Environ Sci Pollut Res DOI 10.1007/s11356-015-5976-3
RESEARCH ARTICLE
Biodegradation of chlorimuron-ethyl and the associated degradation pathway by Rhodococcus sp. D310-1 Chunyan Li 1 & Hailian Zang 1 & Qi Yu 1 & Tongyang Lv 1 & Yi Cheng 1 & Xiaosong Cheng 2 & Keran Liu 1 & Wanjun Liu 1 & Pianpian Xu 1 & Chuanzeng Lan 1
Received: 18 August 2015 / Accepted: 14 December 2015 # Springer-Verlag Berlin Heidelberg 2016
Abstract Chlorimuron-ethyl is a typical long-term residual sulfonylurea herbicide, and strategies for its removal have attracted increasing attention. Microbial degradation is considered the most acceptable dissipation method. In this study, we optimized the cultivation conditions (substrate concentration, pH, inoculum concentration, and temperature) of the chlorimuron-ethyl-degrading bacterium Rhodococcus sp. D310-1 using response surface methodology (RSM) to improve the biodegradation efficiency. A maximum biodegradation rate of 88.95 % was obtained. The Andrews model was used to describe the changes in the specific degradation rate as the substrate concentration increased. Chlorimuron-ethyl could be transformed with a maximum specific degradation rate (qmax), half-saturation constant (KS), and inhibition constant (K i ) of 0.4327 day − 1 , 63.50045 mg L − 1 , and 156.76666 mg L−1, respectively. Eight biodegradation products (2-amino-4-chloro-6-methoxypyrimidine, ethyl 2sulfamoyl benzoate, 2-sulfamoyl benzoic acid, o-benzoic sulfimide, 2-[[(4-chloro-6-methoxy-2-pyrimidinyl)
Responsible editor: Gerald Thouand Electronic supplementary material The online version of this article (doi:10.1007/s11356-015-5976-3) contains supplementary material, which is available to authorized users. * Chunyan Li [email protected]
1
College of Resource and Environment, Northeast Agricultural University, Harbin 150030, Heilongjiang, China
2
College of First Clinical Medicine, Harbin Medical University, Harbin 150001, Heilongjiang, China
carbamoyl] sulfamoyl] benzoic acid, ethyl 2-carbonyl sulfamoyl benzoate, ethyl 2-benzenesulfonyl isocyanate benzoate, and N,N-2(ethyl formate)benzene sulfonylurea) were identified, and three possible degradation pathways were proposed based on the results of high performance liquid chromatography HPLC, liquid chromatography tandem mass spectroscopy (LC-MS/MS), and Fourier transform infrared spectroscopy (FTIR) analyses and the relevant literature. This systematic study is the first to examine the chlorimuron-ethyl degradation pathways of the genus Rhodococcus. Keywords Chlorimuron-ethyl . Rhodococcus sp. D310-1 . Response surface methodology . Kinetics . LC-MS
Introduction Chlorimuron-ethyl is a typical long-term residual sulfonylurea herbicide that is widely used in modern agriculture for pre- and post-emergence broadleaf weed control in soybean and maize (Tan et al. 2013). Due to the long period of the use of chlorimuron-ethyl in soil, its residues have inevitably caused phytotoxicity, damaged subsequent susceptible crops, altered the soil microbial community structure, and decreased soil enzyme activity (Zhao and He 2007). In addition, the residual chlorimuron-ethyl that enters the water through runoff and leaching endangers aquatic organisms (Fenoll et al. 2013). Consequently, new strategies for decreasing or eliminating chlorimuronethyl residues in the environment are needed. Chlorimuron-ethyl residues in soil and water can be reduced using physical, chemical, and biological methods (Brusal et al. 2001; Luo et al. 2008; Xu et al. 2009). Among these methods, microbial degradation plays an important role in removing chlorimuron-ethyl residues
Environ Sci Pollut Res
