Biobegradation and metabolic mechanism of cyprodinil by strain Acinetobacter sp. from a contaminated-agricultural soil in China

Ecotoxicology and Environmental Safety 159 (2018) 190–197

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Biobegradation and metabolic mechanism of cyprodinil by strain Acinetobacter sp. from a contaminated-agricultural soil in China Xiaoxin Chenb, Sheng Hea, Xiaolu Liua, Jiye Hua, a b

T

⁎

College of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, PR China College of Chemistry and Environmental Science, Hebei University, Baoding City, Hebei Province, 071002, PR China

A R T I C LE I N FO

A B S T R A C T

Chemical compounds studied in this article: Cyprodinil (PubChem CID: 86367)

Using sequential soil and liquid culture enrichments with cyprodinil as the sole carbon source, a Gram-negative cyprodinil-degrader from cyprodinil-polluted agricultural soil was isolated. The sequencing analysis of 16 S rRNA indicated that the strain showed 99% homology to Acinetobacter sp. The strain could effectively degrade cyprodinil at the neutral condition. At the initial concentrations of 10, 20, 50, 100, 150 and 200 mg L−1 in minimal medium, cyprodinil was degraded by 10, 20, 49.3, 64.2, 57 and 24 mg L−1 within 14 days, respectively. Two metabolites (4-cyclopropyl-6-methyl-2-pyrimidpyridine amine and monohydroxylated para-substitution) were identified using high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS/MS). A biodegradation pathway involving imines hydrolysis and monohydroxyl substitution on benzene ring was proposed on basis of the identified metabolites. Acinetobacter sp. would have a potential application in bioremediation of cyprodinil-contaminated soil, and the strain might have important implications in detoxification and bioremediation of pyrimidine analogues.

Keywords: Cyprodinil Acinetobacter sp. Isolation Bioavailability Biodegradation pathway

1. Introduction Cyprodinil [4-cyclopropyl-6-methyl-N-phenylpyrimidin-2-amine], as a kind of absorption-type and broad-spectrum fungicide, has been developed by Syngenta crop protection Co. Ltd. for the control of various germs in agricultural fields (Munitz et al., 2013). Due to it's low mammalian toxicity, broad insecticidal scope and high potency, cyprodinil has been registered and used in many countries such as the United States, China and European Union etc. since its first introduction in 1980s. Although cyprodinil exhibits low acute toxicity to mammals, due to its wide and long-term applications, the parent pesticide and its transformation products (TP) may accumulate in soils or be discharged into a broad range of environmental compartments. Therefore, the removal of cyprodinil from soils is one important aspect of ecological restoration. As we all know, microorganisms play an important role in removing toxic substances from ecological system. Microbial bioremediation has been considered as a cost-effective tool for the detoxification of xenobiotics in ecological ecosystem (Li et al., 2012). It has been reported that biodegradation, as an ideal pesticide-degradation way, has acquired a predominant significance in environmental remediation (Li et al., 2012; Seo et al., 2007; Chen et al., 2014; Kwak

et al., 2014; Vecino et al., 2015; Ishag et al., 2016; Cycon et al., 2017; Papale et al., 2017). In recent years, various bacterial strains have been isolated for biodegradation of pesticides and their TPs with fruitful effects (Li et al., 2012; Chen et al., 2014; Kwak et al., 2014; Papale et al., 2017; Cai et al., 2015; Deng et al., 2015). Therefore, biodegradation may be an expectable strategy for detoxification of cyprodinil and its TPs from the environment. To the best of our knowledge, the previous studies about cyprodinil were mainly focused on its residues analysis, bound residue formation, photo-degradation in aqueous solution, and biosynthesis of reference standards by microorganisms (Munitz et al., 2013; Dec et al., 1997; Kang et al., 2002; Fenoll et al., 2011; Chen et al., 2016). Although biodegradation of cyprodinil has been reported by Schocken et al. (Schocken et al., 1997), almost no literature is available about the dominant bacteria capable of degrading cyprodinil and its metabolic mechanism at the biochemical levels. Hence, it is significant to screen monoclonal bacteria capable of degrading cyprodinil, and to make a deeper investigation about biodegradation pathways at molecular and genetic level. The current study is not only beneficial to understand the biodegradation mechanism of cyprodinil at the molecule and gene, but also facilitates the bioremediation of cyprodinil-contaminated

Abbreviations: CEs, Crude enzymes; ESI, Electro spray ionization; HPLC-QTOF-MS/MS, High performance liquid chromatography tandem quadrupole trap-time-of-flight mass spectrometry; MSM, Mineral salt medium; MRM, Multiple reaction monitoring; RRLC-QqQ-MS/MS, Rapid resolution liquid chromatography tandem triple quadrupole mass spectrometry; PCR, Polymerase chain reaction; TPs, Transformation products; WDG, Water dispersible granule ⁎ Corresponding author. E-mail addresses: [email protected] (X. Chen), [email protected] (S. He), [email protected] (X. Liu), [email protected] (J. Hu). https://doi.org/10.1016/j.ecoenv.2018.04.047 Received 4 January 2018; Received in revised form 19 April 2018; Accepted 21 April 2018 Available online 21 May 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.

