Novel hydrolytic de-methylthiolation of the s-triazine herbicide prometryn by Leucobacter sp. JW-1

STOTEN-21300; No of Pages 9 Science of the Total Environment xxx (2016) xxx–xxx

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Novel hydrolytic de-methylthiolation of the s-triazine herbicide prometryn by Leucobacter sp. JW-1 Junwei Liu a, Rimao Hua a, Pei Lv a, Jun Tang a, Yi Wang a, Haiqun Cao a, Xiangwei Wu a,⁎, Qing X. Li b a b

College of Resources and Environment, Anhui Agricultural University, Key Laboratory of Agri-food Safety of Anhui Province, Hefei 230036, PR China Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East–West Road, Honolulu, HI 957822, USA

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Leucobacter sp. JW-1 was isolated from prometryn-contaminated sludge. • Strain JW-1 can efficiently degrade 9 triazine herbicides. • Biodegradation of prometryn started by novel hydrolytic de-methylthiolation. • Prometryn-degrading enzyme has high temperature tolerance.

a r t i c l e

i n f o

Article history: Received 30 September 2016 Received in revised form 30 October 2016 Accepted 1 November 2016 Available online xxxx Editor: Jay Gan Keywords: Biodegradation Prometryn Leucobacter sp. De-methylthiolation Triazine herbicide

a b s t r a c t s-Triazine herbicides have been widely used in recent decades and caused serious concern over contamination of groundwater, surface water and soil. A novel bacterial strain JW-1 was isolated from activated sludge and identified as Leucobacter sp. based on comparative morphology, physiological characteristics and comparison of the 16S rDNA gene sequence. JW-1 was capable of using methylthio-s-triazine prometryn as a sole source of carbon and energy in pure culture. Favorable conditions for prometryn degradation were found at pH 7.0–9.0 and temperature of 37–42 °C. The degradation half-life of prometryn at 50 mg L−1 was remarkably as short as 1.1 h, and increased to 6.0 h when the initial concentration increased to 400 mg L−1. The strain JW-1 could degrade 100% of ametryn, 99% of simetryn, 41% of propazine, 43% of atrazine, 28% of simazine, 12% of terbutylhylazine, 10% of prometon and 13% of atraton at 50 mg L−1 of each herbicide in 2 days. Prometryn was converted to 2hydroxypropazine and methanthiol via a novel hydrolysis pathway. 2-Hydroxypropazine was then transformed to N-isopropylammelide and the final product cyanuric acid via two sequential deamination reactions. In addition to biodegradation by Leucobacter sp. JW-1, the hydrolytic de-methylthiolation would be valuable in biocatalysis. © 2016 Elsevier B.V. All rights reserved.

⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (X. Wu).

http://dx.doi.org/10.1016/j.scitotenv.2016.11.006 0048-9697/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Liu, J., et al., Novel hydrolytic de-methylthiolation of the s-triazine herbicide prometryn by Leucobacter sp. JW-1, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.006

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1. Introduction Triazine herbicides are widely used to control broadleaf and grassy weeds during the past three decades (Fan and Song, 2014). Triazine herbicides have unfortunately caused serious contamination of water sources, which led to limiting their use in Europe (Fenoll et al., 2014). Atrazine, simazine, prometryn, and terbutryn (Evgenidou et al., 2007) have been classified as priority substances by the European Parliament and the Council of the European Union in water policy Directive, 2013/39/UE. Triazine herbicides and their metabolites have been detected frequently in soil, surface water and groundwater (Arias-Estévez et al., 2008; Papadopoulou-Mourkidou et al., 2004; Schuler and Rand, 2007) and exceeded the maximum limit level for drinking water, which is 3 μg L− 1 in USA and 0.1 μg L− 1 in Europe (Mahia et al., 2007; Vryzas et al., 2011). Previous studies indicated that triazine herbicides might cause damages to the central nervous system, endocrine system, immune system, and reproductive process of rats, pigs and amphibians (Bohn et al., 2011; Fan and Song, 2014). Triazine herbicides are weak bases with low sorption by soils, and can persist a relatively long period of time in the environment (Jiang et al., 2006; Wu et al., 2010; Zhou et al., 2012), where they are degraded dominantly by microorganisms (Houot et al., 1998). A number of degradingbacterial strains such as Pseudomonas sp. ADP (Mandelbaum et al., 1995), Arthrobacter nicotinovorans HIM (Aislabie et al., 2005), Nocardioides sp. DN36 (Satsuma, 2010), Rhodococcus sp. FJ1117YT (Fujii et al., 2007), Rhizobium sp. (Smith et al., 2005), and Acinetobacter sp. A6 (Singh et al., 2004) have been isolated from s-triazine herbicidepolluted sites. Their characteristics of degrading s-triazine herbicides in soil and water have been extensively investigated (Wang et al., 2014; Zhou et al., 2012). The genetic pathways for detoxification of s-triazine herbicides such as atrazine have been well documented. The atrazine metabolic reactions involved are dechlorination by enzymes encoded by the atzA/TrzN genes, and displacement of N-alkylamino groups by atzB and atzC genes, via the sequential formation of metabolites hydroxyatrazine, N-isopropylammelide, and cyanuric acid. Cyanuric acid is then mineralized to carbon dioxide and ammonia by the enzymes encoded by the TrzD/atzD, atzE and atzF genes (Kannika et al., 2004; Martinez et al., 2001). Soil microorganisms, especially bacteria, play a key role in the processes of removing or detoxifying organic pollutants from contaminated sites (Wang et al., 2016). Therefore, the biodegradation of triazine herbicides has received much research attention. Prometryn (2,4-bis (isopropylamino)-6-(methylthio)-s-triazine) is a selective, systemic, pre- and post-emergence triazine herbicide used extensively to control annual grasses and broadleaf weeds in a wide range of crops, especially cotton and cereals. Recently, prometryn has been used widely to control broad-leaved weeds and annual grasses in rice, cotton, sugarcane, and soybeans in China (Zhou et al., 2009). Due to its low sorption by soils, moderate water solubility and long half-life in soils, prometryn has high potential to contaminate surface water, groundwater, and soil. The concentration levels of prometryn ranged from 0.19 to 4.40 μg L− 1 in the surface waters of Greece (Vryzas et al., 2011) and exceeded 1.00 μg L−1 in groundwater of the Axios river basin (Papadopoulou-Mourkidou et al., 2004). The halflives of prometryn in soil varied from a few days to months, depending on climates, soil properties, application volumes, and agricultural practices (Dumas et al., 2008; Fenoll et al., 2014; Navarro et al., 2004; Rose et al., 2007; Suzuki and Otani, 2004), and thus prometryn is considered a moderately persistent chemical. Prometryn is listed as a reproductive and developmental toxic compound in the US EPA Toxics Release Inventory List, and in the prioritization list of the European Union as an endocrine disruptor (Database, 2013; Perez-Barcena et al., 2014). Therefore, it is necessary to develop a biodegradation method for cleanup of prometryn pollution. Numerous bacterial strains that can degrade chloro-s-triazines such as atrazine and simazine have been isolated from soils, and

