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Sep 14, 2011 - Abstract Organophosphorous pesticides are widely used in agriculture to control major insect pests. Chlorpyrifos is one of the major ...
World J Microbiol Biotechnol (2012) 28:1301–1308 DOI 10.1007/s11274-011-0879-z

SHORT COMMUNICATION

Biodegradation of chlorpyrifos by bacterial consortium isolated from agriculture soil Chitrambalam Sasikala • Sonia Jiwal Pallabi Rout • Mohandass Ramya



Received: 14 December 2010 / Accepted: 31 August 2011 / Published online: 14 September 2011 Ó Springer Science+Business Media B.V. 2011

Abstract Organophosphorous pesticides are widely used in agriculture to control major insect pests. Chlorpyrifos is one of the major organophosphorous pesticides which is used to control insects including termites, beetles. The widespread use of these pesticides is hazardous to the environment and also toxic to mammals, thus it is essential to remove the same from the environment. From the chlorpyrifos contaminated soil nine morphologically different bacterial strains, one actinomycete and two fungal strains were isolated. Among those isolates four bacterial strains which were more efficient were developed as consortium. The four bacterial isolates namely Pseudomonas putida (NII 1117), Klebsiella sp., (NII 1118), Pseudomonas stutzeri (NII 1119), Pseudomonas aeruginosa (NII 1120) present in the consortia were identified on the basis of 16S rDNA analysis. The intracellular fractions of the consortium exhibited more organophosphorus hydrolase activity (0.171 ± 0.003 U/mL/min). The degradation studies were carried out at neutral pH and temperature 37°C with chlorpyrifos concentration 500 mg L-1. LC-mass spectral analysis showed the presence of metabolites chlopyrifosoxon and Diethylphosphorothioate. These results highlight an important potential use of this consortium for the cleanup of chlorpyrifos contaminated pesticide waste in the environment.

C. Sasikala  M. Ramya (&) Department of Genetic Engineering, School of Bioengineering, SRM University, Kattankulathur 603203, India e-mail: [email protected] S. Jiwal  P. Rout Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur 603203, India

Keywords Biodegradation  Organophosphorous pesticides  Chlorpyrifos  Chlorpyrifos oxon Abbreviations CP Chlorpyrifos TCP 3,5,6-trichloro-2-pyridinol OPH Organophosphorous hydrolase OPs Organophosphates MSM Minimal salt medium

Introduction Pesticides constitute the key control strategy for crop disease and pest management and have been making significant contribution in India towards improving the crop yield per hectare. However, the widespread use of these pesticides has resulted in the problems caused by their interaction with the biological systems in the environment (Singh and Walker 2006) as the residues of applied pesticides stay in the environment for variable periods of time. Organophosphates (OPs) pesticide account for about 38% of total pesticides used globally. OPs are a group of highly toxic chemicals that exhibit broad-spectrum activity against insects and are widely used against major agricultural pests. However, continuous and excessive use of OPs has also caused not only nerve (this class of pesticide has acute neurotoxicity due to their ability to suppress acetylcholine-esterase) and muscular diseases in human and animals but contamination of ecosystems in different parts of the world (Zhang et al. 2008). Chlorpyrifos (O, O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) is a broad spectrum moderately toxic

