Effects of Two Surfactants and Beta-Cyclodextrin on Beta

Nov 29, 2015 - College of Light Industry, Textile & Food Engineering, Sichuan University, 610065, Chengdu, Sichuan, P. R. China. J. Agric. Food Chem. ...
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Effects of Two Surfactants and Beta-Cyclodextrin on BetaCypermethrin Degradation by Bacillus licheniformis B‑1 Jiayuan Zhao, Yuanlong Chi, Fangfang Liu, Dongying Jia, and Kai Yao* College of Light Industry, Textile & Food Engineering, Sichuan University, 610065, Chengdu, Sichuan, P. R. China ABSTRACT: The biodegradation efficiency of beta-cypermethrin (β-CY) is low especially at high concentrations mainly due to poor contact between this hydrophobic pesticide and microbial cells. In this study, the effects of two biodegradable surfactants (Tween-80 and Brij-35) and β-cyclodextrin (β-CD) on the growth and cell surface hydrophobicity (CSH) of Bacillus licheniformis B-1 were studied. Furthermore, their effects on the solubility, biosorption, and degradation of β-CY were investigated. The results showed that Tween-80 could slightly promote the growth of the strain while Brij-35 and β-CD exhibited little effect on its growth. The CSH of strain B-1 and the solubility of β-CY were obviously changed by using Tween-80 and Brij-35. The surfactants and β-CD could enhance β-CY biosorption and degradation by the strain, and the highest degradation was obtained in the presence of Brij-35. When the surfactant or β-CD concentration was 2.4 g/L, the degradation rate of β-CY in Brij-35, Tween-80, and β-CD treatments was 89.4%, 50.5%, and 48.1%, respectively. The half-life of β-CY by using Brij-35 was shortened by 69.1 h. Beta-CY content in the soil with both strain B-1 and Brij-35 decreased from 22.29 mg/kg to 4.41 mg/kg after incubation for 22 d. This work can provide a promising approach for the efficient degradation of pyrethroid pesticides by microorganisms. KEYWORDS: beta-cypermethrin, biodegradation, surfactants, beta-cyclodextrin, Bacillus licheniformis B-1

1. INTRODUCTION

CY degradation by microorganisms in the presence of a surfactant or β-CD would be of great significance. The selected surfactants should be suitable to promote contact between hydrophobic organics and microorganisms21 as well as environmental compatibility.22 Some nonionic surfactants have been proved to be less toxic to microorganisms and are available to be degraded in the environment.13,23 In most studies reported, β-CD has been applied in HO biodegradation20 and soil bioremediation24 as an environment-friendly compound owing to its structure consisting of a hydrophobic internal cavity and a hydrophilic external surface. In this study, the effects of two biodegradable surfactants (Tween-80 and Brij-35) and β-CD on the degradation of β-CY by Bacillus licheniformis B-1 were investigated. The objectives of this study are to clarify the mechanisms of the contact between β-CY and microbial cells in the presence of the surfactants and β-CD, and to explore a potential approach for pesticide degradation by microorganisms.

1

As one kind of hydrophobic organic (HO), pyrethroid pesticides including β-cypermethrin (β-CY) have become one of the dominant insecticides for insect pest control in agriculture and animal industry,2 and their residues would enter the food supply chain and finally accumulate in human and animal bodies.3,4 Considerable attention has been attracted due to the fact that β-CY can cause toxic effects on the reproductive, immune, genetic, and nervous systems of human and mammal bodies.5−8 Therefore, it is critically necessary to remove β-CY residues from the environment. Recently, the degradation of β-CY by microorganisms has been regarded as a potential eco-friendly approach because it is safe and applicable.9−12 The degradation of HOs by microorganisms has not been always efficient since the main limitation was found to be their low frequency of contact.13,14 Regarding the biodegradation of pyrethroid pesticides, many studies have been focused on the way of screening and isolating novel efficient microorganisms in order to promoting their degradation.15−18 Microorganisms such as Ochrobactrum lupine DG-S-01,16 Pseudomonas aeruginosa CH7,15 and Bacillus cereus ZH-317 have been isolated. Bacillus licheniformis B-1 also has an ability to degrade cypermethrin.11 However, most of these microorganisms could not efficiently degrade high concentrations of pyrethroid pesticides.11,17,19 Recently, Zhang et al. reported that the degradation of β-CY by Pseudomonas aeruginosa could be improved by the addition of rhamnolipid,15 one kind of surfactant. In addition, β-cyclodextrin (β-CD) also has been used to improve the biodegradation of HO.20 However, quite a few studies have been reported on the effects of surfactants and β-CD on β-CY biodegradation so far. Therefore, research on β© XXXX American Chemical Society

