B-1, the half-life of CY by using these two strains was shortened from 71.90 to 35.71 h, and the yield coefficient of 3-PBA was decreased from 0.8938 to 0.2651. As in the soil environment, the CY content by using these two strains within a period of 25 days declined from 22.71 to 5.33 mg kg−1 and the 3-PBA content was 1.84 mg kg−1. Compared with those by using only strain B-1, the half-life of CY by using these two strains was shortened from 19.86 to 11.34 days and the yield coefficient of 3-PBA was decreased from 0.5302 to 0.2056. This work could develop a promising approach for the simultaneous degradation of CY and its metabolite 3-PBA in agricultural soil. KEYWORDS: cypermethrin, 3-phenoxybenzoic acid, simultaneous degradation, Bacillus licheniformis B-1, Sphingomonas sp. SC-1, cooperation

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INTRODUCTION Cypermethrin (CY), used as an important pyrethroid pesticide, has been extensively used for pest control in fruits and vegetables or as grain protectant against stored-product insect pests,1−3 especially when the use of organophosphate and organochlorine is restricted due to their high toxicity.2 However, CY often remains in agricultural soil and agricultural products owing to excess spraying,4,5 and CY residues would be accumulated in human or mammal bodies through frequent contact or the food chain,6 which might cause toxic effects on the reproductive system,5 immune system,7 and nervous system.8 Therefore, to remediate CY-contaminated regions, biodegradation is generally considered as an effective method to remove CY residues, owing to its characteristics of high efficiency, easy application, and environmental friendliness.9−11 As reported, most CY-degrading organisms could not mineralize CY, but transformed it into 3-phenoxybenzoic acid (3-PBA).12 3-PBA could disrupt the normal secretion of reproductive hormones13 and lead to sperm DNA fracture and sperm concentration decrease in the male body.14 Moreover, 3PBA is more likely to spread in the environment due to its stronger hydrophilicity compared with CY15 and thus could cause some food safety problems. 16,17 Therefore, the biodegradation of 3-PBA has also attracted increasing attention.15 To the best of our knowledge, there are many research sutdies focused on the degradation of either CY18,19 or 3PBA20,21 separately. Jilani et al. found that Pseudomonas IES-Ps1 had a good degradation ability on CY and could degrade 51% of CY (80 mg L−1) in 48 h.22 Chen et al. reported that © 2014 American Chemical Society

Stenotrophomonas sp. ZS-S-01 could degrade 82.9% of 3-PBA (250 mg L−1) in 9 days.23 However, no information about the simultaneous degradation of CY and 3-PBA is available. In fact, CY and 3-PBA generally coexisted in the CY degradation process. Therefore, the simultaneous degradation of these two compounds would be of important practical application value. Our previous studies proved that Bacillus licheniformis B-1 (strain B-1) and Sphingomonas sp. SC-1 (strain SC-1) had good degradation capacities on CY and 3-PBA, respectively.21,24 In this study, these two strains were used for the cooperative degradation of CY and 3-PBA. The purpose is to enhance CY degradation and remove the metabolite 3-PBA simultaneously. Figure 1 shows the proposed degradation pathway of CY and 3PBA by using strains B-1 and SC-1. CY is first degraded into 3PBA through hydrolysis by strain B-1,12 and then the 3-PBA is converted into muconic acid through benzene ring cleavage by strain SC-1.21 The metabolite of muconic acid has been proved to be of no harm to the human body.25

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MATERIALS AND METHODS

Materials. Cypermethrin (CY, 99.7%) was purchased from the National Standard Substances Center (Beijing, China). 3-Phenoxybenzoic acid (3-PBA, 98%) was purchased from Sigma-Aldrich Chemical Co. (Shanghai, China). Chromatographic grade acetonitrile was purchased from Meridian Medical Technologies (Beijing, China). Acetonitrile, ethyl acetate, ethyl alcohol, KH2PO4, K2HPO4, MgSO4, Received: January 22, 2014 Accepted: July 28, 2014 Published: July 28, 2014 8256

