International Journal of Advanced Engineering Technology E-ISSN

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1 Research Article ISOLATION, CHARACTERIZATION AND GROWTH RESPONSE STUDY OF ENDOSULFAN DEGRADING BACTERIA FROM CULTIVATED SOIL Sumit Kumar* Address for Correspondence Biotechnology Engineering Department, V.V.P. Engineering College, Saurashtra University, Rajkot , Gujarat, India ABSTRACT In this work, five l isolates, viz. RCE-2, GCE-3, GCE-4, GCE-5 and JCE-4 were obtained by enrichment from soil samples collected from the cultivated fields of Rajkot district of Gujarat State. The isolates were capable of growing in the presence of endosulfan, in the concentration range of mg/l. The highest value of MIC (30 mg/l) was reported for l isolate GCE-4. The viable cell count for all five isolates was in the order of 10 7 per gram of soil in presence of endosulfan (10 mg/l). The optimum growth temperature for the l isolates varied between 25 o C and 30 o C, however, for majority of the isolates it was 30 o C. The optimum growth ph for the all these isolates was 7.0. The growth of l isolates in presence of endosulfan as a sole source of carbon and energy showed that these isolates are capable of degradation and detoxification of endosulfan contaminated site. For large scale culture of these isolates for bioremediation purpose, the optimization of temperature and ph is an important requirement. KEYWORDS: Isolation, Characterization, Endosulfan, Bacteria, Growth response INTRODUCTION Pesticides are substances which are used to control, destroy, repel or attract pests in order to minimize their detrimental effects. Pesticides are used in many situations such as livestock farming, cropping, horticulture, forestry, home gardening, homes, hospitals, kitchens, road-sides, recreational and industrial areas. By 1991, there were approximately 23,400 pesticide products registered with the U.S. Environmental Protection Agency (EPA). The agriculture industry used 77%, industrial, commercial and government organizations used 12% and private households used the remaining 11%. In studies of laboratory rodents exposed to extremely high doses, about half of all man-made pesticides are found to be carcinogenic. On a list of most common POISONS, pesticides were found to be number two. According to the Environmental Protection Agency (EPA), 60% of herbicides, 90% of fungicides and 30% of insecticides are known to be carcinogenic [1-3]. Chlorinated organochlorine pesticides are one of the major groups of chemicals responsible for environmental contamination. Endosulfan (6,7,8,9,10,10-hexachloro- 1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3- benzodioxathiepine-3-oxide), an organochlorine insecticide.it is a contact and stomach poison, used to control chewing and sucking insects, such as Colorado beetle, flea beetle, cabbage worm aphids and leaf hopper. This insecticide is used to control insect pests on a wide range of crops, including cereals, cotton, coffee, fruits, oil seeds and vegetables. Endosulfan residues have been detected in the atmosphere, soils, sediments, surface water and foods. Technical-grade endosulfan is a mixture of two stereo isomers, alpha and beta endosulfan, in a ratio of 70:30. Both the isomers are extremely toxic to aqueous organisms. Because of its widespread usage and potential transport, endosulfan contamination is frequently found in the environment at considerable distances from the point of its original applications. Detoxification of endosulfan through biological means is receiving serious attention as an alternative to existing methods, such as incineration and landfill. Many studies have reported about the isolation of and fungi capable of degrading endosulfan [4-8]. Endosulfan is ubiquitous and environmentally persistent, as a result the presence of endosulfan residues were traced in surface water, groundwater, atmosphere, and water bodies by many researchers (Sujatha et al., 1999; Bhattacharya et al., 2002; Berrakat et al., 2002; Cerejeira et al., 2003; Golfinopoulos et al., 2003). Many countries imposed ban on endosulfan production and/or usage, whereas in India it is still one of the priority pollutant for pest control. Because of its abundant usage and potential transport, endosulfan has been detected in the soil, sediments, atmosphere, surface and rain waters, and foods. It is extremely toxic to fish and aquatic invertebrates (Sunderam et. al., 1992) and has been implicated in mammalian gonadal toxicity (Sinha et. al., 1997), genotoxicity (Chaudhari et. al. 1999), and neurotoxicity (Paul & Balasubramaniam, 1997). The health and environmental concerns have led to an interest in degradation and detoxification of endosulfan in the environment. Microorganisms play an important role in the conversion of cyclodiene insecticides in soil to nontoxic products. In the natural environment microorganisms may provide some protection against toxicity of endosulfan. Pure culture of a range of soil microorganisms have been reported to transform endosulfan to a nontoxic diol metabolite in unsealed