and has received a great deal of attention due to its low cost, high efficiency, and environmentally friendly chara c t e r i s t i c s ( M o h a n a e t a l . 20 0 7 ) . A n u m b er o f chlorimuron-ethyl-degrading microorganisms, including Aspergillus niger (Sharma et al. 2012), Stenotrophomonas maltophilia (Li et al. 2011), H a n s s c h l e g e l i a s p . C H L 1 ( Ya n g e t a l . 2 0 1 4 ) , Pseudomonas sp. LW3 (Ma et al. 2009), and Klebsiella jilinsis 2N3 (Zhang et al. 2010), have been isolated from chlorimuron-ethyl-contaminated environments. Unfortunately, most studies have focused on the screening and characterization of chlorimuron-ethyl-degrading microorganisms and the evaluation of the degradation capacity of these organisms (Zhang et al. 2009). Only a few reports have focused on the biodegradation pathways of chlorimuron-ethyl, and these studies have observed degradation only at the sulfonylurea bridge. For instance, Ma et al. (2009) isolated the chlorimuron-ethyl-degrading bacterium Pseudomonas sp. LW3 and identified two products resulting from the cleavage of the sulfonylurea bridge using liquid chromatography-mass spectrometry (LCMS). Generally, cleavage of the sulfonylurea bridge is considered the most common degradation pathway for sulfonylurea herbicides, yielding the corresponding sulfonamide and heterocyclic amine (Braschi et al. 2000; Zhang et al. 2013). The biodegradation pathways of chlorimuron-ethyl have yet to be clearly described. Furthermore, identification of the products formed during chlorimuron-ethyl degradation in these organisms has not been systematically discussed. In previous study, we isolated Rhodococcus sp. D310-1 from chlorimuron-ethyl-contaminated environments. This bacterium can grow in mineral medium with chlorimuronethyl as the sole carbon source and nitrogen (Xiong et al. 2013). However, the ability of this strain to degrade chlorimuron-ethyl and the pathway of degradation were not studied in detail. In this study, cultivation conditions (i.e., substrate concentration, pH, inoculum concentration, and temperature) were optimized by employing response surface methodology (RSM) using the Box–Behnken design (BBD) to improve biodegradation efficiency. In addition, the kinetics of chlorimuron-ethyl degradation by Rhodococcus sp. D310-1 were investigated for the first time based on the Andrews model. Furthermore, the products of chlorimuronethyl degradation by Rhodococcus sp. D310-1 were determined and preliminarily identified using high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectroscopy (LC-MS/MS), and Fourier transform infrared spectroscopy (FTIR), and possible degradation pathways were proposed. The results provide highly valuable information for future studies on the microbial degradation of other sulfonylurea herbicides and will contribute considerably to environmental remediation efforts.
Materials and methods Bacterial strain Rhodococcus sp. D310-1 (GenBank accession number GU138102.1) was isolated from activated sludge samples obtained from factories producing sulfonylurea herbicides in China through enrichment culture and stored in our laboratory (Xiong et al. 2013). Chemicals and media Chlorimuron-ethyl (98.70 % purity) was supplied by the Jiangsu Institute of Ecomones Co., Ltd., China. Chromatographic pure grade acetonitrile, methanol, glacial acetic acid, and acetone (Dikma Technologies Inc., CA, USA) were filtered through 0.22 μm Millex-GP PES filters (Merck Millipore, Billerica, USA) before use. All other chemicals employed in this study were of analytical reagent grade and were obtained from Tianjin Kemiou Chemical Reagent Co., Ltd.. The composition of the mineral salt medium (MSM) was as follows (L−1): CaSO4 0.04 g, K2HPO4 0.1 g, NaCl 0.1 g, FeSO4·7H2O 0.001 g, MgSO4·7H2O 0.2 g, and (NH4)2SO4 0.1 g, at pH 6.0, supplemented with 100 mg L−1 chlorimuronethyl as the sole carbon source. Luria-Bertani (LB) medium was made as follows (L−1): liquid medium containing 5 g yeast extract, 10 g NaCl, and 10 g tryptone, at pH 6.0. Chlorimuron-ethyl degradation Rhodococcus sp. D310-1 was pre-cultivated in LB medium at 28 °C and 160 rpm until the exponential phase was reached. Then, 10 mL of culture medium was centrifuged at 8000 rpm for 5 min. The harvested cells were washed three times with 0.1 M phosphate buffer (pH 7.0) and adjusted to OD600 nm = 2.00 ± 0.1 with MSM. The resuspended cells (2 %, v/v) were