Introduction

Pyrethroid insecticides are synthetic derivatives of pyrethrins, which are natural insecticides that are produced from chrysanthemum plants (Soderlund et al. 2002). Pyrethroid insecticides act as neurotoxins (Narahashi et al. 1998), which have been used in agriculture and home formulations for over 40 years and account for about 25 % of the worldwide insecticide market (Shafer et al. 2005). Recently, with the restriction on the use of organophosphates and carbamates, pyrethroid insecticides have generally been regarded as the replacement with 160 t used for agriculture and 258 t used for nonagriculture pest control in California in 2007 (Weston and Lydy 2010). However, the accumulation and widespread use of pyrethroid insecticides have increased the public concern on potential human health risks (Mohapatra et al. 2007). Toxicological studies performed in water-only systems have found that these pesticides cause lethal and sublethal effects even at extremely low concentrations, with LC50 being generally less than 1 ng mL−1 (Coats et al. 1989). In addition, pyrethroid insecticides have been found to be unaffected by secondary treatment systems at municipal wastewater treatment facilities in California (Weston and Lydy 2010).

Beta-cypermethrin (beta-cp) [cyano-(3-phenoxyphenyl) methyl 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane-1-carboxylate] is one of the most popular pyrethroid insecticides and used worldwide, which occupies more than 50 % of the total production of pyrethroids market in China (Zhang et al. 2007). Even though beta-cp was considered safe for humans, a large number of studies show that beta-cp may have reproductive toxicity (Yuan et al. 2010), endocrine disruption effects (McKinlay et al. 2008), immune toxicity (Jin et al. 2011), and genotoxic effects (Ansari et al. 2011) on nontarget organisms. Moreover, beta-cp is regarded as a possible human carcinogen by the Environmental Protection Agency (EPA) of USA (Shukla et al. 2002). The reported half-life of beta-cp in soil ranges from 94.2 to 1,103 days (Zhang et al. 2011). Hence, disposal of the residues of beta-cp from the environment is of great economic and social significance.

Biodegradation is considered to be the most significant process determining the fate of pyrethroid insecticides in nature (Fenlon et al. 2011) and has received increasing attention as an effective, safe, and cheap approach to clean up contaminated environments (Arora et al. 2012b; Cycoń et al. 2011). Up to now, several strains such as Pseudomonas aeruginosa (Zhang et al. 2011), Streptomyces sp. (Lin et al. 2011), Stenotrophomonas sp. (Chen et al. 2011a), and Serratia marcescens (Cycoń et al. 2014) have been reported to degrade pyrethroid insecticides. Several degrading enzymes involved in pyrethroids degradation have recently been purified (Guo et al. 2009; Wang et al. 2009; Zhai et al. 2012; Fan et al. 2012; Chen et al. 2013a). However, there is no information about beta-cp degradation by Bacillus subtilis. Moreover, the biodegradation pathway of beta-cp has rarely been characterized (Liu et al. 2014).

The present study isolated a novel Bacillus subtilis BSF01 that was able to efficiently degrade beta-cp and other pyrethroid insecticides. Biodegradation conditions for beta-cp were optimized. What’s more, degradation products were determined, and the biodegradation pathway was proposed in this study. These results highlight the promising potentials and advantages of Bacillus subtilis BSF01 for bioremediation of the pyrethroid-contaminated environments.

Materials and methods

Chemicals and media

Technical grade beta-cypermethrin (95 % purity), cypermethrin (92.9 % purity), deltamethrin (98 % purity), cyhalothrin (95 % purity), and beta-cyfluthrin (95 % purity) were obtained from Jiangsu Yangnong Chemical Group Co., Ltd., China. Chromatographic grade acetonitrile was purchased from Oceanpak Alexative Chemical Co., Ltd, Sweden. All the other chemicals and solvents used in this experiment were of analytical grade. Each pyrethroid insecticide was dissolved in acetone or methanol as stock solutions (10,000 mg L−1). The solutions were sterilized by filtration with 0.45 μm of filter membrane prior to use.

