INTRODUCTION:

For decades, the use of pesticides to increase food crops’ protection and production has been the major breakthrough in agricultural sector. The leading pesticides used in India include monochrotophos (10700 Million Tonnes (MT)–highest consumed), acephate (6400 MT), endosulfan (5600 MT) and chlorpyrifos (5000 MT–fourth highest consumed)¹. The diversified application of highly toxic organophosphorous pesticides such as parathion, methamidophos and chlorpyrifos has a profound effect on the ecological niche affecting the non-target microbial flora and fauna².

However, the extensive use of chlorpyrifos contaminates air, ground water, river and lake by releasing chlorpyrifos-oxon and 3,5,6–trichloro-2-pyridinol (TCP), the two potential transformation products showing deleterious effect on beneficial arthropods, fishes, birds and plants. It is noteworthy that there have been reports of delayed seedling, fruit deformities and abnormal cell division in plants upon prolonged exposure to chlorpyrifos³. Furthermore, this pesticide easily enters the human food chain and affects the normal functioning of central nervous system by blocking the acetylcholinesterase enzyme thereby shutting down neurotransmission. Human birth defects with impairment of male reproductive system have also been associated with exposure to chlorpyrifos and its products (NCAP 2000). There is an increased awareness over their persistence in the form of residue in food grains, dairy products, vegetables, fruits and in the environment, wobbling the soil microbiologists and environmentalists to adapt strategies for their judicial use accounting for bioremediation. Even though pesticide can be degraded by biotic and abiotic pathways; biodegradation by microorganism is the primary mechanism of pesticide breakdown and detoxification in many soils. Recently, microbiological transformation is preferred wherein various microbes are employed to effectively mineralize the chemical pesticides into a less harmful and non-toxic metabolites despite other treatment methods. Soil microbes are considered the most potent in decomposing various xenobiotic pesticides either completely or partially⁴. The main objective of the present study focuses on the isolation and identification of Pseudomonas sp. from agricultural field and to assess the time bound transformation potential of chlorpyrifos at different concentrations.

MATERIALS AND METHODS:

Sample collection:

The soil samples collected from a cultivated paddy field exposed to an organo-phosphorous insecticide at FIPPAT (Padappai) Kancheepuram District, Tamilnadu were used for biodegradation experiments. In the study site, soil samples were collected randomly from 10 to 15 cm depth from four corners and from the centre as well. The soil samples were thoroughly mixed, sieved and collected in polythene bags. The samples were transferred to the laboratory within 4 hours and processed.

Screening of potential bacterium degrading bacteria:

Chlorpyrifos (technical grade purchased from local supplier) was used throughout the experiment, provided the key component in it interacts with the micro organisms as in the environment. The heterotrophic microbial load was enumerated by dilution plate method using 10^-4 and 10^-5 as inoculum and CFU / mL was calculated. The isolates were then screened for insecticide tolerance by the enrichment culture technique on nutrient agar medium⁵ and their cultural characteristics studied using standard method⁶ and maintained in Nutrient agar (Hi Media, India) slants at 4ºC.

Analysis of degradation:

A loopful of the bacterial culture was inoculated separately into Kings B medium (Hi Media, India) incubated at 30ºC overnight in a shaker at 140 rpm and centrifuged. The pelleted cells were inoculated into a freshly prepared media and turbidity adjusted to ~0.5 McFarland standard. Chlorpyrifos dilutions of various concentrations (25, 50, 100 and 200 ppm) were prepared using sterile distilled water, added to the broth culture and incubated in a shaker at 30ºC. An aliquot of the samples was withdrawn at different time interval viz. 24, 48, 72 h upto 240 h measuring the optical density using a UV-spectrophotometer (UV-1601, Shimadzu) until a constant λ_max for all time periods and validated.