inoculated into 100 mL of MSM containing chlorimuron-ethyl in a 250-mL Erlenmeyer flask. The cultures were incubated in the dark at 28 °C and 160 rpm in a temperature-controlled rotary shaker (HZQ-C, Harbin Donglian Electronic Technology Development Co., China). The control experiments were carried out under the same conditions without bacteria. Treatment methods for all samples and the conditions for detection of the chlorimuron-ethyl degradation rate were the same as described in BDetection of the products of degradation by Rhodococcus sp. D310-1^ section. Optimization of chlorimuron-ethyl cultivation conditions RSM, which can be used to design an experiment and evaluate the relationship between the responses and the independent variables, was used to optimize important parameters and their
Environ Sci Pollut Res
interactions for improving the chlorimuron-ethyl degradation rate of strain D310-1 (Boyaci 2007; Kirmizakis et al. 2014). The statistical software Design-Expert V. 8.0.6 (Stat-Ease, Inc. Minneapolis, USA) was employed for the experimental design and data analysis. To further improve the biodegradation efficiency of strain D310-1, BBD was used to consider four factors (inoculum size, substrate concentration, pH, and temperature) at three levels. The biodegradation rate was the only response variable. The experimental parameters and the coded levels of the four independent variables are illustrated in Table 1. A second-order polynomial equation (Eq. S1 shown in the Supplementary Information (SI)) is a typical model that depicts the response surface (Yaacob et al. 2013). To validate the predicted results of the optimized model, the biodegradation experiments were tested in triplicate under optimized reaction conditions. The processing of all samples and detection of the degradation rate were performed as described in BDetection of the products of degradation by Rhodococcus sp. D310-1^ section. Kinetics of chlorimuron-ethyl degradation by Rhodococcus sp. D310-1 Degradation kinetic experiments with different initial concentrations of chlorimuron-ethyl (10, 25, 50, 100, 150, 200, 250, 300, 400, 500 mg L−1) by strain D310-1 were performed under the optimum cultivation conditions of the predicted by the BBD model. Samples of non-inoculated medium containing the same concentrations of chlorimuron-ethyl served as controls. The experimental samples and controls were collected every 24 h for 8 days. The chlorimuron-ethyl degradation rate was detected by HPLC as described in BDetection of the products of degradation by Rhodococcus sp. D310-1^ section. The specific chlorimuron-ethyl degradation rate can be expressed as follows:
q¼
1 dS d ðS0−S Þ ⋅ ¼ X dt X ⋅dt
ð1Þ
The Andrews model (Andrews 1968; Li et al. 2012; Wang et al. 2013) is normally used to describe substrate inhibition kinetics, and this model is as follows: q¼
qmax S S þ K S þ S 2 =K i
Sm ¼
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi KS Ki
ð2Þ ð3Þ
where q is the specific chlorimuron-ethyl degradation rate, day−1; X is the dry cellular concentration, mg L−1; ds/dt is the rate of change in the chlorimuron-ethyl concentration; qmax is the maximum specific chlorimuron-ethyl degradation rate, day−1; KS is the half-saturation constant, mg L−1; Ki is the inhibition constant, mg L−1; S is the chlorimuron-ethyl
concentration, mg L−1; S0 is the initial concentration of chlorimuron-ethyl, mg L−1; Sm is the critical inhibitory concentration of chlorimuron-ethyl, which indicates a turning point in the degradation rate (Acuña-Argüelles et al. 2003). Detection of the products of degradation by Rhodococcus sp. D310-1 Chlorimuron-ethyl degradation products were collected after 24 h of incubation with strain D310-1 under optimum cultivation conditions in MSM. A total of six collections were performed at 24-h intervals. The samples of non-inoculated media under the same conditions served as controls. All samples were processed as follows, and all samples were tested in triplicate. Extraction method Ten milliliters of the