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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida a
a
Vijayalakshmi Pradeep & Usha Malavalli Subbaiah a
Department of Microbiology, CPGS, Jain University, Bangalore, India Published online: 31 Mar 2015.
Click for updates To cite this article: Vijayalakshmi Pradeep & Usha Malavalli Subbaiah (2015) Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 50:5, 346-360, DOI: 10.1080/03601234.2015.1000180 To link to this article: http://dx.doi.org/10.1080/03601234.2015.1000180
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Journal of Environmental Science and Health, Part B (2015) 50, 346–360 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2015.1000180
Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida VIJAYALAKSHMI PRADEEP and USHA MALAVALLI SUBBAIAH
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Department of Microbiology, CPGS, Jain University, Bangalore, India
The present study was undertaken with the objective of studying repeated batch and continuous degradation of chlorpyrifos (O,Odiethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate) using Ca-alginate immobilized cells of Pseudomonas putida isolated from an agricultural soil, and to study the genes and enzymes involved in degradation. The study was carried out to reduce the toxicity of chlorpyrifos by degrading it to less toxic metabolites. Long-term stability of pesticide degradation was studied during repeated batch degradation of chlorpyrifos, which was carried out over a period of 50 days. Immobilized cells were able to show 65% degradation of chlorpyrifos at the end of the 50th cycle with a cell leakage of 112 £ 103 cfu mL¡1. During continuous treatment, 100% degradation was observed at 100 mL h¡1 flow rate with 2% chlorpyrifos, and with 10% concentration of chlorpyrifos 98% and 80% degradation was recorded at 20 mL h¡1 and 100 mL h¡1 flow rate respectively. The products of degradation detected by liquid chromatography–mass spectrometry analysis were 3,5,6-trichloro-2-pyridinol and chlorpyrifos oxon. Plasmid curing experiments with ethidium bromide indicated that genes responsible for the degradation of chlorpyrifos are present on the chromosome and not on the plasmid. The results of Polymerase chain reaction indicate that a ~890-bp product expected for mpd gene was present in Ps. putida. Enzymatic degradation studies indicated that the enzymes involved in the degradation of chlorpyrifos are membrane-bound. The study indicates that immobilized cells of Ps. putida have the potential to be used in bioremediation of water contaminated with chlorpyrifos. Keywords: Pseudomonas putida, chlorpyrifos degradation using calcium alginate-immobilized cells, plasmid curing, PCR analysis, membrane-bound degradative enzymes.
Introduction Chlorpyrifos (O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate), is an organophosphate pesticide and has been used widely in the cultivation of corn, wheat, tea, rice, sugarcane, cotton, flowers, vegetables, fruit trees, livestock etc.[1] Throughout the world, it is used in controlling a variety of mites and sucking and chewing insect pests.[2] It is effective in controlling a range of insects, including flea beetles, fire ants, corn rootworms, cutworms, grubs, cockroaches, flies, termites and lice.[3] It is available in various formulations like pellet, dust, spray, emulsifiable concentrate, and flowable, granular and wettable powder.[4] Due to persistent usage and broad spectrum of utility of chlorpyrifos, it causes widespread contamination of the Address correspondence to Usha Malavalli Subbaiah, Department of Microbiology, CPGS, Jain University, 18/3, 9th cross, 3rd Block, Jayanagar, Bangalore-11, India; Email: [email protected] Received October 29, 2014. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lesb.
natural environment; which has lead to serious damage to non-target organisms.[5–7] Contamination of this kind has led to the insecticides and their transformation products being transported to long distances.[8] In soil, there is a variation in the reported half-life of chlorpyrifos ranging from 10 to 120 days.[9,10] There is moderate toxicity shown by chlorpyrifos towards humans.[11] Due to poisoning of this pesticide, respiratory system, cardiovascular system and central nervous system may get affected.[12] Biodegradation of pesticides can be carried out using free and immobilized cells. Cell immobilization has been used for the biological removal of pesticides due to the possibility of maintaining catalytic activity over long duration of time.[13–15] The major advantages with the utilization of immobilized cells in comparison with suspended ones include the retention of higher concentrations of microorganisms in the reactor, protection offered to the cells against toxic substances and prevention of suspended bacterial biomass in the effluent.[16–18] In addition to these advantages, in general, immobilization of microbial cells provides higher efficiency of degradation and good stability of operation.[19–21] Many kinds of materials have been used as carriers for the immobilization of microorganisms for wastewater treatment
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Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida which include activated pumice, polyacylamide hydrazide, alginate, network polymers, activated carbon and calcium (Ca) alginate.[22–26] Although there are reports on the biodegradation of organophosphate pesticides using immobilized cells[27–30] and immobilized enzymes,[13,31,32] reports, particularly on chlorpyrifos degradation using immobilized cells of bacteria, are very few. Chlorpyrifos degradation has been reported by Xie et al.[33] using an immobilized enzyme from Fusarium spp. Enzymatic degradation of chlorpyrifos as well as involvement of mpd gene in the degradation of chlorpyrifos has been studied by several researchers.[2,34–39] The batch degradation study using free cells and Caalginate-immobilized cells of Ps. putida JQ701740 isolated from an agricultural field was carried out by the authors and reported in our earlier publication where immobilized cells showed better degradation potential at higher pesticide concentrations compared with free cells.[40] The objective of the present study was to investigate the repeated batch and continuous degradation of chlorpyrifos using Ca-alginate-immobilized cells of Ps. putida, isolated from a soil amended with the pesticide. The current investigation also involves the study of genes and enzymes involved in the degradation of chlorpyrifos.
Materials and methods Pesticide and other chemicals Commercial-grade insecticide chlorpyrifos (20% EC) was procured from a pesticide selling shop in Bangalore, India. Other chemicals were procured from Hi-Media Pvt. Ltd., Mumbai, Maharashtra, India. Chlorpyrifos, analytical standard, was procured from IIHR, Bangalore. Other chemicals and solvents used for experimental purpose in this study were of analytical grade. These were procured from Sd Fine Chemicals Ltd., Mumbai, and Himedia Laboratories Pvt. Ltd., Mumbai. Double distilled deionised water was used in chlorpyrifos estimation procedure.
Bacterial culture Ps. putida, isolated from an agricultural field with a previous history of pesticide application, and identified based on nucleotide sequence and deposited in the gene bank with the accession number JQ701740, was used in the present study.
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with 0.01-M Phosphate buffer (pH 7.0) were used for immobilization experiments. Immobilization in Ca-alginate Ca-alginate entrapment of Ps. putida was performed according to the method of Bettman and Rehm.[42] Sodium alginate (3% w/v) was dissolved in distilled water and autoclaved at 121 C for 15 min. Fresh bacterial pellet (3% w/v) of Ps. putida was mixed in 100-mL sterilized sodium alginate solution. This mixture was extruded drop by drop into a cold sterile 0.2-M calcium chloride solution using a sterile syringe. Gel beads of approximately 2-mm diameter were obtained. The beads were hardened by resuspending in a fresh 0.2-M calcium chloride solution for 2 h with gentle agitation. Finally, these beads were washed with sterile distilled water and stored in 0.2-M calcium chloride at 4 C until further use. Repeated batch degradation of chlorpyrifos Repeated batch degradation studies were performed to observe long-term stability of Ca-alginate-immobilized Ps. putida cells degrading chlorpyrifos. After each cycle of incubation for 24 h at 150 rpm shaking speed and at 37 C, the spent medium was decanted and beads were washed with sterile distilled water and transferred into a fresh sterile minimal mineral salt medium[43] containing 2% chlorpyrifos. The remaining amount of chlorpyrifos in the media after incubation was estimated by spectrophotometric analysis, as described by Khan et al.[44] At intervals of five days, the stability of beads was monitored and cell leakage was recorded as cfu mL¡1 values by plating 1 mL of spent medium on nutrient agar medium. Continuous degradation of chlorpyrifos Continuous treatment of chlorpyrifos was carried out in a continuous flow reactor. The reactor was filled with Caalginate-immobilized Ps. putida for degradation of chlorpyrifos. Degradation process was carried out by continuous supply of sterile minimal mineral salts medium containing chlorpyrifos with the help of peristaltic pump (Miclins PP10-4C, India). Flow rates ranged from 20 to 100 mL h¡1 with varying concentrations of chlorpyrifos (2 to 10%). Design of bioreactor for continuous treatment
Growth of the culture Ps. putida was grown in mineral salts medium[41] containing 2.5% chlorpyrifos under optimized conditions. After incubation, the bacterial cells were harvested by centrifugation at 10,000 rpm for 15 min. These cells after washing
A cylindrical glass column (4 £ 50 cm, volume 650 mL) as shown in Figure A1 with inlet and outlet facilities was used for continuous degradation of pesticides. The bottom of the column was packed with glass wool (4-cm diameter) followed by a porous glass frit. Then the reactor was packed with the Ca-alginate-immobilized culture to a
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height of 30 cm for the degradation of the chlorpyrifos. The reactor was attached to a reservoir containing minimal mineral salts medium[43] with chlorpyrifos. The medium, after pesticide degradation, was continuously removed from the side arm situated just above the packed bed. The detention time (dt) of degradation was calculated by the following formula: Detention timeðdtÞ : void volume/flow rate mL h ¡ 1 ; Degradation rateðRÞ D ðCi ¡ Ce / £ D; where
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Ci D Ce D DD
concentration of the pesticide in the influent, Concentration of the pesticide in the effluent, Dilution rate D Flow rate (mL h¡1)/void volume of the reactor (mL).