The mineral salt medium (MSM) containing 2.0 g (NH4)2SO4, 0.2 g MgSO4·7H2O, 0.01 g CaCl2·2H2O, 0.001 g FeSO4·7H2O, 1.5 g Na2HPO4·12H2O, and 1.5 g KH2PO4 per liter of water and Luria–Bertani medium (LBM) containing 5-g yeast extract, 10 g tryptone, and 10 g NaCl per liter of water were used for the isolation and cultivation of degrading strains. Both media were adjusted to pH 7.0 and autoclaved to sterilize at 121 °C for 20 min (Guo et al. 2009; Chen et al. 2012; Liu et al. 2014).

Screening and isolation of beta-cp-degrading strains

Activated sludge samples were collected from pesticide-manufacturing wastewater treatment system of Guangxi Research Institute of Chemical Industry, China. One gram of the sludge was suspended with sterile water. The samples were inoculated on the LB agar plates and incubated at 30 °C for 2 days after immersing in the 80 °C water bath for 10 min. The well-isolated colonies were transferred into 250-mL Erlenmeyer flasks containing 50-mL sterilized LBM and incubated at 30 °C on a rotary shaker at 230 rpm. The final cultures were gradually diluted and inoculated on MSM agar plates containing 100 mg L−1 of beta-cp. The eugenic colonies were picked out as the potential strains and their degrading abilities of beta-cp were monitored by high-performance liquid chromatography (HPLC). One strain named BSF01 that showed the highest degrading efficiency of beta-cp was selected for further studies.

Identification of the beta-cp-degrading strain

Morphology of strain BSF01 was observed by electron microscope (Olympus, Japan) and scanning electron microscope (XL-30ESEM, Philips Optoelectronics Co., Ltd., Holland). Physio-biochemical tests were examined as described before (Holt et al. 1994). Genomic DNA was extracted according to protocol of E.Z.N.A.® Bacterial DNA Kit (OMEGA BioTek, USA). 16S rDNA gene was amplified using the universal primers, B1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and B4 (5′-ACGGHTACCTTGTTTACGACTT-3′). The PCR cycling conditions consisted of initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 45 s, and extension at 72 °C for 1.25 min, with the last cycle followed by a 10-min extension at 72 °C. The PCR products were cloned into the vector pMD20-T (TaKaRa Biotechnology Co. Ltd., China) and transformed into Escherichia coli DH5α cells (Chen et al. 2011b, c). Clones were sequenced by Shanghai Yingjun Technology Co. Ltd., China, after purification. The resulting sequence (GenBank accession No. JF706263) was compared with the sequences in the GenBank database by BLAST. Multiple sequence alignments were conducted with the selection of high homology sequences using CLUSTALX 1.8.1. The phylogenetic tree was analyzed and constructed with the neighbor-joining method by MEGA 4.0 version (Tamura et al. 2007).

Relationship between the growth of strain BSF01 and the biodegradation of beta-cp

The degradation of beta-cp was performed in 250-mL Erlenmeyer flasks with 50-mL sterile MSM containing 50 mg L−1 beta-cp. Inocula were prepared by inoculating the individual colony into 50 mL of MSM at 32 °C with shaking at 150 rpm. The bacterial cells in the late-exponential growth phase were harvested by centrifugation at 4,000g for 2 min and washed with 0.9 % sterile saline and resuspended in 50 mL saline (Anwar et al. 2009). One percent of this suspension (the total biomass amount was 0.12 g dry wt L−1) was used as the inocula for the subsequent studies. The experiment was conducted in triplicate at 32 °C on a rotary shaker at 150 rpm for 7 days. The noninoculated served as controls. The growth of the strain was determined by counting the colony-forming units (CFU) per milliliter of serial dilutions, and the residual beta-cp concentration was measured by HPLC.