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organophosphorous insecticide (Xu et al. 2008). It is a broad-spectrum organophosphate insecticide and acaricide, and is widely used for pest control on grain, cotton, fruit, and vegetable crops, as well as, lawns and ornamental plants (Fang et al. 2006). It has high soil-absorption coefficient, but low water solubility (2mgL-1) (Racke 1993). The environmental fate of chlorpyrifos has been studied extensively and its degradation may involve a combination of photolysis, chemical hydrolysis and microbial degradation (Xu et al. 2008). Chlorpyrifos was resistant to biodegradation and remained effective for up to 5–17 years (Baskaran et al. 1999). It was suggested that the accumulation of 3,5,6-trichloro-2-pyridinol (TCP) which is the hydrolytic product of chlorpyrifos has anti-microbial properties and this prevents the proliferation of CPdegrading microorganisms (Racke 1993). Bioremediation is considered as an efficient and cheap biotechnological approach to clean up the polluted environment (Xu et al. 2008; Karpouzas and Singh 2006). A chlorpyrifos-degrading bacterium Bacillus pumilus strain has been recently isolated from soil for bioremediation purpose (Anwar et al. 2009). Chlorpyrifos was reported to be co-metabolically degraded in liquid media by the pure cultures of Flavobacterium sp. (Mallick et al. 1999) and Escherichia coli clone (Richnis et al. 1997). However, these organisms do not utilize chlorpyrifos as an energy source. Kulshrestha and Kumari (2011) reported the pure fungal strain, Acremonium sp., utilized chlorpyrifos (83.9%) as a source of carbon and nitrogen. Similarly two soil fungal isolates Aspergillus niger and Trichoderma viride were evaluated for the degradation of chlorpyrifos (Mukherjee and Gopal 1996). A blue green microalga was reported to degrade 80 ppm of chlorpyrifos using alkaline phosphatase as an enzyme source (Thengodkar and Sivakami 2010). Phytoremediation using hairy root Chenopodium amaranticolor showed accelerated process of biodegradation and metabolized chlorpyrifos to polar products (Garg et al. 2010). However fewer studies were there on the degradation of Chlorpyrfos using bacterial consortium. In the present study, a consortium consist of Klebsiella sp., Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas putida was isolated from chlorpyrifos contaminated agricultural soil. Aim of this work was to study the possible application of isolated consortia in the degradation of chlorpyrifos.

Materials and methods Chemicals The pesticide used in the present study was commercialgrade insecticide chlorpyrifos (50% E.C, Dow AgroSciences

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India Private Limited) obtained from the local market (Chennai, TamilNadu, India). All other chemicals and reagents used in this present study were of analytical grade and purchased from Hi-Media Pvt Ltd Mumbai, India. Enrichment, isolation and selection of bacterial strains Soil sample was collected from paddy field of Kancheepuram district (Walajabad), Tamilnadu, India, where chlopyrifos is sprayed extensively. Soil sample was stored at 20°C for further use. Soil enrichment was carried out in Minimal Salt Medium (pH 7.0) containing (gL-1) K2HPO4, 1.5; KH2PO4, 0.5; (NH4)2SO4, 0.5; NaCl, 0.5; MgSO4, 0.2; CaCl2, 0.05; FeSO4, 0.02. Kenknight and Munaier’s media (pH7.2) containing (gL-1) KH2PO4,0.1 g; MgSO4, 0.1; NaNO30.1; KCl, 0.1; Dextrose,10; Agar,15 was used for the isolation of actinomycetes. Nutrient agar and potato agar medium was used for the isolation of bacteria and fungi. About 1 g of soil was added to an Erlenmeyer flask (250 mL) containing 100 mL MSM supplemented with chlorpyrifos (200mgL-1) as the sole carbon source and incubated at 37°C on a rotary shaker at 120 rpm for 7 days. After 7 days 5 mL culture was recovered from each replicate and transferred to fresh MSM containing chlorpyrifos as the only carbon source and incubated for 7 days. One week following the last transfer, 10 fold dilutions of cultures was prepared and 100lL of each dilution was spreaded on nutrient agar plates, Kenknight and Munaier’s media and potato dextrose agar containing 200 mgL-1 Chlopyrifos. Isolated colonies were purified by repeated streaking. After purification all isolates were tested for growth and chlorpyrifos utilization by inoculating them in MSM containing chlorpyrifos (200mgL-1) as sole carbon and energy source. Taxonomic identification of the isolates Total genomic DNA was extracted from the bacterial strains and the actinomycete as described by Wilson 1990. The 16S rDNA gene was amplified using the universal primers 8f (CACGGATCCAGACTTTGATYMTGGCTC AG, forward) and 1512r (50GTGAAGC TTACGGYT AGCTTGTTACGACTT-30, reverse) (Weisberg et al. 1991). The PCR reaction was performed in a Thermocycler (Mastercycler gradient, Eppendorf, USA) with the following cyclic profile (Initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min and final extension at 72°C for 10 min Cho et al. 2009). The PCR products consisting of the amplified 16S rDNA gene fragment were purified with EZ-10 Spin Column PCR Products Purification Kit (Biobasic Inc., USA) and sequenced using an Automated DNA sequencer AB1 3130

