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Journal of Hazardous Materials 297 (2015) 17–24

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Rapid biodegradation of organophosphorus by Stenotrophomonas sp. G1

Shuyan Deng a, Yao Chen a, Daosheng Wang b, Taozhong Shi a, Xiangwei Wu a, Xin Ma a, Xiangqiong Li a, Rimao Hua a,∗, Xinyun Tang b, Qing X. Li c

a Key Laboratory of Agri-food Safety of Anhui Province, Lab of Quality & Safety and Risk Assessment for Agro-products on Storage and Preservation (Hefei), Ministry of Agriculture, School of Resource and Environment, Anhui Agricultural University, Hefei 230036, China b School of Life Science, Anhui Agricultural University, Hefei 230036, China c Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East–West Road, Honolulu, HI 957822, USA

highlights graphical abstract

• Stenotrophomonas sp. G1 was iso- lated from contaminated sludge. • Strain G1 is closest to Stenotrophomonas acidaminiphila. • Strain G1 can efficiently degrade 8 organophosphorus pesticides (OPs). • Intracellular methyl hydrolase is responsible for the OP degradation. • Three factors were orthogonally opti- mized for degradation of methyl parathion.

article info abstract

Article history: Organophosphorus have been widely used, which are highly poisonous and cause serious Received 19 September 2014 concerns over food safety and environmental pollution. A bacterial strain being capable of degrading O,O- Received in revised form 14 April 2015 dialkyl phosphorothioate and O,O-dialkyl insecticides, designated as G1, was isolated from Accepted 17 April 2015 sludge collected at the drain outlet of a chlorpyrifos manufacture plant. Physiological and biochemical Available online 20 April 2015 characteristics and 16S rDNA gene sequence analysis suggested that strain G1 belongs to the genus Stenotrophomonas. At an initial concentration of 50 mg/L, strain G1 degraded 100% of methyl parathion, Keywords: methyl , , and , 95% of parathion, 63% of chlorpyrifos, 38% of , and 34% Biodegradation of triazophos in 24 h. Orthogonal experiments showed that the optimum conditions were an inoculum Bioremediation ◦ Chlorpyrifos volume of 20% (v/v), a substrate concentration of 50 mg/L, and an incubation temperature in 40 C. p- Methyl parathion hydrolase Nitrophenol was detected as the metabolite of methyl parathion, for which intracellular methyl parathion Organophosphorus pesticides hydrolase was responsible. Strain G1 can efficiently degrade eight organophosphorus pesticides (OPs) and Stenotrophomonas sp. G1 is a very excellent candidate for applications in OP pollution remediation. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ Corresponding author. Tel.: +86 551 65786320; fax: +86 551 65786296. E-mail address: [email protected] (R. Hua).

http://dx.doi.org/10.1016/j.jhazmat.2015.04.052 0304-3894/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 18 S. Deng et al. / Journal of Hazardous Materials 297 (2015) 17–24