characterized regarding their metabolites (Gebendinger and Radosevich, 1999; Mandelbaum et al., 1995; Radosevich et al., 1995; Satsuma, 2010; Vail et al., 2015; Wang et al., 2016). However, only a few strains capable of degrading methylthio-s-triazines such as prometryn and ametryn have been documented (Fujii et al., 2007). Those species include Nocardioides sp. DN36 (Satsuma, 2010), A. nicotinovorans HIM (Aislabie et al., 2005), and Arthrobacter aurescens TC1 (Shapir et al., 2005). Topp et al. (2000) reported that prometryn was converted to corresponding hydroxyl derivatives by whole cells or purified hydrolase of Nocardioides sp. C190 under anaerobic conditions. A. aurescens TC1 can grow in a medium containing ametryn or prometryn as the sole nitrogen source, and recombinant triazine hydrolase (TrzN) from the strain could transform prometryn to hydroxyl derivatives (Shapir et al., 2005; Strong et al., 2002). However, the metabolic pathway of prometryn has not been well understood. Leucobacter sp. can degrade polyethylene glycol 400, ɛ-caprolactam and ciprofloxacin (Liao et al., 2016; Marchal et al., 2008; Sanuth et al., 2013). To our best knowledge, no report of pesticide biodegradation by Leucobacter sp. strains has been found in the literature. In the present study, a new bacterial strain, Leucobacter sp. JW-1, was isolated from activated sludge and characterized for efficient degradation of triazine herbicides. The objectives of this study were to study the degradation of prometryn by strain JW-1 and to elucidate the detailed metabolic pathways of prometryn. 2. Materials and methods 2.1. Chemicals and reagents Prometryn (98%), propazine (98.5%), 2-hydroxypropazine (98.5%), prometon (99%), atraton (99%), terbumeton (98.5%), metribuzin (99%), cyanuric acid (99%), methanthiol (99%), dimethyl disulfide (99%) were purchased from Dr. Ehrenstorfer GmbH, Augsburg, Germany. Ametryn (97%), simetryn (96%), atrazine (97%), simazine (97%), and terbutylhylazine (97%) were obtained from Binnong Technology Co. Ltd., Binzhou, China. The 2× Taq Master Mix, pMD 18-T cloning vector and DNA marker were purchased from Takara Bio, Dalian, China. All other chemicals and solvents were of analytical grade. 2.2. Sludge sample and culture media Activated sludge used in this study were sampled at the outlet of aeration tank of the WWTP in a prometryn manufacture plant, located in Binzhou City, Shandong province, China. Sludge samples were sealed on-site in glass bottles and stored in a cooler at 4 °C, and then transported to the laboratory immediately for the follow-on experiments. Mineral salt medium (MSM) contained 0.4 g of MgSO4·7H2O, 0.2 g of K2HPO4, 0.2 g of (NH4)2SO4, 0.08 g of CaSO4, 0.002 g of FeSO4·7H2O in 1000 mL of deionized water at pH 7.0. Mineral salt peptone medium (MSP) contained 0.5 g of peptone in MSM and beef extract-peptone medium were prepared for enrichment, isolation and purification of prometryn-degrading bacteria (Deng et al., 2015). 2.3. Enrichment and isolation of strain JW-1 One gram of sludge samples was added to a 100-mL Erlenmeyer flask containing 20 mL of sterile MSM supplemented with 20 mg L−1 of prometryn as sole carbon and energy sources. The mixture was incubated at 30 °C and 150 rpm on a rotary shaker. After incubation for 5 d, 1 mL of culture was transferred into 20 mL of sterile fresh MSM supplemented with 40 mg L−1 of prometryn and incubated for 5 d under the same conditions described as above. The culture was successively acclimated 3 times in a sterile fresh MSM with increasing concentrations of prometryn, ranging from 40 to 100 mg L−1. Subsequently, the culture was sampled, diluted, spread on MSP agar plates containing 200 mg L− 1 of prometryn as prometryn-selective medium, and