Estimation of chlorpyrifos Amount of chlorpyrifos in the media after incubation was estimated by spectrophotometric analysis according to Khan et al. (2007).[44] Diazotized p-aminobenzoic acid was prepared by adding 10 mL of p-aminobenzoic acid (5% in ethanol) to 1 mL of 1% sodium nitrite (in 10 v/v hydrochloric acid in water) and the solution was kept in a brown bottle. To 1 mL of the extract taken in a 25-mL graduated tube, 1 mL of 5 mol L¡1 sodium hydroxide was added and kept for 20 min at 30 C for complete hydrolysis. To this, 1 mL of diazotized p-aminobenzoic acid was added, shaken thoroughly and kept at 0–5 C for 15 min for the development of a wine red colour. The solution was then made up to 25 mL with water and absorbance was measured at 520 nm against a reagent blank containing all the reagents except the sample, which was replaced with ethyl acetate. Percentage of degradation was calculated using the given formula: Percent of degradation D [ðC0 ¡ Ct Þ/C0 ] £ 100; where C0 D Ct D
initial concentration and concentration at time “t.”
Identification of degradation products After degradation of chlorpyrifos, using Ca-alginateimmobilized Ps. Putida, the products were extracted from large amount of spent medium using two volumes of ethyl acetate, dried and finally mixed with methanol and sent to Indian Institute of Science, Bangalore, India, for liquid chromatography–mass spectrometry (LC-MS) analysis.
LC-MS analysis conditions Liquid chromatography–mass spectrometry analysis of chlorpyrifos and its degradation products was outsourced from Indian Institute of Science, Bangalore. High-Performance Liquid Chromatography (HPLC) analyses were performed using Thermo Finnigan Survey. The column used was a BDS HYPERSIL C18 (reverse phase) with length: 250 mm; ID: 4.6 mm and particle size: 5 mm. Detection was done with UV at 254 and 280 nm. The detector used was HPLC PDA/UV detector; with ambient temperature and injection volume of 10 mL. An isocratic eluent with acetonitrile:water in the ratio 70:30 was used. The flow rate was 0.2 mL min–1 with a run time of 60 min. HPLC grade acetonitrile was used. Mass spectroscopy was performed using Thermo LCQ Deca XP MAX. The software used was Xcalibur. Conditions used for MS were as follows: probe/source voltage of 4.5 kV; mode of ionization: Cve mode; mass range: 50 to 500 m/z; sheath gas flow (arbitrary units): 40.00; auxiliary/sweep gas flow (arbitrary units): 20.00; source type: Electro Spray Ionization (ESI); sample tray temperature: 5 C; column oven temperature: 40 C; capillary temperature: 275 C; capillary voltage: 16 V; and nebulisation gas flow: helium at 1 mL min¡1 approximately. The helium in the mass analyzer cavity was maintained at 0.1 Pa (10¡3). Plasmid isolation and plasmid curing To know the number of plasmids present and to find whether the genes responsible for the production of enzymes involved in the degradation of chlorpyrifos are present on the genomic DNA or on the extra chromosomal DNA, plasmid isolation and plasmid curing experiments were performed. The Ps. putida degrading chlorpyrifos was grown in Luria Bertani (LB) broth for 24 h. Plasmid DNA was extracted using the alkaline lysis method[45] from cell pellet of the culture. The extracted plasmid DNA was observed by agarose gel electrophoresis. Ps. putida degrading chlorpyrifos was inoculated into 100 mL nutrient broth medium and incubated for 24 h under a shaking speed of 150 rpm. After 24 h of incubation, 1 mL of broth was inoculated into fresh nutrient broth with 300 mg mL¡1 of ethidium bromide. Plasmid DNA was extracted[45] and subjected to 1% agarose gel electrophoresis. The procedure was repeated for six days. Plasmid-cured culture was checked for its efficiency in degrading chlorpyrifos by plating 1 mL of culture on mineral salts medium.[41] Genomic DNA isolation and Polymerase chain reaction (PCR) analysis Ps. putida culture degrading chlorpyrifos was subjected to PCR analysis, which was outsourced from Bhat Bio-tech India Pvt Ltd, Bangalore. The genomic DNA was isolated
Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida
For the amplification of mpd gene, initial denaturation at 94 C for 2 min followed by 35 cycles of denaturation at 94 C for 1 min, annealing at 52 C for 1 min and extension at 72 C for 1 min was used. Final extension was carried out at 72 C for 10 min. PCR product, 10 mL, was analyzed on 1.5% agarose gel electrophoresis. The kit that was used for the analysis was from Bhat Bio-tech India Pvt Ltd, Bangalore.
1 mL of freshly prepared lysozyme solution (10 mg mL¡1) in 10-mM Tris-HCl, pH 8.0.[47] The supernatant, bufferwash and cell lysate were refrigerated until further use. Enzymatic degradation of chlorpyrifos was studied with all the three fractions, i.e. supernatant, buffer wash and cell lysate. The protein content of each fraction was determined by the Bradford’s method.[48] Enzymatic degradation of chlorpyrifos was carried out according to the procedure given by Cho et al.[46] The reaction mixture was taken in triplicates with 1 mL of 50mM phosphate citrate buffer (pH 8) containing 200 mL of supernatant, 0.5 mM of chlorpyrifos and 10% methanol. The mixture was incubated at 37 C for 5 min. After the incubation, decrease in the amount of chlorpyrifos was estimated spectrophotometrically at 520 nm as described by Khan et al.[44] The amount of degraded chlorpyrifos was calculated. Similar experiments were carried out with 200 mL of buffer wash and cell lysate separately and the degradation of chlorpyrifos was recorded in both cases. The buffer wash was subjected to ammonium sulfate precipitation and dialysis as it showed better results compared with other fractions. Ammonium sulfate was added to the buffer wash of Ps. putida to give 30% saturation and the solution was stirred at 4 C for 30 min. The precipitate was removed by centrifugation at 15,000 £ g for 20 min and the supernatant fluid was brought to 50% ammonium sulfate saturation. After that the solution was stirred at 4 C for 30 min and centrifuged at 15,000 £ g for 20 min, the precipitate was redissolved in 50-mM phosphate citrate buffer of pH 8.0.[49] Enzymatic degradation of chlorpyrifos was performed using the ammonium sulfate-precipitated buffer wash fraction. The ammonium sulfate-precipitated buffer wash fraction of Ps. putida was dialysed overnight against 100 vol of 50-mM phosphate citrate buffer (pH 8) at 4 C. The dialysed sample was used for enzymatic degradation of chlorpyrifos. The protein content of ammonium sulphateprecipitated and dialysed buffer wash fraction was determined by the Bradford’s method.[48]
Enzymatic degradation of chlorpyrifos
Results
100 95
Degradation (%)
90 85 80 75 70 65 60
5
10
15
20
25
30
35
40
45
50
Cycle
Fig. 1. Repeated batch degradation of chlorpyrifos by Ca-alginate-immobilized Ps. putida.
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from Ps. putida using the genomic DNA extraction kit. The pellet from 1.5 mL of overnight culture was resuspended in 500 mL of lysis buffer and incubated at 37 C for 1 h to lyse the cells. Genomic DNA was then extracted by phenol/chloroform. DNA from the aqueous phase was precipitated with isopropanol, washed with 70% ethanol and air-dried. The DNA pellet was dissolved in 50 mL of nuclease-free water. Genomic DNA, 1 mL, was used to analyze on 0.5% agarose gel electrophoresis. Amplification of the mpd gene was performed using the following primer pairs[34]: mpd Forward primer: 50 -GCGCTGCAGCACCGCAGGTG-3’ mpd Reverse primer: 50 -CGCAAGCTTTCATCATCACTTGGGGTTGACGACCGA-30
Experiment was conducted to find out whether the enzymes involved in the degradation of chlorpyrifos by Ps. putida were extracellular, membrane-bound or intracellular. Ps. putida was grown in 100 mL of mineral salts medium[41] under optimized conditions. The cells of the culture from the medium were harvested during mid-logarithmic phase by centrifugation at 10,000 rpm for 20 min at 4 C. Supernatant of the culture was stored separately. The cell pellet of Ps. putida was washed twice with 1 mL of 50mM phosphate citrate buffer (pH 8.0).[46] Buffer wash of the culture was collected separately. The cell pellet obtained was resuspended in 1 mL of buffer. The cells were lysed using
Repeated batch degradation From Figure 1, it can be observed that Ca-alginate-immobilized Ps. putida could be reused without affecting its degradation efficiency of 100% up to five cycles. At the end of the 10th cycle, there was reduction in degradation efficiency to 92%. Further increase in the number of cycles resulted in a gradual decrease in degradation efficiency. By the end of the 50th cycle, 65% degradation efficiency was retained by Ca-alginate-immobilized Ps. putida. A cell leakage of 112 £ 103cfu mL¡1 was recorded at the end of the 50th cycle.