1. Introduction 2. Materials and methods

2.1. Sludge sample and cultivation media Some organophosphorus pesticides (OPs) are highly toxic and are still widely used for insect control. Wide use of OPs A sludge sample was collected at the outlet of wastewater causes serious concerns over food safety and environmental drainage system in a chlorpyrifos manufacture plant, Nantong pollution. In the recent years, various bacterial strains were iso- City, Jiangsu Province, China. Mineral salt medium (MSM), beef lated and reported for biodegradation of OPs. Serratia sp. SPL-2 extract-peptone medium, and chlorpyrifos-selective medium were can degrade [1]. Pseudomonas aeruginosa Is-6 can prepared for isolation and screening of chlorpyrifos-degrading bac- degrade , , methyl parathion, , teria [24]. and [2]. Biodegradation of chlorpyrifos by soil bacterial communities comprising seven different isolates of Pseudomonas, 2.2. Reagents and pesticides Agrobacterium and Bacillus has been investigated [3]. Bacillus cereus, Bacillus subtilis, Brucella melitensis, Klebsiella species, Pseudomonas N,O-Bis (trimethylsilyl) trifluoroacetamide was purchased from aeroginosa, Pseudomonas fluorescence, and Serratia marcescens are Aladdin Reagents Co., Ltd., Shanghai, China. The rTaq DNA capable of degrading 46–72% of chlorpyrifos as a sole carbon polymerase and DNA marker were purchased from Takara Biotech- source in an aqueous medium after an incubation of 20 days [4]. nology Co., Ltd., Dalian, China. Chlorpyrifos (48%) emulsifiable Many factors can affect bacterial degradation of OPs, which include concentrate was purchased from Jiangsu Baoling Chemical Co., Ltd., -holding capacity of soil, temperature, pH, and even aging Nantong, China. Chlorpyrifos (95%) was purchased from Shandong of pesticides in soils prior to inoculation. In a study of Enter- Kunfeng Biochemical Co., Ltd., Binzhou, China. Parathion (99.4%), obacter sp., aging of chlorpyrifos resulted in approximately 10% p-nitrophenol (99%), methyl parathion (98.9%), acephate (99%), of it being persistent in soil [5]. The diazinon-degrading Serratia methamidophos (99%), isocarbophos (99%), malathion (97.4%), and marcescens can degrade chlorpyrifos, , and parathion profenofos (96.9%) were purchased from Beijing Helishun Technol- [6]. Stenotrophomonas sp. SMSP-1 can completely degrade methyl ogy Co., Ltd., Beijing, China. Phoxim (98%), methyl paraoxon (98%), parathion, fenitrothion and ethyl parathion, and degrade 37.8% of triazophos (98%), chlorpyrifos (99%), and diazinon (98%) were pur- and 59.7% of phoxim, but cannot degrade chlorpyrifos chased from Dr. Ehrenstorfer GmbH, Germany. All other reagents at 20 mg/L [7]. Stenotrophomonas sp. PF32 can efficiently degrade were of analytical grade. methyl parathion as well as phoxim, fenthion, sumithion, tri- azophos, and chlorpyrifos. Strain PF32 degraded about 99% of 2.3. Screening, isolation, and purification of 100 mg/L methyl parathion, fenthion in 24 h, 99% of 100 mg/L fen- chlorpyrifos-degrading bacteria itrothion at 18 h, 99% of 100 mg/L phoxim in 30 h, 97% of 100 mg/L triazophos in 42 h, and 97% of 100 mg/L chlorpyrifos in 48 h [8]. The sludge sample in 5 mL was transferred into 95 mL of liquid Stenotrophomonas maltophilia MHF ENV20 can degrade chlorpyri- MSM containing 100 mg/L chlorpyrifos emulsifiable concentrate, fos with a half-life of 96 h and its metabolite trichlorophenol as followed by incubation at 28 ◦C on a rotary shaker at 120 r/min. well as diethyl salt [9]. Stenotrophomonas sp. YC-1 After cultivation for 5 days, an aliquot of culture solution was can also utilize chlorpyrifos as a sole carbon source, but cannot fur- sampled, diluted and spread on the chlorpyrifos-selective medium ther degrade its metabolite trichlorophenol. YC-1 can completely agar plates containing 500 mg/L of chlorpyrifos. After incubation degrade 100 mg/L of chlorpyrifos in 24 h [10]. Stenotrophomonas at 28 ◦C, single colonies with a clearing zone were picked asepti- sp. DSP-4 can completely degrade 100 mg/L chlorpyrifos in 24 h cally for further plate cultivation until single colonies of uniform [11]. morphology were obtained. The substrate specificity varies among bacterial species. Methyl parathion hydrolase is an important organophosphorus 2.4. Degradation of chlorpyrifos and other OPs hydrolase that exists in many bacterial species. To date, the hydrolase has been found in methyl parathion-degrading Pseu- The isolates showing a clearing zone were transferred into liq- daminobacter salicylatoxidans mp-1, Achromobacter xylosoxidans uid beef extract-peptone medium and grown to a stationary phase. mp-2, Ochrobactrum tritici mp-3, B. melitensis mp-7, Plesiomonas sp. Cells were harvested by centrifugation of 20-mL culture solution for M6, Sphingopyxis sp. DLP-2, Pseudomonas stutzeri HS-D36 [12–15], 5 min, washed twice with normal saline, and then re-suspended ␮ chlorpyrifos-degrading Cupriavidus sp. DT-1 [16] and phoxim- in 20 mL of MSM. An aliquot of 50 L of chlorpyrifos solution degrading Delftia sp. XSP-1 [17]. Different bacteria species have (5 mg/mL in petroleum ether) was added to the bottom of test tube. different specificity of substrate utilization. Methyl parathion is After complete volatilization of petroleum ether, 4.5 mL of MSM and 0.5 mL of bacterial cell suspension were added, followed by the optimum substrate for methyl parathion hydrolase purified ◦ from Pseudomonas sp. WBC-3 [18], which can degrade chlorpyri- incubation at 37 C. Samples were taken for determination of chlor- fos, ethyl parathion, and sumithion [19,20]. Burkholderia sp. FDS-1 pyrifos degradation. Strain G1 was selected for further degradation can degrade methyl parathion, parathion and sumithion, but not studies. chlorpyrifos, methamidophos, phoxim and triazophos [21]. Methyl After growth in liquid beef extract-peptone medium for 24 h, parathion hydrolase is an intracellular in some strains G1 cells were harvested by centrifugation, washed twice with nor- [21,22] and cell membrane bound in the others [23]. The amino mal saline, re-suspended in MSM, and finally adjusted to OD 1.0 at acid sequence of methyl parathion hydrolase from G1 shared 100% 600 nm determined by UV-2001 (Shimadzu Corp., Kyoto, Japan). homology with that of WBC-3. An aliquot of 0.05 mL of an OP solution was added into a test In the present study, Stenotrophomonas sp. G1 was iso- tube. After complete volatilization of the , 9 mL of MSM lated and characterized for efficient degradation of OPs. Factors and 1 mL of cell suspension were added into the test tube with OP that affect degradation were also studied, which included sub- at a final concentration of 50 mg/L. Each sample was prepared in triplicate. The control group was not inoculated with bacterial cells. strate specificity, biodegradation conditions, and localization of ◦ catabolic . Strain G1 may be significant for bioremediation The mixture was incubated at 37 C on a rotary shaker at 120 r/min. applications. Samples were taken at specific time intervals for further analysis. S. Deng et al. / Journal of Hazardous Materials 297 (2015) 17–24 19

