CARBOFURAN DEGRADING BACTERIA ISOLATED FROM DIFFERENT AREAS OF PUNJAB AS POTENTILA BIOREMEDIATOR OF AGRICULTURAL SOIL.
Abstract: Eight bacterial strains (BCTL-202 -BCTL-209) having specific resistance ability to degrade and utilize carbofuran insecticide for their growth were isolated from different areas of Punjab. The optimum pH and temperature of the selected bacterial isolates were 6.9 - 7 and 30 - 40degC respectively. The growth pattern of the isolates were studied in different media viz, LB, M9+ glucose (50mg/100ml), M9+carbofuran (5mg/l). In LB medium typical growth pattern were observed as compared with those in the M9, in which prolong log or stationary phase were observed. These isolated strain belong to genus Pseudomonas, Cupriavidus, Planococcus, Marinococcus, Sporosarcina, Bacillus, Enterococcus and Micrococcus.
They were also tested for sensitivity to four heavy metal, Hg+2, Co+2, Cr+3 and Cu+2. All the strains showed resistance against these metals. The antibiotic sensitivity of the isolates was also checked against nine antibiotics. The isolates were resistant against ampiciline, furazolidone and fusidic acid. The bacterial isolates have the potentiality of being used for environmental cleanup of agricultural soil.
Key words: Insecticide degrading bacteria and carbofuran.
The economy of Pakistan is largely based on agriculture. It contributes about 25% to the national economy and is main source of income in rural areas, which accounts for 70% of total population. The well being of the economy depends largely on the production, processing and distribution of major products such as cotton, wheat, edible oil, sugar, milk and meat. The total pesticide consumption in 1962 was 5000 metric tons which reached up to 70,000 metric tons in2000, even these insecticides which are banned in the developing countries were still in use in Pakistan. The indiscriminate and unplanned use of agrochemicals have caused serious environmental problem .
The residues of these agrochemicals directly or indirectly gain entry into the food chain and prove health hazardous to animal and plants . The pesticide/insecticide does not readily disappear from the environment; soil microorganism may be responsible for the removal of insecticide. Several factors are involved in insecticide degradation like transformation, photochemical mechanism, physical mechanism, chemical mechanism, microbial degradation and bioremediation. Bioremediation can be defined as any process that uses microorganisms, fungi, green plants or their enzymes to return the natural environment altered by contaminants to its original condition. Bioremediation may be employed to attack specific soil contaminants, such as degradation of chlorinated hydrocarbons by bacteria .
Biotransformation of various pollutants is a sustainable way to clean up contaminated environments. These bioremediation and biotransformation methods harness the naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds including hydrocarbons, polychlorinated biphenyls, polyaromatic hydrocarbons and metals.
Major methodological breakthroughs in recent years have enabled detailed genomic, metagenomic, proteomic, bioinformatic and other high throughput analysis of environmentally relevant microorganisms providing unprecedented insights into biotransformation and biodegradative pathways and the ability of organisms to adapt to changing environmental conditions . Bioaugmentation refers to the introduction of a group of natural microbial strain or a genetically engineered variant so as to achieve bioremediation.
The pesticide undergoes a variety of chemical reaction in soil and most probably in the segments of environment. Microbial degradation is an important step in the disappearance and in most cases detoxification of pesticides. Many soil applied pesticides are degraded more rapidly following repeated application at the same site . Degradation of pesticides is the breaking down of toxic chemicals into nontoxic compounds and, in some cases, back into their original elements. There are some strictly chemical reactions which take place in soil that aid in degradation, however, the most common type of degradation occurs through the activity of microorganisms, especially the fungi and bacteria .
There is nothing mysterious about microbial degradation of pesticides. Microorganisms simply supply a medium and an energy source for rather simple chemical reactions to take place. They in return obtain food, essential elements, or energy to carry on their life functions. The soil fumigant methyl bromide, the herbicide dalapon, and the fungicide chloroneb are examples of pesticides which are degraded by microorganisms .