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membrane filter. Then, the collected filtrate was used for the determination of CY and/or 3-PBA contents. Determination of CY Content. The content of CY was determined by an LC-20AT high-performance liquid chromatograph (HPLC, Shimadzu, Kyoto, Japan) equipped with an LC-20AT pump (Shimadzu), a CTO-20A column oven (Shimadzu), a Kromasil C18 column (250 mm × 4.60 mm, 5.0 μm; Sweden), and an SPD-M20A UV detector (Shimadzu). A series of CY acetonitrile solutions were prepared, and their contents ranged from 2.5 to 500 mg L−1. After pretreatment as described under Pretreatment, 20 μL of the solution was injected manually, and elution was performed at 35 °C using 90% (v/v) aqueous acetonitrile solution with a flow rate of 1.0 mL min−1. The detection wavelength was 210 nm.26 Linear regression was taken between CY contents (X, mg L−1) and their corresponding peak areas (Y), and then the calibration curve equation of CY was obtained as follows: Y = 57585X + 99879, R2 = 0.9996, which was used for the calculation of CY content. Determination of 3-PBA Content. The content of 3-PBA was determined by HPLC. A series of 3-PBA acetonitrile solutions with contents ranging from 1.0 to 200 mg L−1 were prepared. HPLC detection was performed with a mobile phase of 60% (v/v) aqueous acetonitrile solution and a flow rate of 0.6 mL min−1. Other chromatographic conditions were the same as those used for the determination of CY content. The calibration curve equation of 3-PBA was obtained as follows: Y = 127025X + 84767, R2 = 0.9998, where X and Y represent the 3-PBA content (mg L−1) and its corresponding peak area, respectively. It was used for the calculation of 3-PBA content. Degradation of CY by B. licheniformis B-1. CY-degrading tests by using B. licheniformis B-1 were conducted in 250 mL Erlenmeyer flasks, which contained 30 mL of LB medium with 100 mg L−1 CY. Strain B-1 inoculum was first shaken by a vortex mixer for 30 s, and then 1.5 mL of the inoculum was transferred into the medium (inoculated). The flask was shaken at 180 rpm and 30 °C. Uninoculated LB medium with 100 mg L−1 CY served as control. Microbial biomass, CY degradation (%), and 3-PBA content (mg L−1) in media were measured every 12 h. Microbial biomass was expressed by the optical density value measured at 600 nm (OD600), and CY degradation was calculated according to eq 1, where C is the content of CY in inoculated medium and Ck is the content of CY in control.

Figure 1. Proposed degradation pathway of CY and its metabolite 3PBA by the cooperation of Bacillus licheniformis B-1 and Sphingomonas sp. SC-1.

NaCl, NaOH, Na2SO4, (NH4)2SO4, and Tween 80 were of analytical grade and purchased from Kelong Chemical Co. (Chengdu, China). Microorganisms and Media. The CY-degrading strain B-1 and the 3-PBA-degrading strain SC-1 were obtained from the soil in a tea garden (Ya’an, China) and from the sludge in a pesticide factory (Chengdu, China), respectively. Both were transferred to the laboratory by using sterile paper bags and domesticated by using enrichment culture technique. After several enrichment cultures, they were isolated by diluting the final cultures serially and spreading them on enrichment medium agar plates. On the basis of the analysis of morphological, physiological, and biochemical characteristics and 16S rDNA sequence (GenBank accession no.: strain B-1, HQ009796; strain SC-1, JN857975), strains B-1 and SC-1 were identified as B. licheniformis24 and Sphingomonas sp.,21 respectively. Both were stored in a 15% glycerol solution at −80 °C before experiments. Several kinds of media were prepared. Luria−Bertani (LB) medium, containing 5.0 g L−1 yeast extract, 10.0 g L−1 peptone, and 10.0 g L−1 NaCl, was used for CY degradation tests. Mineral salt (MS) medium, containing 0.2 g L−1 MgSO4, 0.5 g L−1 KH2PO4, 0.5 g L−1 NaCl, 1.5 g L−1 (NH4)2SO4, and 1.5 g L−1 K2HPO4 was used for 3-PBA degradation tests. LB-MS medium, containing both LB and MS, was used for CY+3-PBA degradation tests. All media were adjusted to pH 7.0−7.5, and 0.2% Tween 80 (v/v) was added as an emulsifying agent before sterilization at 121 °C for 20 min. Inoculum Preparation. Strain B-1 (or strain SC-1) was thawed and inoculated into a 100 mL Erlenmeyer flask, which contained 30 mL of LB-MS medium with 100 mg L−1 CY (or 3-PBA). Then the flask was placed in a rotary shaker (Multitron-Pro, Infors, Switzerland) at 180 rpm and 30 °C, and the activated strain was obtained at 16 h of cultivation. After being centrifuged (ST40R, Thermo, German) at 10000 rpm and 4 °C for 10 min, the strain was collected and suspended in sterile N-saline (0.9% NaCl) to achieve a cell density of about 1.0 × 108 cells mL−1. Then, the bacterial suspension was used as inoculum. Determination of the Content of CY and 3-PBA by HPLC. Pretreatment. The broth culture was shaken by a vortex mixer for 30 s first, and 5 mL of broth culture was transferred to a 100 mL flask. Then, 5 mL of acetonitrile was added into the flask, and the flask was shaken by a vortex mixer for 30 s. The CY or 3-PBA in broth culture was extracted under ultrasonic (40 kHz and 300 W; B-3510E, Branson, America) for 30 min.26 After centrifugation at 8000 rpm and 25 °C for 20 min, the supernatant was collected and filtered through a 0.22 μm