Table 1 Orthogonal experiment parameters and first-order kinetic data of methyl parathion degradation by strain G1.

Number of test Factor Rate constant (k)a Half-life (h)a

Initial inoculum (v/v,%) Initial substrate concentration (mg/L) Incubation temperature (◦C)

1 10 50 20 0.153 4.5 2 10 100 30 0.103 6.7 3 10 200 40 0.095 7.3 4 20 50 30 0.377 1.8 5 20 100 40 0.220 3.2 6 20 200 20 0.056 12.4 7 30 50 40 0.415 1.7 8 30 100 20 0.117 5.9 9 30 200 30 0.065 10.7

a Data represent average values of triplicate per treatment.

2.5. Orthogonal experimental design for methyl parathion in triplicate. Cellular location of methyl parathion hydrolase was degradation estimated with enzymatic assays [25]. Activity of methyl parathion hydrolase was determined as the number of moles of p-nitrophenol The preliminary degradation screening showed very fast degra- generated in unit time. dation of methyl parathion by strain G1. Multiple culturing parameters were optimized for methyl parathion degradation. An 2.8. Identification and characterization of strain G1 L9 (33) orthogonal array table (Table 1) was designed by SPPS19.0 software (IBM SPSS, New York, USA) to optimize the parameters G1 was preliminarily characterized via morphological obser- of initial inoculum volume, initial substrate concentration, and vation, Gram staining, physiological and biochemical tests [26] incubation temperature. In a 10-mL degradation system, methyl and 16S rDNA sequence alignment. Bacterial genomic DNA was parathion solution, MSM, and cell suspension were added into a extracted according to the SDS-proteinase K method, and 16S test tube according to the orthogonal array table. The mixture was rDNA gene fragment was amplified through polymerase chain reac- incubated on a rotary shaker at 120 r/min, and samples were taken tion (PCR) using universal primers for bacteria, upstream primer at specific time intervals (0, 0.5, 2, 6, 10, and 24 h) for analysis. Each fD1: 5-AGAGTTTGATCCTGGCTCAG-3, and downstream primer group was in triplicate. The control was not inoculated with bac- rD1 5-ACGGCTACCTTGTTACGACTT-3 [27]. PCR conditions were as terial cells, data of the orthogonal experiment were analyzed with follows: 94 ◦C denaturation for 10 min, followed by 30 cycles of SPSS19.0 software. 96 ◦C for 30 s, 56 ◦C for 30 s, and 72 ◦C for 45 s, and a final step of 72 ◦C for 10 min. 2.6. Determination of metabolites of methyl parathion The PCR product was sequenced by Shanghai San- gon BioTech Co., Ltd. The sequence was aligned with that An aliquot of 2 mL of bacterial suspension was inoculated into in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). All 16S 8 mL of MSM with methyl parathion at 50 mg/L. The mixture was rDNA sequences of type strains showing 97% or higher incubated at 37 ◦C on a rotary shaker at 120 r/min. Samples were homology with G1 were downloaded from the database taken at specific time intervals for analysis. Each group was pre- (http://rdp.cme.msu.edu/seqmatch/seqmatch intro.jsp). All pared in triplicate. sequences were aligned in Clustal X [28]. Phylogenetic trees were constructed with Mega 4.0 [29]. The 16S rDNA gene sequence 2.7. Localization of methyl parathion hydrolase was deposited in the GenBank under accession number of JN688160. Stenotrophomonas sp. G1 (=CCTCC M 2,010,231) was After 24 h of cultivation, G1 cells were centrifuged at 9390 × g deposited in China Center for Type Culture Collection in Wuhan and 4 ◦C for 10 min. The supernatant was passed through a 0.22-␮m University, China. membrane filter to obtain crude extracellular enzyme and stored at 4 ◦C. The cell precipitate was washed twice with normal saline and 2.9. Sample extraction and analysis re-suspended in Tris–Cl buffer solution (50 mmol/L, pH 8.0) with an equal volume of the supernatant. One half of the suspension Methamidophos, acephate, isocarbophos, and malathion sam- was stored at 4 ◦C as bacterial cell suspension, and the other half ples (10 mL) were extracted twice with 2 × 20 mL of acetonitrile was used for periplasmic extraction by osmotic shock. The hyper- and the two extracts were combined and concentrated with a gen- tonic solution was 20% sucrose solution containing 1 mmol/L EDTA tle stream of pure nitrogen to near dryness. The residues were and prepared with 50 mmol/L Tris buffer; and the hypotonic solu- then dissolved in ethyl acetate to a constant volume. Methami- tion was 5 mmol/L MgCl2 solution chilled at 4 ◦C. The periplasmic dophos, acephate, isocarbophos, and malathion were detected with extract was passed through a 0.22-␮m membrane filter to obtain a GC2010 gas chromatograph-flame photometric detector with crude periplasmic enzyme and stored at 4 ◦C. a filter (Shimadzu Corp., Kyoto, Japan). The chro- After periplasmic extraction, bacterial cells were re-suspended matograph column was CP-19 (30 m × 0.25 ␮m × 0.25 ␮m). The in Tris–Cl buffer solution and sonicated (4 ◦C, 400 W) for 8 min. The temperatures of injection port and detector were 240 ◦C and 300 ◦C, suspension was then centrifuged at 9390 × g and 4 ◦C for 10 min respectively. The temperature of column was programmed at an and the supernatant was passed through a 0.22-␮m filter to obtain initial temperature of 100 ◦C for 1 min, raised to 200 ◦C at a rate of crude intracellular enzyme and stored at 4 ◦C. 20 ◦C/min and held for 1 min, and then raised to 260 ◦C at a rate ◦ Bacterial cell, crude extracellular enzyme, crude intracellular of 50 C/min and held for 1 min. In the detector, the H2 flow was enzyme and crude periplasmic enzyme solutions were warmed 85.0 mL/min, and the air flow was 90.0 mL/min. The chromatograph ◦ to 37 C and then added into Tris–Cl buffer solution with methyl column flow rate of N2 was 30 mL/min. parathion at 50 mg/L. After incubation for 10 min at 37 ◦C, samples Methyl parathion, parathion, methyl paraoxon, profenofos, were taken for detection of p-nitrophenol. Each group was prepared diazinon, triazophos, phoxim, and p-nitrophenol samples were 20 S. Deng et al. / Journal of Hazardous Materials 297 (2015) 17–24 diluted with methanol and passed through a 0.22-␮m filter. They Table 2 were analyzed on an Agilent 1200 high performance liquid chro- Degradation of 12 organophosphorus pesticides by strain G1. a a matograph (Agilent, Santa Clara, California, USA). The column was Pesticides Structures Degradation (%) T1/2(h) XDB-C18 (5 ␮m, 4.6 × 250 mm). The mobile phase flow rate was 6h 24h 48h 1.0 mL/min. Methyl parathion, parathion, methyl paraoxon, and p-nitrophenol were eluted with 80% aqueous methanol (v/v) and detected at 280 nm. Phoxim and profenofos were also eluted with 80% aqueous methanol, but detected at 282 nm and 210 nm, respec- Chlorpyrifos 23b 63 16.7 tively. Methyl paraoxon were eluted with 40% aqueous methanol and detected at 280 nm. Diazinon was eluted with 70% aqueous methanol and detected at 246 nm. Triazophos was eluted with 75% aqueous methanol and detected at 227 nm. Chlorpyrifos samples were shaked with 5 mL acetonitrile fol- lowed by filtration through a 0.22-␮m filter. Chlorpyrifos was analyzed on a e2695 high performance liquid chromato- Diazinon 81 100 6.8 graph (Waters, USA) at 300 nm. The column was XDB-C18 (5 ␮m, 4.6 × 250 mm). The mobile phase was a mixture of acetonitrile and buffer (85% phosphoric acid/ water, 1:1000, v/v) (80:20, v/v). The flow rate was 1.0 mL/min. The retention times for chlorpyrifos and 3,5,6-trichloro-2-pyridinol (TCP) were 9.5 and 3.4 min, respec- tively.