Please cite this article as: Liu, J., et al., Novel hydrolytic de-methylthiolation of the s-triazine herbicide prometryn by Leucobacter sp. JW-1, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.006

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incubated for 3 d at 30 °C. Individual colonies were picked aseptically, purified until single colonies of uniform morphology were obtained, and tested for their ability to degrade prometryn. A prometryndegrading strain JW-1 was isolated from colonies formed on the plates. 2.4. Identification and characterization of strain JW-1 The strain JW-1 was identified on the basis of morphological observation, physiological and biochemical tests and 16S rDNA gene alignment. After JW-1 grew on agar plates for 3 d at 30 °C, the cells were fixed by glutaraldehyde and stained with citric acid, followed by morphological examination using a transmission electron microscope (TEM) (HT-7700, Hitachi, Japan). The strain JW-1 was physiologically and biochemically characterized according to the methods described by Dong and Cai (2001). The genomic DNA of the strain JW-1 was extracted according to the SDS-proteinase K method, and the fragment of 16S rDNA gene was amplified by polymerase chain reaction (PCR) using universal bacterial primers of 16S rDNA, the forward primer: 5′TGGCGAACGGGTGAGTAATACAT-3′, and the reverse primer: 5′GCGGTTAGGCTAACTACTTCTGG-3′. PCR was performed with a thermal cycler (BioRad C1000, USA) and PCR procedure was set as: 94 °C initial denaturation for 5 min, followed by 30 cycles consisting of denaturation for 30 s at 92 °C, 55 °C for 30 s, 72 °C for 2 min, and a final extension for 10 min at 72 °C. The reaction mixture (25 μL) contained 12.5 μL of 2 × Taq Master Mix (1.25 U Taq polymerase, 3 mmol MgCl2, 100 mmol KCl, and 0.4 mmol dNTPs), 0.5 μL of primer (10 mmol L−1 each), 0.5 μL of DNA template and double-deionized H2O to a final volume of 25 μL. The PCR products were purified with a San Prep DNA Gel extraction Kit (Shanghai Sangon biotech, China), ligated into pMD-18T vector (Takara Bio, Dalian, China) according to the instructions, and subsequently cloned into E. coli DH5α competent cell. Sequence analysis of the 16S rDNA gene was accomplished by Invitrogen Biotechnology Co. Ltd., Shanghai, China. Similarity analyses of the 16S rDNA gene sequences were conducted using the BLAST function of NCBI GenBank (Altschul et al., 1990). Phylogenetic trees were constructed with MEGA 6.0 (Hall, 2013). 2.5. Inoculum preparation The strain JW-1 was cultured in 250-mL Erlenmeyer flasks containing 100 mL beef extract-peptone medium at 30 °C and 150 rpm on a rotary shaker. At the exponential growth phase (40 h), the bacterial cells were centrifuged at 3380 ×g for 10 min to remove medium, then immediately washed twice with 0.9% of aseptic sodium chloride solution, and resuspended with MSM, and finally adjusted to value of 0.6 (~1.2 × 109 cfu mL−1) at a wavelength of 600 nm (OD600) determined by a UV-1800 spectrometer (Shimadzu Corp., Japan). 2.6. Degradation of prometryn by strain JW-1 To assess degradation capacity of JW-1, MSM was supplemented with prometryn as the sole source of carbon and energy. Each flask was inoculated with 0.3 mL of bacterial cell suspensions (OD600, 0.6). All flasks were incubated in a rotary shaker at 37 °C and 150 rpm in the dark. At certain time intervals, the whole culture was sampled for determination of prometryn concentration. Each treatment was carried out in triplicate, and the control experiment without JW-1 was performed under the same conditions. The effect of pH on degradation of prometryn in liquid MSM (50 mg L−1) was tested under pH 5.0, 6.0, 7.0, 8.0, and 9.0 that was adjusted with 0.2 mol L−1 of NaH2PO4 and Na2HPO4 solution. Temperature effect on prometryn degradation (50 mg L− 1) was tested at 20, 25, 30, 37, 42, and 47 °C. The removal efficiency and possible toxicity of prometryn to the strain JW-1 were measured at the concentrations of 50, 200, and 400 mg L−1 in MSM at pH 7 and 37 °C.