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Continuous degradation of chlorpyrifos As shown in Figure 2, Ps. putida was able to show 100% degradation with 2% concentration of pesticide upto 100 mL h¡1 flow rate (dt: 36 min). At a flow rate of 20 mL h¡1 (dt: 324 min) 100% degradation of chlorpyrifos was recorded up to 8% concentration of pesticide. At a flow rate of 40 mL h¡1 (dt: 252 min) Ps. putida could show 100% degradation up to 6% concentration of pesticide. With 10% concentration of chlorpyrifos at 20 mL h¡1 flow rate (dt: 324 min), 98% degradation was observed. At a flow rate of 100 mL h¡1 (dt: 36 min) with 10% concentration of chlorpyrifos, Ps. putida was still able to show 80% degradation of chlorpyrifos.
A chromatogram of chlorpyrifos standard is shown in Figure A2. The chromatograms of chlorpyrifos control and chlorpyrifos test are shown in Figures A3 and A4. The mass spectra of chlorpyrifos control (RT: 8.21–9.12) and that of chlorpyrifos test (RT: 8.22–8.96) are shown in Figures A5 and A6 respectively. The mass spectrum of chlorpyrifos test (RT: 5.6–6.7) is shown in Figure A7. The retention time for chlorpyrifos standard was found to be 8.4 (Fig. A2). Compared with the chromatogram of chlorpyrifos control (Fig. A3), the peak at retention time 8.54 corresponding to chlorpyrifos reduced by more than 60% in the chromatogram of chlorpyrifos test (Fig. A4). The diminished or reduced peak clearly indicates degradation of chlorpyrifos. The same can be observed from the respective m/z values of mass spectrum of chlorpyrifos control (RT: 8.21–9.12) (Fig. A5) and mass spectrum of chlorpyrifos test (RT: 8.22–8.96) (Fig. A6). Intensity change of chlorpyrifos can be observed from the chromatogram and m/z values at retention time 8.54 as listed in Table A1. In the mass spectrum of test sample (RT: 8.22–8.96) (Fig. A6), the m/z value (mass-to-charge ratio) of 350.76 is absent because of change in mass due to 100 95
Degradation (%)
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LC-MS analysis and identification of degradation products
2%
90
4% 6%
85
8% 10%
80 75
20
40
60
Flow rate (ml h-1)
80
100
Fig. 2. Continuous degradation of different concentrations of chlorpyrifos ranging from 2 to 10% by Ps. putida at different flow rates ranging from 20 to 100 mL h¡1; experiments were done in triplicates; error bars indicate §SD.
Fig. 3. Agarose gel electrophoresis of plasmid from Ps. putida subjected to curing. Note: lane 0: indicates control sample (not treated with ethidium bromide), lanes 1—6: indicate ethidium bromide-treated bands of plasmid extracted from day 1 to 6.
addition of oxon, which shows the confirmation of the predicted product. The base peak seen in the mass spectrum of chlorpyrifos control (RT: 8.21–9.12) (Fig. A5) is 172.23, and this is the most stable fragment. In the mass spectrum of chlorpyrifos test sample (RT: 8.22–8.96) (Fig. A6) the base peak is with the m/z value of 200.25, which indicates that addition of oxon takes place to chlorpyrifos, i.e. the original substrate molecule. Due to the influence of oxon, the base peak is of higher m/z value. In the chromatogram of the chlorpyrifos test sample (RT range: 5.6–6.7) (Fig. A4), the chromatogram is merged because of the presence of chlorpyrifos oxon, whereas in the control (Fig. A3), the chromatogram has distinguished peaks. This clearly indicates the presence of oxon in the test sample. The m/z values corresponding to the stable oxon (MW 335) molecule are seen in the MS of chlorpyrifos test sample (RT range: 5.6–6.7). The m/z values corresponding to another degradation product, 3,5,6-trichloro-2-pyridinol (TCP) (MW 197), are also reported in the RT range of 5.6– 6.7 in the mass spectrum of chlorpyrifos test sample (Fig. A7). TCP is comparatively more stable. Hence, chlorpyrifos oxon and TCP could be two predicted products of chlorpyrifos degradation. The m/z values of chlorpyrifos degradation products are listed in Table A2.
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Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida
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Fig. 5. 1.5% Agarose gel electrophoresis of products of mpd PCR. Note: M: DNA molecular weight marker; 1: Ps. putida 2: no template control.
Plasmid isolation and plasmid curing As shown in Figure 3, Ps. putida had a single band of plasmid on plasmid isolation and gel electrophoresis. Curing experiment on Ps. putida was carried out to determine whether the chlorpyrifos degrading genes are present either on plasmid or chromosome. With increase in incubation period, the thickness of the plasmid band on agarose gel decreased. On day 4, the band thickness reduced and by day 5, the plasmid band disappeared (Fig. 3), indicating that the plasmid was cured. Plasmid DNA-cured cells of Ps. putida were able to grow on mineral salts medium supplemented with the pesticide as the sole source of carbon. This indicated that the genes responsible for the degradation of chlorpyrifos are present on the chromosome and not on the plasmid.
Genomic DNA isolation and PCR analysis Fig. 4. 0.5% Agarose gel electrophoresis of genomic DNA from Ps. putida.
Genomic DNA from Ps. putida degrading chlorpyrifos was isolated as shown in Figure 4. The culture was subjected to PCR analysis. The results of PCR analysis
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Pradeep and Subbaiah
Fig. 6. Proposed pathway for degradation of chlorpyrifos.
indicate that a ~890-bp product expected for mpd gene was present in Ps. putida (Fig. 5). Enzymatic degradation of chlorpyrifos The results of the enzymatic degradation of chlorpyrifos with supernatant, buffer wash and cell lysate of Ps. putida are presented in Table 1. As seen in Table 1, a negligible degradation in chlorpyrifos was recorded with cell-free supernatant. A slightly higher degradation of chlorpyrifos was recorded with crude cell lysate, and the buffer wash of cells showed maximum degradation of chlorpyrifos. This indicated that the enzymes involved in the degradation of chlorpyrifos are membrane-bound. When buffer wash of Ps. putida was subjected to ammonium sulfate precipitation, there was an increase in chlorpyrifos degradation. Degradation of the pesticide increased further after dialysis of ammonium sulfate-precipitated sample (Table 2).
Discussion In the present investigation, Ps. putida was able to show 100% degradation with 2% concentration of chlorpyrifos up to 100 mL h¡1 flow rate. At a flow rate of 100 mL h¡1 with 10% concentration of chlorpyrifos, Ps. putida was still able to show 80% degradation. In similar studies, a tezontle-packed up-flow reactor (TPUFR) with an immobilized bacterial consortium for the biological treatment of methyl-parathion and tetrachlorvinphos was evaluated by Ya~ nez-Ocampo et al.[29] They could evaluate four flow rates (0.936, 1.41, 2.19 and 3.51 L h¡1) and four hydraulic residence time periods (0.313, 0.206, 0.133 and 0.083 h) in the reactor. With an operating time of 8 h and a flow of 0.936 L h¡1, they obtained 75% efficiency in the removal of methyl-parathion and tetrachlorvinphos. Calcium-alginate-immobilized cell systems were also developed by Ha et al.[28] for the detoxification and biodegradation of organophosphate insecticide, coumaphos, and its
Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida Table 1. Enzymatic degradation of chlorpyrifos with supernatant, buffer wash and cell lysate of Ps. Putida. Enzyme Fraction Supernatant Buffer wash Cell lysate
Degradation of Chlorpyrifos (%)
Protein Content (mg mL¡1)
7.16 § 0.76 30 § 2 15.33 § 1.52
0.23 § 0.026 0.426 § 0.032 0.756 § 0.03
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Note: Values are a mean of three replicates § SD.
hydrolysis products, chlorferon and diethlythiophosphate (DETP). In the current investigation, repeated batch degradation studies were conducted to observe the long-term stability of Ca-alginate-immobilized cells of Ps. putida. It was observed that Ca-alginate-immobilized Ps. putida could be reused without changing its degradation efficiency of 100% up to five cycles. Even after the 50th cycle, the degradation efficiency of 65% was recorded. A cell leakage of 112 c_ 103 cfu mL¡1 was recorded at the end of the 50th cycle from encapsulated beads with Ps. putida. In similar immobilization studies, a recombinant strain of Escherichia coli containing the opd gene for organophosphate hydrolase (OPH), capable of active hydrolysis of organophosphate neurotoxins, including chlorpyrifos, was cultivated by Kim et al.[50] They harvested and utilized the cells in lab-scale experiments in the form of either freely suspended cells in batch degradation or cells immobilized within a macroporous gel matrix, polyvinyl alcohol (PVA) cryogel. The immobilized cells retained their activity over a four-month period of use and storage, demonstrating both sustained catalytic activity and long-term mechanical stability. Similarly, Richins et al.[13] demonstrated that bi-functional fusion proteins comprising OPH moieties were highly effective in degrading organophosphate nerve agents. This enabled purification and immobilization of proteins on different cellulose materials in a single step. Repeated hydrolysis of paraoxon was achieved in an immobilized enzyme reactor with 100% degradation efficiency over 45 days. Table 2. Enzymatic degradation of chlorpyrifos by partially purified enzyme fractions. Enzyme Fraction Buffer wash of Ps. Putida After ammonium sulfate precipitation After dialysis
Degradation (%)
Protein Content (mg mL¡1)
30 § 2
0.926 § 0.032
42.66 § 1.52
0.753 § 0.03
50 § 2
0.666 § 0.015
Note: Values are a mean of three replicates § SD.