2. MATERIALS AND METHODS 2.1. Materials. Beta-cypermethrin (99.7%) and acetonitrile of chromatographic grade were obtained from the National Standard Substances Center (Beijing, China) and Meridian Medical Technologies (Beijing, China), respectively. Acetonitrile, ethyl acetate, ethyl alcohol, KH 2 PO4 , K 2 HPO 4 , MgSO4 , NaCl, NaOH, Na 2 SO4 , (NH4)2SO4, Brij-35, β-CD, and Tween-80 were of analytical grade and purchased from Kelong Chemical Co. (Chengdu, China). Received: May 5, 2015 Revised: November 20, 2015 Accepted: November 28, 2015

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DOI: 10.1021/acs.jafc.5b04485 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

80, Brij-35, and β-CD were used: 0.3, 0.6, 1.2, and 2.4 g/L. The inoculum (1.5 mL) was separately transferred into four 250 mL flasks containing 30 mL of LB-CY medium with different concentrations of the surfactant or β-CD. The flasks were shaken at 180 rpm and 30 °C for 16 h followed by centrifugation at 8000 rpm and 4 °C for 10 min. The cells obtained were washed twice with a PUM buffer (19.7 g/L K2HPO4, 7.26 g/L KH2PO4, 1.8 g/L CO(NH2)2, and 0.2 g/L MgSO4· 7H2O) or normal saline, and resuspended in the buffer or normal saline to fit an optical density of 1.0 (A0) at 600 nm by using a UV− visible spectrometer (TU-1901, Puxi, Beijing, China). Microbial suspension resuspended by PUM buffer or normal saline was used to measure the CSH of strain B-1 and biosorption of β-CY, respectively. Treatment without surfactants and β-CD served as a control. Hexadecane (500 μL) was vortexed with the bacterial suspension (5 mL) for 2 min. The optical density of the aqueous phase was measured (A1) at 600 nm after 10 min. The CSH (%) of the strain was calculated as (A0−A1)/A0 × 100. The bacterial suspension (1.5 mL) was added to a 250 mL flask with 30 mL of LB-CY medium and 0.03% NaN3. The flask was shaken at 180 rpm and 30 °C for 24 h. The culture was centrifuged at 8000 rpm for 10 min, and β-CY content in its supernatant was detected. The biosorption of β-CY (mg/L) was calculated as C0 − Ci, where C0 and Ci were β-CY contents in the supernatants of the control and samples, respectively. 2.8. Degradation of β-CY by Strain B-1 in the Presence of Tween-80, Brij-35, or β-CD. The degradation of β-CY by B. licheniformis B-1 was conducted in four 250 mL flasks, which contained 30 mL of LB-CY medium with different concentrations of Tween-80, Brij-35, or β-CD (0.3, 0.6, 1.2, 2.4 g/L). Then, the flask was shaken for 30 s by a vortex mixer, and 1.5 mL of inoculum was added to it. After being incubated at 180 rpm and 30 °C for 72 h, the content of β-CY in the culture was measured by HPLC. Uninoculated LB-CY medium served as a control. Its degradation rate was calculated according to eq 2, where C and C0 are the contents of β-CY in inoculated medium and the control, respectively.