In a survey of soils from commercial fields, there was evidence that enhanced biodegradation of the compound has been induced by normal field applications. Factors responsible for the enhanced degradation are micro- organisms present in the soil, able to degrade applied pesticide . Several herbicides are prone to degradation, including members of the thiocarbamate group, the ureas, linuron and monolinuron. The metamitron (herbicide) degrading bacteria Rhodococcus sp. was isolated from the treated soils by enrichment culture. The herbicide was completely degraded within 7 days at 25o C in laboratory tests.
A pure culture of Arthrobacter sp. was shown to degrade metamitron, in the presence of alternative sources of carbon and nitrogen, within 2 weeks of incubation in the dark. They also isolated bacteria from liquid cultures of treated soils which were capable of degrading carbofuran as sole carbon and nitrogen sources . Carbofuran degrading bacteria in soils without a history of previous treatment was noted , .
Degradation is more rapid in alkaline soils than in acidic soils. Breakdown products in soil include carbofuran phenol,3-hydroxycarbofuran, and 3-ketocarbofuran, all of which appear to be relatively nonpersistent. Carbofuran does not bioaccumulate to any noteworthy extent. One of the more significant metabolites is 3-ketocarbofuran .
The present study was conducted with an aim to isolate local strains of insecticide degrading bacteria and evaluates their characteristics for purposes of bioremediation of insecticide contaminated soil in general, but carbofuran in particular, contaminating fields. These isolates will have the potential to clean-up the environment from such persistent pollutants. In future these bacterial strains may be conveniently used in a microbe based system. The present study will provide a baseline data for future studies in biodegradation and bioremediation of carbofuran insecticide.
MATERIAL AND METHODS
Soil samples collected from different agricultural areas of Punjab, where farmers are using insecticide, pesticides and weedicides indiscriminately. The soil samples were collected from Lahore (Ravi, Jalo, Vegetable stores and Packages industry), Faisalabad and Raheemyar Khan. A total of 15 soil samples from different polluted areas have been collected and screened insecticide degrading bacteria. The samples were collected in sterile autoclaved glass bottles; screw capped, labeled and readily brought to the laboratory for analysis. Total nine bacterial isolates separated from these samples and given the name BCTL-202, BCTL-203, BCTL-204, BCTL-205, BCTL-206, BCTL-207, BCTL-208 and BCTL-209.
For isolation of insecticide degrading bacteria, one gram of each soil sample were suspended in 9 ml of distilled water and kept at room temperature. Next day, 50ul of the supernatant was spread on insecticide containing M9 agar medium (0.6g Na2HPO4 2H20, 0.3g KH2PO4, 0.05 g NaCl and 0.1g NH4Cl dissolved in 100 ml of distilled H20, pH adjusted at (7.2- to 7.4). After adding 1.5g agar, the medium was autoclaved. Then 200ul of 1M MgSO4.7H2O and 10ul of 1M CaCl2 solution was added after cooling till 40 C in which glucose replaced by carbofuran insecticide at a final concentration 0.5ml/100ml. The samples were spread on M9 medium containing different concentration and were incubated at 37oC overnight. The isolated insecticide- resistant colonies were streaked on M9 medium plates with higher concentration (100mg, 200mg, 400mg, 600mg, 800mg and 1000mg/100ml) of above mentioned insecticide.
This procedure was repeated by increasing concentration of insecticide in the medium each time, till the isolate failed to grow even after seven days of incubation. This concentration was considering minimum inhibitory concentration (MIC).
In the present study technical grade of Carbofuran [(RS)-a- cyano-3-phenoxbenzyl (IRS, 3RS;1RS, 3SR)-3-(2-dichloro- vinyl)-2, 2-dimethylcyclopropane-carboxylate. Roth: (RS) a- cyano-3-phenoxbenzyl (1RS)-cis-trans-3-(2, 2- dichlorovinyl) -2, 2- dimethylcyclopropane-carboxylate] was used. Carbofuran synthetic pyrethroid which is used to control a wide range of insects, especially Lepidoptera but also coleopteran, Diptera and other classes, in fruits, vienes, vegetables, cucurbits, cereals, cotton, coffee, cocoa, rice, oil seeds, beet, ornamentals, etc. Physical and Biochemical Characterization A single isolated colony of insecticide degrading bacteria was picked up and streaked on the Luria Bertani (LB) agar plates (1g NaCl, 1 g Tryptone and 0.5 g yeast-extract in 100 ml of distilled water at pH 7.0 to 7.2, followed by addition of 1.5g agar) and incubated at 370C for 24 hours. This process was repeated three times for getting pure.