CY degradation (%) = (1 − C /C k ) × 100

(1)

Degradation of 3-PBA by Sphingomonas sp. SC-1. 3-PBAdegrading tests by using Sphingomonas sp. SC-1 were done in 250 mL Erlenmeyer flasks, which contained 30 mL of MS medium with 300 mg L−1 3-PBA. Strain SC-1 inoculum (1.5 mL) was inoculated to the medium, and the flask was shaken at 180 rpm and 30 °C. Uninoculated MS medium with 300 mg L−1 3-PBA was used as control. Microbial biomass and 3-PBA degradation (%) in media were measured every 2 h. 3-PBA degradation was calculated according to eq 2, where C1 is the content of 3-PBA in inoculated medium and Ck1 is the content of 3-PBA in control.

3‐PBA degradation (%) = (1 − C1/C k1) × 100

(2)

Degradation of CY and 3-PBA by Cooperation of Strains B-1 and SC-1. Effect of Inoculation Proportion of Strains B-1 and SC-1. Mixed inocula of strains B-1 and SC-1 at different biomass ratios (10:0, 8:2, 7.5:2.5, 6.7:3.3, 5:5, 4:6, 3.3:6.7, 2.9:7.1, 2.5:7.5, 2:8, 0:10) were prepared, and the cell density of the mixed inocula were all 1.0 × 108 cells mL−1. Each inoculum (1.5 mL) was separately inoculated to 30 mL of LB-MS media with 100 mg L−1 CY. Uninoculated LB-MS medium with 100 mg L−1 CY served as control. All of them were cultivated by shaker at 30 °C and 180 rpm for 72 h. Then, the CY degradation (%) and 3-PBA content (mg L−1) in media were measured. Effect of Inoculation Method. Strains B-1 and SC-1 at the biomass ratio of 3.3:6.7 were inoculated into 30 mL LB-MS medium with 100 mg L−1 CY by using three different methods described below. Uninoculated LB-MS medium with 100 mg L−1 CY served as control. 8257

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CY degradation (%) and 3-PBA content (mg L−1) in media were measured after 72 h of cultivation. Method 1: Strains B-1 and SC-1 were inoculated to the medium, simultaneously. Method 2: Strain B-1 was first inoculated to the medium. At 24 h of cultivation, its biomass was determined as N0. Strain SC-1 at the biomass of 6.7N0/3.3 was inoculated to the medium and cultivated for a further 48 h. Method 3: Strain SC-1 was first inoculated to the medium. At 24 h of cultivation, its biomass was determined as N1. Strain B-1 at the biomass of 3.3N1/6.7 was inoculated to the medium and cultivated for a further 48 h. Effect of Cultivation Time. Degradation tests for a cultivation period of 96 h were done in 30 mL of LB-MS medium with 100 mg L−1 CY. Strain B-1 was first inoculated to the medium. At 24 h of cultivation, its biomass was determined as N, and strain SC-1 at the biomass of 6.7N/3.3 was inoculated and cultivated for a further 72 h. Uninoculated LB-MS medium containing 100 mg L−1 CY served as control. Microbial biomass, CY degradation (%), and 3-PBA content (mg L−1) in media were measured every 12 h. Effect of Initial CY Content. Degradation tests at different initial CY contents (10, 25, 50, 100, 150 mg L−1) in LB-MS media were conducted. Strain B-1 was first inoculated to the medium. At 24 h of cultivation, its biomass was determined as N, and strain SC-1 at the biomass of 6.7N/3.3 was inoculated and cultivated for a further 48 h. Then the CY degradation (%) and 3-PBA content (mg L−1) in media were measured. Uninoculated LB-MS medium containing a certain content of CY served as control. Degradation of CY and Its Metabolite 3-PBA in Soil Environment. The soil was collected from the top 0−10 cm of a vegetable farmland in Sichuan University. The soil was sieved (mesh size = 2 mm) and air-dried overnight. The characteristics of the soil (g−1, dry weight) were detected as follows: organic matter, 54.9 mg; total bacteria, 4.58 × 108 CFU; total actinomycetes, 1.33 × 108 CFU; total fungi, 1.67 × 107 CFU; and pH 7.86. A certain amount of CY was added to the soil to achieve a final content of 20 mg kg−1, and then the soil was placed overnight. Three soil samples with a weight of about 1000 g were used. One sample was inoculated with strain B-1 (1.0 × 108 cells g−1 soil, dry weight) and called B sample. Another sample was first inoculated with strain B-1 (3.3 × 107 cells g−1 soil, dry weight). At 24 h of cultivation, strain SC-1 (6.7 × 107 cells g−1 soil, dry weight) was inoculated and called BS sample. The third sample was uninoculated and used as control. The moisture contents of these three soil samples were kept at 40.0 ± 5.0% (w/w) by supplying water manually, and the average temperature was 22 °C. The contents (mg L−1) of CY and 3-PBA in soil were measured every 2 days. As for the extraction of CY and 3-PBA in the soil, the soil was dried to constant weight, and 5.0 g of soil was mixed with 10 mL of acetonitrile in a 100 mL flask under ultrasonic (40 kHz and 300 W) for 60 min. Ten milliliters of ethyl acetate was then added, and the supernatant was collected. The supernatant was purified by passing through an anhydrous Na2SO4 column.26 After recovery of the organic solvent at 50 °C in a vacuum, the CY and 3-PBA solution was obtained by redissolving into 1 mL of acetonitrile. It was filtered through a 0.22 μm membrane filter and used for the determination of CY and 3-PBA contents.