2.2. Microorganisms and Media. The strain Bacillus licheniformis B-1 was obtained from the soil in a tea garden (Ya’an, China). It was stored in 15% (v/v) of glycerol solution at −80 °C before experiments. Luria−Bertani (LB) medium containing 5.0 g/L yeast extract, 10.0 g/L peptone, and 10.0 g/L NaCl was prepared. LB-CY medium consisted of LB medium and β-CY (100 mg/L). The pH value of each medium was adjusted to 7.0−7.5, and 0.2% (v/v) of ethyl alcohol was added into the medium as a hydrotropic agent before sterilization at 121 °C for 20 min. 2.3. Inoculum Preparation. Strain B-1 was thawed and inoculated into a 100 mL Erlenmeyer flask with 30 mL of LB-CY medium. Then, it was incubated with shaking (180 rpm) at 30 °C for 16 h. After being centrifuged (10000 rpm, 4 °C) for 10 min, the strain was collected and suspended in sterile normal saline (0.9% of NaCl) to achieve an absorbance of about 0.8 at 600 nm. Then, the bacterial suspension was used as an inoculum. 2.4. Determination of β-CY Content. 2.4.1. Pretreatment. Five milliliters of LB-CY medium and acetonitrile were transferred into a 100 mL flask and shaken for 30 s by a vortex mixer. Then, the flask was placed under ultrasonication (40 kHz and 300 W) for 30 min.25 After the mixture was centrifuged at 8000 rpm for 20 min, the supernatant was collected and filtered through a 0.22 μm membrane filter. Finally, the obtained filtrate was used to determine its β-CY content by HPLC. 2.4.2. HPLC Method. The content of β-CY was determined on an LC-20AT high-performance liquid chromatograph (HPLC, Shimadzu, Kyoto, Japan) equipped with an LC-20AT pump (Shimadzu), a CTO20A column oven (Shimadzu), a Kromasil C18 column (250 mm × 4.60 mm, 5.0 μm; Sweden), and an SPD-M20A detector. A series of βCY acetonitrile solutions were prepared with its contents being from 2.5 mg/L to 500 mg/L. After pretreatment as described above, 20 μL of the solution was manually injected into the HPLC system, and the elution process was performed at 35 °C using 90% (v/v) of aqueous acetonitrile solution at a flow rate of 1.0 mL/min. The detection wavelength was 210 nm.25 Linear regression was taken between β-CY contents (X, mg/L) and their corresponding peak areas (Y), and the calibration curve equation was obtained as follows: Y = 57585X + 99879, R2 = 0.9996. 2.5. Growth of Strain B-1 in the Presence of Tween-80, Brij35, or β-CD. A series of LB-CY media with different concentrations (0.3, 0.6, 1.2, 2.4 g/L) of Tween-80, Brij-35, or β-CD were prepared, respectively. A volume of 30 mL of LB-CY medium with a surfactant or β-CD was mixed with 1.5 mL of inoculum in a 250 mL flask. The flask was shaken (180 rpm) at 30 °C for 40 h. LB-CY medium without surfactants and β-CD served as a control. Microbial biomass was expressed by optical density value measured at 600 nm, and relative biomass was calculated. Furthermore, in order to quantify the effects of two surfactants and β-CD on the growth of strain B-1 during incubation, a Logistic growth dynamic model (eq 1) was used to describe the specific growth rate in the presence of a surfactant or β-CD at 1.2 g/L:26

N = N0 e μm t /(1 − (N0/Nm)(1 − e μm t ))

degradation rate (%) = (1 − C /C0) × 100

(2)

The determination of β-CY contents in different phases was performed using a modified method described by Rodrigues et al.28 Five milliliters of the culture was centrifuged at 8000 rpm and 4 °C for 10 min. The supernatant and sediment were used to measure the content of β-CY in liquid phase and solid phase, respectively. The content of β-CY in liquid phase was determined by the following method. The supernatant mixed with equal volume of acetonitrile was centrifuged at 8000 rpm and 4 °C for 10 min, and the content of β-CY in the supernatant obtained was detected by HPLC. The content of βCY in solid phase was measured as follows. The sediment of strain B-1 cell suspended with 5 mL of normal saline was mixed with equal volume of acetonitrile followed by a centrifugation at 8000 rpm and 4 °C for 10 min. The resulting supernatant was collected for HPLC detection. First order degradation kinetic model (eq 3) was used to describe βCY degradation by strain B-1 in LB-CY medium with 1.2 mg/L of the surfactant or β-CD. The content of β-CY in the culture was measured every 12 h. The degradation rate constant (k) and half-life (t1/2) were calculated by eq 3 and eq 4, respectively:

(1)

where N, N0, Nm, μ, μm, and t represent biomass, initial biomass, maximum biomass, specific growth rate, maximum specific growth rate, and incubation time, respectively. 2.6. Measurement of β-CY Solubility in the Presence of Tween-80, Brij-35, or β-CD. Two milligrams of β-CY was separately mixed with 10 mL of Tween-80, Brij-35, or β-CD solution with a concentration of 0.5, 1.0, 1.5, 2.0, or 2.5 g/L under ultrasonication (40 kHz, 300 W) for 30 min. Then, the mixture was incubated with shaking (120 rpm) at 30 °C for 24 h. The undissolved β-CY was separated by centrifugation at 8000 rpm for 10 min.25 The supernatant was used for HPLC detection. Treatment without surfactants and βCD was used as a control. The linear fitting was done based on the contents (X) of a surfactant or β-CD and solubility (Y) of β-CY. 2.7. Measurement of Cell Surface Hydrophobicity and Biosorption of β-CY. The cell surface hydrophobicity (CSH) of strain B-1 and biosorption of β-CY were measured by the microbial adhesion to the hydrocarbon method14 and the method described by Zhang et al.,27 respectively. The following concentrations of Tween-

C t = C0 e−kt

(3)

t1/2 = ln 2/k

(4)

where C0, Ct, k, and t are the initial content (mg/L) of β-CY, its content at time t, degradation rate constant (h−1), and incubation time (h), respectively. 2.9. Degradation of β-CY in Soil with Strain B-1 and Brij-35. 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 airdried overnight.24 The characteristics of the soil (g−1, dry weight) were detected as follows:24 organic matter 64.2 mg, total bacteria 5.0 × 107 B

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Journal of Agricultural and Food Chemistry CFU, total actinomycetes 1.5 × 107 CFU, total fungi 8.35 × 105 CFU, and pH 7.65. A certain amount of β-CY was added to the soil to achieve a final content of 20 mg/kg, and then the soil was left overnight. Three soil samples (A, B, and C) with weight about 1000 g were used in the experiment. In sample A, only Brij-35 (0.3 g per gram soil, wet weight) was added. In sample B, only strain B-1 (1.0 × 108 cells per gram soil, wet weight) was inoculated. In sample C, both Brij-35 and strain B-1 were added as the above concentrations. The moisture contents in these three soil samples were kept at 40.0 ± 5.0% (w/w) by adding water manually, and the average temperature was 26 °C. The contents (mg/kg) of β-CY in soil samples were measured every 2 days by the method of Liu et al.11 2.10. Statistical Analysis. Each experiment was carried out in triplicate, and the results obtained were expressed as the means of three replicates with standard deviations. All statistical analyses were performed by using SPSS v 17.0 (SPSS Inc., Chicago, IL, USA).

Table 2. Parameters of Logistic Growth Dynamic Model of Strain B-1 at 1.2 g/L of Tween-80, Brij-35, or β-CD treatment

N0

Nm

tm (h)

μm (h−1)

Tween-80 Brij-35 β-CD control

0.006 0.005 0.006 0.005

2.089 2.067 2.158 2.157

4.0 5.7 4.4 4.4

0.34 0.23 0.31 0.31

3.2. Effects of Two Surfactants and β-CD on the Solubility of β-CY. When the concentrations of surfactants are higher than their critical micelle concentrations, their molecules could form micelles with HOs. So, the HOs dissolved in aqueous solution and existed in the form of liquid phase.13,28 Therefore, the surfactant was an important factor to improve the biodegradation of β-CY by affecting its solubility. The solubility of β-CY in the presence of Tween-80, Brij-35, or βCD is shown in Figure 1. The solubility of β-CY in the aqueous