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2.7. Degradation of other triazine herbicides An aliquot of 0.3 mL of bacterial cell suspensions (OD600, 0.6) was inoculated in 20 mL of MSM containing 50 mg L−1 of ametryn, simetryn, atrazine, simazine, terbutylhylazine, propazine, prometon, atraton, terbumeton, metribuzin or cyanuric acid. The target analytes in culture mixtures were extracted for analysis after incubation for 2 and 4 d at 37 °C on a rotary shaker at 150 rpm. Each treatment was performed in triplicate and the control group was not inoculated with bacterial cell suspensions. 2.8. Determination and identification of prometryn metabolites An aliquot of 2 mg of prometryn was added into 20 mL of MSM in 100-mL Erlenmeyer flasks with 100-mL headspace vials inoculated with 0.3 mL of bacterial cell suspensions. All flasks were incubated at 37 °C and 150 rpm of shaking. At 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 120 h, 20 mL of methanol was added to the Erlenmeyer flasks. The mixture was ultrasonicated for 2 min, and additional methanol was added to adjust the volume to 50 mL (methanol/ water = 3/2, v/v). The solution was filtered through a 0.22-μm membrane and analyzed by an Agilent 1260 series high performance liquid chromatograph (HPLC) equipped with an ultraviolet detector (Agilent Technologies, California, USA). Additionally, the above extracts at 4 h, 48 h, and 60 h were simultaneously analyzed by an Accela liquid chromatograph-LTQ Orbitrap XL high resolution mass spectrometer (LCHRMS) (Thermo Fisher Scientific, Massachusetts, USA). The samples in headspace vials were frozen at −20 °C for 4 h, and then 40 mL of isooctane was injected in the vials. After the samples were melted, swirled for 2 min, and the supernatant was analyzed using a Varian GC-450 gas chromatograph with a pulsed flame photometric detector (PFPD) (Varian, California, USA). Prometryn, 2-hydroxypropazine, and cyanuric acid were analyzed with HPLC equipped with an ultraviolet detector and a column (Agilent Eclipse XDB-C18, 5 μm, 4.6 × 250 mm). The mobile phase flow rate was 1.0 mL min−1, and the column temperature was held at 30 °C. The column was eluted with 20% acetonitrile aqueous solution for 5 min, increased linearly to 90% acetonitrile in 5 min and held for 8 min, followed by a gradual decrease to 20% acetonitrile over 2 min and held for 5 min prior to the next injection. The sample injection volume was 20 μL. The detection wavelength was set at 224 nm. The LC-HRMS system consisted of a Hypsil Gold C18 column (5 μm, 4.6 × 250 mm) that was maintained at 30 °C. The elution gradient program was the same as the HPLC analysis except holding 90% acetonitrile for 18 min instead of 5 min. The flow rate was 0.2 mL min−1. The mass spectrometer was operated with electrospray ionization (ESI) source with voltage of 4 kV. Nitrogen was used as the sheath gas and aux gas, with flow rates of 20 and 5 arbitrary units, respectively. The vaporizer temperature was 275 °C in positive mode and 84 °C in negative mode, and the capillary temperature was 275 °C. The tube lens voltage was 95 V in positive mode and 40 V in negative mode. Methanthiol and dimethyl disulfide from prometryn degradation were measured using a GC-450 gas chromatograph equipped with pulsed flame photometric detector (PFPD) with a sulfur filter. The separation column was Varian CP-SIL 19CB (1 μm, 30 m × 0.53 mm). GC injector and detector temperatures were 130 and 300 °C, respectively. Carrier gas (nitrogen) flow rate was set at 4 mL min−1. The oven temperature started at 30 °C for 1 min, increased at a rate of 15 °C min−1 to 100 °C, and then held for 1 min. 2.9. Quantitative analysis of prometryn and other triazines Ametryn, prometryn, simetryn, atrazine, simazine, terbutylhylazine, propazine, prometon, atraton, terbumeton, and metribuzin in samples were extracted and analyzed according to the same procedure as prometryn metabolites, except isocratic elution using 60% aqueous

Please cite this article as: Liu, J., et al., Novel hydrolytic de-methylthiolation of the s-triazine herbicide prometryn by Leucobacter sp. JW-1, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.006

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incubation for 2 h at 37 °C, the samples were collected for determination of prometryn. Each treatment was carried out in triplicate, and the controls without the added crude enzymes was performed under the same conditions. An aliquot of 2.7 mL PBS containing 50 mg L−1 of prometryn was warmed to the temperatures at 20, 25, 30, 37, 42, 47, 52, 57, and 62 °C. Intracellular crude enzymes (0.3 mL) was added to the PBS, and incubated for 30 min. Prometryn concentration was measured using the method described above. 2.11. Statistical analysis The degradation rate was calculated as Fig. 1. Phylogenetic tree of strain JW-1 constructed from the 16S rDNA sequences according to the Neighbor-Joining method. Bootstrap values obtained with 1000 repetitions are indicated at branch points.

