International Journal of Pharma and Bio Sciences ISSN … by Dhanuka Agritech limited, Gurgoan in Haryana, India was used in this study as target substrate. It contains 62.5% v/v Methyl

Int J Pharm Bio Sci 2014 Jan; 5(1): (B) 592 - 603

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Research Article Microbiology

International Journal of Pharma and Bio Sciences ISSN

0975-6299

OPTIMIZATION OF PROCESS PARAMETERS FOR PSEUDOMONAS DIMINUTA,

P. PUTIDA AND P. AERUGINOSA FOR BIODEGRADATION OF METHYL

PARATHION

JYOTI SHARMA*1, A.K. GOEL2 AND K.C. GUPTA1

1

V.R.G. Gov. P.G College, Department of Zoology & Microbiology, Morar, Gwalior, Pin - 474002, India. 2

Biotechnology Division, Defence Research & Development Establishment, Jhansi Road, Gwalior - 474002, India.

ABSTRACT

Pseudomonas diminuta, P. putida and P. aeruginosa were established as a biodegrading agent for Methyl Parathion (MP) at 1200 mg/L, under basic growth parameters for mesophiles. In this study, the effects of process parameters, such as temperature, pH, salinity, carbon and nitrogen sources on the growth of those bacteria were studied and optimized to establish higher degradation of the target chemical. The growth was assessed in BMM containing MP at various temperatures such as 25°C, 30°C, 37°C and 40°C; at various pH such as 7.0, 8.0, 8.5, 9.0 and 9.5; at various salinity such as 0.1%, 0.5%, 1%, 2.0% and 3.5%; and glucose (1 gm/L) and yeast extract (0.5 gm/L) separately. The higher growth was recorded at 37°C and pH 8.5. Salinity and addition of supplements did not support the bacterial growth. These species may be used to treat MP polluted soils by providing optimized conditions suggested by the present study.

KEYWORDS: Methyl parathion, Biodegradation, Pseudomonas diminuta, Pseudomonas putida, Pseudomonas aeruginosa.

*Corresponding author

JYOTI SHARMA

V.R.G. Gov. P.G College, Department of Zoology & Microbiology,

Morar, Gwalior, Pin - 474002, India.



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INTRODUCTION

India is an agriculture based country, where more than 60-70% population dependents on agriculture1 and this livelihood is the major determinant of Indian economy. To feed the ever-increasing population, there is a mounting pressure on production efficiency of the crops. In India 15-20% agricultural products are destroyed by pests every year2 thus, farmers are forced to use large quantity of pesticides to increase the crop production. Extensive usages of these persistent chemicals have led to environmental pollution and short and long term health hazards1. Organophosphorus pesticides are used extensively worldwide to control wide ranges of insect species. Methyl parathion (MP), a highly toxic chemical, is used for protecting cotton from pests3. It is also highly toxic to non target organism including human4. Researchers have studied several organophosphorus pesticide-degrading bacteria and have isolated them from agricultural soil. These microbes use pesticides as a source of carbon and energy and degrade them5,6,7,8,9. Bioremediation process depends upon the physical and chemical characteristics of the substrate such as nutrient status, pH, temperature10,11,12 and biotic factors such as inoculum density13. Optimization of these factors enhance growth of the bacteria and quicker the degradation process. The degradation of lipids and detergents in effluent water has been enhanced through optimization of pH, temperature and agitation speed14. Optimization of pH and incubation temperature enhanced biodegradation of alpha and beta endosulfan in soil slurry by Pseudomonas aeruginosa15. Complete phenol biodegradation by optimizing pH, temperature, initial concentration of phenol, additional carbon sources and additional nitrogen sources16. Many authors have observed that organophosphorus pesticide degradation was highest in alkaline pH5,17,18,19,20. But some researchers authors that microbial degradation ability of pesticides was highest in acidic pH13,21. In our previous study, the Pseudomonas diminuta, P. putida and P. aeruginosa were

isolated from Gwalior and nearby places of Madhya Pradesh, India, which degraded 1200 mg/L MP after 48 hrs at 30°C, pH 7.0 and at 200 rpm22. The present study was carried out to optimize pH, temperature, salinity and additive carbon and nitrogen sources for maximum degradation of MP through those bacteria in Laboratory condition.