culture medium was centrifuged at 12, 000 rpm for 10 min, and then 5 mL of the supernatant was extracted with 10 mL dichloromethane for chlorimuron-ethyl extraction from liquid cultures. After the sample was shaken for 2 min then stationary for 10 min, each sample was extracted with CH2Cl2 three times, and then the organic phases were combined. The dichloromethane phase was dried over anhydrous Na2SO4, collected, and concentrated to near-dryness in a 100-mL flat bottom flask with a rotary evaporator (RE52C, Shanghai, China). The chlorimuron-ethyl and biodegradation products were dissolved in 1.5 mL of acetonitrile. The samples were filtered through 0.22 μm Millex-GP PES filters for HPLC analysis (Ma et al. 2009; Zhang et al. 2010). HPLC conditions Chlorimuron-ethyl concentrations were analyzed using a Waters 600 reverse-phase HPLC with an autosampler, a Waters 2487 dual wavelength detector, and a C18 column (250 × 4.6 mm i.d., 5-μm particle size). The compounds were separated using a mobile phase of methanol/water/glacial acetic acid (70/30/0.5, v/v/v) at a flow rate of 0.8 mL min−1. The injection volume was 20 μL. Ultraviolet wavelength detection was performed at 254 nm. The column temperature was 25 °C. Empower Software (Waters, MA, USA) was employed to record and analyze the experimental data (Polati et al. 2006; Wang et al. 2012). The biodegradation rate formula is shown in the SI. MS conditions The samples were analyzed using a Thermo Finnigan LCQ Deca LC/MSn system (Thermo LCQ DECA Xp MAX, Thermo Finnigan, MA, USA) equipped with a liquid chrom a t o g r a p h , a Wa t e r s X b r i d g e ™ C 1 8 c o l u m n
Environ Sci Pollut Res Table 1 Levels of various factors affecting chlorimuron-ethyl biodegradation Factors
Name
Units
Low (−1)
Mid (0)
High (1)
A
Inoculum size
% (v/v)
B C D
Substrate concentration pH Temperature
mg L−1
1 50
2 100
3 150
°C
5.5 25
6.0 28
6.5 31
multiple regression analysis and fitting the polynomial quadratic model to the experimental data, the regression model for the four-factor system was achieved (Boyaci 2007; Wang et al. 2013). The second-order polynomial equation can be written as follows: Y ¼ 81:91−0:31A−3:26B−2:99C þ 2:50D þ 0:71AB −2:66AC−0:59AD−5:27BC−2:85BD−2:71CD 2
2
2
−2:03A −9:07B − 9:52C þ 0:53D (2.1 mm × 150 mm, 5 m), and a thermostat (30 °C). The MS apparatus was equipped with an electrospray ionization (ESI) source in both positive and negative ion modes. The ESI-MS interface parameters were set as follows: spray voltage, 5.0 kV; capillary temperature, 350 °C; drying gas (nitrogen) flow rate, 1.0 L min−1; nebulizer gas pressure, 25.0 psi; and mass range, m/z 80 to 500. The Xcalibur® data system (Thermo Finnigan, MA, USA) was employed to acquire and analyze the experimental data (Peng et al. 2012; Ma et al. 2009). The chlorimuron-ethyl products of degradation by Rhodococcus sp. D310-1 were analyzed by LC-MS/MS using the following procedures: First, a full scan mass spectrum was obtained using positive and negative modes simultaneously. Then, a base peak that met the preset intensity threshold was selected from the mass spectrum. The MS/MS scan mass spectrum with an appropriate mass range for the selected peak was acquired under a relative collision energy of 40 or 50 % (Benzi et al. 2013; Gao et al. 2014). FTIR analysis The degradation products were dealt with as described in BExtraction method^ section, after which the dichloromethane solvents containing degradation products were evaporated to dryness using a freeze dryer (ALPHA1-4, Martin Christ, Germany). The freeze-dried samples were dispersed in KBr (1:20 weight ratio) and pressed for 3 min to obtain a translucent pellet. A blank KBr pellet was obtained as a background reference. Infrared absorption spectra were recorded using the FTIR spectrometer (ALPHA-T FTIR spectrometer, 179 BRUKER, Germany) in transmission mode and working with 128 scans at a resolution of 4 cm−1 and a scanning wavenumber range from 4000 to 400 cm−1 (Chlopek et al. 2009; Mathur et al. 2010; Hou et al. 2014).