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To monitor cyprodinil degradation, 5 g sample was taken from each treatment at the given time intervals (0, 1, 2, 3, 5, 7, 11 and 19 days), and transferred into 50 mL PTFE centrifuge tube, to which 10 mL acetonitrile, 4 g MgSO4 and 1 g NaCl were added and vortexed for 1 min. After then, the centrifugal procedure was performed at 4000 rpm for 3 min. The supernatant was filtered into an auto-sampler with 0.22 µm syringe filter, and then it was analyzed using RRLC-QqQ-MS/ MS analyzer. Each treatment was performed in triplicates. Meanwhile, the control test without bacterial was conducted as follows. Another 50 g of the contaminated-soil was sterilized with an autoclave at 121 °C for 30 min. After then, the sterilized soil (50 g) and the uncontaminated-fresh soil (50 g) were added to 150 mL sterilized MSM, respectively, subsequently subjected to the same experiments. A sharp decline of cyprodinil was observed at the incubation of 11 days, then 1 mL of soil suspension was transferred to 50 mL flasks containing 5 mL fresh sterile MSM supplemented with cyprodinil (50 mg L−1) and further incubated for 7 days under the same conditions. The successive enrichment subcultures every 7 days were performed in the same MSM supplemented with different cyprodinil concentrations (50, 100, 200, 500 and 800 mg L−1). This step was repeated five more times to eliminate any impurities of organic matter originating from the soil sample and to attempt to isolate a mixed culture of cyprodinil-degrading microorganisms (Kandil et al., 2015; Howell et al., 2014). After five successive subcultures, 100 μL of serial dilutions (10−5, 10−6 and 10−7) of the enrichment culture were spread onto beef-protein medium agar plates, respectively. These plates were incubated in a light-free incubator at 30 °C for 48 h. On basis of morphological features, three different strains were obtained from the soil after successive enrichments. The discrete unique bacterial colonies were picked and further incubated in beef-protein broth for at 30 °C for 48 h. These isolated strains were preserved at − 80 °C in beef-protein broth supplemented with 20% sterile glycerol for further cyprodinildegrading assay.

environment. The main objectives in this study were (1): to isolate and purify a cyprodinil-degrader from polluted-agricultural soils using sequential soil and liquid culture enrichments with cyprodinil as the sole carbon source, as well as identify the monoclonal bacterium based on the 16 S rRNA sequencing analysis; (2): to investigate the pesticide-degrading activity in MSM supplemented with cyprodinil as the sole carbon source; (3): to identify the metabolites using HPLC-QTOF-MS/MS technique and to elucidate the metabolic mechanism at biochemical level. 2. Material and methods 2.1. Chemicals and reagents Cyprodinil standard (98.2% purity) was provided by Chengdu Kelilong Biochemical Co., Ltd. (Chengdu, China). 50% cyprodinil water dispersible granule (WDG) was provided by Syngenta crop protection Co. Ltd., China. The HPLC-grade acetonitrile and formic acid were purchased from the Dikma Co., Ltd. (Beijing city, China). All other reagents and chemicals were analytical-grade. The beef extract, peptone, agarose, acetonitrile and acetone were provided by Aladdin Reagents Co., Ltd., Shanghai, China. Sodium chloride, anhydrous magnesium sulfate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, ferrous sulfate, ammonium nitrate, manganese sulfate, calcium chloride and zinc sulfate were purchased from MYM Biological Technology Co., Ltd. (Beijing city, China). Syringe filter (nylon, 0.22 µm) was provided by Peak Sharp Company, P. R. China. The stock solutions (1000 mg L−1, 5000 mg L−1 and 20 g L−1) were prepared by directly dissolving cyprodinil in acetone and stored at 4 ℃ in dark. Before experiment, the mineral salt medium (MSM) was prepared with the following ingredients: 1 g K2HPO4, 1 g NaH2PO4·12H2O, 1 g NH4NO3, 0.25 g MgSO4, 0.02 g NaCl, 0.01 g FeSO4·7H2O, 0.01 g MnSO4·H2O and 0.01 g ZnSO4·7H2O were dissolved in 1 L distilled water. The beef-protein medium was used for both bacterial purification and amplification culture, which contained beef extract (5 g), peptone (5 g) and NaCl (5 g) into 1 L distilled water, subsequently adjusted pH to 7. All of the suspensions were sealed with sterilized filter membrane and autoclaved for 30 min at 121 °C and 0.1 MPa, allowed to cool at room temperature, and kept in a refrigerator at 4 °C until used.