3. RESULTS AND DISCUSSION 3.1. Influence of Two Surfactants and β-CD on the Growth of Strain B-1. The biomass of the strain is an important factor for the degradation of hydrophobic organics (HOs).13 So, the influence of two surfactants and β-CD on the growth of strain B-1 was studied. As shown in Table 1, the Table 1. Relative Biomass of Strain B-1 in the Presence of Different Concentrations of Tween-80, Brij-35, or β-CD treatment

concn (g/L)

Tween-80

0.3 0.6 1.2 2.4 0.3 0.6 1.2 2.4 0.3 0.6 1.2 2.4

Brij-35

β-CD

rel biomass 1.016 1.053 1.048 1.082 0.950 0.985 0.956 0.863 1.007 0.988 0.973 0.991

± ± ± ± ± ± ± ± ± ± ± ±

0.014 0.009 0.015 0.009 0.005 0.003 0.001 0.001 0.018 0.006 0.009 0.062

Figure 1. β-CY solubility in the presence of different concentrations of Tween-80, Brij-35, or β-CD.

solution was enhanced by these surfactants and β-CD, and was associated with their concentrations. This is because nonionic surfactants such as Tween-80 and Brij-35 can form micelles with β-CY in solution,15,22,31 and because β-CD has the ability to form water-soluble inclusion complexes by incorporating suitably sized low polarity molecules in their cavities.32 Linear fitting was carried out on the relationship between the concentration of Tween-80, Brij-35, or β-CD and β-CY solubility, and the linear equations and solubility coefficients (k) are shown in Table 3. The correlation coefficients (R2)

relative biomass of strain B-1 in the media with different concentrations of Tween-80 was more than 1.0, indicating that Tween-80 might be used as a carbon source by the strain to stimulate its growth. This result is in accordance with that reported by González et al.29 In addition, both Li et al. and Kolomytseva et al. observed that Tween-80 could be preferentially used as a carbon source by microorganisms in LB medium.22,30 The relative biomass in β-CD treatment was approximately equal to 1.0, suggesting that β-CD could not be utilized by the strain and did not affect its growth. However, the relative biomass in Brij-35 treatment significantly decreased at 2.4 g/L. The reason for this may be that high concentrations of Brij-35 may increase the contact between β-CY and living cells, and the growth of the strain was inhibited when its cells contacted high contents of cypermethrin.17 The maximum specific growth rate (μm) of strain B-1 in Tween-80 treatment was higher than that in β-CD or Brij-35 treatment at 1.2 g/L (Table 2). This is in accordance with the results in Table 1. Therefore, Tween-80 could be utilized by strain B-1 as an additional carbon source, but β-CD and Brij-35 could not be used by the strain and exhibited little effect on its growth.

Table 3. Linear Equation Parameters of β-CY Solubility in the Presence of Tween-80, Brij-35, or β-CD treatment

equation

k

R2

Tween-80 Brij-35 β-CD

Y = 11.31X + 2.34 Y = 5.15X + 2.47 Y = 4.99X + 0.02

11.31 5.15 4.99

0.9715 0.9695 0.9879

were between 0.9695 and 0.9879, suggesting that the solubility of β-CY could be effectively and linearly enhanced in the presence of the surfactant or β-CD (Figure 1). Similar results were also found in promoting the solubility of hydrophobic phenanthrene and pyrene.22,33,34 The highest solubility coefficient (11.31) appeared in Tween-80 treatment, indicating that it was the most efficient compound to enhance the solubility of β-CY. This is because the solubility of β-CY in C