A well isolated bacterial colony was inoculated into 50 ml of LB in 250 ml flask and incubated overnight at 37oC on a shaking water bath at 25 rpm. This overnight culture was used for Gram staining according to Benson  and characterization of bacterial isolates. For biochemical characterization, the isolates were tested for oxidase, catalase, starch hydrolysis, citrate utilization, urease, motility, nitrate reduction, antibiotic resistance, acid release from sugar, sporulation and indole tests according to Benson , whereas Voges- Proskauer's and methyl red test was performed according to Cheesbrough . The isolates were also tested for tyrosine decomposition, according to Collee and Marr .
Determination of Optimum Growth Conditions
For determination of optimum pH, 5ml LB broth was taken in three sets, each of 9 test tubes (dia. 2 cm), and pH was adjusted at 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0. These tubes were inoculated with 50 l of log phase bacterial isolates and incubated at 37oC for 24 hours. Their absorbance was taken at 600 nm. A graph was plotted between pH (along X-axis) and respective absorbance (along Y-axis) for the determination of optimum pH. For determination of optimum temperature, 5 ml LB broth in three sets, each of four test-tubes was inoculated with 50 l of log phase culture of bacterial isolates and incubated at 30oC, 35 oC, 37oC and 40oC. After 6-8 hour incubation, their absorbance was taken at 600nm. A graph was plotted between temperature and respective absorbance. Determination of antibiotic resistance The five bacterial isolates were spread on five LB agar plates separately.
Then sensitivity discs of ampicillin, spectinomycine, chloramphenicol, Erythromycin, lincomycin, kanamycine, fusidic acid, furazolidone and vanacomcin were placed on these plates and the plates were incubated overnight. The appearance of clear area around antibiotic discs showed the sensitivity of isolates to those antibiotics, while the growth of the isolates around the discs showed their resistance against those antibiotics. The zone of inhibition was also measured for each disc and interpreted according to BBL and Difco chart by Benson .
Determination of heavy metal resistance
All isolates were checked for their ability to resist heavy metal. For this purpose small filter paper disc (9mm diameter) were autoclaved and loaded with 1% solution of copper, chromium, mercury and cobalt and allowed to dry for one hour. These discs were placed on the surface of the inoculated nutrient agar plates at 37oC for 24 hours. The clear zone of inhibition around the disc indicated that the organism were inhibited by the heavy metal, whereas growth around the disc indicated that the given organism were resistant to the heavy metal.
The soil samples were collected from four different agricultural areas of the Punjab (Lahore, Faisalabad, Khariyan and Raheemyar Khan). This region is known for crop such as wheat, rice, maize, sugarcane, cotton and different vegetables and fruits. For the protection of these crops from insects, farmers are using insecticide indiscriminately. We have taken the soil samples from these areas and checked the degradability of this insecticide with bacteria. The pH of the region, ranged between 6.7- 7 and temperature 25 to 37degC respectively. These strains were capable of utilizing carbofuran as a sole source of carbon in the M9 medium. Three strains were collected from Lahore Packages industry (BCTL-203), vegetable market (BCTL-204) River Ravi (BCTL-205), Jalo (BCTL-206) and Chandraey (BCTL-208) were able to grow on M8 media. One strain each from Faisalabad (BCTL-202), Kharian (BCTL-209) and Rahimyar Khan (BCTL-207) respectively, was capable of utilizing carbofuran as sole source of carbon.
The technical grade carbofuran (88% concentrated) 5 mg/liter used for this study. The colonies appeared on carbofuran treated plates (Table-I).
Table I: Numbers of colonies appeared on carbofuran (0.5ml/l) treated plates
Sr.###Isolated strains###No.###of No.###of
Morphological and physical characterization
All selected strains were gram positive cocci except BCTL-208 and BCTL 209 which were negative. All the bacterial strains were cocci except BCTL-202 is rod shaped. The strains of BCTL-203 was of pink color, BCTL- 206 was of bright yellow and BCTL-207 was of pale yellow and BCTL-208 was orange color while all other were of creamy color. BCTL-202, 203, 204, 205, 206, 207 and 208 strains had colonies which were round regular elevate shiny and with smooth margin. Some colonies have oily appearance and buttery consistency. Most of these colonies were small in size. However BCTL-205 and BCTL-209 gave dry, irregular colonies with wavy margin (Table-II).