Degradation ð%Þ ¼

acetonitrile (v/v). After direct filtration of the samples through a 0.22μm membrane, cyanuric acid was eluted with 5% aqueous methanol containing 5 mmol L−1 of K2HPO4 and 2 mmol L−1 of KH2PO4 buffer solution at a flow rate of 0.8 mL min−1 and detected at 213 nm (Stamper et al., 2005). The average recoveries of prometryn fortified MSM at 0.05, 1, 50, and 400 mg L−1 varied from 95.3% to 105.1% with a relative standard deviation (RSD) ≤4.0% (Table S1). These data indicated that the method was satisfactory for the analysis of prometryn and other triazines. Degradation percentages of other analytes were quantitatively relative to their respective controls.

residual amount of control treatment‐residual amount of treatment  100% residual amount of control treatment

Assuming the degradation follows the first-order of kinetics, the degradation half-life (T1/2) was calculated as T1/2 = (ln2) / k, where k is the first-order reaction rate constant (Cycon et al., 2014). The means values and standard errors of the data were calculated using Microsoft Excel software (Microsoft, USA). The data were statistically analyzed with SPSS 16.0 software package (SPSS Inc., USA). Statistically significant differences between the different treatments were analyzed using one-way analysis of variance at 5% significance level. 3. Results and discussion

2.10. Degradation of prometryn by crude enzymes 3.1. Isolation, identification and characterization of strain JW-1 Extracellular crude enzymes were extracted from the JW-1 culture medium by removing the cell pellets after centrifugation. The intracellular crude enzymes were obtained from the microbial cells using the following steps. After 40 h of cultivation, JW-1 culture was centrifuged at 3380 ×g and 4 °C for 10 min. Ammonium sulfate was added into supernatant to saturation, and the extracellular crude enzyme precipitate was salted out overnight at 4 °C. The precipitate was added in phosphate buffer solution (PBS, 0.05 mol L−1 NaH2PO4–Na2HPO4, pH 7.0), washed twice with PBS, and resuspended in the PBS solution. The cells were then sonicated at 4 °C (150 W) for 8 min by an ultrasonic cell disrupter (Scientz JY92-IIN, Scientz Biotechnology Co., Ltd. Ningbo city, China). The suspension was centrifuged at 9390 ×g and 4 °C for 10 min to obtain intracellular crude enzymes and stored at −20 °C. Extracellular and intracellular crude enzymes were warmed at 37 °C and then added into PBS containing 50 mg L− 1 of prometryn. After

Single strain colonies with distinct clearing zones were collected on MSP agar medium containing prometryn as the prometryn-selective plates for further plate cultivation until single colonies of uniform morphology. The strain JW-1 capable of utilizing prometryn was isolated from prometryn-contaminated activated sludge. JW-1 was a Gram-positive, rod-shaped, amphitrichate bacterium with a size of 1.57– 1.97 μm × 0.48–0.58 μm (Fig. S1). JW-1 colonies on a plain agar plate manifested a light yellow color in a smooth, opaque and concave shape. The strain JW-1 was positive in oxidase, catalase, nitrate reduction, and pyruvate decarboxylase, but negative in urease and arginine dihydrolase. JW-1 cannot hydrolyze starch and casein. It can survive at glucose inosine, L-(+)-lactic acid, Tween 40, and 8% of NaCl, but cannot utilize levulose, sucrose, and gelatin (Table S2). The characteristics of JW-1 matched those of the genus of Leucobacter. The partial length of

Fig. 2. Degradation kinetics of prometryn at varying pH values of MSM (a), temperature (b), and initial prometryn concentration (c) inoculated with the strain JW-1.

Please cite this article as: Liu, J., et al., Novel hydrolytic de-methylthiolation of the s-triazine herbicide prometryn by Leucobacter sp. JW-1, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.006

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Table 1 Pseudo-first-order kinetic degradation of prometryn by strain JW-1 at different pH, temperature, and initial concentration. pH

Temperature (°C)

Prometryn concentration (mg L−1)

Inoculation volume (mL)a

Reaction rate constant (h−1)

R2

T1/2 (h)b

5 6 7 8 9 7 7 7 7 7 7 7 7 7

30 30 30 30 30 20 25 30 37 42 47 37 37 37

50 50 50 50 50 50 50 50 50 50 50 50 200 400

0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3

0.11 0.14 0.25 0.26 0.22 0.06 0.15 0.24 0.50 0.54 0.37 0.66 0.17 0.12

0.97 0.98 0.97 0.98 0.98 0.99 0.99 0.99 0.97 0.97 0.79 0.97 0.99 0.93

6.44 4.97 2.74 2.63 3.11 11.6 4.63 2.89 1.39 1.28 1.91 1.05 4.02 6.01

a b

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.02 0.04 0.02 0.02 0.02

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.19 a 0.36 b 0.06 c 0.06 c 0.08 c 0.00 a 0.31 b 0.12 c 0.03 e 0.05 e 0.18 d 0.03 a 0.35 b 0.83 c

Added to 20 mL of medium. Different letters indicate significant differences by one-way AVONA on the same column (P b 0.05).