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0.6 g/L, it could dramatically reduce CSH, then the CSH value almost kept constant (2.0%). In addition, the biosorption of βCY in Tween-80 and Brij-35 treatments was almost equal when their concentrations were higher than 0.6 g/L (Figure 2B). The CSH was determined as the percentage of cells adhering to hexadecane. Compared with the control, Tween-80 could decrease the CSH of strain B-1 but increase β-CY biosorption at 0.3 g/L. This might be due to β-CY being different from hexadecane and the contacts between the strain and these two compounds not being totally equal.28 As the concentrations of Tween-80 increased, LPS on the cell surface gradually released, thus resulting in a continuous increase in CSH.28 Increased CSH was also beneficial to the biosorption of β-CY because the hydrophobic cell surface could easily touch it. But CSH decreased when the concentrations of Tween-80 were higher than 1.2 g/L, which contributed to the adsorption of the surfactant onto the cell surface.27 Tween-80 molecules adsorbed on cell surface benefited β-CY biosorption because some of them could form micelle with the pesticide. As the concentrations of β-CD increased, the CSH value of strain B-1 increased from 1.33% to 39.48%, and the biosorption of β-CY also increased from 0.28 mg/L to 0.40 mg/L. This might be due to loss of hydrophilic LPS on the cell surface with an increase in β-CD concentration.33 The addition of Brij-35 could increase the hydrophilicity of the strain, which promoted the contact between the cells and β-CY in the micellar phase.28 3.4. Effects of Two Surfactants and β-CD on the Biodegradation of β-CY. The effects of Tween-80, Brij-35, and β-CD at different concentrations on the degradation of βCY are shown in Figure 3. β-CY biodegradation could be

Tween-80 micellar solutions is linearly dependent on the concentrations of the surfactant.21 However, the solubility of βCY in the presence of β-CD was the lowest in the treatments. At 2.5 g/L, the maximum solubility of β-CY in Tween-80 treatment reached 32.07 mg/L, which was almost two times that in Brij-35 treatment (16.02 mg/L), three times that in βCD treatment (12.68 mg/L), and two hundred times that (0.18 mg/L) in control. Therefore, Tween-80, Brij-35, and β-CD can increase the solubility of β-CY. 3.3. Effects of Two Surfactants and β-CD on Cell Surface Hydrophobicity of Strain B-1 and Biosorption of β-CY. The contact between microbes and HOs is strictly correlated with their cell surface hydrophobicity (CSH). The degree of contract can be expressed as biosorption of HOs. A surfactant or β-CD can change microbial CSH by releasing lipopolysaccharides (LPS) from their cell surface or absorbing molecules of a surfactant on the cell surface,14,24,28 thus affecting the contact between the cells and HOs. The contact beween β-CY and strain B-1 is important for its degradation because the cells degrade β-CY mainly through extracellular enzyme. The CSH values of strain B-1 and the biosorption of βCY significantly changed when different concentrations of Tween-80, Brij-35, and β-CD were used (Figure 2). Tween-80 exhibited much higher enhancement on CSH than β-CD. The highest CSH value (69.89%) occurred at 1.2 g/L of Tween-80 (Figure 2A). After that, CSH in Tween-80 treatment slightly decreased. However, Brij-35 showed a different effect on the CSH of the strain. At concentrations ranging from 0.3 g/L to

Figure 3. Degradation rate of β-CY in the presence of different concentrations of Tween-80, Brij-35, or β-CD.

enhanced by the surfactants and β-CD, and it went up as the concentrations increased. The most efficient compound for promoting β-CY degradation was Brij-35 followed by Tween80 and β-CD. At 2.4 g/L, the maximum degradation rates in Brij-35, Tween-80, and β-CD treatments were 89.4%, 50.5%, and 48.1%, respectively. Compared with the control, the maximum degradation rate of β-CY in Brij-35 treatment was increased by 47.0%. The biodegradation of β-CY might be subject to the effects of the surfactants and β-CD on the growth and CSH of strain B-1, and the solubility and biosorption of β-CY. The biodegradation of β-CY in the surfactants and β-CD treatments was higher than that in the control. This is because the surfactants and β-CD could not only change the solubility of β-

Figure 2. Cell surface hydrophobicity of strain B-1 (A) and biosorption of β-CY (B) in the presence of different concentrations of Tween-80, Brij-35, or β-CD. D