2.3. Sequence analysis of 16S rRNA The dominant cyprodinil-degrading strain was identified and characterized by a 16 S rRNA gene sequence analysis (Tian et al., 2016). The monoclonal strain was cultured in beef-protein medium and incubated for 72 h at 28 °C. The genomic DNA was extracted following the protocol of the TIANamp Bacteria DNA kit (TIANGEN Biotech, Beijing). For polymerase chain reaction (PCR), 1 μL of the extracted DNA was added to a reaction mixture of 50 μL, containing 1 U of Taq polymerase (SBS company), the dNTP mixture (250 μM for each type), 10 pmol upstream primer 27 F (5 ´-AGAGTTTGATYMTGGCTCAG-3 ´) and downstream primer 1492 R (5 ´-TACGGYTACCTTGTTACGACTT-3 ´), 2 mM magnesium chloride and Tris-HCl buffer (pH 8.5). The amplification was carried out by a period of initial denaturation of 3 min at 96 °C, 35 cycles of denaturation at 90 °C for 45 s, annealing at 58 °C for 60 s, and extension at 72 °C for 90 s, with a period of final extension of 10 min. The genes fragments were sequenced by Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). A BLAST search of 16 S rRNA nucleotide sequences was carried out by the National Center for Biotechnology Information (NCBI) sequence database. The sequences were aligned based on the multiple sequence alignment program CLUSTRALW. A phylogenetic tree was constructed by the neighbor-joining method to expyridine amine interrelationships among strains using Molecular Evolutionary Genetic Analysis (MEGA, version 7.0). In each case, bootstrap values were calculated based on 1000 replications.

2.2. Enrichment and isolation of cyprodinil-degraders Field plot (30 m2) was exposed to high-dosage cyprodinil (135 mg kg−1) in open-field conditions, and repeated semimonthly over six months in Changping district, Beijing city, China. The initial concentration of field soils before contamination was below 0.005 mg kg−1. The cyprodinil-polluted soils were randomly collected from subsurface zone (the first 10–20 cm of top soil) and mixed thoroughly to make a composite sample (2 kg). These soil samples were immediately transported to the Laboratory and sieved through a 40-mesh. These soils with the moisture content of 18% were stored at 4 °C for no more than two weeks. 50 ± 0.01 g of the contaminated-soil sample was added to 150 mL sterilized MSM supplemented with 0.5 mL 50 g L−1 cyprodinil suspension, and the soil suspension samples containing 150 mg kg−1 cyprodinil was incubated on a dark rotary shaker at 30 °C at 200 rpm for 19 days for the first period. The residue of cyprodinil was below 0.005 mg kg−1 at the end of the first period. After then, a second cyprodinil suspension of 0.5 mL 50 g L−1 was added to the liquid culture systems and started for the second soil enrichment with the initial concentration of 135 mg kg−1. This process was implemented to increase the chance of creating a favorable condition for cyprodinil-degrading bacteria while building a growth-limiting culture for other soil microorganisms after they consume soil organic matter and nutrients, thus ensuring the adaptability of cyprodinil-degrading bacteria to cyprodinil and its degradation byproducts (Li et al., 2012; Liu et al., 2012; Kandil et al., 2015; Carles et al., 2017).

2.4. Optimization of growth condition of cyprodinil-degrading strain In this study, the effects of pH, substrate concentration and incubation temperature on cyprodinil-degradation ability were investigated, respectively. A series of 5-mL MSM degradation system containing cyprodinil 191

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(50 mg L−1) and cell suspension were aseptically added into a test flask. To investigate the effects of pH on biodegradation of cyprodinil, the MSM of cyprodinil (50 mg L−1) was adjusted to 4, 5, 6, 7, 8 and 9, respectively. To investigate the effect of cultural temperature on cyprodinil-biodegradation ability, the MSM of cyprodinil (50 mg L−1) was incubated on a light-free rotary shaker at 25, 30 and 38 °C, respectively. Samples were taken at specific time intervals. Samples were pretreated and subjected to RRLC-QqQ-MS/MS as described above after filtration with a 0.22 µm membrane. The same bacterial culture supernatants lacking cyprodinil were used as a negative control, and a noninoculated culture containing the same amount of cyprodinil served as a blank control. Taking into account the interaction effect of pH, temperature and substrate concentration on cyprodinil biodegradation, the growth conditions were further optimized through L9 (33) orthogonal assay on basis of the result of single factors. Three levels were selected for each factor: pH (6.7, 7.0 and 7.3), temperature (27, 30 and 33 °C), and substrate concentrations (40, 50 and 60 mg L−1). According to the orthogonal design as shown Table S1, the MSM supplemented with cyprodinil was incubated on a light-free rotary shaker at 200 rpm for 24 h. Samples were pretreated and analyzed via RRLC-QqQ-MS/MS as described above.