2.10. Data calculation Methyl paraoxon 92 100 1.6

Degradation (%)

(Residual amount in blank control − residual amount in sample ) = × 100 (Residual amount in blank control)

−kt According to the first-order kinetic equation Ct = C0 × e , the degradation half-life (T ) was obtained as T = (ln2)/k. 1/2 1/2 Parathion 60 95 100 5.8

3. Results and discussion

3.1. Biodegradation of chlorpyrifos and other OPs by G1

Chlorpyrifos emulsifiable concentrate was used to make the chlorpyrifos-selective plates for efficiently observing the distinct clearing zones. The result showed that it is a rapid and effective Methyl parathion 94 100 1.6 method for isolating degrading bacteria. A bacterial strain with a distinct clearing zone, designated G1, was obtained through isolation and purification on chlorpyrifos-selective medium plate. Factors affecting chlorpyrifos degradation by strain G1 were tested in liquid MSM medium (chlorpyrifos at 50 mg/L) (Fig. 1). The chlor- pyrifos concentration remained unchanged in the non-inoculated control during the experimental period, whereas 42.6% of 50 mg/L chlorpyrifos was degraded in 20 h of incubation at 37 ◦C. Its metabo- Phoxim 78 100 7.2 lite TCP was also detected at the concentration equivalent to the concentration of chlorpyrifos degraded, but not detected in the G1-free control during the experimental period. The relation of decrease in chlorpyrifos concentrations and increase in TCP con- centrations suggested that strain G1 cannot degrade TCP. The result is similar with the study of Stenotrophomonas sp. YC-1 [10]. The of TCP is low to moderate for aquatic and terrestrial biota, and relatively negligible to mammals [30]. Strain G1 could quickly Profenofos 15 38 78 22.5 degrade diazinon, phoxim, methyl paraoxon, parathion, and methyl parathion (Table 2), could also degrade profenofos and triazophos at slightly lower rate, but could not degrade acephate, isocar- bophos, malathion, and methamidophos. Such substrate specificity may relate to the structures of the pesticides. All the OPs, which can be degraded by strain G1, are phosphotriesters, and some of the metabolites indicate that the OPs are hydrolyzed at a phosphotriester bond. The metabolic specificity of OP-degrading bacteria is determined by the chemical resemblance among the OPs [31]. Enterobacter strain B-14 degraded the diethylthiophospshate- containing parathion, diazinon, , and S. Deng et al. / Journal of Hazardous Materials 297 (2015) 17–24 21

Table 2 (Continued)

a a [11] [8] [7] Reference [9] Pesticides Structures Degradation (%) T1/2(h) [10]