16S rDNA gene sequence of JW-1 is 1312 bp. The 16S rDNA gene sequence of JW-1 has been deposited in the GeneBank under the accession number of KT439069. The isolate JW-1 was closely related to Leucobacter sp. (Fig. 1). Upon alignment with other 16S rDNA gene sequences in the GenBank, the results demonstrated a high degree of similarity (99%) to other members of Leucobacter sp. A phylogenetic tree including the isolate JW-1 was presented in Fig. 1, and the strain JW-1 is denoted here as Leucobacter sp. JW-1, which has been deposited in China General Microbiological Culture Collection Center (CGMCC), Beijing, China under the CGMCC number of 11754.

3.2. Effect of pH on biodegradation of prometryn The biodegradation of prometryn at 50 mg L−1 in MSM of pH 5.0, 6.0, 7.0, 8.0, and 9.0 is shown in Fig. 2a. In the controls without JW-1, the removal percentage of prometryn was b0.72% during the whole experimental period. T1/2 values were 6.4, 5.0, 2.7, 2.6, and 3.1 h at pH 5.0, 6.0, 7.0, 8.0, and 9.0, respectively (Table 1). The T1/2 values at pH 7.0 and 9.0 were significantly shorter (p ≤ 0.05) than those at pH 5.0 and 6.0. These results indicated that the degradation of prometryn by JW-1 favored the neutral or weakly alkaline solution, whereas the conditions

Table 2 Degradation of 11 triazine compounds by strain JW-1. Compound

Structure

Degradation percentage (%) 2d

4d

Ametryn

100.0 ± 0.0

100.0 ± 0.0

Simetryn

99.4 ± 0.1

100.0 ± 0.0

Propazine

40.9 ± 2.0

46.2 ± 4.0

Atrazine

42.9 ± 1.5

52.0 ± 6.9

Simazine

28.2 ± 2.3

29.3 ± 3.1

Terbutylhylazine

12.4 ± 1.9

15.0 ± 0.8

Prometon

10.0 ± 0.4

14.5 ± 1.2

Atraton

13.1 ± 1.5

13.1 ± 3.3

Terbumeton

1.8 ± 4.3

3.7 ± 2.2

Metribuzin

0.4 ± 2.2

−1.6 ± 1.4

Cyanuric acid

3.4 ± 0.5

1.5 ± 0.1

Please cite this article as: Liu, J., et al., Novel hydrolytic de-methylthiolation of the s-triazine herbicide prometryn by Leucobacter sp. JW-1, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.006

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of stronger acidity or alkaline inhibited the degradation. Similar to our results, Swissa et al. (2014) reported that the optimum pH for degradation of atrazine by Raoultella planticola was at 7.0. Neutral pH was also the most favorable for atrazine degradation by bacterial consortium (Dehghani et al., 2013). However, Wang et al. (2011) found that the optimal pH range for atrazine degradation by Arthrobacter sp. HB-5 was within 6.0 and 9.0. A broad optimum pH range for atrazine degradation by Arthrobacter sp. DAT1 was within 5.0 and 10.0 (Wang and Xie, 2012). 3.3. Effect of temperature on biodegradation of prometryn Degradation percentages of prometryn at 50 mg L− 1 in MSM at pH 7.0 by JW-1 varied largely between 20 and 47 °C. Negligible loss of prometryn was observed in the controls (b 1%). T1/2 values of prometryn were 12, 4.6, 2.9, 1.4, 1.3, and 1.9 h at 20, 25, 30, 37, 42, and 47 °C, respectively (Table 1). More than 90% of prometryn was

degraded within the temperatures between 30 and 47 °C after 10 h of incubation (Fig. 2b). This result indicated that the degradation efficiency of prometryn by the strain JW-1 increased with temperature (within the range of 20 and 42 °C) and then decreased when temperature reached 47 °C (Table 1). The ANOVA analysis showed that T1/2 of prometryn at 37 and 42 °C were significantly shorter than those at 20, 25, and 47 °C, and that the optimum temperature range was within 37 and 42 °C for prometryn degradation in pure culture (Table 1). It is interesting to note that JW-1 could not tolerate 47 °C (data not shown), but could still efficiently degrade prometryn. It is noteworthy that degradation of prometryn was very quick, and the cell growth could not be measured during this period of time. In general, the doubling time of JW-1 cells was approximately 12 h. Such efficient degradation at relatively high temperature promoted us to further investigate the degradation of prometryn by JW-1 crude enzymes.

Fig. 3. HPLC and GC chromatograms of standard compounds, control, and strain JW-1culture extracts after incubation 4 h and 60 h. (a), Prometryn and 2-hydroxypropazine, (b), cyanuric acid, and (c), methanthiol and dimethyl disulfide.

