| Abamectin is a commercial preparation of avermectin B1a, which is extensively used against a broad spectrum of endo-parasites and ecto-parasites in animals, and against different phytophagous pests of field crops, ornamentals, vegetables, fruits etc and also used in controlling fire ants. Abamectin has caused great environmental pollution by posing potential risk to non-target soil and dung invertebrates leading to the instability of an ecosystem. Moreover, the abamectin residue can easily run-off from the sites of application to nearby water systems, and the compound is reported to be very toxic to aquatic organisms even at very low concentrations. So, there is a need to develop remediation technologies to cleanup the abamectin contaminated environment. Microbial remediation has always been the method of choice to decontaminate the polluted environment due to its high efficiency and low cost.A novel abamectin-degrading bacterium designated as GB-01 was isolated from the soil of a citrus orchard, which has been exposed to the application of abamectin for many years, using continuous enrichment culture technique. The bacterium GB-01 was able to grow in mineral salt medium (MSM) using abamectin as sole carbon source for growth. The colony morphology and microscopic characters of strain GB-01 were examined under a light microscope, as well as under a Transmission Electron Microscope (TEM). Biochemical tests for the identification were carried out according to the Bergey’s Manual of Determinative Bacteriology. The isolate was a gram negative bacterium, which formed smooth colony with an average diameter of approximately 0.6-1 mm. Cells of strain GB-01 were straight rods, having rounded ends, with dimensions of 0.4-0.7μm in width and 1.2-1.7μm in length, and occasionally formed pairs or short chains. On the basis of morphological, phenotypical and biochemical characteristics, the strain GB-01 was identified as a Burkholderia species. Polyphasic taxonomic approach was used for the complete identification of strain GB-01. Biolog GN2 MicroPlating, API 20NE, ID 32 GN, and API 50 CH commercial kits were used to examine the physiological and biochemical characteristics. Chemotaxonomic characteristics i.e. G+C mol% and whole cellular fatty acid profile, were investigated. PCR amplification of 16S rRNA and recA genes was performed. Alignment with different gene sequences from GenBank was achieved using the software Clustal X 1.8.3 with default settings. Phylogenesis was analyzed using MEGA version 4.0 software and distances were calculated using the Kimura 2 parameter distance model. Phylogenetic trees were built using the neighbor-joining method. Each dataset was bootstrapped for 1000 replicates. The decisive evidence was collected from DNA-DNA hybridization with closely related species. Polyphasic taxonomic analysis revealed that strain GB-01 was a member of Burkholderia cepacia complex (Bcc), being an atype strain of Burkholderia diffusa species.The optimum growth conditions for strain GB-01 were recorded as pH 7.0 and temperature 30℃. Biodegradation assays were conducted in liquid broth cultures and soil microcosms. In liquid cultures, the strain GB-01 was able to utilize more than 90%of abamectin, supplemented into MSM medium as a sole carbon source, at initial concentration of 50 mg L-1 and 100 mg L-1 in 30 and 36 h respectively. The inoculum density was maintained to 1.5 optical densities at 600nm (OD600), and the cells were inoculated at the level of 2% (v/v,2 ml inoculum per 100 ml of culture medium) in all of the shake flask culture experiments. The optimum conditions for biodegradation activity were also recorded as temperature 30℃and pH 7 in broth culture. However, no significant difference of biodegradation activity was observed at temperature 35℃, and pH 8 as compared to optimum conditions. The biodegradation capability was decreased sharply below pH 5 or above pH 9, and similar was the observation at temperature 10℃and 45℃Initial concentrations of abamectin in MSM medium significantly affect the biodegradation activity of strain GB-01. The longer degradation cycle was observed with abamectin concentrations higher than 100 mg L-1, while the strain was unable to degrade abamectin at initial concentration of 200 mg L-1. Induction of degrading cells with abamectin (2 mg L-1) during the log phase of growth for inoculum production considerably enhanced the biodegradation efficiency of strain GB-01.The degradation products of abamectin in the culture medium extracts were isolated and identified. Two novel metabolites were detected as the catabolic products of abamectin by strain GB-01 using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). These degradation products were identified and characterized on the basis of their mass spectral data and characteristic fragmentation patterns. Based upon these findings, a plausible partial biodegradation pathway of abamectin was proposed.In soil microcosms, the significant abamectin degradation activity of strain GB-01 demonstrated its potential to be used as a bioremediation tool in actual field conditions. The optimum temperature for abamectin degradation in soil microcosm was 30-35℃, while a neutral to slightly alkaline soil pH i.e.7-8 considerably enhanced the degradation rate. These findings indicated the bioremediation potential of strain GB-01 to be used in different soil regions. An inoculum quantity of 108 cfu (colony forming unit) per gram of dw (dry weight) soil exhibited the maximum degradation activity, and strain was able to degrade abamectin (50 mg Kg-1) under optimum temperature and pH conditions. The efficient degradation of 50 mg Kg-1 abamectin in soil microcosms indicated the potential of strain GB-01 to meet over use or spill situations in field conditions. Inoculation frequencies showed significant effects on the biodegradation activity, and successive inoculations at defined intervals resulted in rapid degradation rate as compared to single inoculation. Induced inoculum showed the rapid degradation rate, while flooded conditions did not significantly affect the biodegradation activity of strain GB-01 in soil microcosms.In summery, abamectin biodegradation by Burkholderia diffusa GB-01 strain was studied from a number of angles. This organism has several interesting and useful traits such as the ability to degrade high concentrations of abamectin, an unusual and effective degradation pathway and demonstrated the ability to remove abamectin from contaminated soil. To our knowledge, this is the first report of aerobic degradation of abamectin by a Burkholderia sp. isolated from indigenous soil microbial community. Our results indicated that Burkholderia diffusa GB-01 strain can be used as an effective bioremediation tool for abamectin contaminated soils. In addition, this is the first study, in which the bioremediation potential of Burkholderia diffusa species isolated from environmental source is reported. |
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