Results and discussion Optimization of the culture conditions using RSM The experimental design and results obtained with RSM optimization are shown in Table S1. Through performing
2
where Y is the biodegradation rate; A, B, C, and D are independent factors; AB, AC, AD, BC, BD, and CD are interaction factors; and A2, B2, C2, and D2 are quadratic terms. The results of the analysis of variance (ANOVA) for the RSM are shown in Table S2. The accuracy of the model was evaluated using a determination coefficient of R2 = 0.9808, which indicates that the model has a very high correlation (Walia et al. 2015). Song et al. (2013) and Hou et al. (2014) stated that the use of a regression model was statistically acceptable when p < 0.05 and lack of fit >0.05. They also reported that a model with a p value < 0.0001, F value = 51.19, and a lack of fit value = 0.3057 was statistically acceptable. Table 2 indicates that B, C, D, AC, BC, BD, CD, A2, B2, and C2 are significant terms (p < 0.05) (Schenone et al. 2015). Among these variables, the p values for the substrate concentration, pH, and temperature were below 0.01, which had significant impacts on the degradation rate; thus, the substrate concentration, pH, and temperature had significant effects on the degradation rate. According to the values of PAC, PBC, PBD, and PCD (0.0027,
RESEARCH ARTICLE
Biodegradation of chlorimuron-ethyl and the associated degradation pathway by Rhodococcus sp. D310-1 Chunyan Li 1 & Hailian Zang 1 & Qi Yu 1 & Tongyang Lv 1 & Yi Cheng 1 & Xiaosong Cheng 2 & Keran Liu 1 & Wanjun Liu 1 & Pianpian Xu 1 & Chuanzeng Lan 1
Received: 18 August 2015 / Accepted: 14 December 2015 # Springer-Verlag Berlin Heidelberg 2016
Abstract Chlorimuron-ethyl is a typical long-term residual sulfonylurea herbicide, and strategies for its removal have attracted increasing attention. Microbial degradation is considered the most acceptable dissipation method. In this study, we optimized the cultivation conditions (substrate concentration, pH, inoculum concentration, and temperature) of the chlorimuron-ethyl-degrading bacterium Rhodococcus sp. D310-1 using response surface methodology (RSM) to improve the biodegradation efficiency. A maximum biodegradation rate of 88.95 % was obtained. The Andrews model was used to describe the changes in the specific degradation rate as the substrate concentration increased. Chlorimuron-ethyl could be transformed with a maximum specific degradation rate (qmax), half-saturation constant (KS), and inhibition constant (K i ) of 0.4327 day − 1 , 63.50045 mg L − 1 , and 156.76666 mg L−1, respectively. Eight biodegradation products (2-amino-4-chloro-6-methoxypyrimidine, ethyl 2sulfamoyl benzoate, 2-sulfamoyl benzoic acid, o-benzoic sulfimide, 2-[[(4-chloro-6-methoxy-2-pyrimidinyl)
Responsible editor: Gerald Thouand Electronic supplementary material The online version of this article (doi:10.1007/s11356-015-5976-3) contains supplementary material, which is available to authorized users. * Chunyan Li [email protected]
1
College of Resource and Environment, Northeast Agricultural University, Harbin 150030, Heilongjiang, China
2
College of First Clinical Medicine, Harbin Medical University, Harbin 150001, Heilongjiang, China
carbamoyl] sulfamoyl] benzoic acid, ethyl 2-carbonyl sulfamoyl benzoate, ethyl 2-benzenesulfonyl isocyanate benzoate, and N,N-2(ethyl formate)benzene sulfonylurea) were identified, and three possible degradation pathways were proposed based on the results of high performance liquid chromatography HPLC, liquid chromatography tandem mass spectroscopy (LC-MS/MS), and Fourier transform infrared spectroscopy (FTIR) analyses and the relevant literature. This systematic study is the first to examine the chlorimuron-ethyl degradation pathways of the genus Rhodococcus. Keywords Chlorimuron-ethyl . Rhodococcus sp. D310-1 . Response surface methodology . Kinetics . LC-MS
Introduction Chlorimuron-ethyl is a typical long-term residual sulfonylurea herbicide that is widely used in modern agriculture for pre- and post-emergence broadleaf weed control in soybean and maize (Tan et al. 2013). Due to the long period of the use of chlorimuron-ethyl in soil, its residues have inevitably caused phytotoxicity, damaged subsequent susceptible crops, altered the soil microbial community structure, and decreased soil enzyme activity (Zhao and He 2007). In addition, the residual chlorimuron-ethyl that enters the water through runoff and leaching endangers aquatic organisms (Fenoll et al. 2013). Consequently, new strategies for decreasing or eliminating chlorimuronethyl residues in the environment are needed. Chlorimuron-ethyl residues in soil and water can be reduced using physical, chemical, and biological methods (Brusal et al. 2001; Luo et al. 2008; Xu et al. 2009). Among these methods, microbial degradation plays an important role in removing chlorimuron-ethyl residues
