Characterization of Carbofuran-Degrading Bacteria
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African Journal of Environmental Science and Technology Vol. 6(2), pp. 104-114, February 2012 Available online at http://www.academicjournals.org/AJEST DOI: 10.5897/AJEST11.067 ISSN 1996-0786 ©2012 Academic Journals Full Length Research Paper Characterization of methomyl and carbofuran degrading-bacteria from soils of horticultural farms in Rift Valley and Central Kenya Kevin Mbogo Omolo1*, Gabriel Magoma1, Kamau Ngamau2 and Tsanuo Muniru3 1Biochemistry Department, Jomo Kenyatta University of Agriculture and Technology, P. O. Box 62000-00200, Nairobi, Kenya. 2Horticulture Department, Jomo Kenyatta University of Agriculture and Technology P. O. Box 62000-00200, Nairobi, Kenya. 3Department of Pure and Applied Sciences, Pwani University College, P. O. Box 175 Kilifi, Kenya. Accepted 30 May, 2011 The use of pesticides is very critical in protecting the farmers’ investment in seeds, fertilizer and labour since they provide a sure cover from damage by pests. The use of pesticides is therefore inevitable and the environmental pollution due to pesticides and their residues will continue to be a challenge. In this study, bacterial strains capable of degrading methomyl (S-methyl-N-[(methylcarbamoyl) oxy]- thioacetimidate) and carbofuran (2, 3-dihydro-2, 2-dimethyl-7-benzofuranyl methylcarbamate) were isolated from soils sampled from horticultural farms with a history of pesticide usage. High pressure liquid chromatography was used to monitor biodegradation of both methomyl and carbofuran using reference standards and acetonitrile and water as mobile phases. Partial 16S rDNA sequence analysis indicated that the carbofuran-degrading strains were closely related to members of the genus Pseudomonas and Alcaligenes while the methomyl degrading strains were closely related to members of the genus Flavobacterium and Alcaligenes. The morphological and biochemical characteristics of the isolates also confirmed the phylogenetic signature. The study established that the activities of the esterase and phosphatase enzymes correlated well with biodegradative capability and recommends possible application of the isolates in the in vivo bioremediation of pesticide contaminated soils. Key words: Pesticides, carbofuran, methomyl, biodegradation, bacteria. INTRODUCTION Xenobiotic compounds are widely distributed in the for pest management and vector control thus the diverse environment as a result of their widespread use as environmental contamination with pesticides (Ngowi et pesticides, solvents, fire retardants, pharmaceuticals, and al., 2007). Carbamates, synthetic organic chemicals, are lubricants. Several of these chemicals cause highly poisonous pesticides that have found wide usage considerable environmental pollution and human health in agricultural farms as insecticides, fungicides, problems due to their persistence and toxicity (Rossberg herbicides, nematicide and acaricides (WHO, 1986). The et al., 1986). Agricultural modernization due to increased non-target toxicity of carbamates extends from human market demand for agricultural products has greatly beings to both aquatic and terrestrial organisms with high facilitated the industrial production and use of pesticides sensitivity in fish and earthworms (WHO, 1986). Some of the carbamates used in horticultural farming include carbofuran (in form of Furadan), carbaryl, aldicarb and methomyl. Many researchers have reported the *Corresponding author. E-mail: [email protected]. Tel: biodegradation of various pesticides under different +254-723-623-791. physiological conditions and isolated many bacterial Omolo et al. 105 species. It has thus become increasingly possible to contained 0.1 g/L cycloheximide (Sigma-Aldrich, Steinheim, isolate microorganisms that are capable of degrading Germany) to suppress fungal growth (Maarten et al., 2007). xenobiotic and recalcitrant compounds from environments polluted with toxic chemicals (Gibson and Morphological and biochemical characterization of the isolate Harwood, 2002). This study focused on the isolation and characterization of methomyl and carbofuran degrading Cell morphology was determined by a phase contrast Zeiss bacterial strains in selected agricultural soils. Axiophot microscope (Carl Zeiss, Oberkochem, Germany) supplemented by the classical gram staining method (Bartholomew, 1962). Cell motility was assesed by direct microscopic observation from cultures in the growth phase (in SIM agar; pH 7.3). MATERIALS AND METHODS Biochemical tests for nitrate reduction, indole production, citrate utillization, hydrogen sulphide production and acid production were Sampling done according to the procedures described by Cappuccino and Sherman (2002). Soil samples were collected from horticultural