6h 24h 48h

Triazophos 13 34 69 29.2 \\ \\ \\ 99/30 59.7/48

Acephate 0 0 0 >48 \ –– – \\ 69/48 78/48 100/24 100/24 100/24

Isocarbophos 0 0 0 >48 99/24 97/42 \\ 100/48 37.8/48 \\ \\\\\\\

Malathion 0 0 0 >48 \\ \ 99/40 \ ++ \\\ \\\\\\

Methamidophos 0 0 0 >48

a Data represent average values of triplicate determinations. b Date is 4 h degradation rate. a c \ 100/48 100/48 b − Pesticide (degradation/time, %/h) Chlorpyrifos Methyl parathion Fenitrothion Parathion Ethyl parathion Fenthion Profenofos Phoxim Diazinon Methyl paraoxon sp. 50 100/40 100/12 Stenotrophomonas Substrate concentration (mg/L) Enzyme Methyl parathion hydrolase

Fig. 1. Degradation of chlorpyrifos and generation of 3,5,6-trichloro-2-pyridinol (TCP) by strain G1.  The residue of chlorpyrifos in the controlled group; ᮀ residue Means the pesticide did not be tested. of TCP in controlled group;  residue of TCP with inoculation treatment;  residue Means can not degrade the pesticide. of chlorpyrifos with inoculation treatment. + Means strain can degrade the− pesticide, but the degradation rate was\\ not mentioned in the reference. c a MHF ENV20 100 50/96 SMSP-1 Organophosphorus pesticidehydrolase 20 Strain G1 PF32 Not be reported 100 96/48 99/24 99/18 YC-1 100 100/24 100/10 100/10 100/10 DSP-4 100 100/24 + b Table 3 Substrate ranges of other strains belongs to 22 S. Deng et al. / Journal of Hazardous Materials 297 (2015) 17–24

Fig. 2. Degradation of methyl parathion and generation of p-nitrophenol by strain G1 in MSM under the optimum conditions.  The residue of methyl parathion with inoculation treatment; ᮀ the residue of methyl parathion in blank control;  the residue of p-nitrophenol with inoculation treatment. Vertical bars represent the standard errors of the means.

Fig. 3. Amount of p-nitrophenol accumulated enzymatically in different cellular components of strain G1 within 10 min. (1) The treatment with crude periplasmic enzyme; (2) the treatment with crude extracellular enzyme; (3) the treatment with bacterial cell suspension; (4) the treatment with crude intracellular enzyme. Vertical bars represent the standard errors of the means. Different lowercase letters indicate statistically significant difference between groups (p < 0.001). isazofos when provided as the sole source of carbon and phos- other Stenotrophomonas sp. degraders cannot, although all of them phorus, but not , , ethoprop, and cadusafos, have methyl parathion hydrolase (Table 3). which have different side chains [32]. Burkholderia sp. strain In practice, a variety of different OPs is often used to control KR100 was able to degrade the OPs with dimethyl phosphoroth- insect pests. The OPs residues are one of the food safety and envi- ionate side chain (fenitrothion), diethyl phosphorothionate side ronmental problems worldwide. Many efforts have been focused on chain (chlorpyrifos), dimethyl phosphorodithionate side chains the isolation of bacterial species, which most isolates can degrade (dimethoate and malathion), and dimethyl phosphate side chain one or a couple of OPs [1,4,5,10,11], but few can degrade many () [33]. Pseudomonas sp. WBC-3, from which the OPs [7,8]. Therefore, a bacterial isolate that is capable of degrading amino acid sequence of methyl parathion hydrolase shared multiple OPs is needed for bioremediation applications. By having 100% homology with that from strain G1, can degrade methyl a broad degradation spectrum of various OPs, strain G1 possesses a parathion, ethyl parathion, chlorpyrifos, and sumithion [19,20]. great potential to provide versatile genes and enzyme systems for Strain G1 can degrade the organophosphorus insecticides that bioremediation of toxic OPs in a mixture. Strain G1 may be used in a S. Deng et al. / Journal of Hazardous Materials 297 (2015) 17–24 23