Optimal conditions for beta-cp biodegradation

Response surface methodology (RSM) was explored to optimize the biodegradation conditions of strain BSF01. The Box–Behnken design consisting of 15 experimental runs with three replicates at the center point was used to optimize the independent variables which significantly influenced the beta-cp biodegradation. Based on preliminary study, three critical factors and their optimal ranges were selected in this experiment: temperature (22–42 °C), medium pH (5–9), and inocula (total biomass amount 0.02–0.22 g dry wt L−1) (Table 1). The dependent variable was the degradation of 50 mg L−1 beta-cp in MSM by day 7. Empirical model equation (Eq. (1)) was created that correlated the relationship between the independent variables and the dependent variable by statistic analysis system (SAS) software version 9.0 (Chen et al. 2011b).

$$ {Y}_{\mathrm{i}}={b}_0+\varSigma {b}_{\mathrm{i}}{X}_{\mathrm{i}}+\varSigma {b}_{\mathrm{i}\mathrm{j}}{X}_{\mathrm{i}}{X}_{\mathrm{j}}+\varSigma {b}_{\mathrm{i}\mathrm{i}}{X_{\mathrm{i}}}^2 $$
(1)

where Y i is the predicted response, X i and X j are the variables, b 0 is the constant, b i is the linear coefficient, b ij is the interaction coefficient, and b ii is the quadratic coefficient.

Table 1 Box–Behnken experimental design and the response of dependent variable for beta-cp degradation
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Effect of different initial beta-cp concentrations on its biodegradation

Biodegradation experiments of different initial beta-cp concentrations were performed in 250-mL Erlenmeyer flasks with 50-mL sterile MSM containing 25–400 mg L−1 beta-cp. The MSM was incubated in triplicate under the optimal conditions with noninoculated as controls. Andrews equation (Eq. (2)) was used to describe the specific degradation rate (q) at different initial beta-cp concentrations (Chen et al. 2014).

$$ q=\frac{q_{\max }S}{S+{K}_s+\left({S}^2/{K}_i\right)} $$
(2)

where q max is the maximum specific degradation rate, K i is the inhibition constant, K s is the half-rate constant, and S is the beta-cp concentration. The kinetic parameters were calculated using Matrix Laboratory (MATLAB) software version 7.8 according to Chen et al. (2014).

Biodegradation kinetics of various pyrethroid insecticides

Sterile MSM was supplemented with beta-cp, cypermethrin, deltamethrin, cyhalothrin, or beta-cyfluthrin at 50 mg L−1 and inoculated at the optimal degradation conditions for 7 days. The experiment was performed in triplicate with noninoculated as controls. The first-order kinetic model (Eq. (3)) was applied to explore the biodegradation kinetics of various pyrethroid insecticides (Chen et al. 2013b).

$$ {C}_t={C}_0\times {\mathrm{e}}^{-kt} $$
(3)

where C 0 is the initial concentration of pyrethroid insecticides at time zero, C t is the concentration of pyrethroid insecticides at time t, and k and t are the rate constant (day−1) and degradation period in days, respectively.

The half-life (T 1/2) of different pyrethroid insecticides was calculated by Eq. (4) (Chen et al. 2013b).

$$ {T}_{1/2}=\frac{ \ln 2}{k} $$
(4)

where k is the rate constant (day−1).

Identification of beta-cp metabolites and analysis of biodegradation pathway

The cell-free filtrates of the cultures grown in MSM for 7 days containing 50 mg L−1 of beta-cp were collected at an interval of 1 day. The noninoculated culture containing the same amount of beta-cp was used as control. The cultures were extracted with an equal volume of dichloromethane after acidification to pH 2 with 2 M HCl. The solvent phase was concentrated using rotary evaporator (Heidolph, Germany). The extracts were dissolved in methanol and were subjected to gas chromatography–mass spectrometry (GC-MS).

Chemical analysis

Thirty milliliters of cell-free medium was extracted using 60 mL of acetone/petroleum ether (1:1, v/v) in an ultrasonic bath. After partitioning, the supernatants were passed through a 0.22 mm polytetrafluoroethylene (PTFE) membrane filter (Millipore, USA), and the filtrates were concentrated using rotary evaporator (Heidolph, Germany). The pyrethroid residues were determined by an Agilent 1260 HPLC system with an Agilent ZORBAX SB-C18 reversed phase column (4.6 nm × 150 mm, 5 μm) at 235-nm wavelengths. A mixture of acetonitrile and water (85:15, v/v) was used as the mobile phase at a flow rate of 1.0 mL min−1 with the injection volume of 10 μL.