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In the present investigation, Ps. putida was efficient in degrading chlorpyrifos. Through LC-MS analysis, chlorpyrifos oxon and 3,5,6-trichloropyridinol were the predicted products of chlorpyrifos degradation by Ps. putida. It was predicted that chlorpyrifos is degraded through a hydrolytic degradation pathway. In similar studies, Singh et al.[37] investigated the ability of the strain Enterobacter strain B-14 to mineralize chlorpyrifos. Strain B-14 was able to degrade chlorpyrifos to DETP and TCP and utilized DETP as a sole source of carbon. Sasikala et al.[51] showed the presence of metabolites – chlopyrifos oxon and diethylphosphorothioate through LC-MS analysis. Based on our degradation studies and the degradation products predicted by LC-MS analysis, the predicted pathway of degradation of chlorpyrifos is shown in Figure 6. In our present investigation, growth of plasmid-cured Ps. putida was observed on mineral salts medium containing chlorpyrifos as a sole source of carbon. This indicated that genes responsible for the degradation of chlorpyrifos are present on the chromosome and not on the plasmid. Genetic studies based on plasmid curing and electroporation-mediated transformation on a chlorpyrifos degrading bacterium Ps. putida MAS-1 was performed by Ajaz et al.[52] It was observed that after curing with acridine orange, the property to grow on the nutrient agar containing 10 mg mL¡1 chlorpyrifos was lost by the bacterium. Isolation of plasmid bearing chlorpyrifos degrading determinants/genes was carried out. It was then transferred into E. coli DH5a. However, it was observed that the transformants could not resist and grow in the medium with chlorpyrifos. The conclusion drawn from the experiment was that degradation of chlorpyrifos by Ps. putida MAS-1 was due to the combined action of plasmid and chromosomal genes. In most of the studies, opd genes were found to be plasmid-based and had similar DNA sequences.[37] However, Horne et al.[53] isolated an opd gene from Agrobacterium radiobacter, which was located on the chromosome but had a similar sequence to the opd gene from another bacteria. In our present investigation, Ps. putida degrading chlorpyrifos was subjected to PCR analysis. A ~890-bp product expected for mpd gene involved in the degradation of chlorpyrifos was amplified from Ps. putida. In similar studies, an effective chlorpyrifos-degrading bacterium, named strain YC-1, was isolated from the sludge of the wastewater treating system of an organophosphorus pesticide manufacturer by Yang et al.[35] and the entire mpd functional gene of strain YC-1 was amplified by PCR with a pair of primers.[54] In the current study, higher enzymatic degradation of chlorpyrifos by Ps. putida was recorded with the buffer wash of cells compared with the crude cell lysate and the cell-free supernatant. This clearly indicated that the enzymes involved in the degradation of chlorpyrifos are membrane-bound. According to Steiert,[55] in the Flavobacterium spp. and P. diminuta the native Parathion hydrolase is membrane-bound, whereas in S. lividans, the
354 opd gene is expressed as a secreted soluble enzyme. However, when organophosphorus hydrolase activity of individual isolates and consortium were checked by Sasikala et al.[51] in the presence and absence of chlorpyrifos, the extracellular fractions failed to show enzyme activity.
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Conclusions Thus, it can be concluded that Ca-alginate-immobilized cells of Ps. putida were efficient in degrading chlorpyrifos, and the Ca-alginate-immobilized cells were stable for a long duration. Chlorpyrifos was degraded by hydrolytic pathway and chlorpyrifos oxon and 3,5,6-trichloro-3-pyridinol were the predicted products of degradation. Further, it was demonstrated that the genes involved in the degradation of chlorpyrifos were present on the chromosome and the enzymes involved in its degradation were membrane-bound.
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Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida [32] Mansee, A.H.; Chen, W.; Mulchandani, A. Detoxification of the organophosphate nerve agent coumaphos using organophosphorus hydrolase immobilized on cellulose materials. J. Ind. Microbiol. Biotechnol. 2005, 32, 554–560. [33] Xie, H.; Zhu, L.; Ma, T.; Wang, J.; Wang, J.; Su, J.; Shao, B. Immobilization of an enzyme from a Fusarium fungus WZ-I for chlorpyrifos degradation. J. Environ. Sci. 2010, 22(12), 1930–1935. [34] Zhang, X.Z; Cui Z.L.; Hong, Q.; Li, S.P. High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800. Appl. Environ. Microbiol. 2005, 71(7), 4101–4103. [35] Yang, C.; Liu, N.; Guo, X.; Qiao, C. Cloning of mpd gene from a chlorpyrifos-degrading bacterium and use of this strain in bioremediation of contaminated soil. FEMS Microbiol. Lett. 2006, 265, 118–125. [36] Li, X.; He, J.; Li, S. Isolation of a chlorpyrifos-degrading bacterium, Sphingomonas sp. strain Dsp-2, and cloning of the mpd gene. Res. Microbiol. 2007, 158 (2), 143–149. [37] Singh, B.K.; Walker, A.; Morgan, J.A.; Wright, D.J. Biodegradation of chlorpyrifos by Enterobacter strain B-14 and its use in bioremediation of contaminated soils. Appl. Environ. Microbiol. 2004, 70(8), 4855–4863. [38] Cho, C.M.; Mulchandani, A.; Chen, W. Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos. Appl. Environ. Microbiol. 2004, 70(8), 4681–4685. [39] Xie, H.; Zhu, L.S.; Wang, J.; Wang, X.G.; Liu, W.; Qian, B.; Wang Q. Enzymatic degradation of organophosphorus insecticide chlorpyrifos by fungus WZ-I. Huan. Jing. Ke. Xue. 2005, 26(6), 164–168. [40] Vijayalakshmi, P.; Usha, M.S. Degradation of chlorpyrifos by free cells and calcium-alginate immobilized cells of Pseudomonas putida.Adv. Appl. Sci. Res. 2012, 3(5), 2796–2800. [41] Chaudhry, G.R.; Ali, A.N.; Wheeler, W.B. Isolation of a methylparathion degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp. Appl. Environ. Microbiol. 1988, 54(2), 288–293. [42] Bettman, H.; Rehm H. Degradation of phenol by polymerentrapped microorganisms. Appl. Microbiol. Biotechnol. 1984, 20 (5), 285–290. [43] Manohar, S.; Karegoudar T.B. Degradation of naphthalene by cells of Pseudomonas sp. Strain NGK 1, immobilized in alginate, agar and polyacrylamide. Appl. Microbiol. Biotechnol. 1998, 49, 785–792. [44] Khan S.; Rai M.K.; Gupta, V.K.; Janghel, E.K.; Rai J.K. A new spectrophotometric determination of chlorpyrifos in environmental samples. J. Chem. Soc. Pak. 2007, 29(1), 37–40. [45] Sambrook, J.; Russell, D.W. Molecular Cloning: A Laboratory Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2001. [46] Cho, C.M.; Mulchandani, A.; Chen, W. Bacterial cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents. Appl. Environ. Microbiol. 2002, 68(4), 2026–2030. [47] Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular Cloning, A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989; p. 1.29. [48] Bradford, M.M. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 1976, 72, 248–252. [49] Rowland, S.S.; Speedie. M.K.; Pogell B.M. Purification and characterization of a secreted recombinant phosphotriesterase (parathion hydrolase) from Streptomyces lividans.Appl. Environ. Microbiol. 1991, 57(2), 440–444.
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Appendix
Fig. A1. Reactor packed with Ca-alginate-immobilized Ps.
putida.
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Fig. A2. HPLC chromatogram of chlorpyrifos analytical standard.
Fig. A3. HPLC chromatogram of chlorpyrifos control; RT value (8.54) of chlorpyrifos is ticked.
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Fig. A4. HPLC chromatogram of chlorpyrifos test; RT value (8.53) of chlorpyrifos is ticked.
Fig. A5. Mass spectrum of chlorpyrifos control in the RT range: 8.21–9.12. Fragment ions corresponding to chlorpyrifos
based on the NIST spectrum of chlorpyrifos are ticked.
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Fig. A6. Mass spectrum of chlorpyrifos test in the RT range: 8.22–8.96; Fragment ions corresponding to chlorpyrifos based on the NIST spectrum of chlorpyrifos are ticked.