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XL (Gentic Analyser, Applied Biosystems, USA). The determined sequence was compared with those available in the GenBank database using the NCBI BLAST pro gram. (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The nucleotide sequence analyses of the sequences were performed at BlastN site at NCBI server (http://www.ncbi.nlm.nih. gov/BLAST). Fungal strains were identified by staining with methylene blue followed by microscopic observation. Assay for organophosphorus hydrolase The isolated bacterial strains and consortium were cultured separately in LB medium overnight with chlorpyrifos (100 mg/L). The cells were harvested by centrifugation at 8,000 rpm for 10 min and resuspended in 50 mmol/L Tris Cl (pH 8.0) buffer. The cells were disrupted by sonication for 10 times for a period of 10 s at 15 s interval and the crude cell lysate and extracellular supernatant used for enzymatic analysis. Cells grown without chlorpyrifos were used as uninduced controls. The assay was performed according to Mulbry and Karns 1989. 100 lL of enzyme solution was added to 900 lL of 50 mmol/L Tris Cl (pH 9.0) assay buffer containing 5 lL of 10 mg/mL methyl parathion as a substrate, incubated at 37°C for 10 min. The reaction was terminated by addition of 1 mL of 10% CCl3COOH (Trichloroacetic acid). And then 1 mL of 10%Na2CO3 was added for displaying color. The absorbance was measured at 410 nm. According to the amount of p-Nitrophenol (PNP) the product of hydrolysis, enzyme activity can be calculated. One unit of Organophosphorus activity is defined as, the amount of enzyme liberating 1 lmol of p-Nitrophenol per minute at 37°C. Biodegradation of chlorpyrifos Shake flask studies were carried to work out the chlorpyrifos degrading capacity of the isolated strains. Seed culture of each isolated strains were grown in nutrient broth containing chlorpyrifos (500 mg/L). Following 24 h of incubation, 1% inoculum of the cultures were inoculated in MSM (200 mL) containing 500 mg/L chlorpyrifos and incubated at 37°C and 150 rpm on a rotary shaker. MSM flask without inoculum was kept as control. Extraction of samples (pesticide residues) for LC–MS analyses Samples were recovered from culture flasks at respective time intervals (0, 15, 20 and 30 days) and centrifuged at 7,2009g for 10 min to obtain cell free medium. The supernatant thus obtained was added to the separating funnel and chlorpyrifos residues were extracted from

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supernatant using equal volume of dichloromethane (Anwar et al. 2009). After partitioning, organic layer of dichloromethane was evaporated by solvent evaporator to obtain a powdery residue of the organic compound. The residues were dissolved in HPLC grade acetonitrile (1 mL) and analyzed in LC–MS (SHIMADZU) as per the following conditions: column: Atlantis C18 (150 9 4.6 mm), detector: programmable variable wavelength UV detector, flow rate: 1 mL/min, mobile phase: acetonitrile ? water (90:10), injection volume: 20lL chlorpyrifos was detected with a programmable variable-wavelength UV detector at 254 nm. The retention time for chlorpyrifos was found to be 18.57 min. The Mass spectroscopy (MS) was performed using a Finnegan model MS (Themo electron corporation, USA). The ion trap detector with atmospheric pressure electro-spray ionization (API-ESI) source was used for quantification in negative ionization mode. The operating conditions were Spray Voltage (kV): 5.02, Capillary Voltage (V):16.96 Capillary Temperature (°C): 275.00, Dynode (kV): -14.86, Multiplier (V):821.20. Soil microcosm studies 100 g of finely grounded agricultural soil was added to glass petriplates. Chlorpyrifos (500 mg/kg) was added to the soil and the soil was thoroughly mixed to ensure uniform concentration of chlorpyrifos. Bacterial isolates were grown in nutrient broth medium. Following 24 h incubation, the cells were harvested by centrifugation at 4,6009g for 5 min (Anwar et al. 2009) and resuspended in MSM. Colony forming units of this suspension were quantified by the dilution plate count technique. The cells were inoculated to the contaminated soil at a concentration of 2 9 108cfu/g and incubated at 37°C for 20 days (Kumar et al. 2008). Soil sample (25 g) was recovered and extracted with acetonitrile: water (90:10, v/v; 25 mL) (Singh et al. 2004). The extract was analyzed by LC–MS.