2.6. Biodegradation activity test of crude enzymes (CEs) To verify biodegradation activity of crude enzymes on cyprodinil, the following procedure was performed (Feng et al., 2016). The monoclonal strain was cultured in beef-protein medium and incubated for 72 h at 28 °C. The centrifugation of broth was carried out at 4 °C and 12,000 rpm for 15 min. The supernatant that might contain ectoenzyme was used to test the biodegradation activity of ectoenzyme on cyprodinil. Meanwhile, the cells at the bottom of the centrifuge tube were washed three times with 10 mL phosphate buffer solution (PBS) (50 mM, pH 7), and then they were resuspended in 5 mL PBS. After then, the sonic disruption over intervals up to five minutes for four cycles resulted in increasing amounts being liberated under ice bath conditions. The crushed mixture after sonic disruption was centrifuged at 4 °C and 13,000 rpm for 25 min. The supernatant (acellular extract) containing endoenzymes were preserved at − 20 °C until used. A series of 5 mL-phosphate buffer solution (pH 7) supplemented with cyprodinil (50 mg L−1) were inoculated with 200 μL acellular extract (or ectoenzyme extract), then incubated at 30 °C and 200 rpm on a light-free rotary shaker. The control test with no inocula was conducted under the same conditions. A whole bottle sample was taken for each treatment at intervals of 0, 2, 4, 8, 12 and 16 h. 50 μL concentrated hydrochloric acid was added to each sample to terminate the enzymatic reaction. The samples were pretreated using the method in part 2.5 and analyzed using RRLC-QqQ-MS/MS analyzer. Each treatment was performed in triplicate.

2.5. Biodegradation of cyprodinil in MSM The individual isolates were transferred into the fresh and sterile MSM (15 mL) supplemented with cyprodinil (100 mg L−1) medium and grown to a stationary phase for 15 days. After that bacterial suspension was used to the next inoculation. The biodegradation studies were conducted in a series of 50 mL sterile glass flasks. An appropriate aliquot of cyprodinil stock solution (5000 mg L−1 in acetone, filter sterilized beforehand) was added into the bottom of 50 mL flask. After complete volatilization of solvent, the sterile MSM (5 mL) was added and reconstituted by ultrasonic to reach the final concentration of cyprodinil (50 mg L−1) as the sole carbon source, subsequently inoculated aseptically with a total of 1 mL inoculum (OD600 = 1) to each glass flask. The degradation solution was incubated at 30 °C and cyprodinil residue was detected at an interval of 2 days. After confirmation of cyprodinil-degrading ability, the MSM (5 mL) supplemented with cyprodinil (10, 20, 50, 100, 150 and 200 mg L−1) as the sole carbon source was inoculated with the dominant degrading-strain and incubated on a rotary shaker at 30 °C and 200 rpm under dark conditions. The pH of broth was measured with a precision pH meter (Sartorius Group, Germany). The control test was conducted under the same manner as the glass flask. A whole bottle sample was taken for each treatment at given time intervals for determination of cyprodinil. To extract the target analyte in MSM, 5 mL enrichment broth was transferred into 50 mL PTFE centrifuge tube, to which 10 mL acetonitrile and 2 g NaCl were added and vortexed for 1 min. After then, the centrifugal procedure was performed at 4000 rpm for 3 min. The supernatant was filtered into an auto-sampler via with 0.22 µm syringe filter, and then it was analyzed using RRLC-QqQ-MS/MS analyzer. Each treatment was performed in triplicate. To detect cyprodinil parent, the rapid resolution liquid chromatography tandem triple quadrupled mass spectrometer (RRLC-QqQ-MS/ MS) (Agilent 6420, USA) equipped with a reversed phase C18 column (50 mm × 3 mm I.D., 2.7 µm) was employed for liquid chromatography separation at 30 °C. An electro spray ionization interface was operated in positive ion mode (ESI+). The sample injection volume was 5.0 μL. The mobile phase was the mixture of 0.2% formic acid solution (A) and acetonitrile (B) in the volume ratio of 20:80 (v: v) with the flow rate of 0.35 mL min−1. The MS parameters were as follows: gas temperature of 350 °C; gas flow rate of 11 L min−1; nebulizer gas pressure of 45 psi; column temperature of 30 °C. The capillary voltages were controlled at 4000 V. The multiple reaction monitoring (MRM) mode was selected for quantification of cyprodinil parent on basis of the quantitative fragment ion (m/z 93), while the scan mode was used to monitor metabolites.

2.7. Analysis of cyprodinil degradation products To identify the metabolites of cyprodinil by the dominant bacteria, the samples from Section 2.5 were analyzed by high performance liquid chromatography tandem mass spectrometry equipped with quadrupoletime-of-flight mass spectrometer (HPLC-QTOF-MS/MS, Agilent 6465) with scan and product iron (PI) modes. An Agilent Poroshell120 EC-C18 (2.1 ×100 mm, 2.7 µm) was used to separate the transformation products with an equal gradient. The sample injection volume was 2 μL. The mobile phase was a mixture of acetonitrile (A) and 0.2% aqueous of formic acid (B) in volume ratio 40:60 (v: v) and the flow rate was 0.35 mL min−1. The acquisition time was 10 min. The parameters of MS detection were as follows: drying (sheath) gas temperature of 325 (350 °C); drying (sheath) gas flow rate of 8 (11) L min−1; nebulizer gas pressure of 35 psi; capillary voltage (+) of 3500 V; fragmentor of 120 V. The scan and PI modes were used to obtain adequate mass spectrometry data from mass analyzer over a range of m/z 50–500. 2.8. Theoretical calculation of partition coefficient (LogP) of metabolites It is important to predict the migration of pesticides and their metabolites in soil and groundwater because this reflects potential environmental risks of these compounds (Jilani and Khan, 2006; Adams et al., 2008). Actually, the distribution between non-aqueous (octanol) and aqueous (water) phase octanol-water partition coefficient (logP) of a compound reflects the preference of the molecule to reside in either the organic or aqueous phase. The lower logP of a compound indicates a higher solubility and mobility in water. Therefore, the logP might be able to reflect potential environmental risk of compounds to groundwater to a certain extent. If the metabolites of a pesticide in soil or other environment showed lower logP, they might produce higher potential risks to groundwater due to the stronger diffusion and leaching. Usually, the partition coefficient between two immiscible solvents is expressed as the equilibrium distribution between the concentrations of a solute in each solvent. In addition, the partition coefficient is related to the change in energy associated with the solute-solvent interactions, which is expressed as the free energy difference (ΔG) of solute in each solvent (Adams et al., 2008). In this study, the partition coefficients of pyraclostrobin parent and major metabolites were 192