Extraction and concentration:

The extraction procedure was carried upon by the addition of ethyl acetate and sodium chloride (3:1) to the broth culture and incubated in a shaker for 24 h. The upper ethyl acetate layer was collected in a small vial prior incubation and concentrated using nitrogen gas. It was further characterized by performing High Performance thin layer chromatography (HPTLC) on Silica gel 60 F₂₅₄plate using n-hexane and acetone (7:3) as the mobile phase. The plates were developed in a saturation twin trough chamber and detected on exposure to UV light. Quantitative evaluation was performed with TLC scanner 2 using CAMAG software².

RESULTS AND DISCUSSION:

Isolation and Identification:

Of the 55 isolates, Pseudomonas aeruginosa with its characteristic bluish green color pigment on Kings B medium (Table 1) constituted the major soil isolate (n = 8) involved in pesticide degradation followed by Rhizobium sp. (n=5), Azotobacter sp., Bacillus sp. (n = 4), Serratia sp. (n=2), Micrococcus and E. coli. Fungi such as Aspergillus niger, Rhizopus sp., Fusarium sp. and Penicillium sp. have also been identified along with bacteria. The heterotrophic bacterial load was enumerated using a colony counter and an estimate of 1.05x10¹¹CFU / mL was determined. All the isolates were Gram negative organisms that were involved in the process of pesticide depletion. The isolate Pseudomonas aeruginosa was designated PF1 based on significant growth in chlorpyrifos incorporated nutrient agar medium with a distinct zone around the streaked area upto 200ppm concentration⁷. The spectrophotometric analysis of the chlorpyrifos degradation showed a distinct peak with its maximum absorbance at 261.8 nm.

Table 1 showing the cultural characteristics of the potential strain Pseudomonas aeruginosa

Biochemical test	Result
Gram staining	Gram -ve rods
Motility	Actively motile
Indole	̶
Methyl red test	̶
Vogues-Proskauer test	̶
Citrate	+
Catalase	+
Oxidase	+
Triple Sugar Iron Agar	K/K No gas, No H₂S
Urease test	̶
Carbohydrate fermentation
a. Maltose	̶
b. Mannitol	̶
c. Glucose	̶
d. Lactose	̶

Bio-degradation profile:

The bio-degradation of chlorpyrifos in nutrient broth at different concentrations viz. 25, 50, 100, 200ppm using Pseudomonas aeruginosa PF1 showed 48 per cent degradation of chlorpyrifos at 25ppm within 24 h and continued upon increase in the incubation period. Figure 1 clearly elucidates the correlation between the concentration of chlorpyrifos and time taken for degradation wherein a significant decline in the concentration of chlorpyrifos was observed till 48 h. There was no catabolic activity in 72 h and 120 h of incubation showing only 83 and 84 per cent (1 per cent difference) degradation respectively. After prolonged incubation up to 240 h, an overall 94 per cent in catabolic activity with little or no change observed thereafter.

Fig. 1: Bio-degradation of Chlorpyrifos by P. aeruginosa at 25 ppm concentration

The degradation pattern of chlorpyrifos at 50ppm concentration showed an active catabolic activity with 44 and 61 per cent degradation in 24 and 48 h respectively (fig. 2). The stationary phase could be observed during 72 h with 66 per cent as a limiting factor. Further incubation resulted in 84 per cent degradation till 240 h and remained unchanged thereafter. Similarly at 100ppm concentration (fig. 3), there was a gradual increase in the degradation pattern with 29, 47 and 53 per cent at 24, 48 and 72 h respectively followed by assimilatory phase. On further incubating till 120 h, there was a sudden rise in catabolic activity with 78 per cent degradation followed by 84 per cent degradation observed during 240 h.