Fig. A7. Mass spectrum of chlorpyrifos test showing m/z values corresponding to TCP and chlorpyrifos oxon (RT: 5.6–6.7) according to NIST standards. The fragment ions of TCP and chlorpyrifos oxon are ticked. TCP and chlorpyrifos oxon are the predicted products of chlorpyrifos degradation.
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Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida
Fig. A8. (a) NIST Mass spectra of chlorpyrifos (contributor: Mass Spectrometry Center, University of Utah EPA-PTSEL
2900), (b) NIST mass spectra of TCP (contributor: NIST Mass Spectrometry Data Center, 1990), (c) NIST mass spectra of chlorpyrifos oxon (contributor: NIST Mass Spectrometry Data Center, 1990).
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Table A1. m/z values of chlorpyrifos in the mass spectrum of chlorpyrifos control and test. M/z values corresponding to chlorpyrifos (according to the NIST standard – Figure A8 (a) in the mass spectrum of chlorpyrifos control RT: 8.21–9.12 (Fig. A5) 350.76, 314.09, 212.76, 197.25, 125.09, 97.74
M/z values corresponding to chlorpyrifos (according to the NIST standard – Figure A8(a) in the mass spectrum of chlorpyrifos test RT: 8.22–8.96 (Fig. A6) 314.10, 212.66, 197.37, 125.98, 97.27
Table A2. m/z values of products of chlorpyrifos degradation in the mass spectrum of chlorpyrifos test (RT: 5.6–6.7). M/z values of chlorpyrifos degradation products in the mass spectrum of chlorpyrifos test sample (RT: 5.6–6.7) (Fig. A7) M/z values corresponding to TCP (according to the NIST standard of TCP – Figure A8 (b))
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60.10, 98.25, 107.31, 169.37, 197.27, 199.39, 200.59
M/z values corresponding to chlorpyrifos oxon (according to the NIST standard of chlorpyrifos oxon – Figure A8 (c)) 335, 333.34, 298.44, 226.68, 226.00, 200.59, 199.39, 169.37
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida a
a
Vijayalakshmi Pradeep & Usha Malavalli Subbaiah a
Department of Microbiology, CPGS, Jain University, Bangalore, India Published online: 31 Mar 2015.
Click for updates To cite this article: Vijayalakshmi Pradeep & Usha Malavalli Subbaiah (2015) Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 50:5, 346-360, DOI: 10.1080/03601234.2015.1000180 To link to this article: http://dx.doi.org/10.1080/03601234.2015.1000180
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Journal of Environmental Science and Health, Part B (2015) 50, 346–360 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2015.1000180
Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida VIJAYALAKSHMI PRADEEP and USHA MALAVALLI SUBBAIAH
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Department of Microbiology, CPGS, Jain University, Bangalore, India
The present study was undertaken with the objective of studying repeated batch and continuous degradation of chlorpyrifos (O,Odiethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate) using Ca-alginate immobilized cells of Pseudomonas putida isolated from an agricultural soil, and to study the genes and enzymes involved in degradation. The study was carried out to reduce the toxicity of chlorpyrifos by degrading it to less toxic metabolites. Long-term stability of pesticide degradation was studied during repeated batch degradation of chlorpyrifos, which was carried out over a period of 50 days. Immobilized cells were able to show 65% degradation of chlorpyrifos at the end of the 50th cycle with a cell leakage of 112 £ 103 cfu mL¡1. During continuous treatment, 100% degradation was observed at 100 mL h¡1 flow rate with 2% chlorpyrifos, and with 10% concentration of chlorpyrifos 98% and 80% degradation was recorded at 20 mL h¡1 and 100 mL h¡1 flow rate respectively. The products of degradation detected by liquid chromatography–mass spectrometry analysis were 3,5,6-trichloro-2-pyridinol and chlorpyrifos oxon. Plasmid curing experiments with ethidium bromide indicated that genes responsible for the degradation of chlorpyrifos are present on the chromosome and not on the plasmid. The results of Polymerase chain reaction indicate that a ~890-bp product expected for mpd gene was present in Ps. putida. Enzymatic degradation studies indicated that the enzymes involved in the degradation of chlorpyrifos are membrane-bound. The study indicates that immobilized cells of Ps. putida have the potential to be used in bioremediation of water contaminated with chlorpyrifos. Keywords: Pseudomonas putida, chlorpyrifos degradation using calcium alginate-immobilized cells, plasmid curing, PCR analysis, membrane-bound degradative enzymes.
Introduction Chlorpyrifos (O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate), is an organophosphate pesticide and has been used widely in the cultivation of corn, wheat, tea, rice, sugarcane, cotton, flowers, vegetables, fruit trees, livestock etc.[1] Throughout the world, it is used in controlling a variety of mites and sucking and chewing insect pests.[2] It is effective in controlling a range of insects, including flea beetles, fire ants, corn rootworms, cutworms, grubs, cockroaches, flies, termites and lice.[3] It is available in various formulations like pellet, dust, spray, emulsifiable concentrate, and flowable, granular and wettable powder.[4] Due to persistent usage and broad spectrum of utility of chlorpyrifos, it causes widespread contamination of the Address correspondence to Usha Malavalli Subbaiah, Department of Microbiology, CPGS, Jain University, 18/3, 9th cross, 3rd Block, Jayanagar, Bangalore-11, India; Email: [email protected] Received October 29, 2014. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lesb.
natural environment; which has lead to serious damage to non-target organisms.[5–7] Contamination of this kind has led to the insecticides and their transformation products being transported to long distances.[8] In soil, there is a variation in the reported half-life of chlorpyrifos ranging from 10 to 120 days.[9,10] There is moderate toxicity shown by chlorpyrifos towards humans.[11] Due to poisoning of this pesticide, respiratory system, cardiovascular system and central nervous system may get affected.[12] Biodegradation of pesticides can be carried out using free and immobilized cells. Cell immobilization has been used for the biological removal of pesticides due to the possibility of maintaining catalytic activity over long duration of time.[13–15] The major advantages with the utilization of immobilized cells in comparison with suspended ones include the retention of higher concentrations of microorganisms in the reactor, protection offered to the cells against toxic substances and prevention of suspended bacterial biomass in the effluent.[16–18] In addition to these advantages, in general, immobilization of microbial cells provides higher efficiency of degradation and good stability of operation.[19–21] Many kinds of materials have been used as carriers for the immobilization of microorganisms for wastewater treatment
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Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida which include activated pumice, polyacylamide hydrazide, alginate, network polymers, activated carbon and calcium (Ca) alginate.[22–26] Although there are reports on the biodegradation of organophosphate pesticides using immobilized cells[27–30] and immobilized enzymes,[13,31,32] reports, particularly on chlorpyrifos degradation using immobilized cells of bacteria, are very few. Chlorpyrifos degradation has been reported by Xie et al.[33] using an immobilized enzyme from Fusarium spp. Enzymatic degradation of chlorpyrifos as well as involvement of mpd gene in the degradation of chlorpyrifos has been studied by several researchers.[2,34–39] The batch degradation study using free cells and Caalginate-immobilized cells of Ps. putida JQ701740 isolated from an agricultural field was carried out by the authors and reported in our earlier publication where immobilized cells showed better degradation potential at higher pesticide concentrations compared with free cells.[40] The objective of the present study was to investigate the repeated batch and continuous degradation of chlorpyrifos using Ca-alginate-immobilized cells of Ps. putida, isolated from a soil amended with the pesticide. The current investigation also involves the study of genes and enzymes involved in the degradation of chlorpyrifos.
Materials and methods Pesticide and other chemicals Commercial-grade insecticide chlorpyrifos (20% EC) was procured from a pesticide selling shop in Bangalore, India. Other chemicals were procured from Hi-Media Pvt. Ltd., Mumbai, Maharashtra, India. Chlorpyrifos, analytical standard, was procured from IIHR, Bangalore. Other chemicals and solvents used for experimental purpose in this study were of analytical grade. These were procured from Sd Fine Chemicals Ltd., Mumbai, and Himedia Laboratories Pvt. Ltd., Mumbai. Double distilled deionised water was used in chlorpyrifos estimation procedure.
Bacterial culture Ps. putida, isolated from an agricultural field with a previous history of pesticide application, and identified based on nucleotide sequence and deposited in the gene bank with the accession number JQ701740, was used in the present study.