Please cite this article as: Liu, J., et al., Novel hydrolytic de-methylthiolation of the s-triazine herbicide prometryn by Leucobacter sp. JW-1, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.006

J. Liu et al. / Science of the Total Environment xxx (2016) xxx–xxx

The optimal temperature range for atrazine degradation by Arthrobacter sp. DAT1 was at 25–35 °C, whereas degradation was inhibited at temperature N 35 °C (Wang and Xie, 2012). Wang et al. (2011) also reported that the optimal temperature range for atrazine degradation by Arthrobacter sp. HB-5 was within 20–40 °C. The higher temperature could reduce the activity of enzymes related to the degradation of atrazine. Kang et al. (2012) found that the temperature greatly reduced the activity of nicosulfuron degrading enzyme of Bacillus subtilis YB1. In the present study, the number of JW-1 cells did not increase significantly within 20–47 °C due to both the short incubation time and slow growth. However, the degradation efficiency of prometryn increased with increasing culture temperature. This suggests that temperature might be related to the activity of degrading enzyme, which is addressed below. 3.4. Effect of prometryn concentration on its biodegradation The effect of prometryn concentration on its degradation in MSM was examined at pH 7.0 and 37 °C (Fig. 2c). The prometryn concentration remained unchanged in the absence of JW-1 cells during the experimental period. Compared to the controls without the inoculation, degradation of prometryn was enhanced obviously in the presence of JW-1 cells. After incubation for 8 h, the degradation rates of prometryn were approximately 6.2, 18, and 21 mg L−1 h−1 at the concentrations of 50, 200, and 400 mg L−1, respectively. These degradation rates were proportional to prometryn between 50 and 400 mg L−1 with R2 values of 0.950–0.997, suggesting that the degradation was subjected to pseudo-first-order kinetics. The T1/2 values of prometryn estimated from the pseudo-first-order kinetics were 1.1, 4.0, and 6.0 h at the concentrations of 50, 200 and 400 mg L−1, respectively (Table 1). Zhao et al. (2015) reported that the degradation T1/2 values of thifensulfuron-methyl by Ochrobactrum sp. ZWS16 were 4.0, 4.4, 4.1, 5.3, and 5.6 d at the concentrations of 5.0, 10, 100, 200, and 400 mg L−1. The bacteria consortium (Klebsiella sp. A1 and Comamonas sp. A2) showed high atrazine-degradation efficiency with approximately 83.3% of 5 g L−1 atrazine being degraded after 24 h (Yang et al., 2010). The degradation rate of atrazine by Pseudomonas sp. ADP was 5.0 mg L− 1 h− 1 at the concentration of 100 mg L− 1 within 24 h (Mandelbaum et al., 1995). The degradation rate of prometryn by JW1 was 14.9 mg L−1 h− 1 at the concentration of 400 mg L− 1 within 24 h, which was approximately 3 times that of atrazine at 100 mg L−1 by the strain ADP.

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and Nocardioides sp. DN36 (Satsuma, 2010) can also degrade various types of s-triazine herbicides. 3.6. Identification and biodegradation of prometryn metabolites During the degradation of prometryn by the strain JW-1, five metabolites were identified. After incubation for 4 h and 60 h, four metabolites derived from prometryn (100 mg L−1) were found in HPLC and GC analysis. Meanwhile, at 8, 48 and 96 h, the three metabolites from prometryn (100 mg L−1) were identified with the precursor ions m/z 212.14954 (ESI+, M + 1), 171.08692 (ESI+, M + 1) and 128.01038 (ESI−, M − 1) (Fig. S2a–c) by LC-HRMS. Compared to the reference standards and the exact mass of metabolites, these metabolites were identified to be 2-hydroxypropazine, cyanuric acid, methanthiol, dimethyl disulfide (Fig. 3a–c) and N-isopropylammelide (Fig. S2b). The degradation kinetics of prometryn was monitored, along with of the quantification of metabolites 2-hydroxypropazine, methanthiol, dimethyl disulfide, and cyanuric acid (Fig. 4). Dimethyl disulfide is the auto-oxidation product of methanthiol. The prometryn concentration remained unchanged in the control during the experimental period. The results showed that 0.414 mmol L−1 of prometryn (100 mg L−1) was almost completely degraded by the strain JW-1 within 8 h to 2hydroxypropazine (0.204 mmol L−1), methanthiol (0.355 mmol L−1), dimethyl disulfide (0.017 mmol L−1) and cyanuric acid (0.188 mmol L−1). The concentrations of 2-hydroxypropazine and methanthiol reached the maximum after 8 h of incubation, and methanthiol and dimethyl disulfide concentration was 0.390 mmol L−1, which was equivalent to 94.2% of the initial sulfur content in the prometryn (0.414 mmol L−1). Methanthiol is a gas at an ambient temperature, which is difficult to be accurately quantified. By 96 h, 98.1% of the prometryn initially added at 0.414 mmol L−1 was converted to cyanuric acid which appeared to be quite stable until N 120 h. N-isopropylammelide was not quantitatively measured due to lack of the authentic standard. A degradation pathway of prometryn was proposed in Fig. 5, in which prometryn undergoes de-methylthio hydrolysis to form 2-hydroxypropazine and methanthiol, and the former product precedes the subsequent deamination to form cyanuric acid. Nocardioides sp. C190 can degrade atrazine via hydrolytic dechlorination to yield hydroxyatrazine, and via deamination to yield Nethylammelide, but the strain C190 could not metabolize Nethylammelide to cyanuric acid (Topp et al., 2000). Being similar to