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bacteria widespread in water, soil and living organisms. A distinguishing feature of Acinetobacter spp. was that it can use various carbon sources for growth and even can be cultured on relatively simple media (Barbe et al., 2004). Morphological characterization of the dominant strain was studied by microscopic examination (Wang et al., 2016). The strain C could grow rapidly on beef-protein plates with a diameter of 4–5 mm, circular, yellow, and had a ridged surface and regular edge without spore, based on the morphology analysis. The strain C could effectively degrade cyprodinil at pH values of 5–9 and at temperatures of 20–38 °C. The biodegradation rate increased with increasing pH and reached its maximum at pH 7. The neutral condition was favorable for cyprodinil-biodegradation, whereas the higher or lower pH inhibited its biodegradation. This result was similar to that of Paichongding by Sphingobacterium sp. reported by Cai et al. (2015). The higher or lower temperatures were unfavorable to cyprodinil-degradation owing to a decrease of enzyme activity to a certain extent. In general, the degradation of cyprodinil by strain C could occur over a wider range of temperature or pH, and the monoclonal strain was capable of degrading cyprodinil with a maximum concentration of 200 mg L−1. The above results suggested that strain C has strong adaptability and survivability even though in a hostile environment for a long period. The orthogonal assay indicated that the effects of three factors on cyprodinil-degradation ability followed the order of temperature > substrate concentration > pH (Table S1). No obvious interaction of the three factors was observed at the designed levels. The optimum temperature, pH and substrate concentration were 30 °C, 7.0 and 50 mg L−1, respectively.

simulated by the Basis Set 6–31 (d, p) of DFT/b3lyp of Gaussian-09 Software as follows.

Log P =

−ΔGtransfer × 1000 RT ln(10)

=

(ΔGhydration − ΔGsolvation ) × 1000 RT ln(10)

Where, R and T were ideal gas constant (8.314 J K−1 mol−1) and temperature (298 K), respectively. ΔGhydration (kJ mol−1) and ΔGsolvation (kJ mol−1) were the Gibbs free energies of a compound in aqueous solution and n-octanol, respectively. 3. Results and discussion 3.1. Identification of cyprodinil-degrading strain Three different strains (named A, B and C) were isolated from soils after successive enrichments, in which the strain C has the largest amount and higher degradation activity. This result indicated that strain C was the dominant strain. The 16 S rRNA gene sequences of the dominant strain were compared by using BLAST similarity searches, and the closely related sequences were obtained from GenBank. On basis of the comparative analysis of 16 S rRNA gene sequence, the phylogenetic tree of dominant bacteria was constructed using the neighbor-joining algorithm as shown in Fig. 1. The phylogenetic analysis indicated that 16 S rRNA gene sequence of the isolate was closely similar to that of Acinetobacter junii strain ATCC 17,908 with a homology of 99%. The Acinetobacter sp. was a genus of Gram-negative

Fig. 1. Phylogenetic tree of strain Acinetobacter sp. based on the 16 S rRNA sequence analysis. 193

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8 10 12 14 16 18 20

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Fig. 2. (a, b). Biodegradation kinetics of cyprodinil by microorganisms during soil enrichment.