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Journal of Agricultural and Food Chemistry CY and the CSH of strain B-1 but also enhance the biosorption of the pesticide. The degradation rate of β-CY in Brij-35 treatment was higher than that reported in a previous study.16 Noticeably, Brij-35 demonstrated a higher enhancement on the degradation of β-CY than Tween-80 and β-CD, although it was not the most efficient compound for increasing the solubility of β-CY and the CSH of strain B-1. This may be ascribed to Brij35 not obviously affecting the growth of strain B-1. Also, since hydrophilic cell surface easily contacted with β-CY in the micellar phase (Figure 2B), its degradation in Brij-35 treatment could also be promoted. Although Tween-80 could efficiently increase the CSH of strain B-1 and the biosorption and solubility of β-CY, its biodegradation was confined because the cells of the strain preferentially used Tween-80 rather than the pesticide as a carbon source. β-CD showed no apparent enhancement on β-CY degradation, which might be related to its smallest effect on increasing the CSH of the strain and the biosorption and solubility of β-CY. The degradation rate of β-CY increased as incubation time was prolonged at 1.2 g/L Tween-80 (Figure 4A), Brij-35 (Figure 4B), and β-CD (Figure 4C), of which Brij-35 showed the most efficient effect on its degradation. The half-life (t1/2), degradation rate constant (k), and regression equations of β-CY by strain B-1 in Tween-80, Brij-35, and β-CD treatments are shown in Table 4. Compared with the control, the half-lives of β-CY in Brij-35, Tween-80, and β-CD treatments were shortened by 69.1 h, 42.6 h, and 26.0 h, respectively. The half-life of β-CY in Brij-35 treatment was shorter than that (35.7 h) of cypermethrin without surfactants and β-CD by using both strain B-1 and Sphingomonas sp. SC-1 reported by Liu et al.11 3.5. Changes of β-CY Contents in Different Phases during Its Degradation. In order to understand the possible mechanism for the surfactants and β-CD to promote β-CY degradation by strain B-1, the changes of β-CY contents in solid phase (undissolved) and liquid phase (aqueous and micellar phase) were analyzed during the incubation period (Figure 4). The degradation of β-CY in the presence of Tween-80 increased as its contents in liquid phase decreased, and an opposite trend was observed in solid phase due to the gradual precipitation of the dissolved β-CY (in micellar phase) into solid phase (Figure 4A). However, the reductions of β-CY contents in two phases corresponded with the trend of its degradation in the presence of Brij-35, and its contents in solid and liquid phases within 72 h decreased from 37.5 mg/L and 60.0 mg/L to 4.6 mg/L and 13.6 mg/L, respectively (Figure 4B). In Figures 4C and 4D, the contents of β-CY in solid phase within 72 h decreased as degradation rate increased in β-CD treatment and in the control, decreasing from 89.6 mg/L and 95.4 mg/L to 45.1 mg/L and 52.3 mg/L, respectively. It can be assumed from the above results that strain B-1 may degrade β-CY through different mechanisms with or without a surfactant or β-CD. In the absence of surfactants and β-CD, the strain can directly use β-CY partly dissolved in aqueous phase. In the presence of the surfactant, hydrophobic cell surface with the hemimicelles of the surfactant molecules can adsorb micellar β-CY22,35 while hydrophilic cell surface may directly contact with β-CY in micellar and aqueous phases. Although Brij-35 was not used by the strain B-1, the degradation of β-CY was significantly enhanced because the increased contact between β-CY in liquid phase and hydrophilic cell surface was almost constant by the addition of Brij-35 (Figure 5). In addition, when β-CY in liquid phase was consumed by strain B-

Figure 4. Contents of β-CY in liquid phase and solid phase and its degradation by strain B-1 in the presence of 1.2 g/L of Tween-80 (A), Brij-35 (B), and β-CD (C), and without surfactants and β-CD (D).