Environ Sci Pollut Res
and has received a great deal of attention due to its low cost, high efficiency, and environmentally friendly chara c t e r i s t i c s ( M o h a n a e t a l . 20 0 7 ) . A n u m b er o f chlorimuron-ethyl-degrading microorganisms, including Aspergillus niger (Sharma et al. 2012), Stenotrophomonas maltophilia (Li et al. 2011), H a n s s c h l e g e l i a s p . C H L 1 ( Ya n g e t a l . 2 0 1 4 ) , Pseudomonas sp. LW3 (Ma et al. 2009), and Klebsiella jilinsis 2N3 (Zhang et al. 2010), have been isolated from chlorimuron-ethyl-contaminated environments. Unfortunately, most studies have focused on the screening and characterization of chlorimuron-ethyl-degrading microorganisms and the evaluation of the degradation capacity of these organisms (Zhang et al. 2009). Only a few reports have focused on the biodegradation pathways of chlorimuron-ethyl, and these studies have observed degradation only at the sulfonylurea bridge. For instance, Ma et al. (2009) isolated the chlorimuron-ethyl-degrading bacterium Pseudomonas sp. LW3 and identified two products resulting from the cleavage of the sulfonylurea bridge using liquid chromatography-mass spectrometry (LCMS). Generally, cleavage of the sulfonylurea bridge is considered the most common degradation pathway for sulfonylurea herbicides, yielding the corresponding sulfonamide and heterocyclic amine (Braschi et al. 2000; Zhang et al. 2013). The biodegradation pathways of chlorimuron-ethyl have yet to be clearly described. Furthermore, identification of the products formed during chlorimuron-ethyl degradation in these organisms has not been systematically discussed. In previous study, we isolated Rhodococcus sp. D310-1 from chlorimuron-ethyl-contaminated environments. This bacterium can grow in mineral medium with chlorimuronethyl as the sole carbon source and nitrogen (Xiong et al. 2013). However, the ability of this strain to degrade chlorimuron-ethyl and the pathway of degradation were not studied in detail. In this study, cultivation conditions (i.e., substrate concentration, pH, inoculum concentration, and temperature) were optimized by employing response surface methodology (RSM) using the Box–Behnken design (BBD) to improve biodegradation efficiency. In addition, the kinetics of chlorimuron-ethyl degradation by Rhodococcus sp. D310-1 were investigated for the first time based on the Andrews model. Furthermore, the products of chlorimuronethyl degradation by Rhodococcus sp. D310-1 were determined and preliminarily identified using high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectroscopy (LC-MS/MS), and Fourier transform infrared spectroscopy (FTIR), and possible degradation pathways were proposed. The results provide highly valuable information for future studies on the microbial degradation of other sulfonylurea herbicides and will contribute considerably to environmental remediation efforts.
Materials and methods Bacterial strain Rhodococcus sp. D310-1 (GenBank accession number GU138102.1) was isolated from activated sludge samples obtained from factories producing sulfonylurea herbicides in China through enrichment culture and stored in our laboratory (Xiong et al. 2013). Chemicals and media Chlorimuron-ethyl (98.70 % purity) was supplied by the Jiangsu Institute of Ecomones Co., Ltd., China. Chromatographic pure grade acetonitrile, methanol, glacial acetic acid, and acetone (Dikma Technologies Inc., CA, USA) were filtered through 0.22 μm Millex-GP PES filters (Merck Millipore, Billerica, USA) before use. All other chemicals employed in this study were of analytical reagent grade and were obtained from Tianjin Kemiou Chemical Reagent Co., Ltd.. The composition of the mineral salt medium (MSM) was as follows (L−1): CaSO4 0.04 g, K2HPO4 0.1 g, NaCl 0.1 g, FeSO4·7H2O 0.001 g, MgSO4·7H2O 0.2 g, and (NH4)2SO4 0.1 g, at pH 6.0, supplemented with 100 mg L−1 chlorimuronethyl as the sole carbon source. Luria-Bertani (LB) medium was made as follows (L−1): liquid medium containing 5 g yeast extract, 10 g NaCl, and 10 g tryptone, at pH 6.0. Chlorimuron-ethyl degradation Rhodococcus sp. D310-1 was pre-cultivated in LB medium at 28 °C and 160 rpm until the exponential phase was reached. Then, 10 mL of culture medium was centrifuged at 8000 rpm for 5 min. The harvested cells were washed three times with 0.1 M phosphate buffer (pH 7.0) and adjusted to OD600 nm = 2.00 ± 0.1 with MSM. The resuspended cells (2 %, v/v) were inoculated into 100 mL of