The metabolites of beta-cp were identified by an Agilent 6890N/5975 GC-MS system according to Chen et al. (2011b).

Results

Isolation and characterization of the beta-cp-degrading strain

In this study, one strain that was able to utilize beta-cp as the sole carbon source for growth was isolated from activated sludge and named as BSF01, which degraded 89.4 % of 50 mg L−1 beta-cp within 7 days. Strain BSF01 was an obligate aerobic, Gram-positive, rod-shaped bacterium with spores of 1.0 to 1.5 μm in length and 0.6 to 0.9 μm in width (Fig. 1). Colonies grown on LBM agar plates were rough, opaque, dirty white, or slightly yellow, with winkles when cultured for a long time. It was positive in tests such as amylohydrolysis, gelatin liquefaction, Voges–Proskauer (V-P), nitrate reduction, and utilization of d-glucose and d-mannitol while it was negative in utilization of l-arabinose and d-xylopyranose. PCR amplification of 16S rDNA gene from strain BSF01, a single fragment of 1,417 bp (Fig. S1), with GenBank accession number JF706263, was obtained and completely sequenced. Analysis of the 16S rDNA gene sequences demonstrated that strain BSF01 grouped among Bacillus species (Fig. 2). In conclusion, strain BSF01 was identified as Bacillus subtilis based on the morphology, physio-biochemical characteristics, and 16S rDNA gene analysis. This strain was deposited in China Center for Type Culture Collection (collection number: CCTCC AB 2014103).

Fig. 1
figure 1

Morphological characteristics of strain BSF01 under scanning electron microscope (×10,000)

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Fig. 2
figure 2

Phylogenetic tree based on the 16S rDNA sequence of strain BSF01 and related strains. Numbers in parentheses represent the sequences accession number in GenBank. Numbers at the nodes indicate bootstrap values from the neighborhood-joining analysis of 1,000 resampled data sets. Bar represents sequence divergence

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Utilization of beta-cp for growth by strain BSF01

The relationship between the growth of strain BSF01 on beta-cp and the biodegradation of beta-cp was shown in Fig. 3. The degradation rate of beta-cp increased rapidly when the strain was at logarithmic (1–2 days) and stationary (3–4 days) phase. However, the degradation rate tended to slow down when the strain arrived at the decline phase (after 5 days of incubation). An 89.4 % of 50 mg L−1 beta-cp was degraded by strain BSF01 within 5 days. In contrast, there was no significant change in beta-cp concentration in the noninoculated controls. Additionally, strain BSF01 grew well on MSM agar plates with beta-cp as the sole carbon source. These results drew a conclusion that strain BSF01 may be used in bioremediation of beta-cp-contaminated environments.

Fig. 3
figure 3

Utilization of beta-cp for growth in MSM by strain BSF01. Degradation kinetics of beta-cp (black square), noninoculated control (white square), and growth of strain BSF01 (black triangle). Values are the means of three replicates with standard deviation

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Optimization of beta-cp-degrading conditions by strain BSF01

The Box–Behnken design was applied to determine the effects of important variables containing temperature (X 1), pH (X 2), and inocula (X 3) according to previous single-factor experiments. The experimental design and the response of dependent variables for beta-cp degradation are presented in Table 1. Data from Table 1 were processed by response surface regression procedure of SAS software, and results were obtained by fitting with the second-order polynomial model equation (Eq. (5)):

$$ \begin{array}{l}{Y}_{BSF01}=90.3+4.3125{X}_1-0.6375{X}_2-0.425{X}_3-9{X_1}^2-1.55{X}_1{X}_2\\ {}+0.725{X}_1{X}_3-3.55{X_2}^2+0.275{X}_2{X}_3-2.425{X_3}^2\end{array} $$
(5)

where Y BSF01 is the predicted beta-cp degradation (%) by strain BSF01 and X 1, X 2, and X 3 are the coded values for the temperature, pH, and inocula, respectively.