Results Isolation and characterization of chlorpyrifos degrading bacteria From the enrichment culture, 9 morphologically different bacterial strains, two fungal and one actinomycete strains were isolated. These strains were further purified and checked for their chlorpyrifos utilization ability by inoculating them in minimal salt medium containing chlorpyrifos (200 mgL-1) as sole carbon and energy source. After a week of incubation, culture samples were extracted and

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analysed by HPLC. The obtained colonies were purified and subjected to molecular identification. Identification of the isolates The nine bacterial isolates and one actinomycete strain was identified by 16S rDNA gene amplification using thermocycler and were sequenced. Sequence analysis of 16S rDNA gene using BLAST showed that the bacterial strains were Staphylococcus sp., Micrococcus sp., Rhizobium sp., Comamonas aquatica, Staphylococcus hominis, Klebsiella sp., Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas putida, and actinomycetes strain was Streptomyces radiopugnans. The fungal strains were identified as Aspergillus niger, Tricophyton sps. The percentage of chlorpyrifos degradation was represented in Table 1. Four bacterial strains namely Klebsiella sp., Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas putida which showed highest degradation ability ([40%) were chosen and tested further. The sequences of four isolates were submitted to genbank and the corresponding genbank accession numbers are HM135446, HM135447, HM135448 and HM135449. These strains were deposited at National Institute for Interdisciplinary Science and Technology (CSIR), Trivandrum, India with the following deposition numbers Pseudomonas putida (NII 1117), Klebsiella sp., (NII 1118), Pseudomonas stutzeri (NII 1119), Pseudomonas aeruginosa (NII 1120). Organophosphorus hydrolase analysis Organophosphorus hydrolase activity of individual isolates and consortium were checked in the presence and absence

of chlorpyrifos. The extracellular fractions failed to show the enzyme activity. Table 2 depicts the intracellular enzyme activity of individual isolates and consortium. The developed consortium exhibited more amount of enzyme activity in the induced condition (0.171 ± 0.003U/mL) than the uninduced condition (0.127 ± 0.004 U/mL/min). Biodegradation studies The dichloromethane extracted samples were analyzed by liquid chromatography. As shown in Fig. 1a, the chlorpyrifos sample at 0 h showed a major peak with the retention time of 18.57 min (m/z 349.97) (Fig. 2a). After 15 days of incubation the sample was subjected to similar studies, one new peak was observed while other peaks disappeared at a retention time of 15.78 min (Fig. 1b) with the m/z value of 340.32 (Fig. 2b). This mass value is similar to the mass value of chlorpyrifos-oxon [O, O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate] (Tang et al. 2001). When the spectrum was taken for the same sample after 20 days, it was quite interesting to note that the peak at m/z 340.32 was disappeared instead new peaks with m/z 279.06 and 350.04 (Fig. 2c) at the retention time of 19.68 and 20.79 min (Fig. 1c) were observed. Comparing this spectrum with the spectrum at 0th hour shows that the chlorpyrifos is still present in the medium, since still a peak at m/z ratio of 350.04 is visible in the spectrum. At the end of 30 days, the formation of diethylphosphorothioate at retention time 10.43 min (Fig. 1d) with m/z 157.13 (Fig. 2d) was observed, another peak with m/z 352.29 at the retention time of 23.36 min was observed in the same spectrum. The metabolites found during chlorpyrifos degradation were listed in (Table 3). Comparing this spectrum with the spectrum at 0th hour showed that the chlorpyrifos

Table 1 Chlorpyrifos degradation efficiency of bacterial, fungal and actinomycetes strains obtained by enrichment culture S. no

Organisms

% of degradation of chlorpyriofs

Table 2 Intracellular organophosphorus hydrolase activity of individual isolates and the consortium

1

Staphylococcus sp

18 ± 0.9

Micrococcus sp.,

13 ± 1.1

S. no

Organism

2 3

Rhizobium sp.,

24 ± 1.3

Uninduced enzyme (unit/mL/min)

Induced enzyme (unit/mL/min)

4

Comamonas aquatica

21 ± 1.5

1

0.128 ± 0.004*

Staphylococcus hominis

23 ± 2.1

Pseudomonas aeroginosa

0.072 ± 0.008***

5 6

Pseudomonas aeroginosa

42 ± 2.4

2

Pseudomonas putida

0.093 ± 0.004***

0.129 ± 0.005***

7

Pseudomonas putida

45 ± 2.1

3

0.085 ± 0.005*

Pseudomonas stutzeri

50 ± 1.2

Pseudomonas stutzeri

0.066 ± 0.005*

8 9

Klebsiella sp.

41 ± 2.3

4

Klebsiella sp.

