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Biodegradation of Methyl Parathion Using Pseudomonas stutzeri (MTCC 2643)
Pesticides applied in agricultural fields for crop protection result in the contamination of the environment. They also affect the flora and fauna as well as the quality of air, water and soil. Hence their remediation is of concern. Among different strategies available, microbial remediation is cost effective and ecofriendly. The present work is designed to test the efficiency of Pseudomonas stutzeri, a bacterial strain obtained from MTCC, IMTECH, Chandigarh, India. After treating with 50, 100, 150 and 200 ppm of methyl parathion, parameters like, orthophosphate released, pH and turbidity changes were monitored upto thirty hours. Degradation of 200 ppm concentration was confirmed by UV-Visible spectrophotometry and HPLC analysis. This strain effectively degraded methyl parathion and it can be used in the remediation of contaminated agricultural fields.
Increase in population and speedy development of industrialization cause accretion of extensive assortment of chemicals in the environment. Thus, there is a need for remarkable effort to develop new technologies to reduce or remove these toxic chemical components from their accumulated area 1. Management strategies like land-filling, recycling, pyrolysis and incineration are very much effective in remediation of polluted soils. At the same time, they also cause harmful effects on the environment by leading to the development of toxic intermediates. Moreover, these methods are more luxurious and sometimes it is very difficult to implement these methods especially in large agricultural fields for example in pesticide polluted regions 2, 3, 4, 5.
Bioremediation is one of the most promising strategies which utilizes the microorganisms for the removal of pesticides from the polluted areas. It is an alternative treatment method which is cheaper, economic, flexible and environmental friendly in nature. Subsequently, it was noticed that microbes have the capacity to convert and/or degrade the xenobiotics 6, 7, 8, 9. Researchers isolated a variety of microorganisms from contaminated environment with the ability of degrading pollutants. Therefore, more research investigations have been carried out in biotransformation of organic pollutants in nature to empathize microbial environment, physiology and evolution with reference to their bioremediation ability. Microbial degradation with reference to its biochemical and genetic aspects has created more interest among the researchers which results in identification of several microorganisms with the potential to degrade pesticides along with their genes/enzymes responsible for that process. Thus, microorganisms offer prospective resources for biodegradation 10, 11, 12.
Organophosphorus pesticides including parathion, methyl parathion, malathion, monocrotophos, and dimethoate are most commonly used pesticides in agriculture all over the world. When organophosphates are dispersed in the surrounding ecosystem, their activity is determined by several environmental factors and microbial degradation. Microbial degradation is the most important factor responsible for the removal of these pesticides in the environment 13. Hence in the present study an attempt has been made to find out the methyl parathion degrading ability of Pseudomonas stutzeri strain (MTCC 2643) obtained from IMTECH, Chandigarh, Punjab, India.
The pesticide used in the present study belongs to the class of organophosphates which is commercially available as methyl parathion. It is selected on the basis of its wide application and present market trends.
Pesticide, especially methyl parathion applied soil samples were collected from agricultural fields in Thiruppuvanam, Sivaganga District, Tamil Nadu, India (Figure 1) in sterile containers and immediately brought to the laboratory for analysis.
Pseudomonas stutzeri strain (MTCC 2643) obtained from IMTECH, Chandigarh, India was inoculated into minimal broth containing different concentrations of commercial grade (raw pesticide) methyl parathion (50, 100, 150 and 200 ppm). The flasks were incubated at room temperature and the samples were then subjected for the estimation of orthophosphate.
One ml of sample was taken in a flask and 1 ml of ammonium molybdate and 3 drops of stannous chloride solution were added and kept for 10 minutes for the development of blue colour and the absorbance was recorded in a colorimeter at 650 nm. Distilled water blank was subjected in a similar manner.
Similarly the standard phosphorus solution of different strengths was processed and standard curve was plotted between absorbance and the concentrations of standard phosphorus solution. The orthophosphate content of the sample was deduced by comparing its absorbance with the standard curve.
pH was analyzed every 6 hours up to 30 hours of treatment for the samples from different concentrations of methyl parathion using pH meter and readings were recorded.
Growth was measured as turbidity at 600 nm in 6 hours interval for 30 hours.
The efficiency of pesticide degrading ability of the bacterial strain was tested by providing different carbon sources like fructose, glycerol, lactose, maltose and sucrose of 1% concentration in minimal medium containing 200 ppm concentration of methyl parathion. The flasks were incubated at 37°C and orthophosphate released was estimated every 6 hours up to 30 hours.