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with 0.01-M Phosphate buffer (pH 7.0) were used for immobilization experiments. Immobilization in Ca-alginate Ca-alginate entrapment of Ps. putida was performed according to the method of Bettman and Rehm.[42] Sodium alginate (3% w/v) was dissolved in distilled water and autoclaved at 121 C for 15 min. Fresh bacterial pellet (3% w/v) of Ps. putida was mixed in 100-mL sterilized sodium alginate solution. This mixture was extruded drop by drop into a cold sterile 0.2-M calcium chloride solution using a sterile syringe. Gel beads of approximately 2-mm diameter were obtained. The beads were hardened by resuspending in a fresh 0.2-M calcium chloride solution for 2 h with gentle agitation. Finally, these beads were washed with sterile distilled water and stored in 0.2-M calcium chloride at 4 C until further use. Repeated batch degradation of chlorpyrifos Repeated batch degradation studies were performed to observe long-term stability of Ca-alginate-immobilized Ps. putida cells degrading chlorpyrifos. After each cycle of incubation for 24 h at 150 rpm shaking speed and at 37 C, the spent medium was decanted and beads were washed with sterile distilled water and transferred into a fresh sterile minimal mineral salt medium[43] containing 2% chlorpyrifos. The remaining amount of chlorpyrifos in the media after incubation was estimated by spectrophotometric analysis, as described by Khan et al.[44] At intervals of five days, the stability of beads was monitored and cell leakage was recorded as cfu mL¡1 values by plating 1 mL of spent medium on nutrient agar medium. Continuous degradation of chlorpyrifos Continuous treatment of chlorpyrifos was carried out in a continuous flow reactor. The reactor was filled with Caalginate-immobilized Ps. putida for degradation of chlorpyrifos. Degradation process was carried out by continuous supply of sterile minimal mineral salts medium containing chlorpyrifos with the help of peristaltic pump (Miclins PP10-4C, India). Flow rates ranged from 20 to 100 mL h¡1 with varying concentrations of chlorpyrifos (2 to 10%). Design of bioreactor for continuous treatment
Growth of the culture Ps. putida was grown in mineral salts medium[41] containing 2.5% chlorpyrifos under optimized conditions. After incubation, the bacterial cells were harvested by centrifugation at 10,000 rpm for 15 min. These cells after washing
A cylindrical glass column (4 £ 50 cm, volume 650 mL) as shown in Figure A1 with inlet and outlet facilities was used for continuous degradation of pesticides. The bottom of the column was packed with glass wool (4-cm diameter) followed by a porous glass frit. Then the reactor was packed with the Ca-alginate-immobilized culture to a
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height of 30 cm for the degradation of the chlorpyrifos. The reactor was attached to a reservoir containing minimal mineral salts medium[43] with chlorpyrifos. The medium, after pesticide degradation, was continuously removed from the side arm situated just above the packed bed. The detention time (dt) of degradation was calculated by the following formula: Detention timeðdtÞ : void volume/flow rate mL h ¡ 1 ; Degradation rateðRÞ D ðCi ¡ Ce / £ D; where
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Ci D Ce D DD
concentration of the pesticide in the influent, Concentration of the pesticide in the effluent, Dilution rate D Flow rate (mL h¡1)/void volume of the reactor (mL).
Estimation of chlorpyrifos Amount of chlorpyrifos in the media after incubation was estimated by spectrophotometric analysis according to Khan et al. (2007).[44] Diazotized p-aminobenzoic acid was prepared by adding 10 mL of p-aminobenzoic acid (5% in ethanol) to 1 mL of 1% sodium nitrite (in 10 v/v hydrochloric acid in water) and the solution was kept in a brown bottle. To 1 mL of the extract taken in a 25-mL graduated tube, 1 mL of 5 mol L¡1 sodium hydroxide was added and kept for 20 min at 30 C for complete hydrolysis. To this, 1 mL of diazotized p-aminobenzoic acid was added, shaken thoroughly and kept at 0–5 C for 15 min for the development of a wine red colour. The solution was then made up to 25 mL with water and absorbance was measured at 520 nm against a reagent blank containing all the reagents except the sample, which was replaced with ethyl acetate. Percentage of degradation was calculated using the given formula: Percent of degradation D [ðC0 ¡ Ct Þ/C0 ] £ 100; where C0 D Ct D
initial concentration and concentration at time “t.”
Identification of degradation products After degradation of chlorpyrifos, using Ca-alginateimmobilized Ps. Putida, the products were extracted from large amount of spent medium using two volumes of ethyl acetate, dried and finally mixed with methanol and sent to Indian Institute of Science, Bangalore, India, for liquid chromatography–mass spectrometry (LC-MS) analysis.
LC-MS analysis conditions Liquid chromatography–mass spectrometry analysis of chlorpyrifos and its degradation products was outsourced from Indian Institute of Science, Bangalore. High-Performance Liquid Chromatography (HPLC) analyses were performed using Thermo Finnigan Survey. The column used was a BDS HYPERSIL C18 (reverse phase) with length: 250 mm; ID: 4.6 mm and particle size: 5 mm. Detection was done with UV at 254 and 280 nm. The detector used was HPLC PDA/UV detector; with ambient temperature and injection volume of 10 mL. An isocratic eluent with acetonitrile:water in the ratio 70:30 was used. The flow rate was 0.2 mL min–1 with a run time of 60 min. HPLC grade acetonitrile was used. Mass spectroscopy was performed using Thermo LCQ Deca XP MAX. The software used was Xcalibur. Conditions used for MS were as follows: probe/source voltage of 4.5 kV; mode of ionization: Cve mode; mass range: 50 to 500 m/z; sheath gas flow (arbitrary units): 40.00; auxiliary/sweep gas flow (arbitrary units): 20.00; source type: Electro Spray Ionization (ESI); sample tray temperature: 5 C; column oven temperature: 40 C; capillary temperature: 275 C; capillary voltage: 16 V; and nebulisation gas flow: helium at 1 mL min¡1 approximately. The helium in the mass analyzer cavity was maintained at 0.1 Pa (10¡3). Plasmid isolation and plasmid curing To know the number of plasmids present and to find whether the genes responsible for the production of enzymes involved in the degradation of chlorpyrifos are present on the genomic DNA or on the extra chromosomal DNA, plasmid isolation and plasmid curing experiments were performed. The Ps. putida degrading chlorpyrifos was grown in Luria Bertani (LB) broth for 24 h. Plasmid DNA was extracted using the alkaline lysis method[45] from cell pellet of the culture. The extracted plasmid DNA was observed by agarose gel electrophoresis. Ps. putida degrading chlorpyrifos was inoculated into 100 mL nutrient broth medium and incubated for 24 h under a shaking speed of 150 rpm. After 24 h of incubation, 1 mL of broth was inoculated into fresh nutrient broth with 300 mg mL¡1 of ethidium bromide. Plasmid DNA was extracted[45] and subjected to 1% agarose gel electrophoresis. The procedure was repeated for six days. Plasmid-cured culture was checked for its efficiency in degrading chlorpyrifos by plating 1 mL of culture on mineral salts medium.[41] Genomic DNA isolation and Polymerase chain reaction (PCR) analysis Ps. putida culture degrading chlorpyrifos was subjected to PCR analysis, which was outsourced from Bhat Bio-tech India Pvt Ltd, Bangalore. The genomic DNA was isolated
Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida
For the amplification of mpd gene, initial denaturation at 94 C for 2 min followed by 35 cycles of denaturation at 94 C for 1 min, annealing at 52 C for 1 min and extension at 72 C for 1 min was used. Final extension was carried out at 72 C for 10 min. PCR product, 10 mL, was analyzed on 1.5% agarose gel electrophoresis. The kit that was used for the analysis was from Bhat Bio-tech India Pvt Ltd, Bangalore.
1 mL of freshly prepared lysozyme solution (10 mg mL¡1) in 10-mM Tris-HCl, pH 8.0.[47] The supernatant, bufferwash and cell lysate were refrigerated until further use. Enzymatic degradation of chlorpyrifos was studied with all the three fractions, i.e. supernatant, buffer wash and cell lysate. The protein content of each fraction was determined by the Bradford’s method.[48] Enzymatic degradation of chlorpyrifos was carried out according to the procedure given by Cho et al.[46] The reaction mixture was taken in triplicates with 1 mL of 50mM phosphate citrate buffer (pH 8) containing 200 mL of supernatant, 0.5 mM of chlorpyrifos and 10% methanol. The mixture was incubated at 37 C for 5 min. After the incubation, decrease in the amount of chlorpyrifos was estimated spectrophotometrically at 520 nm as described by Khan et al.[44] The amount of degraded chlorpyrifos was calculated. Similar experiments were carried out with 200 mL of buffer wash and cell lysate separately and the degradation of chlorpyrifos was recorded in both cases. The buffer wash was subjected to ammonium sulfate precipitation and dialysis as it showed better results compared with other fractions. Ammonium sulfate was added to the buffer wash of Ps. putida to give 30% saturation and the solution was stirred at 4 C for 30 min. The precipitate was removed by centrifugation at 15,000 £ g for 20 min and the supernatant fluid was brought to 50% ammonium sulfate saturation. After that the solution was stirred at 4 C for 30 min and centrifuged at 15,000 £ g for 20 min, the precipitate was redissolved in 50-mM phosphate citrate buffer of pH 8.0.[49] Enzymatic degradation of chlorpyrifos was performed using the ammonium sulfate-precipitated buffer wash fraction. The ammonium sulfate-precipitated buffer wash fraction of Ps. putida was dialysed overnight against 100 vol of 50-mM phosphate citrate buffer (pH 8) at 4 C. The dialysed sample was used for enzymatic degradation of chlorpyrifos. The protein content of ammonium sulphateprecipitated and dialysed buffer wash fraction was determined by the Bradford’s method.[48]
Enzymatic degradation of chlorpyrifos
Results
100 95
Degradation (%)
90 85 80 75 70 65 60
5
10
15
20
25
30
35
40
45
50
Cycle
Fig. 1. Repeated batch degradation of chlorpyrifos by Ca-alginate-immobilized Ps. putida.