Figure 2. Microbial biomass (OD600), CY degradation, and 3-PBA content in the CY degradation by B. licheniformis B-1. Results are expressed as the average and standard deviation of three parallel samples.

amount of cyano-3-phenoxybenzyl alcohol was transformed to 3-PBA. From 24 to 72 h, the CY degradation continued to increase, and the 3-PBA content began to increase quickly. These might be because cyano-3-phenoxybenzyl alcohol was gradually oxidized to 3-phenoxybenzaldehyde and then to 3PBA.12 At 72 h, CY degradation and 3-PBA content reached 50.36% and 23.16 mg L−1, respectively. Compared with other CY-degrading organisms, such as Pseudomonas IES-Ps-1 isolated by Jilani and Khan that could degrade 51% CY (80 mg L−1) in 48 h22 and Bacillus sp. ISTDS2 reported by Sundaram and co-workers that could completely degrade CY (50 mg L−1) in 180 h,27 strain B-1 exhibited similar degradation ability on CY. However, the metabolite 3-PBA would be accumulated in the CY degradation process, and strain B-1 possessed poor degradation of 3-PBA. Degradation of 3-PBA by Sphingomonas sp. SC-1. Microbial biomass (OD600) and 3-PBA degradation in the 3PBA degradation process by Sphingomonas sp. SC-1 were determined, and the results are shown in Figure 3. From 0 to 6

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RESULTS AND DISCUSSION Degradation of CY by B. licheniformis B-1. Microbial biomass (OD600), CY degradation, and 3-PBA content in the CY degradation process by B. licheniformis B-1 are shown in Figure 2. In the initial cultivation phase (0−24 h), the OD600 value and CY degradation all exhibited rapid increase trends, and the CY degradation reached 23.35% at 24 h, whereas only 0.72 mg L−1 3-PBA was detected. This might be due to the fact that CY was first degraded into cyano-3-phenoxybenzyl alcohol by esterase produced by strain B-1,12 and only a very small

Figure 3. Microbial biomass (OD600) and 3-PBA degradation in the 3PBA degradation by Sphingomonas sp. SC-1. Results are expressed as the average and standard deviation of three parallel samples.

h, strain SC-1 could be in the lag growth phase, so its OD600 value remained constant. Low 3-PBA degradation was found. From 6 to 16 h, 3-PBA was degraded rapidly and could be completely degraded at 16 h. The possible mechanism of 3PBA degradation by strain SC-1 was described as follows. 3PBA was first degraded to 2-phenoxyphenol under the effects of 8258