3.2. Biodegradation kinetics of cyprodinil in soil The biodegradation kinetics of cyprodinil by microorganisms during soil enrichment was shown in Fig. 2(a, b). Fig. 2(a) indicated that the initial concentration of cyprodinil was 150 mg kg−1 in the first cycle when the cyprodinil suspension of 0.5 mL 50 g L−1 was added into the MSM according to the theoretical calculation dose of 135 mg kg−1. This result was mainly attributed to the fact that the soil sample itself contained a certain amount of cyprodinil, because the soil sample was collected from a six-month cyprodinil-exposure field. Meanwhile, Fig. 2(a) indicated that cyprodinil with an initial concentration of 150 mg kg−1 was degraded to 1.9 mg kg−1 with a removal rate of 98.7% after the incubation of 11 days at the first enrichment. There was a significant increase in degradation efficiency during the second enrichment. The residual level of cyprodinil was below 3.7 mg kg−1 after 7 days incubation with a removal rate of 97.3%, and cyprodinil with an initial concentration of 135 mg kg−1 was degraded to 1.5 mg kg−1 with an incubation of 11 days. However, after 45 days of incubation, almost no apparent degradation of cyprodinil was observed in the uncontaminated fresh soil and the sterilized soil samples under the same conditions (Fig. 2(b)). A slight reduce of cyprodinil in the uncontaminated fresh and sterilized soils might be attributed to the conjugated or bound residues. Furthermore, our previous studies indicated that the half-lives of cyprodinil in three types of soil (Brown sandy loam, Red clay soil and Paekdusan black soil) were in the ranges of 66–257 days under aerobic field conditions, while the half-lives were approximately 46–182 days at the level of 2 mg kg−1 cyprodinil in soil under flooded field conditions. The above statements showed the inoculation of soil with the mixed microflora significantly accelerated the degradation of cyprodinil. In other words, the cyprodinil-degraders could efficiently utilize the persistent pesticide as growth substrate. Moreover, when cyprodinil was added to the soil once again after 19 days, it could be rapidly degraded, showing that these isolates could sustain itself in the natural environment for a long period. Therefore, the high removal rate of cyprodinil and strong survivability endowed these degraders with potential practical value in bioremediation of cyprodinil-contaminated environment.

Fig. 3. Biodegradation kinetics of cyprodinil by the strain Acinetobacter sp. in MSM.

respectively. When the initial cyprodinil was 100, 150 and 200 mg L−1, respectively, approximately 64.2, 57.0 and 24.3 mg L−1 of the added cyprodinil was degraded by the monoclonal isolate after incubation of 14 days. In addition, a lag period in degradation curves was observed at the higher cyprodinil concentration after a faster degradation. The finding suggested that increased concentration of cyprodinil has a slight effect on microbial metabolic activity of the strain with a modest increase in the duration of lag phase, but did not lead to complete inhibition or cell death. The similar result has also been reported by Jilani and Khan (2006). This feature endowed the cyprodinil-degrader with a competitive advantage in variable environments, as they could survive and utilize the pollutants even exposed to a high concentration. As seen from the control test, almost no obvious change in cyprodinil concentration was observed in the uninoculated control containing the same amount of cyprodinil, and none of any metabolites was detected in the negative control lacking cyprodinil. Therefore, this dominant strain might be an ideal candidate for bioremediation of cyprodinilcontaminated environment.

3.3. Biodegradation kinetics of cyprodinil in MSM 3.4. Biodegradation kinetics of cyprodinil by CEs The degradation kinetic of cyprodinil by the dominant strain in MSM was investigated as shown in Fig. 3 and Table S2. The result indicated that the monoclonal strain was capable of rapidly degrading and utilizing the added cyprodinil up to an initial concentration as high as 200 mg L−1. The biodegradation of cyprodinil ranging from 10 to 200 mg L−1 in MSM followed first-order kinetics with half-lives of 0.4–91.2 days. In addition, the degradation rate was the fastest with the substrate concentration of 50 mg L−1 (Table S2). As seen from Fig. 3, at initial concentration of 10, 20 and 50 mg L−1, cyprodinil was degraded by 9.94 mg L−1, 19.6 mg L−1 and 49.3 mg L−1 in just two days,

The degradation kinetic of cyprodinil by CEs was investigated. As shown in Fig. 4, at the initial concentration of 50 mg L−1, approximately 3.7, 9.7, 27.5 and 31.9 mg L−1 of cyprodinil were degraded by the acellular extract containing endoenzymes in just 2, 4, 8 and 12 h, respectively. Within the incubation of 16 h, more than 68.1% of the added cyprodinil was catalytically decomposed by the acellular extract of strain. In addition, the enzymatic metabolites agreed with that of the strain. However, almost no obvious degradation was observed in the degradation system of supernatant containing ectoenzyme and the

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50 45 40 35 30 25 20 15 10 5 0