All the selected strains were motile except BCTL- 209. The moving strains showed rotatory and vibratory movement under microscope. The strains BCTL-202, 203, 205 and 207 were spore forming while other strains were non sporing. All isolated bacterial strain showed good growth on nutrient agar which mean all strains where positive for this test. Prominent growth of all the strains was observed on nutrient broth while heavy growth was observed on glucose medium in all the strains.
Various type of biochemical tests were performed to characterized the bacterial strains. All the strains showed positive oxidase and catalase test except BCTL-202 which does not show active bubbling. All the strains show positive result in urease test and able to produce cytochrome-c oxidases and can split urea, with the production of enzyme urease in bacteria except BCTL-202. In tyrosine decomposition test acetoacetate and fumarate are formed by the decomposition of tyrosine. Only BCTL-208 and BCTL-209 strains of bacteria were able to perform this function,while all the remaining strain shows negative result.
Starch hydrolysis with amylase was observed in BCTL-202, 203 and 204, while all the other strains depict negative results. The methyl red test is used to identify enteric bacteria based on their pattern of glucose metabolism. Two strains BCTL-205 and BCTL-209 show positive result while all other strains show negative result.
Bacteria use citrate as its sole carbon source in citrate utilization tests. If it is positive strain for this test, the pH raises and there will be no acid in the end product. The BCTL-205 and 208 strains showed positive result, while all other strains show negative result. The nitrate reductase test is a test to differentiate between bacteria based on their ability or inability to reduce nitrate (NO3) to nitrite (NO2) using anaerobic respiration. Out of eight two strains BCTL- 202 and 208 show nitrate reduction while other strain show negative result. In Voges-Prokauer(v.p) test we have to check the production of acetylmethylcarbinol (acetoin) from fermentation of glucose. No red color was observed following the application of Voges-Prokauer(v.p) test on strain BCTL- 205 and 208 remaining all showed negative result. The indole test is performed on bacterial isolates to determine the ability of the organism to split indole from the amino acid tryptophan.
Five strains (BCTL-202, BCTL-203, BCTL-206, BCTL-207 and BCTL-209) showed negative test, while all other showed positive test.The carbohydrate utilization tests are designed to detect the change in pH which would occur if fermentation of the given carbohydrate occurred. Acids lower the pH of the medium which will cause the pH indicator (phenol red) to turn yellow. If the bacteria do not ferment the carbohydrate then the media remains red. If gas is produced as a byproduct of fermentation, then the Durham tube will have a bubble in it. All the strains show fermentation ability while show no gas production.
Microbial degradation is an important step in the disappearance and, in most cases detoxification of pesticides. Herbicide biodegradation may prevent the problem of environmental pollution but it can also reduce the effectiveness of a compound in controlling targeted pests. Many soil applied pesticides are degraded more rapidly following repeated application at the same site . Suett and Walker  reported the repeated application of pesticides to soil over many years would have some quantitative and qualitative changes in soil and their microbial population. Felsot  also recognized that major pathway of pesticide degradation is microbial. Biodegradation of xenobiotic can be influenced by soil properties including pH, organic matter contents, seasonal climatic factors such as soil moisture contents and temperature .
In present studies optimum temperature and pH for growth of insecticide degrading bacterial isolates ranged between 6.5-7.5 and 30 to 42degC. Suett  reported that factor such as treatment in insecticides histories, different pH value or cropping histories, exposing to different fertilizer regime etc. can have a significant impact on the soil microbial populations. Rates of biodegradation generally decrease with decreasing temperature, probably due to decrease rate of enzymatic activity .