MATERIALS AND METHODS

(i) Pesticide Commercial grade methyl parathion (MP) manufactured by Dhanuka Agritech limited, Gurgoan in Haryana, India was used in this study as target substrate. It contains 62.5% v/v Methyl Parathion. (ii) Media preparation Culturing of bacteria for the experiments were carried out in a no-carbon no-energy source like Basal mineral medium (BMM); which was prepared by mixing 4.8 g K2HPO4, 1.2 g KH2PO4, 1.0 g NH4NO3, 0.25 g MgSO4.7H2O, 0.04 g CaCl2 and 0.005 g FeSO4.7H2O in one liter of distilled water 23,24. Luria Bertani Agar (LB, Hi-media) was prepared by dissolving 25 gm in one liter distilled water. All the media was sterilized by autoclaving at 121°C, 15 psi pressure for 15 minutes. (iii) Isolation of MP degrading bacteria MP degrading bacteria Pseudomonas diminuta, P. putida and P. aeruginosa were isolated from agricultural soil pretreated with MP from several years (7 to 10 years), from Gwalior and nearby places of Madhya Pradesh in India. These species were isolated by enrichment method in BMM containing MP. These were found capable of degrading higher concentration of MP (1200 mg/L) after 48 hrs of incubation at 30°C, pH 7.0 and at 200 rpm22. Degradation was analyzed by GC-MS (Agilent Technologies, USA) in full scane mode. Isolates were preserved for further studies in LB and 50% v/v glycerol at -80°C.



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(iv) Inoculum preparation Isolates preserved in LB and 50% v/v glycerol at -80°C, were taken out and one loop-full of each bacteria were immediately streaked on LB Agar plates and incubated at 37°C for 24 hrs. After incubation, a single colony of each bacterium was inoculated in 5 mL LB broth and incubated at 37°C, 200 rpm for 24 hrs. After growth was observed, 1.5 mL culture of each bacterium was taken in appendorf and cells were separated by centrifugation at 4°C on 12000 rpm for 15 minutes. After discarding the supernatant, these cells were washed twice with sterile normal saline (0.85%) at 4°C on 12000 rpm for 15 minutes and re-suspended in a small amount of sterile BMM. These suspended cells of each bacterium were then inoculated to 100 mL of BMM containing 50 mg/L MP and incubated at 30°C, pH 7 and maintaining 200 rpm22. Further optimization experiments were carried out using these bacterial cultures in triplicate at 200 rpm to achieve viable experimental data. (v) Optimization of temperature To optimize the temperature condition, 5 mL culture of each bacterium was inoculated in fresh 95 mL BMM, containing 1200 mg/L MP22 and incubated at different temperatures as 25°C, 30°C, 37°C and 40°C, at 200 rpm while maintaining pH at 7.0. BMM without inoculum served as control. Bacterial growth was determined by measuring optical density at 600 nm using a spectrophotometer (BIO-TEK ELx808, USA) at specific time intervals of 2 hrs. When maximal growth was observed for each bacterium, the temperature was noted as optimal for degradation of MP at 1200 mg/L. This temperature was taken for further optimization of pH. (vi) Optimization of pH Optimization of pH was done using the same process as followed above. The cultures in BMM were incubated at optimized temperature at different pH conditions as 7.0, 8.0, 8.5, 9.0 and 9.5 at 200 rpm. BMM without inoculum served as control. Bacterial growth was again observed for each bacterium at every 2 hrs at