Analysis of variance (ANOVA) for beta-cp degradation is tabulated in Table 2. The determination coefficient R 2 of 0.9848 indicated that approximately 98 % of responses were covered by the model, demonstrating that predicted values of the model were in good agreement with the experimental values. In general, this model for beta-cp degradation is highly significant (p < 0.01), indicating that the established quadratic polynomial model for beta-cp degradation by strain BSF01 was adequate and reliable in representing the actual relationship between response and variables.

Table 2 ANOVA for the fitted quadratic polynomial model of beta-cp degradation
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The results of the regression parameter estimate displayed that linear and square terms of temperature (X 1) values showed significant effects (P < 0.05) on the beta-cp degradation by strain BSF01, while the linear and square terms of pH (X 2) and inocula (X 3) were insignificant (P > 0.05). Figure 4 exhibited a three-dimensional response surface which was plotted to intuitively display the effects of temperature and pH with the value of inocula fixed at 0.12 g dry wt L−1. There was a theoretical maximum value of 90.9 % at the stationary point. At the theoretical maximum point, the values of temperature (X 1), pH (X 2), and inocula (X 3) in terms of the uncoded units were 34.5 °C, 6.7, and 0.11 g dry wt L−1, respectively. That is to say that the optimal conditions for beta-cp degradation by strain BSF01 were determined to be temperature at 34.5 °C, pH 6.7, and inocula of 0.11 g dry wt L−1.

Fig. 4
figure 4

Response surface plot showing the effects of temperature and medium pH on beta-cp degradation while fixing the value of inocula amount at a zero level

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Effect of different initial beta-cp concentrations on biodegradation

Kinetic curves of beta-cp degradation at different initial concentrations (25–400 mg L−1) by strain BSF01 are shown in Fig. 5a. At low concentrations of 25, 50, and 100 mg L−1, the degradation rates of beta-cp reached 93.9, 89.4, and 84.7 % after 7 days of incubation, respectively. Approximately 80 % of degradation rate was achieved at higher initial concentrations (>100 mg L−1) with longer lag phase.

Fig. 5
figure 5

Biodegradation of beta-cp in MSM with different initial concentrations by strain BSF01. a Degradation kinetics of beta-cp. Black diamond, 25 mg L−1; black square, 50 mg L−1; black triangle, 100 mg L−1; white triangle, 200 mg L−1; white diamond, 300 mg L−1; white square, 400 mg L−1. Values are means of three replicates with standard deviation. b Relationship between specific degradation rate and initial beta-cp concentration

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Figure 5b shows the relationship between specific degradation rate (q) and initial bata-cp concentration. The kinetic parameters q max, K s, and K i were established to be 2.19 day−1, 76.37 mg L−1, and 54.14 mg L−1, respectively. The value of R 2 was 0.9637 demonstrating that the experimental data were well correlated with Andrews equation. The critical inhibitor concentration was determined to be 64.30 mg L−1, indicating that when the initial concentrations of beta-cp were lower than 64.30 mg L−1, the specific degradation rate (q) gradually increased. The inhibition of beta-cp would be prominent at higher concentrations.

Degradation kinetics of various pyrethroid insecticides by strain BSF01

The degradation kinetics of various pyrethroid insecticides by strain BSF01 is shown in Fig. 6. The strain utilized beta-cp, cypermethrin, deltamethrin, beta-cyfluthrin, and cyhalothrin as the growth substrates with the degradation rates of 89.4, 89.2, 86.9, 86.5, and 76.8 % within 7 days, respectively.

Fig. 6
figure 6

Biodegradation dynamics of various pyrethroid insecticides by strain BSF01 with the initial concentration of 50 mg L−1. Cyhalothrin (white square), beta-cp (white diamond), deltamethrin (black diamond), beta-cyfluthrin (black square), and cypermethrin (black triangle). Values are means of three replicates with standard deviation

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The first-order model (Eq. (3)) was used to represent the biodegradation kinetics of various pyrethroid insecticides by strain BSF01. The degradation kinetic parameters of various pyrethroid insecticides are presented in Table 3. The degradation rate constants (k) of beta-cp, deltamethrin, beta-cyfluthrin, cyhalothrin, and cypermethrin were 0.2995, 0.3811, 0.4476, 0.2320, and 0.5164 day−1, respectively, and the degradation half-lives (T 1/2) were 2.31, 1.82, 1.55, 2.99, and 1.34 days, respectively. The correlation coefficient R 2 ranged from 0.9112 to 0.9867 indicating that the degradation data were well fitted with the model.