0.117 ± 0.005*

0.133 ± 0.008***

10

Aspergillus niger

15 ± 1.2

5

Consortium

0.127 ± 0.004*

0.171 ± 0.003***

11 12

Tricophyton sps Streptomyces radiopugnans

17 ± 1.1 25 ± 0.6

Values are mean of three experiments (±) SEM

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Values are mean of three experiments (±) SEM Significantly different from control cells at * P \ 0.05, *** P \0.001 by t test, Oneway analysis of variance (ANOVA) with Turkey–Kramer comparison test

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Fig. 1 Chromatogram of the degradation study of chlorpyrifos. a 0 h. b 15 days. c 20 days. d 30 days. e 20 days (soil sample)

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was still present in the medium but the percentage of degradation was found to be 70%. It is well established that the most common pathway of hydrolytic degradation of chlorpyrifos involves formation of 3,5,6-trichloro-2-pyridinol which is accelerated under alkaline conditions therefore in the present study we investigated the formation of TCP in soil. Agricultural soil contaminated with chlorpyrifos (500 mg/kg) was extracted after 20 days of incubation. It was found that the consortium formed 3,5,6trichloro-2-pyridinol in soil and degraded 65.87% of Fig. 2 Mass spectra of dichloromethane extract of the culture filtrate at different time intervals. a 0 h. b 15 days. c 20 days. d 30 days. e 20 days (soil sample)

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chlorpyrifos. Under the conditions described above, the retention time for 3,5,6-trichloro-2-pyridinol by LC–MS was about 14.44 min (Fig. 1e) and m/z 201.12 (Fig. 2e).

Discussion In order to isolate potential microorganisms to degrade chlorpyrifos, bacterial consortium was developed from agricultural soil by selective enrichment technique by

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Fig. 2 continued

Table 3 showing the metabolites of chlorpyrifos degradation S. no

Time (days)

Retention time (min)

m/z

Chemical name

1

15

15.78

340.32

Chlorpyrifos-oxon

2

30

10.43

157.13

Diethylphosphorothioate

3

20 soil microcosm

14.44

201.12

3,5,6-trichloro-2pyridinol

providing chlorpyrifos as a sole source of carbon. The number of isolates was lesser in the soil taken for the study. It might be due to the inhibitory effect of chlorpyrifos on the soil microbial population (Chu et al. 2008). Degradation of chlorpyrifos by consortium could be due to the synergistic effect of various bacterial isolates. In the present study the four bacterial isolates namely Pseudomonas putida (NII 1117), Klebsiella sp., (NII 1118), Pseudomonas stutzeri (NII 1119), Pseudomonas aeruginosa (NII 1120) present in the consortia were identified on

the basis of morphological characteristics and 16S rDNA analysis. Previously, a chlorpyrifos degrading bacterial strain Klebsiella sp. was isolated. The isolate was able to utilize chlorpyrifos as a sole source of carbon (Ghanem et al. 2007). A bacterial consortium was developed from pesticide-contaminated soil of Punjab consisting of Pseudomonas aeruginosa, Bacillus cereus, Klebsiella sp and Serratia marscecens, that could successfully degrade chlorpyrifos (Lakshmi et al. 2009). In the present study it was found that the consortium was able to degrade chlorpyrifos in the medium and also was able to in situ bioremediate agricultural soil contaminated with chlorpyrifos. After 21 days of incubation it was found that the consortium formed 3,5,6-trichloro-2-pyridinol in soil and degraded 65.87% of chlorpyrifos. After 30 days of incubation, it was found that the consortium degraded 70.54% of chlorpyrifos in the medium. In medium and soil the products diethylthiophosphorothioate and 3,5,6-trichloro-2-pyridinol was formed as a result of the hydrolysis of the phosphoester linkage in chlorpyrifos. Moreover,

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chlorpyrifos-oxon may be formed as a result of the desulfuration of chlorpyrifos. The degradation mechanism might have involved an inducible enzyme chlorpyrifos hydrolase, an organophosphorus ester-hydrolyzing enzyme, to hydrolyze chlorpyrifos to a non-toxic metabolite (Xu et al. 2008). These results highlight an important potential use of this consortium for the cleanup of contaminated pesticide waste in the environment. Characterizing the pathway of degradation and identifying the genes and enzymes involved in the process represent areas of further investigation. Acknowledgments The authors are thankful to Dr. T. Kumar Mass spectra analyst for the interpretation of mass spectra results and the facilities provided by SRM University to support this work.

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