MSM containing chlorimuron-ethyl in a 250-mL Erlenmeyer flask. The cultures were incubated in the dark at 28 °C and 160 rpm in a temperature-controlled rotary shaker (HZQ-C, Harbin Donglian Electronic Technology Development Co., China). The control experiments were carried out under the same conditions without bacteria. Treatment methods for all samples and the conditions for detection of the chlorimuron-ethyl degradation rate were the same as described in BDetection of the products of degradation by Rhodococcus sp. D310-1^ section. Optimization of chlorimuron-ethyl cultivation conditions RSM, which can be used to design an experiment and evaluate the relationship between the responses and the independent variables, was used to optimize important parameters and their
Environ Sci Pollut Res
interactions for improving the chlorimuron-ethyl degradation rate of strain D310-1 (Boyaci 2007; Kirmizakis et al. 2014). The statistical software Design-Expert V. 8.0.6 (Stat-Ease, Inc. Minneapolis, USA) was employed for the experimental design and data analysis. To further improve the biodegradation efficiency of strain D310-1, BBD was used to consider four factors (inoculum size, substrate concentration, pH, and temperature) at three levels. The biodegradation rate was the only response variable. The experimental parameters and the coded levels of the four independent variables are illustrated in Table 1. A second-order polynomial equation (Eq. S1 shown in the Supplementary Information (SI)) is a typical model that depicts the response surface (Yaacob et al. 2013). To validate the predicted results of the optimized model, the biodegradation experiments were tested in triplicate under optimized reaction conditions. The processing of all samples and detection of the degradation rate were performed as described in BDetection of the products of degradation by Rhodococcus sp. D310-1^ section. Kinetics of chlorimuron-ethyl degradation by Rhodococcus sp. D310-1 Degradation kinetic experiments with different initial concentrations of chlorimuron-ethyl (10, 25, 50, 100, 150, 200, 250, 300, 400, 500 mg L−1) by strain D310-1 were performed under the optimum cultivation conditions of the predicted by the BBD model. Samples of non-inoculated medium containing the same concentrations of chlorimuron-ethyl served as controls. The experimental samples and controls were collected every 24 h for 8 days. The chlorimuron-ethyl degradation rate was detected by HPLC as described in BDetection of the products of degradation by Rhodococcus sp. D310-1^ section. The specific chlorimuron-ethyl degradation rate can be expressed as follows:
q¼
1 dS d ðS0−S Þ ⋅ ¼ X dt X ⋅dt
ð1Þ
The Andrews model (Andrews 1968; Li et al. 2012; Wang et al. 2013) is normally used to describe substrate inhibition kinetics, and this model is as follows: q¼
qmax S S þ K S þ S 2 =K i
Sm ¼
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi KS Ki
ð2Þ ð3Þ
where q is the specific chlorimuron-ethyl degradation rate, day−1; X is the dry cellular concentration, mg L−1; ds/dt is the rate of change in the chlorimuron-ethyl concentration; qmax is the maximum specific chlorimuron-ethyl degradation rate, day−1; KS is the half-saturation constant, mg L−1; Ki is the inhibition constant, mg L−1; S is the chlorimuron-ethyl
concentration, mg L−1; S0 is the initial concentration of chlorimuron-ethyl, mg L−1; Sm is the critical inhibitory concentration of chlorimuron-ethyl, which indicates a turning point in the degradation rate (Acuña-Argüelles et al. 2003). Detection of the products of degradation by Rhodococcus sp. D310-1 Chlorimuron-ethyl degradation products were collected after 24 h of incubation with strain D310-1 under optimum cultivation conditions in MSM. A total of six collections were performed at 24-h intervals. The samples of non-inoculated media under the same conditions served as controls. All samples were processed as follows, and all samples were tested in triplicate. Extraction method Ten milliliters of the culture medium was centrifuged at 12, 000 rpm for 10 min, and then 5 mL of the supernatant was extracted with 10 mL dichloromethane for chlorimuron-ethyl extraction from liquid cultures. After the sample was shaken for 2 min then stationary for 10 min, each sample was extracted with CH2Cl2 three times, and then the organic phases were combined. The dichloromethane phase was dried over anhydrous Na2SO4, collected, and concentrated to near-dryness in a 100-mL flat bottom flask with a rotary evaporator (RE52C, Shanghai, China). The chlorimuron-ethyl and biodegradation products were dissolved in 1.5 mL of acetonitrile. The samples were filtered through 0.22 μm Millex-GP PES filters