Table 3 Degradation kinetics of various pyrethroids by strain BSF01
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Identification of beta-cp metabolites by GC-MS

The beta-cp metabolites were extracted and identified by GC-MS, and the results are summarized in Table 4. Each peak was identified on the basis of its characteristic fragment ion peaks and the National Institute of Standards and Technology (NIST, USA) library database. GC chromatogram of the culture filtrates for beta-cp degradation by strain BSF01 is shown in Fig. S2.

Table 4 Chromatographic properties of metabolites of beta-cp
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It was worth mentioning that two peaks, compound A (32.021 min) and compound B (32.134 min), were first observed during the beta-cp degradation by strain BSF01. These two compounds [A and B] were identified as beta-cp (cis) and beta-cp (trans), respectively, based on the similarity of their fragment retention times and molecular ions with those of corresponding authentic compounds in the NIST library database. Afterward, these two new compounds [A and B] were further metabolized to form 3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanecarboxylate [C], α-hydroxy-3-phenoxy-benzeneacetonitrile [D], 3-phenoxybenzaldehyde [E], 3-phenoxybenzoic acid [F], and 3,5-dimethoxyphenol [G] (Fig. S3). In the noninoculated control containing the same amount of beta-cp, only beta-cp was detected (Fig. S4). Based on analysis of the metabolites, the biodegradation pathway of beta-cp in strain BSF01 was proposed (Fig. 7). Beta-cp was firstly metabolized by hydrolysis of its ester linkage to yield 3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanecarboxylate [C] and α-hydroxy-3-phenoxy-benzeneacetonitrile [D]. Compound D was unstable in the environment and was oxidized to form 3-phenoxybenzaldehyde [E]. Subsequent oxidization of compound E resulted in the formation of 3-phenoxybenzoic acid [F]. Compound F could be further transformed to 3,5-dimethoxyphenol [G], leading to detoxification of beta-cp.

Fig. 7
figure 7

Proposed pathway for the biodegradation of beta-cp by strain BSF01

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Discussion

A number of studies had shown that it was an effective method to isolate degrading microorganisms from long-term pesticide-contaminated soil, wastewater, or activated sludge (Tallur et al. 2008; Chen et al. 2011b). Bacillus subtilis are widely available and have been safely used in a variety of food applications due to their properties of nontoxigenic and nonpathogenic that had been stated by the Food and Drug Administration (FDA) (Gong et al. 2014). It had been found to degrade other xenobiotic compounds, including 4-chloro-2-nitrophenol, dimethylformamide, and red M5B dye (Arora 2012a; Vidhya and Thatheyus 2013; Gunasekar et al. 2013). This study provides the first evidence that Bacillus subtilis participates in efficient degradation of beta-cp.

The relationship between the growth of strain BSF01 and the biodegradation of beta-cp showed that all degradation was associated with bacterial growth, and this observation is reminiscent of a previous finding by Singh et al. (2004). Strain BSF01 could utilize beta-cp as the sole carbon source and degrade it over a wide range of initial beta-cp concentration (25–400 mg L−1), temperature (22–42 °C), and pH (5–9). However, longer lag phase was achieved at higher initial beta-cp concentration, and it might be because of that the greater number of bacteria was needed to initiate rapid degradation of pesticide (Karpouzas and Walker 2000; Anwar et al. 2009). Microorganisms need an acclimatization period to induce the formation of necessary degradative enzymes that may account for prolonged lag phase, which was observed at higher concentration of cypermethrin (Dubey and Fulekar 2013). Moreover, strain BSF01 was capable of degrading various pyrethroid insecticides including beta-cp, cypermethrin, deltamethrin, beta-cyfluthrin, and cyhalothrin, and it performed no significant pyrethroid degradation specificity as Brevibacterium aureum DG-12 reported by Chen et al. (2013b). This may be because that the degrading enzyme(s) from strain BSF01 had no substrate specificity. Another possible reason could be attributed to the fact that all the tested pyrethroid insecticides share a similar structure. Similar result had been reported by Wang et al. (2011).