118.0524, 106.0649, 93.0571, 77.0385 and 67.0288, corresponding to the loss of NH3, (NH3 +CH3), (NH+C3H5), (NH3 +C3H4), (NH+C3H5 +C2H5) and (NH3 +C3H4 +C2H2), respectively. The reasonable fragmentation patterns from accurate mass spectrum were beneficial for verifying the molecular structure of TPs1 (4-cyclopropyl-6-methyl-2pyrimidamine). On the other hand, LC-MS/MS analysis of ATCC isolate 36112 also indicated cyprodinil could be metabolized to a molecular cleavage product, agreeing with the above finding in our study (Schocken et al., 1997). TPs2 was eluted at the retention time of 1.371 min with a protonated molecular ions [M+H]+ of m/z 242.1286. Obviously, TPs2 exhibited 16 Da higher than that of cyprodinil parent with the protonated molecular weight of 226.1339. This result suggested an additional oxygen atom emerged on parent molecule. Moreover, the accurate mass spectrum indicated that the most probable formula was C14H15N3O. Therefore, it was certain that monohydroxylation occurred on cyprodinil molecule. Firstly, the monohydroxylated metabolite was tentatively proposed as the hydroxylation of para-H on the benzene ring. To determine the position of hydroxylation, the mass fragmentation spectra of TPs2 (4-cyclopropyl-6-methyl-N-phenol-2-pyrimidinamine) were further analyzed. The prominent fragment ions at m/z 148.0865 represented an unsubstituted methylcyclopropylamino pyrimidine moiety, which indicated that the hydroxylation substitution occurred on the phenyl ring. In other words, the loss of 94 mass from TPs2 was corresponded to loss of the phenol. Considering the electronic and steric effects, substitution para to the amino group on the phenyl ring was subjected to preference. Furthermore, para-hydroxylation site on benzene ring was a priority based on the mulliken atomic charges analysis of cyprodinil molecular (Chen et al., 2016). Based on the accurate molecular fragment ions, TPs2 was splitted into these fragments of m/z 148.0865, 108.0805, 93.0567 and 65.0382, corresponding to the loss of phenol, (C6H6O+C3H4), (C6H6O+C3H4 +CH3) and (C6H6O+C3H4 +CH3 +C2H4), respectively. The reasonable fragmentation patterns from accurate mass spectrum facilitated to support the molecular structure of TPs2. In conclusion, TPs2 was identified as a monohydroxylated metabolite and generated from the substitution of parahydrogen atom on benzene ring of cyprodinil molecule. The results agreed with that of previous literature reported by Schocken et al. (1997). Herein it was worth emphasizing that TPs1 was the predominant metabolite in 95% yield, and its polarity was far greater than that of parent compound on basis of the retention times.

Endoenzyme Ectoenzyme CK

0

2

4

6

8

10

12

14

16

18

Sampling intervals (hours) Fig. 4. Biodegradation kinetics of cyprodinil by crude enzymes.

uninoculated control containing the same amount of cyprodinil. These findings suggested that the enzyme capable of catalyzing cyprodinil to TPs1 and TPs2 might belong to endoenzyme of the strain instead of ectoenzyme. However, the specific endoenzyme would be further isolated and characterized by complete genome sequencing analysis in the future.

3.5. Identification of metabolites While cyprodinil was degraded by the particular strain, two polar and unknown biodegradation byproducts (designed as TPs1 and TPs2) were increasingly produced (Fig. S1). The tentative structural identification for each metabolite was deduced on basis of the high-resolution MS/MS data including their protonated molecules, MS/MS fragmentation patterns, some reasonable fragment loss regulations, elemental formula and the chemical structure of cyprodinil. The retention times, molecular weights (MWs), characteristic fragments and chemical structure of these compounds were summarized in Table 1. The peak of TPs1 appeared at 0.423 min in the chromatograph, showing a protonated molecular ion [M+H]+ at norminal m/z 150.1024 with the empirical formula of C8H11N3. Based on the accurate mass spectra, the major fragments of TPs1 exhibited at m/z 133.0758, Table 1 Mass spectra data and structures of cyprodinil and TPs using HPLC-QTOF-MS/MS. Compound

Cyprodinil

Structure

Molecular formula

Rt (min)

m/z [M+H]+ Experimental (Theoretical)

Error (mDa)

Fragment

Product ion

C14H15N3

2.509

226.1339

0.012

226.1336 210.1026 133.0758 108.0805 93.0572 77.0384

[M [M [M [M [M [M

+ + + + + +

H]+ H – CH4]+ H – C3H5 – CH3CN]+ H – C3H5 – C6H5]+ H – C3H5 – C6H5 – CH3]+ H – TPs1]+

1.17

150.1024 133.0758 118.0524 106.0649 93.0571 77.0385 67.0288

[M [M [M [M [M [M [M

+ + + + + + +

H]+ H – NH3]+ H – NH3 – CH3]+ H – NH – C3H5]+ H – NH3 – C3H4]+ H – NH – C3H5 – C2H5]+ H – NH3 – C3H4 – C2H2]+

0.78

242.1286 148.0865

[M + H]+ [M + H – C6H6O]+ (loss of phenol) [M + H – C6H6O – C3H4]+ [M + H – C6H6O – C3H4 – CH3]+ [M + H – C6H6O – C3H4 – CH3 – C2H4]+

(226.1339)

TPs1

C8H11N3

0.423

150.1024

(150.1026)

TPs2

C14H15N3O

1.371

242.1286

(242.1288)

195

108.0805 93.0567 65.0382

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CH3

CH3

N N H

H2O enzyme

N

H2N N

TPs 1

Parent

H2 O

Lastly, the stability of these metabolites was also observed in current study. When cyprodinil was almost completely degraded (> 98%) within 11 days during the soil enrichment incubation, the amount of TPs1 and TPs2 was not reduced for an extension of incubation time (30 days). This result suggested that the two metabolites showed stability to a certain extent, and they might not be oxidized or mineralized to CO2 by the strain. That was to say, TPs1 and TPs2 were the final biodegradation products of cyprodinil by the dominant strain. In addition, the strain might exhibit specificity for cyprodinil substrate, and it could not use the transformation products as carbon source. In short, cyprodinil might not be totally degraded by the bacteria Acinetobacter sp. to produce CO2 and H2O etc.