600 nm. When maximal growth was observed for each bacterium, the pH was noted as optimal for degradation of MP at 1200 mg/L. The optimal temperature and pH conditions were taken for further optimization of salinity. (vii) Optimization of salinity The effect of different salinity on bacterial growth at MP concentration of 1200 mg/L was carried out by taking 5 mL of each prepared inoculum and inoculating them in fresh 95 mL BMM, containing 1200 mg/L MP. NaCl solutions of 0.1%, 0.5%, 1.0%, 2.0% and 3.5% were added to the cultures. Separate cultures in BMM without any NaCl solution was also taken to compare bacterial growth in the presence and absence of salinity. BMM without inoculum served as control. These flasks were incubated at optimized temperature and pH conditions. The growth was checked at 600nm at every 2 hrs. When maximal growth was observed for each bacterium, the salinity was noted as optimal for degradation of MP at 1200 mg/L. The optimal temperature, pH and salinity were taken for further optimization of carbon and nitrogen sources. (viii) Optimization of carbon and nitrogen sources The effects of additional carbon and nitrogen sources, such as glucose (Difco) at 1gm/L7, 25

and yeast extract (Difco) at 0.5 gm/L26,27 were also examined for the growth of MP degrading bacteria. From prepared inoculum, 5 mL culture of each bacterium was inoculated in fresh 95 mL BMM, containing 1200 mg/L MP and both of these sources were added separately. A parallel flask was also prepared without additives to observe bacterial growth with or without supplements. Media without inoculum served as control. The flasks were incubated at optimized conditions. The growth was determined at 2 hrs intervals at 600nm.

RESULTS The growth of MP degrading bacterial species P. diminuta, P. putida and P. aeruginosa at different temperatures is depicted in Graphs 1a,



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1b and 1c. Higher and rapid growths of all bacteria were recorded at 37°C after 42 hrs of incubation. The growth of these three species was negatively affected when incubated at 25°C, 30°C and 40°C. At 40°C, all the species showed diminished growth rate comparative to other temperature variables. No growth was observed in control. The optimization of pH was carried out at 37°C. The effect of different pH on the bacterial growth is given in Graphs 2a, 2b and 2c. It was observed that the bacterial species were capable of growing at all pH variables, but rapid and higher growth was observed at pH 8.5 after 36 hrs of incubation at 37°C. In this study, higher growth rate was recorded at alkaline pH. No growth was observed in control. The effect of salinity was observed at optimized temperature and pH conditions of 37°C and 8.5, respectively. Bacterial growth with and without salinity has been represented in Graphs 3a, 3b and 3c. The growth was significantly decreased in the presence of all salinity variables. All three species showed rapid and higher growth in the absence of salinity after 36 hrs of incubation at 37°C and pH 8.5. The growth of P. diminuta was highly negatively affected in the presence of salinity when compared to other two species. No growth was observed in control. The effect of glucose and yeast extract supplement on the growth of all three bacterial species at 37°C and pH 8.5 are depicted in Graphs 4a, 4b and 4c. It was observed that addition of glucose and yeast extract decreased the growth rate of the isolates. Whereas, in the absence of supplements, higher growth rates of all three species were recorded after 36 hrs of incubation. No growth was recorded in control.

DISCUSSION

The Bacillus pumilus T1 isolated from soil, having the ability to degrade methyl parathion at a concentration of 500 ppm at 30ºC28. A consortium was prepared from P. aeruginosa and other four species which degraded 15 mg/L of MP at pH 7-10 and temperature of 40°C29. In previous study, the P. diminuta, P. putida and P. aeruginosa were isolated which were capable of degradation of 1200 mg/L MP at pH 7.0 and temperature of 30°C after 48 hrs of incubation22. However, after optimization of process parameters during the present study P. diminuta, P. putida and P. aeruginosa showed rapid growth and degradation proximate at 36 hrs. MP was completely degraded by P. aeruginosa within 24 to 48 hrs at 37°C4. According to him the maximum growth indicated the highest degradation of pesticides. So it may be presumed that, as those bacteria in the present study showed highest growth at pH 8.5 and temperature of 37°C, there will be complete degradation of 1200 mg/L of MP. The degradation of MP by P. putida were enhanced by the presence of kaolinite and depressed by the presence of goethite30. At low salinity the Burkholderia cepacia, showed the higher degradation of MP17. Parathion was degraded faster in the absence of salinity31. The p-nitrophenol degradation ability of Ochrobactrum sp did not enhance by the addition of glucose in medium19. Though in this study it was fund that an addition of glucose or yeast extract did not support bacterial growth, rather the supplements negatively affected growth rate.