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decarboxylase and hydroxylase produced from strain SC-1. Then 2-phenoxyphenol was converted into catechol owing to cleavage of the ether linkage and further degraded into muconic acid by the role of dioxygenase.21 Chen et al. reported that Stenotrophomonas sp. ZS-S-01 could achieve 82.9% degradation of 3-PBA (250 mg L−1) in 9 days,23 and Ochrobactrum lupine DG-S-01 could completely degrade 100 mg L−1 3-PBA in 14 days.28 Compared with these two strains, strain SC-1 exhibited a stronger degradation capacity on 3-PBA and could completely degrade 300 mg L−1 3-PBA in 16 h. Simultaneous Degradation of CY and Its Metabolite 3-PBA by Cooperation of Strains B-1 and SC-1. Effect of Inoculation Proportion of Strains B-1 and SC-1. Strains B-1 and SC-1 have been proved to possess good degradation performance on CY and 3-PBA, respectively. Therefore, to improve the degradation of CY and decrease the metabolite 3PBA content, simultaneously, the cooperation of these two strains was used in a CY degradation process. The simultaneous degradation of CY and its metabolite 3PBA was performed under different inoculation proportions of strains B-1 and SC-1, as the results show in Figure 4. When

Figure 5. CY degradation and 3-PBA content by using three different inoculation methods. Results are expressed as the average and standard deviation of three parallel samples.

inoculated, was the most effective inoculation method for degrading CY and 3-PBA, simultaneously. It was presumably because (i) the antagonism and competition between strains B1 and SC-1 might be minor29 and (ii) CY was first degraded into 3-PBA by strain B-1 and then the 3-PBA was further degraded by strain SC-1, in accordance with the proposed degradation process of CY shown in Figure 1. Therefore, method 2 was used as the inoculation method in the following experiments. Effect of Cultivation Time. Microbial biomass (OD600), CY degradation, and 3-PBA content in the cultivation period of 96 h were investigated, and the results are shown in Figure 6. In

Figure 4. CY degradation and 3-PBA content by using different inoculation proportions of strains B-1 and SC-1. Results are expressed as the average and standard deviation of three parallel samples.

only strain B-1 was inoculated to the media (B-1/SC-1 = 10:0), 51.08% CY was degraded and 22.81 mg L−1 3-PBA was accumulated. As the inoculation proportion of strains B-1 and SC-1 ranged from 8:2 to 2:8, the 3-PBA content gradually decreased, which indicated that strain SC-1 can effectively degrade 3-PBA even in the coexistence of strain B-1 and CY. When the inoculation proportion was 3.3:6.7, the maximum CY degradation (65.36%) and only 9.6 mg L−1 3-PBA was observed. When only strain SC-1 was used (B-1/SC-1 = 0:10), only 21.08% CY could be degraded, indicating that strain SC-1 possessed poor degradation performance on CY. Therefore, strains B-1 and SC-1 used in a cooperative way can degrade CY and 3-PBA, simultaneously, and the proportion of 3.3:6.7 was appropriate and used in the following experiments. Effect of Inoculation Method. The CY degradation and 3PBA content by using three inoculation methods are shown in Figure 5. The CY degradations by using methods 1, 2, and 3 were 64.78, 75.60, and 53.89%, respectively. There was a statistically significant difference (t test, P < 0.05) among the three methods. The 3-PBA contents by using these three methods were all about 10 mg L−1. Therefore, method 2, in which strain B-1 was first inoculated and then strain SC-1 was

Figure 6. Microbial biomass (OD600), CY degradation, and 3-PBA content in the cultivation period. ↑ represents addition of strain SC-1. Results are expressed as the average and standard deviation of three parallel samples.

the initial cultivation period (0−24 h, only strain B-1 was inoculated to the media), the OD600 value increased quickly and reached 1.899 at 24 h, but only 8.13% CY was degraded. This was presumably because strain B-1 mainly utilized the nutrients in medium except CY to achieve fast growth in the initial phase. After the addition of strain SC-1 (24−96 h), CY degradation began to increase rapidly and reached 78.13% at 96 h, and only 10.87 mg L−1 3-PBA was detected. The possible mechanism of CY degradation by the cooperation of these two strains was as follows: CY was first degraded into 3-PBA by strain B-1, then 3PBA was further degraded by strain SC-1. Meanwhile, the degradation of metabolite 3-PBA contributed to accelerate CY degradation. Compared with those by using only strain B-1 as described under Degradation of CY by B. licheniformis B-1, CY 8259

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its metabolite 3-PBA were completely degraded. When the initial CY content increased from 25 to 100 mg L−1, the CY degradations were all >75% and the 3-PBA contents were ≤10 mg L−1. When the initial CY content reached 150 mg L−1, only 37.13% CY was degraded, and the 3-PBA content was still about 10 mg L−1. This might be because when the CY reach a high content, it could produce a toxic effect on the voltagesensitive Na+ channels of strains B-1 and SC-1,1 prolonging the lag growth phase of these two strains,22 and so their degradation activity on CY or 3-PBA declined. These results indicated that the simultaneous degradation of CY and its metabolite 3-PBA by these two strains could be well achieved when the initial CY content was