Fig. 4. Phylogenetic tree constructed based on complete 16S rDNA gene sequences of strain G1 isolated in this study and closely related reference species retrieved from GenBank database. Trees were constructed with 2000 bootstrap replications. Bootstrap value >50% was given at the node of branches. Scale bar represents 0.2% genetic distance between species. GenBank accession numbers of 16S rDNA sequences are given in the parentheses. A superscript capital letter T indicates type strain. mixture with other bacterial strains that can degrade p-nitrophenol extract, peptone and glucose [34]. However, strain G1 can degrade to avoid the accumulation of p-nitrophenol residues. p-nitrophenol, when maltose or glucose was co-supplemented in MSM. Plesiomonas sp. strain M6 hydrolyzed methyl parathion to O,O-dimethyl phosphorothinate and p-nitrophenol via methyl 3.2. Optimal condition for methyl parathion degradation by parathion hydrolase, but could not degrade p-nitrophenol further, strain G1 however, it was able to use benzoic acid and phenyl acetic acid as sole carbon and energy source [15]. The ANOVA analysis suggested that both initial substrate con- centrations and incubation temperatures had significant effects on 3.3. Localization of methyl parathion hydrolase in G1 T1/2 of methyl parathion. By comparison, the initial substrate con- centration had the greatest effect on T of methyl parathion by 1/2 Our recent study showed that strain G1 contains the gene G1, whereas the initial inoculum volume did not have significant coding methyl parathion hydrolase [24]. Methyl parathion hydro- effects on the experimental results. lase can transform methyl parathion to generate p-nitrophenol. The data were analyzed with Duncan multiple comparison test. Since G1 is a Gram-negative bacterium with periplasmic space, The results showed that initial substrate concentrations and incu- methyl parathion hydrolase potentially being secreted into the bation temperatures had three different subsets, indicating that periplasmic space can be extracted by osmotic shock [35]. Fig. 3 there were significant differences among the three levels. However, shows that a large amount of p-nitrophenol (1.29 × 10−4 mmol/L) there were no great differences among the three levels of initial was generated by the treatment with the crude intracellular inoculum volume. The optimum conditions that had the shortest enzyme for 10 min, whereas only a small amount of p-nitrophenol T of methyl parathion degradation were an initial inoculum of 1/2 (1.70 × 10−5 mmol/L) was generated by the treatment with the 20% (v/v); substrate concentration at 50 mg/L, and incubation tem- ◦ G1 crude extracellular enzyme. A small amount of p-nitrophenol perature at 40 C. (2.35 × 10−5 mmol/L) was generated with the crude periplasmic Under the optimal conditions, methyl parathion was used as enzyme preparations. The result suggested that methyl parathion the sole carbon source, and T was 1.60 h (Fig. 2). The con- 1/2 hydrolase was present in G1 cells as the intracellular enzyme. This centrations of p-nitrophenol as a degradation product increased result is consistent with the previous reports of other strains, such quickly in the first 6 h of incubation, being proportional to as Burkholderia sp. FDS-l and Plesiomonas sp. M6 [21,22]. Methyl methyl parathion degradation. The concentrations of p-nitrophenol parathion hydrolase in Ochrobactrum sp. Yw18 was, however, cell remained approximately constant between 6 h and 24 h of incu- membrane bound [23]. bation. After 12 h, 99.6% of methyl parathion was degraded and the p-nitrophenol concentration stopped increasing but remained at 0.195 mmol/L, which was approximately equal to the initial 3.4. Identification and characterization of strain G1 methyl parathion concentration (0.198 mmol/L). After 24 h, methyl parathion was no longer detected in the degradation system. These Strain G1 is a Gram-negative rod-shaped bacterium. The results observations indicted that strain G1 was able to degrade methyl of physiological and biochemical analysis showed that strain G1 parathion into p-nitrophenol, but could not further metabolize p- is positive in methyl red test, alcohol oxidation, catalase and V.P nitrophenol. During this process, there was no bacterial growth test, but negative in urease Tween 80 test. It can produce ammonia as indicated by a lack of substantial OD600 increase (data not and crystalloid of dextrin, but not levan. Strain G1 can hydrolyze shown), which reflected that strain G1 could not utilize methyl gelatin and esculin, but not starch and pectin. It can survive at 3% parathion as the sole carbon source for growth or completely of NaCl, cannot utilize glucose and sucrose. All characteristics of G1 mineralize it. No bacterial growth was observed, when strain G1 are consistent those of the genus of Stenotrophomonas. The total was cultured with p-nitrophenol and O,O-dimethyl phosphoroth- length of 16S rDNA gene sequence amplified from G1 was esti- ioate as the substrates. Bacillus sp. TAP-1 could degrade triazophos mated to be 1463 bp. The 16S rDNA gene sequence of G1 shared through co- when fed with nutrients such as yeast the highest homology with that of Stenotrophomonas acidaminiphila 24 S. Deng et al. / Journal of Hazardous Materials 297 (2015) 17–24

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