The seed cultures of the strain were grown in nutrient broth and the cells were harvested by centrifugation at 10,000 rpm for 10 minutes and the cells were washed and suspended in 0.1% NaCl. Then 3.5% of sodium alginate was added to the cell suspension and mixed thoroughly without forming any air bubble in the slurry. The slurry containing the cells was extended as drops through a tube (2 mm diameter) into 4% CaCl2 solution. The drops formed into spherical beads of 2 mm size. The gel beads were kept in 4% CaCl2 solution at 5°C for about an hour for complete gelation. Then the beads were washed with sterile distilled water and used for methyl parathion degradation study 14.
The samples from 200 ppm concentration of methyl parathion were centrifuged at six hours interval for thirty hours and the clear supernatant was used for spectral analysis. The clear supernatant was scanned from 200 to 600 nm in a spectrophotometer (Elico SL: 159) and analysed for specific absorption in the spectrum.
The samples from 200 ppm concentration of methyl parathion before and after 30 hours of treatment period were subjected to HPLC analysis by UV detection.
Pesticide contamination as a result of agricultural and industrial activities poses serious threats to the environment and indeed to human life15. In modern days, utilization of living organisms in destruction or transformation of toxic chemical compounds is very much effective when compared to traditional treatment strategies. Microbial degradation process to detoxify pesticide contaminants can be efficiently used to overcome pollution problems. Numerous reports are available indicating the use of microorganisms for pollution control and for the production of chemicals of economic importance16, 17. The pesticide methyl parathion is widely distributed in the environment due to its vast applications and is chemically similar to nerve agents used in chemical warfare18. Thus, degradation of methyl parathion is very much essential with relevant environmental concern.
Degradation of methyl parathion appears to be faster in the presence of sediment and in fresh water than that of salt water. The rate of degradation depends on the presence and acclimation of microbial populations in the body of water. Methyl parathion has a half life in water environment of 175 days and ten days to two months in soil19, 20. In the present study, the biodegradation activity of P. stutzeri MTCC 2643 was tested against methyl parathion. Orthophosphate released levels during the degradation of methyl parathion at different concentrations of methyl parathion (50, 100, 150 and 200 ppm) by P. stutzeri were analysed. P. stutzeri released maximum orthophosphate at 200 ppm concentration of methyl parathion which was selected for further analysis (Figure 2). The level of orthophosphate released in the control was very much less which indicated that P. stutzeri could utilize methyl parathion as a source of phosphorus and carbon 21. Figure 3 shows the effective degradation of methyl parathion by P. stutzeri taking place at the pH range of 6-8. With the increase in treatment period, the pH was shown to decrease at 12 hours treatment period and then decreased. From the decrease in pH, it can be understood that the acidity of the medium increases i.e, during the process of degradation, an acid was formed as a product and thus decrease in the pH was noted in the medium22. Turbidity was shown to increase during the treatment intervals such as 6, 12, 18, 24 and 30 hours (Figure 4) which indicated the growth of the organism. From the increase in the turbidity of the medium, it can be inferred that the organism uses methyl parathion as a source of carbon and phosphorus as it grows on minimal medium with methyl parathion and causing the degradation of methyl parathion. Similar kinds of observations were noticed in the earlier investigations related to biodegradation of pesticides using different bacterial strains 23, 24, 25, 26, 27.
Some microorganisms can use methyl parathion as a carbon source. Methyl parathion positive effects were observed in bacteria and actinomycetes, while fungi and yeasts were less able to utilize this compound. Methyl parathion is susceptible to degradation by hydrolysis to paranitrophenol and dimethyl thiophosphate in soil and water environments by nitro group reduction to methyl amino parathion or both. Hydrolysis is a leading pathway in nonflooded soil, while methyl parathion is degraded essentially by nitro group reduction in predominantly anaerobic ecosystems such as flooded soil. Isolated two mixed bacterial cultures by soil enrichment were capable of utilizing methyl parathion as a sole source of carbon which indicated that mixed cultures are more stable in retaining their ability to completely degrade methyl parathion than that of isolated bacteria28, 29, 30, 31. Bacillus sp. was isolated from methyl parathion treated flooded soil but required yeast extract for the degradation of methyl parathion in mineral salt medium. Pseudomonas strains utilize methyl parathion as both carbon and phosphorous source. Pseudomonas sp. WBc-3 isolated from polluted soil, was capable of complete degradation of methyl parathion and tolerated high concentration of methyl parathion up to 800mg/l in basic medium and upto 2000 mg/l in 0.1% glucose medium32, 33.