2 liquid cultures [9-11]. The objectives of the present study were isolation, characterization, identification and growth response study in terms of temperature and ph, of native strains of that are capable of degrading endosulfan. Since metabolic and physiological characteristics of native isolates are highly adapted under existing environmental conditions, it would be helpful in the degradation and detoxification of the endosulfan polluted site, as this pesticide poses a threat to the aquatic organisms and causes various hazards in land animals and humans. MATERIALS AND METHODS Sample collection for enrichment study The soil samples used in this study were collected from three different talukas (administrative blocks) of Rajkot district of Gujarat State. The samples were collected from agricultural fields growing mainly cotton, groundnut and vegetables. A total of 30 composite soil samples were collected from 30 different agricultural fields having history of repeated application of endosulfan for 6 and more years. The soil samples were collected by using auger up to a depth of 15 cm. The collected samples were air dried, ground, passed through 2 mm sieve and stored in the sealed plastic bags at room temperature. These stored samples were used for microbial enrichment study. Pesticide, chemicals and media The pesticide selected for the present study was a technical grade of endosulfan (Endoin, EC 35%), which is a type of cyclodiene organochlorine. The pesticide was purchased from the local pesticide supplier. This commercial formulation of endosulfan was dissolved in sterile distilled water for amendments to soil samples. The analytical grade chemicals and reagents used in this work were purchased from Hi-media and Qualigens. The culture media used were Nutrient Broth (NB), Luria Bertani (LB) and M9. The LB medium was prepared by mixing tryptone 10g, sodium chloride 10g and yeast extract 5g in 1L distilled water, and the medium ph was adjusted in the range of The M9 medium was prepared by adding di-sodium hydrogen phosphate 6g, potassium dihydrogen phosphate 3g, sodium chloride 0.5g and ammonium chloride 1g in 1L distilled water and the medium ph was adjusted in the range of For semi-solid media, 1.5% agar was added. All the media were autoclaved at C temperature, 15 psi pressure for 20 minutes. After autoclaving, filtered sterilized 10ml glucose (20%), and separately autoclaved 2ml magnesium sulfate (1M) and 0.1ml calcium chloride (1M) were added in the M9 medium. Isolation, MIC and purification of endosulfandegrading The enrichment culture technique was used for the isolation of l strains capable of utilizing endosulfan as a sole source of carbon and energy. Endosulfan mixed media were prepared by thoroughly mixing different volumes of endosulfan with 100 ml media (LB, M9 and N-broth) as a sole carbon source, when the media were about to solidify (45-50 o C). Different concentrations ranging from 5 to 200 mg/l of endosulfan were added and checked for the growth of. One gram of each soil sample was suspended in 9 ml of distilled water and kept at room temperature for 24 hours. On the next day, 500 µl of the supernatant was spread on endosulfan containing N-agar, LB and M9 petridishes, and incubated at room temperature. The petridishes were observed on the next day to till fourth day for the appearance of resistant colonies. The M9 medium plates containing endosulfan concentration ranging from 5 to 200 mg/l were streaked with pesticide resistant colonies and incubated at 37 o C for 7 days. The concentration at which the isolate failed to grow even after 7 days of incubation was considered as minimum inhibitory concentration (MIC) for the given l isolates. A single isolated colony of the endosulfan resistant/degrading was picked up with the help of sterilized wire loop and was streaked on LB agar medium. Each isolated strain was streaked at least 3 to 4 times on LB agar plates for purification. After the purified isolates were obtained, they were restreaked on M9 agar medium containing endosulfan for confirmation of isolates. The single colony of l strain was inoculated in 100ml LB broth, incubated at 37 o C and then