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from Ps. putida using the genomic DNA extraction kit. The pellet from 1.5 mL of overnight culture was resuspended in 500 mL of lysis buffer and incubated at 37 C for 1 h to lyse the cells. Genomic DNA was then extracted by phenol/chloroform. DNA from the aqueous phase was precipitated with isopropanol, washed with 70% ethanol and air-dried. The DNA pellet was dissolved in 50 mL of nuclease-free water. Genomic DNA, 1 mL, was used to analyze on 0.5% agarose gel electrophoresis. Amplification of the mpd gene was performed using the following primer pairs[34]: mpd Forward primer: 50 -GCGCTGCAGCACCGCAGGTG-3’ mpd Reverse primer: 50 -CGCAAGCTTTCATCATCACTTGGGGTTGACGACCGA-30
Experiment was conducted to find out whether the enzymes involved in the degradation of chlorpyrifos by Ps. putida were extracellular, membrane-bound or intracellular. Ps. putida was grown in 100 mL of mineral salts medium[41] under optimized conditions. The cells of the culture from the medium were harvested during mid-logarithmic phase by centrifugation at 10,000 rpm for 20 min at 4 C. Supernatant of the culture was stored separately. The cell pellet of Ps. putida was washed twice with 1 mL of 50mM phosphate citrate buffer (pH 8.0).[46] Buffer wash of the culture was collected separately. The cell pellet obtained was resuspended in 1 mL of buffer. The cells were lysed using
Repeated batch degradation From Figure 1, it can be observed that Ca-alginate-immobilized Ps. putida could be reused without affecting its degradation efficiency of 100% up to five cycles. At the end of the 10th cycle, there was reduction in degradation efficiency to 92%. Further increase in the number of cycles resulted in a gradual decrease in degradation efficiency. By the end of the 50th cycle, 65% degradation efficiency was retained by Ca-alginate-immobilized Ps. putida. A cell leakage of 112 £ 103cfu mL¡1 was recorded at the end of the 50th cycle.
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Continuous degradation of chlorpyrifos As shown in Figure 2, Ps. putida was able to show 100% degradation with 2% concentration of pesticide upto 100 mL h¡1 flow rate (dt: 36 min). At a flow rate of 20 mL h¡1 (dt: 324 min) 100% degradation of chlorpyrifos was recorded up to 8% concentration of pesticide. At a flow rate of 40 mL h¡1 (dt: 252 min) Ps. putida could show 100% degradation up to 6% concentration of pesticide. With 10% concentration of chlorpyrifos at 20 mL h¡1 flow rate (dt: 324 min), 98% degradation was observed. At a flow rate of 100 mL h¡1 (dt: 36 min) with 10% concentration of chlorpyrifos, Ps. putida was still able to show 80% degradation of chlorpyrifos.
A chromatogram of chlorpyrifos standard is shown in Figure A2. The chromatograms of chlorpyrifos control and chlorpyrifos test are shown in Figures A3 and A4. The mass spectra of chlorpyrifos control (RT: 8.21–9.12) and that of chlorpyrifos test (RT: 8.22–8.96) are shown in Figures A5 and A6 respectively. The mass spectrum of chlorpyrifos test (RT: 5.6–6.7) is shown in Figure A7. The retention time for chlorpyrifos standard was found to be 8.4 (Fig. A2). Compared with the chromatogram of chlorpyrifos control (Fig. A3), the peak at retention time 8.54 corresponding to chlorpyrifos reduced by more than 60% in the chromatogram of chlorpyrifos test (Fig. A4). The diminished or reduced peak clearly indicates degradation of chlorpyrifos. The same can be observed from the respective m/z values of mass spectrum of chlorpyrifos control (RT: 8.21–9.12) (Fig. A5) and mass spectrum of chlorpyrifos test (RT: 8.22–8.96) (Fig. A6). Intensity change of chlorpyrifos can be observed from the chromatogram and m/z values at retention time 8.54 as listed in Table A1. In the mass spectrum of test sample (RT: 8.22–8.96) (Fig. A6), the m/z value (mass-to-charge ratio) of 350.76 is absent because of change in mass due to 100 95
Degradation (%)
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LC-MS analysis and identification of degradation products
2%
90
4% 6%
85
8% 10%
80 75
20
40
60
Flow rate (ml h-1)
80
100
Fig. 2. Continuous degradation of different concentrations of chlorpyrifos ranging from 2 to 10% by Ps. putida at different flow rates ranging from 20 to 100 mL h¡1; experiments were done in triplicates; error bars indicate §SD.
Fig. 3. Agarose gel electrophoresis of plasmid from Ps. putida subjected to curing. Note: lane 0: indicates control sample (not treated with ethidium bromide), lanes 1—6: indicate ethidium bromide-treated bands of plasmid extracted from day 1 to 6.
addition of oxon, which shows the confirmation of the predicted product. The base peak seen in the mass spectrum of chlorpyrifos control (RT: 8.21–9.12) (Fig. A5) is 172.23, and this is the most stable fragment. In the mass spectrum of chlorpyrifos test sample (RT: 8.22–8.96) (Fig. A6) the base peak is with the m/z value of 200.25, which indicates that addition of oxon takes place to chlorpyrifos, i.e. the original substrate molecule. Due to the influence of oxon, the base peak is of higher m/z value. In the chromatogram of the chlorpyrifos test sample (RT range: 5.6–6.7) (Fig. A4), the chromatogram is merged because of the presence of chlorpyrifos oxon, whereas in the control (Fig. A3), the chromatogram has distinguished peaks. This clearly indicates the presence of oxon in the test sample. The m/z values corresponding to the stable oxon (MW 335) molecule are seen in the MS of chlorpyrifos test sample (RT range: 5.6–6.7). The m/z values corresponding to another degradation product, 3,5,6-trichloro-2-pyridinol (TCP) (MW 197), are also reported in the RT range of 5.6– 6.7 in the mass spectrum of chlorpyrifos test sample (Fig. A7). TCP is comparatively more stable. Hence, chlorpyrifos oxon and TCP could be two predicted products of chlorpyrifos degradation. The m/z values of chlorpyrifos degradation products are listed in Table A2.
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Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida
351
Fig. 5. 1.5% Agarose gel electrophoresis of products of mpd PCR. Note: M: DNA molecular weight marker; 1: Ps. putida 2: no template control.
Plasmid isolation and plasmid curing As shown in Figure 3, Ps. putida had a single band of plasmid on plasmid isolation and gel electrophoresis. Curing experiment on Ps. putida was carried out to determine whether the chlorpyrifos degrading genes are present either on plasmid or chromosome. With increase in incubation period, the thickness of the plasmid band on agarose gel decreased. On day 4, the band thickness reduced and by day 5, the plasmid band disappeared (Fig. 3), indicating that the plasmid was cured. Plasmid DNA-cured cells of Ps. putida were able to grow on mineral salts medium supplemented with the pesticide as the sole source of carbon. This indicated that the genes responsible for the degradation of chlorpyrifos are present on the chromosome and not on the plasmid.
Genomic DNA isolation and PCR analysis Fig. 4. 0.5% Agarose gel electrophoresis of genomic DNA from Ps. putida.
Genomic DNA from Ps. putida degrading chlorpyrifos was isolated as shown in Figure 4. The culture was subjected to PCR analysis. The results of PCR analysis
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Fig. 6. Proposed pathway for degradation of chlorpyrifos.
indicate that a ~890-bp product expected for mpd gene was present in Ps. putida (Fig. 5). Enzymatic degradation of chlorpyrifos The results of the enzymatic degradation of chlorpyrifos with supernatant, buffer wash and cell lysate of Ps. putida are presented in Table 1. As seen in Table 1, a negligible degradation in chlorpyrifos was recorded with cell-free supernatant. A slightly higher degradation of chlorpyrifos was recorded with crude cell lysate, and the buffer wash of cells showed maximum degradation of chlorpyrifos. This indicated that the enzymes involved in the degradation of chlorpyrifos are membrane-bound. When buffer wash of Ps. putida was subjected to ammonium sulfate precipitation, there was an increase in chlorpyrifos degradation. Degradation of the pesticide increased further after dialysis of ammonium sulfate-precipitated sample (Table 2).