N

enzyme CH3

3.7. Potential risk prediction of metabolite

N HO

N H

The LogP values of cyprodinil and two metabolites were calculated (Table S3). The LogP values of cyprodinil, TPs1 and TPs2 at the same temperature (298 K) were 5.56, 2.97 and 5.48, respectively. The result suggested that the mobility and diffusion of TPs1 in water was obviously higher than that of cyprodinil, whilst that of TPs1 was slightly lower than parent's. In other words, the risk probability of groundwater pollution caused by TPs1 was far higher than that of parent pesticide in respect to the leaching and diffusion perspectives. As seen from the retention time in the reversed C18 column, the polarity of TPs was obviously greater than that of cyprodinil. Therefore, the potential dietary risk of TPs1 might deserve attention with the wide and longterm application of cyprodinil parent.

N

TPs 2

Fig. 5. The proposed microbial degradation pathways of cyprodinil in MSM.

3.6. Biodegradation pathway On basis of the structures of the identified metabolites and cyprodinil parent, a novel microbial biodegradation pathway of cyprodinil was firstly proposed and identified in this study. As shown in Fig. 5, cyprodinil parent might be metabolized undergoing two enzyme-catalyzed reactions: (a) cleavage of amine bond; (b) hydroxylation of hydrogen atom para to amino group on the phenyl ring. Firstly, cyprodinil was mainly catalyzed by Acinetobacter sp. to yield a pyridine amine compound. This result indicated that the strain might contain gene coding of amine hydrolase. In acidic or aqueous solutions, the electrophilic attack of one proton occurred on the nitrogen atom linked to benzene ring of cyprodinil, and thus caused the cleavage of pyridine amine bond. Other strains of Acinetobacter sp. could be searched from UniProtKB database, and they showed similar catalytic mechanisms of amine hydrolases (Adams et al., 2008; Thotsaporn et al., 2004). The available literatures further explained the above conclusion. Besides, only a small part of cyprodinil was transformed into the para-substituted metabolite attributing to the substitution para to the amino group on the phenyl ring. This finding suggested that the strain might contain gene coding of hydroxylase. In aqueous solutions, the nucleophilic attack of one hydroxyl occurred on the carbon atom para to the secondary amine group on the phenyl ring. The hydroxylation position has been corroborated in part 3.5. Several strains of Acinetobacter sp. from UniProtKB database exhibited similar hydrolytic mechanism (Thotsaporn et al., 2004). Furthermore, Schocken et al. (1997) found that cyprodinil could be hydroxylated by the enzyme cytochrome P-450 monooxygenase, in which molecular oxygen could be activated directly so that an oxygen atom was added to the cyprodinil molecule. The above findings indicated that the cyprodinil-degrader might contain the gene coding of hydroxylase and amine hydrolase, and the enzymes expressed by these genes were mainly the endoenzymes of Acinetobacter sp. This finding was similar to the result of Sphingopyxis sp. USTB-05 by Feng et al. (2016). The cleavage of amine bond was beneficial to the detoxification of cyprodinil because amine bond was an important functional group in cyprodinil molecular. Attributed to the same active sites and similar metabolic mechanisms, Acinetobacter sp. expressing amine hydrolase might be able to degrade pyrimidine analogues. Therefore, Acinetobacter sp. might have important implications in detoxification and bioremediation of pyrimidine analogues. However, the cloning and expression of these genes as well as their action sites and mechanism at the genetic level would be further investigated in the future.

4. Conclusions In this study, the bacteria strain C capable of degrading cyprodinil was obtained from the polluted agricultural soil using successive enrichments with cyprodinil as the sole carbon source. The monoclonal strain exhibited a higher degradation activity of cyprodinil with initial concentration as high as 200 mg L−1, while the mixed bacterium was capable of tolerating and degrading cyprodinil of 800 mg L−1, suggesting that it was the most efficient microflora of cyprodinil-degrading up to now. These findings suggested that Acinetobacter sp. might be an ideal candidate for bioremediation of the cyprodinil-contaminated environment, as it could survive for a long-term period and utilize the pollutants even exposed to a high concentration. Two polar metabolites (Pyridine aniline and para-substituted metabolite) suggested that the cyprodinil-degrader itself might have gene coding of amine hydrolase and hydroxylase, thus it would be an ideal candidate for expression and cloning of cyprodinil-degrading genes in further genomics studies. Furthermore, owing to the similar active sites and metabolic mechanisms, Acinetobacter sp. might have important implications in detoxification and bioremediation of pyrimidine analogues. Acknowledgments This work was supported by financial support from National Natural Science Foundation of China (Project NO. 21677009) as well as Natural Science Foundation of Beijing (Project NO. 8162029). The authors gratefully acknowledged the Ministry of Agriculture of People Republic of China for the project support as well as Agilent user Laboratory for technical assistance with high-resolution mass spectra technique. Conflicts of interest The authors declare no competing financial interest and human conflicts. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2018.04.047. 196

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