The basic purpose of RSM is the evaluation of the relationship between the predicted values of the dependent variable and the conditions of dependent variables (Hosseini-Parvar et al. 2009). The use of the Box–Behnken design is popular in industrial research because it is an economical design and requires only three levels for each factor (Khuri and Mukhopadhyay 2010). Zhang et al. (2010) and Chen et al. (2011b) previously used RSM based on the Box–Behnken design to optimize the pyrethroid insecticide degradation conditions at different pH, temperatures, and inocula amounts. They both demonstrated that the RSM was convenient and efficient to investigate the optimum degradation conditions for pyrethroid insecticides by various microorganisms. In the present study, a statistical model based on RSM was applied to optimize the beta-cp degradation conditions by strain BSF01, which was proved to be accurate and reliable within the limits of chosen factors.

Studies on the biodegradation pathway are very important in developing a bioremediation strategy for pyrethroid-contaminated environments because most of the pyrethroid insecticides can produce more toxic metabolites from the biodegradation process (Laffin et al. 2010). It is evident from the results that the particular strain BSF01 not only efficiently degraded beta-cp but also transformed the metabolites (compounds C, D, E, F, and G). This conclusion is in agreement with Zhang’s finding (Zhang et al. 2011). It was generally accepted that ester hydrolysis by carboxylesterases was the main degradation pathway of pyrethroid insecticides which resulted in yielding an acid and an alcohol (Tallur et al. 2008; Zhang et al. 2010). In the present study, beta-cp was metabolized firstly by hydrolysis of its ester linkage to produce 3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanecarboxylate and α-hydroxy-3-phenoxy-benzeneacetonitrile. α-Hydroxy-3-phenoxy-benzeneacetonitrile was unstable and spontaneously transformed to yield 3-phenoxybenzaldehyde. This phenomenon is similar with the degradation of cypermethrin by different microorganisms (Lin et al. 2011; Chen et al. 2011b). 3-Phenoxybenzaldehyde can accumulate in the media or soils, and enhanced biodegradation may not occur owing to its antimicrobial activities (Chen et al. 2011b). In this study, 3-phenoxybenzaldehyde was further metabolized by strain BSF01 to form 3-phenoxybenzoic acid. The intermediates 3-phenoxybenzoic acid and 3-phenoxybenzaldehyde are both the major metabolites of pyrethroid insecticides except cyfluthrin (Laffin et al. 2010). In previous study, Pseudomonas strains were reported to utilize 3-phenoxybenzoic acid as a growth substrate (Halden et al. 1999); however, all these strains could not metabolize beta-cp. In this study, Bacillus subtilis BSF01 was found to degrade not only beta-cp but also its major metabolic intermediates 3-phenoxybenzaldehyde and 3-phenoxybenzoic acid, which are rarely seen in other pyrethroid-degrading strains. These results demonstrate that Bacillus subtilis BSF01 harbors the metabolic pathway for the complete detoxification of beta-cp, indicating that the isolate may be an ideal microorganism for bioremediation of the beta-cp-contaminated environments.

In conclusion, a novel bacterium Bacillus subtilis has been isolated and characterized in the present study, which could utilize beta-cp as the sole carbon source for growth. What’s more, it was capable to degrade a wide range of pyrethroid insecticides including beta-cp, cypermethrin, deltamethrin, cyhalothrin, and beta-cyfluthrin. Furthermore, we presented evidence that the bacterium harbors the metabolic pathway for complete degradation and metabolism of beta-cp. To our knowledge, this is the first report that a pyrethroid-degrading bacterium transforms beta-cp through hydrolysis of ester linkage and cleavage of diaryl bond, which we propose is of vital importance in beta-cp biogeocycle.