Discussion In the present investigation, Ps. putida was able to show 100% degradation with 2% concentration of chlorpyrifos up to 100 mL h¡1 flow rate. At a flow rate of 100 mL h¡1 with 10% concentration of chlorpyrifos, Ps. putida was still able to show 80% degradation. In similar studies, a tezontle-packed up-flow reactor (TPUFR) with an immobilized bacterial consortium for the biological treatment of methyl-parathion and tetrachlorvinphos was evaluated by Ya~ nez-Ocampo et al.[29] They could evaluate four flow rates (0.936, 1.41, 2.19 and 3.51 L h¡1) and four hydraulic residence time periods (0.313, 0.206, 0.133 and 0.083 h) in the reactor. With an operating time of 8 h and a flow of 0.936 L h¡1, they obtained 75% efficiency in the removal of methyl-parathion and tetrachlorvinphos. Calcium-alginate-immobilized cell systems were also developed by Ha et al.[28] for the detoxification and biodegradation of organophosphate insecticide, coumaphos, and its
Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida Table 1. Enzymatic degradation of chlorpyrifos with supernatant, buffer wash and cell lysate of Ps. Putida. Enzyme Fraction Supernatant Buffer wash Cell lysate
Degradation of Chlorpyrifos (%)
Protein Content (mg mL¡1)
7.16 § 0.76 30 § 2 15.33 § 1.52
0.23 § 0.026 0.426 § 0.032 0.756 § 0.03
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Note: Values are a mean of three replicates § SD.
hydrolysis products, chlorferon and diethlythiophosphate (DETP). In the current investigation, repeated batch degradation studies were conducted to observe the long-term stability of Ca-alginate-immobilized cells of Ps. putida. It was observed that Ca-alginate-immobilized Ps. putida could be reused without changing its degradation efficiency of 100% up to five cycles. Even after the 50th cycle, the degradation efficiency of 65% was recorded. A cell leakage of 112 c_ 103 cfu mL¡1 was recorded at the end of the 50th cycle from encapsulated beads with Ps. putida. In similar immobilization studies, a recombinant strain of Escherichia coli containing the opd gene for organophosphate hydrolase (OPH), capable of active hydrolysis of organophosphate neurotoxins, including chlorpyrifos, was cultivated by Kim et al.[50] They harvested and utilized the cells in lab-scale experiments in the form of either freely suspended cells in batch degradation or cells immobilized within a macroporous gel matrix, polyvinyl alcohol (PVA) cryogel. The immobilized cells retained their activity over a four-month period of use and storage, demonstrating both sustained catalytic activity and long-term mechanical stability. Similarly, Richins et al.[13] demonstrated that bi-functional fusion proteins comprising OPH moieties were highly effective in degrading organophosphate nerve agents. This enabled purification and immobilization of proteins on different cellulose materials in a single step. Repeated hydrolysis of paraoxon was achieved in an immobilized enzyme reactor with 100% degradation efficiency over 45 days. Table 2. Enzymatic degradation of chlorpyrifos by partially purified enzyme fractions. Enzyme Fraction Buffer wash of Ps. Putida After ammonium sulfate precipitation After dialysis
Degradation (%)
Protein Content (mg mL¡1)
30 § 2
0.926 § 0.032
42.66 § 1.52
0.753 § 0.03
50 § 2
0.666 § 0.015
Note: Values are a mean of three replicates § SD.
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In the present investigation, Ps. putida was efficient in degrading chlorpyrifos. Through LC-MS analysis, chlorpyrifos oxon and 3,5,6-trichloropyridinol were the predicted products of chlorpyrifos degradation by Ps. putida. It was predicted that chlorpyrifos is degraded through a hydrolytic degradation pathway. In similar studies, Singh et al.[37] investigated the ability of the strain Enterobacter strain B-14 to mineralize chlorpyrifos. Strain B-14 was able to degrade chlorpyrifos to DETP and TCP and utilized DETP as a sole source of carbon. Sasikala et al.[51] showed the presence of metabolites – chlopyrifos oxon and diethylphosphorothioate through LC-MS analysis. Based on our degradation studies and the degradation products predicted by LC-MS analysis, the predicted pathway of degradation of chlorpyrifos is shown in Figure 6. In our present investigation, growth of plasmid-cured Ps. putida was observed on mineral salts medium containing chlorpyrifos as a sole source of carbon. This indicated that genes responsible for the degradation of chlorpyrifos are present on the chromosome and not on the plasmid. Genetic studies based on plasmid curing and electroporation-mediated transformation on a chlorpyrifos degrading bacterium Ps. putida MAS-1 was performed by Ajaz et al.[52] It was observed that after curing with acridine orange, the property to grow on the nutrient agar containing 10 mg mL¡1 chlorpyrifos was lost by the bacterium. Isolation of plasmid bearing chlorpyrifos degrading determinants/genes was carried out. It was then transferred into E. coli DH5a. However, it was observed that the transformants could not resist and grow in the medium with chlorpyrifos. The conclusion drawn from the experiment was that degradation of chlorpyrifos by Ps. putida MAS-1 was due to the combined action of plasmid and chromosomal genes. In most of the studies, opd genes were found to be plasmid-based and had similar DNA sequences.[37] However, Horne et al.[53] isolated an opd gene from Agrobacterium radiobacter, which was located on the chromosome but had a similar sequence to the opd gene from another bacteria. In our present investigation, Ps. putida degrading chlorpyrifos was subjected to PCR analysis. A ~890-bp product expected for mpd gene involved in the degradation of chlorpyrifos was amplified from Ps. putida. In similar studies, an effective chlorpyrifos-degrading bacterium, named strain YC-1, was isolated from the sludge of the wastewater treating system of an organophosphorus pesticide manufacturer by Yang et al.[35] and the entire mpd functional gene of strain YC-1 was amplified by PCR with a pair of primers.[54] In the current study, higher enzymatic degradation of chlorpyrifos by Ps. putida was recorded with the buffer wash of cells compared with the crude cell lysate and the cell-free supernatant. This clearly indicated that the enzymes involved in the degradation of chlorpyrifos are membrane-bound. According to Steiert,[55] in the Flavobacterium spp. and P. diminuta the native Parathion hydrolase is membrane-bound, whereas in S. lividans, the
354 opd gene is expressed as a secreted soluble enzyme. However, when organophosphorus hydrolase activity of individual isolates and consortium were checked by Sasikala et al.[51] in the presence and absence of chlorpyrifos, the extracellular fractions failed to show enzyme activity.
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Conclusions Thus, it can be concluded that Ca-alginate-immobilized cells of Ps. putida were efficient in degrading chlorpyrifos, and the Ca-alginate-immobilized cells were stable for a long duration. Chlorpyrifos was degraded by hydrolytic pathway and chlorpyrifos oxon and 3,5,6-trichloro-3-pyridinol were the predicted products of degradation. Further, it was demonstrated that the genes involved in the degradation of chlorpyrifos were present on the chromosome and the enzymes involved in its degradation were membrane-bound.
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Appendix
Fig. A1. Reactor packed with Ca-alginate-immobilized Ps.
putida.
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Fig. A2. HPLC chromatogram of chlorpyrifos analytical standard.
Fig. A3. HPLC chromatogram of chlorpyrifos control; RT value (8.54) of chlorpyrifos is ticked.
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Fig. A4. HPLC chromatogram of chlorpyrifos test; RT value (8.53) of chlorpyrifos is ticked.
Fig. A5. Mass spectrum of chlorpyrifos control in the RT range: 8.21–9.12. Fragment ions corresponding to chlorpyrifos
based on the NIST spectrum of chlorpyrifos are ticked.
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Fig. A6. Mass spectrum of chlorpyrifos test in the RT range: 8.22–8.96; Fragment ions corresponding to chlorpyrifos based on the NIST spectrum of chlorpyrifos are ticked.
Fig. A7. Mass spectrum of chlorpyrifos test showing m/z values corresponding to TCP and chlorpyrifos oxon (RT: 5.6–6.7) according to NIST standards. The fragment ions of TCP and chlorpyrifos oxon are ticked. TCP and chlorpyrifos oxon are the predicted products of chlorpyrifos degradation.
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Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida
Fig. A8. (a) NIST Mass spectra of chlorpyrifos (contributor: Mass Spectrometry Center, University of Utah EPA-PTSEL
2900), (b) NIST mass spectra of TCP (contributor: NIST Mass Spectrometry Data Center, 1990), (c) NIST mass spectra of chlorpyrifos oxon (contributor: NIST Mass Spectrometry Data Center, 1990).
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Table A1. m/z values of chlorpyrifos in the mass spectrum of chlorpyrifos control and test. M/z values corresponding to chlorpyrifos (according to the NIST standard – Figure A8 (a) in the mass spectrum of chlorpyrifos control RT: 8.21–9.12 (Fig. A5) 350.76, 314.09, 212.76, 197.25, 125.09, 97.74
M/z values corresponding to chlorpyrifos (according to the NIST standard – Figure A8(a) in the mass spectrum of chlorpyrifos test RT: 8.22–8.96 (Fig. A6) 314.10, 212.66, 197.37, 125.98, 97.27
Table A2. m/z values of products of chlorpyrifos degradation in the mass spectrum of chlorpyrifos test (RT: 5.6–6.7). M/z values of chlorpyrifos degradation products in the mass spectrum of chlorpyrifos test sample (RT: 5.6–6.7) (Fig. A7) M/z values corresponding to TCP (according to the NIST standard of TCP – Figure A8 (b))
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60.10, 98.25, 107.31, 169.37, 197.27, 199.39, 200.59
M/z values corresponding to chlorpyrifos oxon (according to the NIST standard of chlorpyrifos oxon – Figure A8 (c)) 335, 333.34, 298.44, 226.68, 226.00, 200.59, 199.39, 169.37