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Graph 1a

Effect of different temperatures 25°C, 30°C, 37°C and 40°C on the growth of Pseudomonas diminuta in BMM at pH 7.0 after 72 hrs of incubation.

Graph 1b

Effect of different temperatures 25°C, 30°C, 37°C and 40°C on the growth of Pseudomonas putida in BMM at pH 7.0 after 72 hrs of incubation.



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Graph 1c

Effect of different temperatures 25°C, 30°C, 37°C and 40°C on the growth of Pseudomonas aeruginosa in BMM at pH 7.0 after 72 hrs of incubation.

Graph 2a

Growth of Pseudomonas diminuta in BMM at different pH 7.0, 8.0, 8.5, 9.0, 9.5 incubated at 37°C for 72 hrs.



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Graph 2b

Growth of Pseudomonas putida in BMM at different pH 7.0, 8.0, 8.5, 9.0, 9.5 incubated at 37°C for 72 hrs.

Graph 2c

Growth of Pseudomonas aeruginosa in BMM at different pH 7.0, 8.0, 8.5, 9.0, 9.5 incubated at 37°C for 72 hrs.



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Graph 3a

Growth of Pseudomonas diminuta at different salinity of 0.1%, 0.5%, 1.0%, 2.0%, 3.5% and without salinity, incubated at 37°C, pH 8.5 for 72 hrs.

Graph 3b

Growth of Pseudomonas putida at different salinity of 0.1%, 0.5%, 1.0%, 2.0%, 3.5% and without salinity, incubated at 37°C, pH 8.5 for 72 hrs.



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Graph 3c

Growth of Pseudomonas aeruginosa at different salinity of 0.1%, 0.5%, 1.0%, 2.0%, 3.5% and without salinity, incubated at 37°C, pH 8.5 for 72 hrs.

Graph 4a

Growth of Pseudomonas diminuta in BMM in presence and absence of supplement nutrients, i.e. 0.5 gm/L yeast extracts, 1.0 gm/L glucose at 37°C, pH 8.5 after 72 hrs of incubation.



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Graph 4b

Growth of Pseudomonas putida in BMM in presence and absence of supplement nutrients, i.e. 0.5 gm/L yeast extracts, 1.0 gm/L glucose at 37°C, pH 8.5 after 72 hrs of incubation.

Graph 4c

Growth of Pseudomonas aeruginosa in BMM in presence and absence of supplement nutrients, i.e. 0.5 gm/L yeast extracts, 1.0 gm/L glucose at 37°C, pH 8.5 after 72 hrs of incubation.

CONCLUSION Present study showed that Pseudomonas diminuta, P. putida and P. aeruginosa were able to tolerate and degrade MP concentration of 1200 mg/L. Temperature and pH were proven

to be crucial factors affecting growth of Pseudomonas species and, in turn, degradation of MP. It was observed that salinity and addition of carbon and nitrogen sources like glucose and



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yeast extract did not influence bacterial growth and degradation of MP. The present study was conducted in laboratory conditions, therefore, in future studies these bacterium species may be tested with industrial waste and natural environments. As it is a known fact that bacterial consortium work better than individual bacterium in biodegradation of xenobiotic compounds, a consortium study of these isolates may offer better MP degradation potential.

ACKNOWLEDGEMENT

We would like to thank Officials of Defence Research and Development Establishment Gwalior (MP), India for providing Laboratory facilities and logistics. We would also like to thank Dr.Jyoti Prasad, Principal, VRG, PG college Morar, Gwalior (MP), India for her moral support for this work. We also extend our thanks to laboratory and support staffs for collection of field samples and sample preparation.

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Documents

International Journal of Pharma and Bio Sciences ISSN … by Dhanuka Agritech limited, Gurgoan in Haryana, India was used in this study as target substrate. It contains 62.5% v/v Methyl