This study is concerned with the isolation and identification of carbofuran resistant bacteria from soil samples collected from agricultural fields. Identification and characterization of bacterial isolate on the basis of morphological, cultural and biochemical characteristics depicts that they belong to genus Pseudomonas and this genus was gram positive cocci. Pseudomonas bacteria were belonging to aromatic hydrocarbons, oil, petroleum products and pesticides degradation [21, 22]. It was also observed that in the presence of high concentration of carbofuran, the bacteria were greatly stressed and consequently their growth was slowed down. The bacteria changed its normally rod shape morphology to that of a coccus at increased insecticide concentration. However, this change was temporary, because the cells recovered the original rod form after a few days.
We reported that out of eight strains one strain was BCTL-202 Bacillus pumilus and it was capable of utilizing toxic xenobiotics insecticide. It has been independently reported by other researchers [23, 24, 25, 26, 27, 28 and 29].
BCTL-203 was Micrococcus roseus bacterial strain and formed colonies which were orange in colour, gram-positive, motile and non-endospore forming. Micrococcus roseus forms bicyclic ketocaretenoids , Pigmentation in M. roseus was found to be increased when the bacteria were grown at 5degC as compared to its pigmentation at 25degC.
The strain BCTL-204 was identified as Marinococcus halophilus and it was gram positive, non-endospore forming, motile bacterium and showed maximum growth at 37@C. Louis and Galinski  also reported that M. halophilus is a gram positive moderate halophilic eubacterium.
The strain BCTL-205 was identified as Sporosarcina ureae.
It was gram-positive endospore forming, motile and carbohydrate oxidizing. Claus et al.,  also reported Sporosarcina ureae as an obligate slightly halophilic bacteria from salt marsh soil.
BCTL-206 strain belong to genus Planococcus koccuri having colonies of bright yellow color, optimum pH 6.5 and non-endospore forming. This strain grows well in the media containing high concentrations of NaCl. Miller,  also reported that increase of NaCl favoured the growth of bacterial culture Planococcus citeus.
BCTL-207 strain was fit in genus Enterococci and was gram positive cocci, motile and releases acids from sugars. Enterococcus is a genus of lactic acid bacteria of the phylum Firmicutes. Members of this genus were classified as Group D Streptococcus until 1984 when genomic DNA analysis indicated that a separate genus classification would be appropriate .
The strain BCTL-208 was identified as Pseudomonas sp. A pentacholorophenol (PCP) mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. In batch culture Pseudomonas sp. Used PCP as its sole source of carbon and energy and was capable of degrading this compound. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. .
BCTL-209 strain was identified as Cupriavidus necator and it was gram negative, non motile and its colonies were off white in colour. Cupriavidus necator to accommodate a non- obligate bacterial predator of various Gram-negative and Gram-positive soil bacteria and fungi .
When microorganisms are exposed to insecticides, they evolve strategies to cope with such unavoidable stresses. It was also noticed during the study that size of bacterial colony significantly reduced in insecticide mixed media. These results are supported by previously conducted studies on insecticide resistant bacterial strains .
Girvan et al.,  reported increased bacterial and fungal counts in the total and cultural communities in soils with different fertilizer and pesticide applications and also noted decrease in heterogeneity of the active bacterial community.
Pesticide addition did not significantly affect bacterial numbers or heterogenecity, but it led to major shifts in the active soil bacterial community structure.
Locally isolated strains are beneficial in two ways. Firstly the local strains are better adapted to the environmental conditions which have been prevalent for very long period. Secondly the variation in genes and genetic elements acquired in response to certain selection pressures or due to spontaneous mutations enhance the survival values of microorganisms for their efficient exploitation in environmental cleanup operations. Microbes and the environment constantly interacting with each other constitute a very ingenious system of evolving each other. Microbes are now at the forefront of the campaign against environment pollution due to their catabolic pathways.
The properties of the bacterial strains confirmed their place as strong candidates for biodegradation of pesticides, especially carbamates. Moreover, these isolates efficiently work in a microbe based reactor. These isolated strains provide a base line data for studies in this field. These bacterial strains can be further investigated for identification of particular gene of enzymes responsible for biodegradation of insecticides.
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|Author:||Mujeeb, Khawaja Abdul; Riaz, Asma; Mushtaq, Sumaira; Abbasi, Muddasir Hassan; Ali, Syed Shahid|
|Date:||Sep 30, 2012|
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