farms in three geographically distinct regions; Naivasha which is in Northwest of ״ ״ ΄ Nairobi located at 0° 43 0 South and 36° 0 East and Gilgil Assay of phosphatase activity ״ ΄ which is located in the following geographical co-ordinates 0° 13 0 South 37° 5΄ The isolates were first grown in a low phosphate media suitable for ״East in Rift Valley and Thika 1° 3΄ 0 ״South, 36° 16΄ 0 East in Central Kenya. Stratified random sampling method was formation of both repressible and constitutive alkaline phosphatase ״0 used to collect soils with the geographical regions forming three from bacteria. The composition of the media included; peptone strata. From each region, plots (farms) were identified by simple water 3 g/L, yeast extract 1 g/L, NaCl 31.2 g/L, MgCl:6H2O 5.39g/L, randomization. Cross-sectional sampling was done on the upper (5 Fe(NH4)2SO4, Trizima base 12.1 g/L, pH 7.4 and each isolate was to 20 cm deep) soil from five locations within a given plot of ~100 cultured in 10 ml of the medium in universal bottles with reciprocal m2. Samples were transferred in glass jars and stored at 25°C until shaking at 27°C for 24 h. used. Aerobic conditions for sampling, medium preparation and Phosphatase activity was estimated in duplicate samples by culture handling were maintained throughout the subsequent monitoring the liberation of p-nitrophenol from p-nitrophenyl enrichment process. phosphate (disodium salt; BDH) using a modified procedure described by Bolton and Dean (1972). One milliliter of cultured medium was incubated with 1 ml of 50 mM p-nitrophenol Media composition and culturing phosphate solution, 1 ml of 0.5 M Tris-HCl buffer pH 8.2 and 1 ml of mineral salts; 0.4 M NaCl, 0.05 M MgCl2, 0.01 M KCl and 0.01 M The organisms were cultivated in phosphate buffered mineral salts CaCl2. The mixture was incubated for 60 min at 28°C before medium (MSM) as described by Chaundry and Ali (1988). The addition of 4 ml of 0.2 M sodium hydroxide. The p-nitrophenolate MSM contained the following constituents, in grams per liter: liberated was measured using spectrophotometer at 405 nm verses K2HPO4, 4.8; KH2PO4, 1.2; NH4NO3, 1.0; MgSO4 7H2O, 0.2; Ca p-nitrophenol standard. (NO3)2 .4H20, 0.4, and Fe2 (SO4)3, 0.001. The media was supplemented with carbofuran or methomyl as the sole carbon source to a final concentration of 2 mM. All the solutions, cultures Assay of esterase activity and media were prepared and maintained using aerobic techniques Esterase activity was assayed using Tween 80 as described by which included covering media with cotton wool and shaking to -1 allow air circulation in the cultures. Cultures were incubated on a Sierra (1957). The media contained (g L ): peptone 10.0, NaCl 5.0, shaker (100 rpm) at 30°C and monitored for the loss of carbofuran CaCl2.2H2O 0.1 and agar 18.0, pH 7.4. To the sterilized culture or methomyl using high pressure liquid chromatography while media, previously sterilized, Tween 80 was added at a turbidity increase due to bacterial growth was monitored using concentration of 1% (v/v). The medium was inoculated with the spectrophotometer (Shimadzu UV240) at OD600. isolates and the presence of halos was observed and the halo diameters recorded. The enzymatic index was then calculated by dividing the halo diameter by colony diameter. Culture enrichment and isolation Chromatographic analysis of carbofuran and methomyl The isolation of methomyl- and carbofuran-degrading bacteria was degradation done using a modified enrichment culture technique described by Chaundry and Ali (1988). Approximately 1 g of each soil sample The decline in carbofuran and methomyl concentration upon was suspended in 10 ml of mineral medium (MSM) containing 2 degradation was monitored using high pressure liquid mM of carbofuran or methomyl as the sole carbon source. Cultures chromatography (HPLC) (VP shimadzu) on a reverse phase C-18 were grown in 100 ml- culture flasks under aseptic conditions at column, 25 cm × 4.6 mm, fitted with a C-18 silica reverse phase 30°C with shaking in a rotary shaker at 100 rpm for 18 days. Using guard column and equipped with a UV detector and retention time wire loop, cultures were then streaked into agar plates containing identified using reference standards at 254n m, 70% acetonitrile mineral salt medium supplemented with 2 mM of methomyl or and 30% water for carbofuran and a wavelength of 235 nm, 30% carbofuran. Single colonies obtained were re-suspended in basal acetonitrile and 70% water for methomyl (Figures 1 and 2, medium (MSM) containing 2 mM of methomyl or carbofuran for 14 respectively). The HPLC pumps were set at a flow rate of 1ml/min days to confirm the ability of the isolates to utilize methomyl or at a temperature of 27°C. carbofuran. The cultures were then streaked into plates containing The mobile phase was