used for further characterization of isolates. The purified l strains were stored under refrigeration after preparing slants. Characterization and identification of For the selected l isolates, colony morphology was observed by growing them on N-agar plates. Morphological characters viz. size, shape, surface, opacity, texture, etc. were determined by visual observation as well as by using light trans-illuminator and microscopy. The Gram staining technique was used for differentiation between gram positive and gram negative l strains. The biochemical tests such as growth on McConkey agar, starch hydrolysis, motility, oxidase, catalase, coagulase, urease, methyl red, citrate utilization, nitrate reduction, Voges-Proskauer, Indole, H 2 S production, etc. were performed as per Cheesbrough (1993) and Benson (1994). The l isolates were identified according to Microbiological Methods (Collins, et. al., 1995) and Bergy s Manual of Systematic Bacteriology (Palleroni, 1984). Determination of viable cell count

3 The population of l isolates in terms of colony forming units (CFUs) was determined using viable plate count technique. The soil samples, from which potential l strains were isolated, were used for determining l population per gram of soil. One gram of soil was properly dissolved in 9ml of sterile distilled water and diluted to 10-3 and 10-5 using the sterile distilled water. From these two dilutions, 0.1 ml portion was used to spread the prepared plates. The plates were incubated at room temperature for 48 hours. A fresh pipette was used for each dilution and the work was performed in the laminar air flow cabinet, under sterile condition. Nutrient agar plates containing endosulfan (20 mg/l) were used for determining the viable count of resistant colonies. The l cells visible to the naked eyes were counted in terms of CFUs. All the plating was performed in triplicates and results were represented as mean. The viable count was obtained from this value by reference to the serial dilution used. Growth response study of endosulfan-degrading Two important parameters viz. temperature and ph, affecting l growth were considered. For determining the effect of temperature and ph on the growth of l isolates, 100 ml of N-broth was taken in 250 ml borosil make Erlenmeyer flasks and autoclaved at 121 o C, 15 psi for 20 minutes. After cooling, each flask was inoculated with purified l isolate in active log phase, in the proportion of 2% of medium. For determining the optimum growth temperature, flasks were incubated at five different temperatures, viz. 15, 25, 30, 37 and 45 o C. While for ascertaining the optimum ph for growth of l isolates, the ph of N-broth was adjusted to 5.0, 6.0, 7.0, 7.5, 8.0 and 9.0 prior to autoclaving and inoculated flasks were incubated at room temperature. After 48 hours of incubation, the absorbance was taken at 600 nm. All the experiments were performed in triplicates and the mean value of respective absorbance was used to plot a graph to determine the optimum temperature and ph of respective l isolates. JCE-4 were isolated. Out of five isolates, one each was isolated from Rajkot and Jetpur taluka, and the remaining three from Gondal taluka Rajkot district of Gujarat State. Three out of five isolates were obtained from Gondal taluka where endosulfan spraying was most frequent. The microbial populations in this area were exposed to endosulfan application for past many years, which resulted in adaptation of the microbes against this pesticide. The Minimum Inhibitory Concentration (MIC) of endosulfan in M-9 media and the time taken for the appearance of first visible colony on the agar plate was worked out for the l isolates. The MIC of l isolates was in the range of mg/l of endosulfan and the growth of isolates was observed within hours of incubation. The l isolate GCE-4 showed highest value of MIC (30 mg/l) and the growth appeared after 72 hours of incubation at room temperature. The MIC of remaining four l isolates was 20 mg/l of endosulfan. The details of the MIC of various l isolates were as given in the Table-1. Table-1: The MIC of endosulfan-resistant l isolates Bacterial isolates MIC (mg/l) Incubation Time (hr.) RCE GCE GCE GCE JCE Characterization and identification of Endosufandegrading On the basis of physical and morphological characteristics, gram staining behaviour and different biochemical tests, the l isolates were identified as Bacillus subtilis (RCE-2), Arthrobacter sp. (JCE-4), Arthrobacter sp. (GCE-3), Pseudomonas putida (GCE-4), and Bacillus pumulus (GCE-5). The details of characterization and identification are as presented in the Table-2 and Table-3. RESULTS AND DISCUSSION Isolation and MIC of endosulfan-degrading In this work, five endosulfan-resistant l isolates, code named as RCE-2, GCE-3, GCE-4, GCE-5 and Table-2: Physical and morphological characterization of the l isolates Bacterial Isolates Size Margin Elevation Surface Opacity Pigmentation Form RCE-2 Medium Undulate Flat Glistering Opaque Light yellow Circular JCE-4 Medium Undulate Flat Glistering Translucent White Circular GCE-3 Medium Undulate Flat Smooth Translucent Light yellow Circular GCE-4 Medium Entire Raised Smooth Opaque White Circular GCE-5 Medium curled Flat Dull Opaque White Irregular