3.5. Biodegradation of other triazine herbicides by strain JW-1 Biodegradation results of 11 triazines herbicides by JW-1 are listed in Table 2. The determination of degradation spectrum of strain JW-1 revealed that at the initial concentration of 50 mg L−1, the isolate JW-1 could degrade 100% of ametryn, 100% of simetryn, 52% of atrazine, 46% of propazine, 29% of simazine, 15% of terbutylhylazine, 14.5% of prometon, 13.1% of atraton after incubation for 4 d. JW-1 could quickly degrade ametryn and simetryn, and also could degrade atrazine, propazine, simazine, terbutylhylazine, prometon and atraton at a slightly slower rate, but could not degrade terbumeton, metribuzin and cyanuric acid. This indicated that such substrate specificity may relate to the structures of triazine herbicides. Therefore, the enzyme reaction is presumably involved in the initial stage of degradation of triazines. Triazine herbicides, which could be biodegraded by JW-1, contain methylthio group (\\SCH3), chloro group (\\Cl) or methoxy group (\\OCH3). The degradation rate for the compounds containing\\SCH3 functional group was faster than those containing \\Cl or \\OCH3 group. These results are consistent with the experimental observation that Nocardioides sp. C190 could degrade nine s-triazine herbicides containing \\Cl and \\OCH3 functional groups, and the degradation rate was faster for methylthio-substituted herbicides than chlorinated analogs (Topp et al., 2000). A. nicotinovorans HIM (Aislabie et al., 2005)

Fig. 4. Degradation of prometryn and its metabolites (2-hydroxypropazine, methanthiol, dimethyl disulfide, and cyanuric acid) by the strain JW-1 in MSM.

Please cite this article as: Liu, J., et al., Novel hydrolytic de-methylthiolation of the s-triazine herbicide prometryn by Leucobacter sp. JW-1, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.006

8

J. Liu et al. / Science of the Total Environment xxx (2016) xxx–xxx

Fig. 5. Proposed degradation pathway of prometryn by the strain JW-1.

JW-1, Arthrobacter sp. HB-5 metabolized atrazine to yield cyanuric acid via dechlorination and deamination (Wang et al., 2011). A. nicotinovorans HIM could degrade atrazine to cyanuric acid, but was unable to mineralize 14C-ring-labelled atrazine (Aislabie et al., 2005). Atrazine was also metabolized to cyanuric acid after dechlorination and deamination reaction by Nocardioides sp. DN36 (Satsuma, 2010) and A. aurescens TC1 (Strong et al., 2002). Similar to the strain JW-1, all above strains degraded s-traizine herbicides (atrazine or prometryn) to cyanuric acid and none of the strains can cleave the s-triazine ring. However, B. subtilis HB-6 could metabolize atrazine to cyanuric acid, cleave the s-triazine ring and mineralize atrazine (Wang et al., 2014).

spectrum renders the strain JW-1 as a promising candidate for biodegradation of triazine herbicides in the environment. JW-1 was tentatively identified as Leucobacter sp. The catabolic pathway of prometryn was proposed to be a multistep process involving de-methylthio hydrolysis, and deamination. Overall, the strain JW-1 exhibited a great potential to biologically remove prometryn contamination in water. However, many physicochemical and biological factors that might influence the survival and activity of the strain JW-1 in soils need to be further investigated in order to be effectively used in the bioremediation of triazine residues. The enzyme responsible for de-methylthiolation of the s-triazine herbicides is quite thermostable and unique in biocatalysis.

3.7. Prometryn-degrading enzyme

Acknowledgments

Intracellular crude enzymes could degrade 99.9% of prometryn (50 mg L−1) in 2 h, whereas a negligible amount of prometryn was decomposed in the presence of extracellular crude enzymes, and the amount was very close to that of enzyme-free controls. These results indicated that the intracellular enzymes involved the biodegradation of prometryn. The percentage of prometryn degradation by the intracellular crude enzymes increased with temperature, and reached the maximum at 42 °C, and then decreased with increasing temperature (Fig. 6). High temperature tolerance and de-methylthiolation of the s-triazine herbicides are two interesting properties of the enzyme, which warrants its isolation and characterization in the future experiments.

This work was supported in part by the National High Technology Research and Development Program of China (2013AA102804B), the National Natural Science Foundation of China (No. 31572033, 31601657), the Natural Science Foundation of Anhui Province of China (1408085MKL36), and Foundation for the Excellent Youth Scholars of Anhui Province (2013SQRL016ZD). We thank Dr. Hui Li in the Michigan State University for helpful discussions. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2016.11.006.

4. Conclusion The strain JW-1 isolated from activated sludge could efficiently degrade prometryn, and manifest a broad spectrum of degradation of s-triazine herbicides ametryn, simetryn, atrazine, propazine, simazine, terbutylhylazine, prometon and atraton without the needs to supplement other carbon and energy sources. This broad degradation

Fig. 6. Effect of temperature on enzymatic degradation of prometryn.

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Please cite this article as: Liu, J., et al., Novel hydrolytic de-methylthiolation of the s-triazine herbicide prometryn by Leucobacter sp. JW-1, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.006