for HPLC analysis (Ma et al. 2009; Zhang et al. 2010). HPLC conditions Chlorimuron-ethyl concentrations were analyzed using a Waters 600 reverse-phase HPLC with an autosampler, a Waters 2487 dual wavelength detector, and a C18 column (250 × 4.6 mm i.d., 5-μm particle size). The compounds were separated using a mobile phase of methanol/water/glacial acetic acid (70/30/0.5, v/v/v) at a flow rate of 0.8 mL min−1. The injection volume was 20 μL. Ultraviolet wavelength detection was performed at 254 nm. The column temperature was 25 °C. Empower Software (Waters, MA, USA) was employed to record and analyze the experimental data (Polati et al. 2006; Wang et al. 2012). The biodegradation rate formula is shown in the SI. MS conditions The samples were analyzed using a Thermo Finnigan LCQ Deca LC/MSn system (Thermo LCQ DECA Xp MAX, Thermo Finnigan, MA, USA) equipped with a liquid chrom a t o g r a p h , a Wa t e r s X b r i d g e ™ C 1 8 c o l u m n
Environ Sci Pollut Res Table 1 Levels of various factors affecting chlorimuron-ethyl biodegradation Factors
Name
Units
Low (−1)
Mid (0)
High (1)
A
Inoculum size
% (v/v)
B C D
Substrate concentration pH Temperature
mg L−1
1 50
2 100
3 150
°C
5.5 25
6.0 28
6.5 31
multiple regression analysis and fitting the polynomial quadratic model to the experimental data, the regression model for the four-factor system was achieved (Boyaci 2007; Wang et al. 2013). The second-order polynomial equation can be written as follows: Y ¼ 81:91−0:31A−3:26B−2:99C þ 2:50D þ 0:71AB −2:66AC−0:59AD−5:27BC−2:85BD−2:71CD 2
2
2
−2:03A −9:07B − 9:52C þ 0:53D (2.1 mm × 150 mm, 5 m), and a thermostat (30 °C). The MS apparatus was equipped with an electrospray ionization (ESI) source in both positive and negative ion modes. The ESI-MS interface parameters were set as follows: spray voltage, 5.0 kV; capillary temperature, 350 °C; drying gas (nitrogen) flow rate, 1.0 L min−1; nebulizer gas pressure, 25.0 psi; and mass range, m/z 80 to 500. The Xcalibur® data system (Thermo Finnigan, MA, USA) was employed to acquire and analyze the experimental data (Peng et al. 2012; Ma et al. 2009). The chlorimuron-ethyl products of degradation by Rhodococcus sp. D310-1 were analyzed by LC-MS/MS using the following procedures: First, a full scan mass spectrum was obtained using positive and negative modes simultaneously. Then, a base peak that met the preset intensity threshold was selected from the mass spectrum. The MS/MS scan mass spectrum with an appropriate mass range for the selected peak was acquired under a relative collision energy of 40 or 50 % (Benzi et al. 2013; Gao et al. 2014). FTIR analysis The degradation products were dealt with as described in BExtraction method^ section, after which the dichloromethane solvents containing degradation products were evaporated to dryness using a freeze dryer (ALPHA1-4, Martin Christ, Germany). The freeze-dried samples were dispersed in KBr (1:20 weight ratio) and pressed for 3 min to obtain a translucent pellet. A blank KBr pellet was obtained as a background reference. Infrared absorption spectra were recorded using the FTIR spectrometer (ALPHA-T FTIR spectrometer, 179 BRUKER, Germany) in transmission mode and working with 128 scans at a resolution of 4 cm−1 and a scanning wavenumber range from 4000 to 400 cm−1 (Chlopek et al. 2009; Mathur et al. 2010; Hou et al. 2014).
Results and discussion Optimization of the culture conditions using RSM The experimental design and results obtained with RSM optimization are shown in Table S1. Through performing
2
where Y is the biodegradation rate; A, B, C, and D are independent factors; AB, AC, AD, BC, BD, and CD are interaction factors; and A2, B2, C2, and D2 are quadratic terms. The results of the analysis of variance (ANOVA) for the RSM are shown in Table S2. The accuracy of the model was evaluated using a determination coefficient of R2 = 0.9808, which indicates that the model has a very high correlation (Walia et al. 2015). Song et al. (2013) and Hou et al. (2014) stated that the use of a regression model was statistically acceptable when p < 0.05 and lack of fit >0.05. They also reported that a model with a p value < 0.0001, F value = 51.19, and a lack of fit value = 0.3057 was statistically acceptable. Table 2 indicates that B, C, D, AC, BC, BD, CD, A2, B2, and C2 are significant terms (p < 0.05) (Schenone et al. 2015). Among these variables, the p values for the substrate concentration, pH, and temperature were below 0.01, which had significant impacts on the degradation rate; thus, the substrate concentration, pH, and temperature had significant effects on the degradation rate. According to the values of PAC, PBC, PBD, and PCD (0.0027,