In pesticide degradation, immobilized cells are more effective than that of free cells employed in conventional methods and also exhibit advantages like high cell concentrations, reuse of cells, elimination of “cell wash” problems at a high dilution rate, high yields and the capacity to retain catalytic activity for longer time 34, 35. In the present study, immobilized cells of P. stutzeri released orthophosphate with a constant, stable and gradual increase but in the case of free cells it seems to decrease after 72 hours (Figure 5). When carbon sources are supplemented in the minimal medium (fructose, glycerol, lactose, maltose, and sucrose), they enhanced the degradation process, where P. stutzeri utilized sucrose, maltose and lactose effectively for methyl parathion degradation (Figure 6).
Spectrophotometry is a prominent method used in pesticide analysis over the years, because of its features like ruggedness, economical, and suitability for a variety of pesticides by using different reagents, detectors and techniques like flow injection, PLS (Partial least square) etc.36. Malik and coworkers37 have reported various methods for the analysis of fungicides spectrophotometrically. Jhangel and Pervez 38 proposed a sensitive spectrophotometric method for the analysis of the acaricide, kelthane. This method showed good sensitivity in sub parts per million levels. In the present study, UV-Visible Spectrophotometric analysis was used to find out the biodegradation of methyl parathion (Figure 7). There was a change in absorption spectrum with the increase in incubation period. This is an evidence for the degradation of methyl parathion 39, 40. Chromatographic analysis is one of the most suitable methods for pesticide analysis. HPLC is a commendable method for the analysis of wide range of pesticides. Several heat unstable and polar pesticides can be better detected with this technique. Several research works using HPLC proved its effective application in pesticide analysis 41, 42. In the present study, the retention time for standard methyl parathion was 3.433 minutes with the peak area of 7.345 mV.s and peak height of 0.444 mV (Figure 8) in HPLC analysis. The retention time for 200 ppm of methyl parathion after degradation by P. stutzeri was 2.433 minutes and 3.367 minute with the peak areas of 6.140 mV.s and 29.749 mV.s and a peak height of 0.213 mV and 1.280 mV which indicated the formation of intermediate compounds during methyl parathion degradation after 30 hours of treatment (Figure 9).
Statistical analysis of degradation rates of methyl parathion samples from two gulf coast estuaries over a three year period indicated that biodegradation occurred in the presence of sediment but was insignificant in water. Under aerobic, photosynthetic conditions, the cyanobacterium, Anabaena sp. capable of degrading methyl parathion was reported which takes place only in the presence of light43. Burkholderiacepacia which has a potential methyl parathion degrading ability from agricultural soil was isolated44. Table 1 exhibits the two way ANOVA for the factors such as orthophosphate released during degradation by P. stutzeri, pH and turbidity with the variables, treatment period and methyl parathion concentration. Variables of tested factors, treatment period and methyl parathion concentration caused significant variations which were statistically significant at 5% level. Pseudomonas stutzeri strain MTCC 2643 was able to degrade methyl parathion up to 200 ppm concentration in thirty hours. All the sugars tested enhanced the release of orthophosphate during degradation.Table 1. Two way analysis of variance for the factors with the variables, treatment period and methyl parathion concentration
|Factor||Source ofVariation||SS||df||MS||CalculatedF - value||Table valueat 5% level||Level ofsignificance|
|Orthophosphate released||Treatment Period||475.8||4||118.9||9.9615||3.0069||Significant P‹ 0.05|
|Methyl Parathion Concentration||1264.9||4||316.2||26.4837||3.0069||Significant P‹ 0.05|
|pH||Treatment Period||0.0894||4||0.022||47.1603||3.0069||Significant P‹ 0.05|
|Methyl Parathion Concentration||0.0905||4||0.022||47.7089||3.0069||Significant P‹ 0.05|
|Turbidity||Treatment Period||1.4417||4||0.360||87.2405||3.0069||Significant P‹ 0.05|
|Methyl Parathion Concentration||0.3199||4||0.800||19.3571||3.0069||Significant P‹ 0.05|