The degradation pattern of chlorpyrifos at 200ppm concentration was clearly shown in fig. 4. This also follows the same trend where degradation could well be appreciated during the initial stages of incubation with 27, 39, 43, 45 and 50 per cent catabolic activity at 24, 48, 72, 120 and 240 h. In this case, 50 per cent degradation was observed only during 240 h whereas at lower concentrations 50 per cent degradation was observed within 72 h of incubation. The increase in concentration of the chlorpyrifos in the media was found directly proportional to the increase in time taken by the isolate for degradation. Various bacterial and fungal species have been reported to be able to grow on diazinon, chlorpyrifos or malathion⁸. Bhagobaty and Malik ⁹ isolated four bacteria from the soil that were able to grow in 1.6 g/L chlorpyrifos among which the most efficient bacterium belong to Pseudomonas sp. which corroborates with our current study.

Fig. 2: Bio-degradation of Chlorpyrifos by P. aeruginosa at 50 ppm concentration

Fig. 3: Bio-degradation of Chlorpyrifos by P. aeruginosa at 100 ppm concentration

HPTLC pattern:

The HPTLC profile of product I (24 h) showed four spots with different R_f values as shown in chart 1. The spots due to negative control were nullified by comparing with peaks in chart 4. (Blank with Pseudomonas species) spot 1 and 2 in product and control were alike and nullified. The remaining three spots were found to be different on comparing with the standard chlorpyrifos as seen from their R_f values and λ_max values. Hence there is a possible biotransformation or biodegradation of chlorpyrifos even though the transformation is not selective. On comparing with the standard, the HPTLC pattern of chlorpyrifos showed two spots the peak at R_f value 0.67. Pseudomonas species was found to be more effective for the effective biotransformation of chlorpyrifos at a concentration of 25ppm within 24 h.

The HPTLC profile of product 2 showed seven spots with different R_f values as shown in chart 2. The spots in negative control and peaks in chart 2 were compared. They are different from their R_f values and maximum wavelength when compared with standard (chart 3) and control (chart 4). The seven spots were found to be different on comparing with standard chlorpyrifos seen in the chart 3. Hence, it was said to be a bio-transformed product. The product obtained from sample 2 does not match with the R_fvalue of standard chlorpyrifos.

The R_f value of Chlorpyrifos was found to be 0.67 as per the literature. The formulated compound chlorpyrifos R_f was compared with the R_f obtained by Ugbeye et al ¹⁰ which was found to be 0.67. Pseudomonas species was found to be more effective in the degradation of chlorpyrifos in 48 h of 25 ppm concentration.

In general, microorganisms have considerable capacity in metabolising the pesticides by various processes such as reduction, oxidation, acetylation, methylation, hydrolysis and biological processes like anaerobic and chemilithotrophic metabolism. Studies conducted in soil have generally reported significantly longer dissipation half–lives under sterilized versus natural conditions and led to the conclusion that microbial activities are important in the degradation of chlorpyrifos in soil¹¹.

However, the most important microbial role in the chlorpyrifos degradation pathway may be further metabolism and mineralization of 3,5,6-trichloro-2-pyrinidinol (TCP) and 3,5,6-trichloro-2-methoxypyridine (TMP) metabolites (Racke 1993). For instance, organophosphorus hydrolase (OPH) one of the important hydrolytic enzymes in detoxification process was found to hydrolyze P–O, P–F and P–S bonds¹². This enzyme was first isolated from Pseudomonas diminuta MG 42 which had the ability to hydrolyse a wide range of organophosphorus compounds¹³. With the advent of synthetic biotechnology, engineering of microbial cells with bioremediation capabilities has been the most sought after tool. Yang et al.¹⁴ engineered P. putida JS444 with altered specificity of MPH enhance the degradation of chlorpyrifos. Thus, microbial assisted biodegradation remains to be a viable option for cleaning up the contaminated sites with its eco-friendliness, high efficiency and cost-effectiveness.

Fig. 4: Bio-degradation of Chlorpyrifos by P. aeruginosa at 200 ppm concentration

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Received on 03.11.2017 Modified on 07.12.2017

Research J. Pharm. and Tech 2018; 11(5):1725-1728.

DOI: 10.5958/0974-360X.2018.00320.7