4 Table-3: Biochemical characterization and identification of endosulfan-resistant l isolates Note: + : Positive, - : Negative, A : Acid production, AG : Acid & Gas production Viable cell count of l isolates The population of each l isolates per gram of soil was determined in terms of CFUs using viable plate count technique. All the plating was performed in triplicates and results were represented as mean. After incubation for 48 hours at room temperature, the viable counts were reported in the order of and 10 7 per gram of soil for untreated and endosulfan (10 mg/l) treated samples respectively. The highest viable count in endosulfan treated samples was reported for isolate JCE-4 and the lowest for GCE-3. The details are as given in Table-4. Table-4: Population of endosulfan-resistant (CFU) per gram of soil Bacterial Isolates Endosulfan free media Endosulfan (10 mg/l) supplemented media RCE x x 10 7 GCE x x 10 7 GCE x x 10 7 GCE x x 10 7 JCE x x 10 7 Effect of temperature on the growth of l isolates As per literature, the maximum growth and activity of microorganisms in soils are reported at 25 o C to 35 o C of temperature. In the present work, the optimum growth temperature was 30 o C for endosulfan-resistant isolates RCE-2, GCE-4, and GCE-5. It was 25 o C for isolates GCE-3 and JCE-4. It is clear that the growth temperature of isolates varied between 25 o C and 30 o C, however, for majority of the isolates it was 30 o C (Figure-1). Therefore, the optimization of growth temperature is important for large scale culture of isolates capable of degrading endosulfan, before their use for remediation purpose. Figure-1: Effect of temperature on the growth of endosulfan-resistant Effect of ph on the growth of l isolates Abiotic and biotic degradation processes of endosulfan may be affected by soil ph. The mobility and bioavailability of endosulfan in the soil may be also influenced by ph of soil. In this study, the optimum growth ph was 7.0 for all the five endosulfan-resistant l isolates (Figure-2). In all cases, the growth of culture declined sharply below ph 5.0 and above ph 9.0. Therefore, the optimization of ph is absolutely essential before

5 large scale culture of the isolates capable of degrading endosulfan. CONCLUSION The present work describes the isolation, characterization, identification and growth response in terms of temperature and ph, of l isolates capable of degrading endosulfan. Five indigenous strains of that are capable of degrading endosulfan were isolated from the cultivated fields having known history of endosulfan application. 7. Rainer Martens: Applied & Env. Microbiol., 31 (6), (1976). 8. Mathava Kumar and Ligy Philip: J. of Env. Sc. & Health Part B, 41, (2006) 9. Mathava Kumar and Ligy Philip: Bioremediation Journal, 10(4): , Tariq Siddique, et. al.: J. Environ. Quality, 32, (2003). 11. Osama El Gialani Elsaid, et. al.: Int. J. of Env. Sciences, 1 (2), (2010). Figure-2: Effect of ph on the growth of endosulfan-resistant For large scale culture of such l isolates, to be used for bioremediation purpose, it is essential to determine the optimum growth temperature and ph. These isolated strains of are highly adapted to the existing environmental conditions and thus could be effectively utilized for bioremediation and metabolic detoxification of endosulfan contaminated site. ACKNOWLEDGEMENTS The author wishes to acknowledge Dr. Sachin Parikh, Principal, VVP Engineering College Rajkot, for providing the necessary infrastructure and Gujarat Council on Science & Technology (GUJCOST) Gandhinagar, for partly providing the financial support for this work under Student Sci-Tech scheme. REFERENCES 1. Hock, W., Day, and Morley: Farm Chemicals Manual, EPA, U.S., Pesticides Industry Sales and Usage: 1996 and EPA, U.S., Pesticides: Topical & Chemical Fact Sheets, Shivaramaiah H.M. & Kennedy I.R.: J. of Env. Sc. & Health Part B, 41, (2006) 5. Goebel H.: Residue Review, 83, 8 28 (1982). 6. Fitt G.P.: Annuals review of Entomology, 39, (1994).