1, the undissolved β-CY in solid phase was quickly changed into dissolved β-CY when Brij-35 was added in the medium. Tween80 could increase the solubility of β-CY and the contact between the pesticide in micellar phase and hydrophobic cell E

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Journal of Agricultural and Food Chemistry Table 4. First Order Kinetic Parameters of β-CY Degradation by Strain B-1 in the Presence of Tween-80, Brij-35, or β-CD after Incubation for 72 h treatment Tween-80 Brij-35 β-CD control

regression equation Ct Ct Ct Ct

= = = =

91.0128 87.1473 90.3237 97.5144

−0.0123t

e e−0.0232t e−0.0095t e−0.0070t

k (h−1)

t1/2 (h)

R2

0.012 0.023 0.010 0.007

56.4 29.9 73.0 99.0

0.9406 0.9864 0.9471 0.9726

Figure 6. Contents of β-CY in soil samples with only Brij-35, only strain B-1, and both strain B-1 and Brij-35. Figure 5. Possible mechanisms of the contact between β-CY and strain B-1 cells in the presence of Tween-80, Brij-35, or β-CD. Control: Little β-CY contacts with the strain cells owing to its low solubility. βCD: A small amount of β-CY contacts with the strain cells because of its slightly increased solubility. Tween-80: More β-CY contacts with the strain cells as a result of its obviously enhanced solubility, but some of the Tween-80 molecules are consumed by the cells. Brij-35: Much more β-CY contacts with the strain cells mainly due to its enhanced solubility and the decreased hydrophobicity of the cell surface.

Table 5. First Order Kinetic Parameters of β-Cypermethrin Degradation in Soil treatment sample A sample B sample C

surface of strain B-1 (Figure 5), thus enhancing its degradation. Nevertheless, the content of β-CY consumed by the cells would be decreased since the strain preferentially utilized Tween-80 during degradation. Furthermore, the contact between β-CY and the cells of strain B-1 was reducing when the concentration of Tween-80 decreased as the time prolonged. Therefore, the efficiency of β-CY degradation in Tween-80 treatment gradually went down as the time prolonged. In the presence of β-CD, poor contact between β-CY and the cells occurred (Figure 5) because it could not efficiently increase the CSH of strain B-1 and the biosorption and solubility of β-CY, thus βCY degradation was not apparently improved. 3.6. Degradation of β-CY in Soil. Brij-35 could more efficiently improve the degradation of β-CY in the media by strain B-1 than Tween-80. Moreover, Tween-80 was not an efficient surfactant for improving the biodegradation of hydrophobic organics (HOs) in soil because native microorganisms could use it as a carbon source.36,37 Franzetti et al. reported that Tween-80 was not suitable for the situ remediation of soil contaminated with HOs due to its high affinity to soil.38 Therefore, only Brij-35 was chosen for β-CY degradation experiment in soil. The contents of β-CY in soil samples with only Brij-35 (sample A), only strain B-1 (sample B), and strain B-1 and Brij-35 (sample C) are shown in Figure 6. After incubation for 22 days, β-CY contents in samples A, B, and C were 12.45 mg/L, 5.80 mg/L, and 4.41 mg/L, respectively. This is because Brij-35 can increase the contact between HOs and microbes in soil.39 Iglesias et al. and Chang et al. found that Brij-35 could improve the biodegradation of hydrophobic phenanthrene and nonylphenol in soil by increasing their solubility.40,41 Furthermore, the degradation rate constant and half-life of βCY from first order degradation kinetic model were calculated and are summarized in Table 5. The half-lives of β-CY in

regression equation −0.0249t

Ct = 19.3463 e Ct = 18.4322 e−0.0629t Ct = 16.2892 e−0.0743t

k (d−1)

t1/2 (d)

R2

0.025 0.063 0.074

27.8 11.0 9.3

0.9689 0.9528 0.9159

samples A, B, and C were 27.8 d, 11.0 d, and 9.3 d, respectively. Chang et al. (2009) also reported that the half-life of nonylphenol by using Brij-35 was shortened from 27.1 to 13.1 d.41 The half-life of β-CY in sample C was longer than that in the media, which might be because the soil environment contained less nutrients and oxygen for the growth of strain B1.11 From the above results, it can be further confirmed that Brij-35 can effectively improve the degradation of β-CY in soil by strain B-1.

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AUTHOR INFORMATION

Corresponding Author

*Tel/fax: +86 28 85404298. E-mail: [email protected]. Funding

This work was financially supported by the National Natural Science Foundation of China (31371775) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (20130181120094). Notes

The authors declare no competing financial interest.

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