Isolation Of Highly Efficient Degradation Bacteria Of Pesticide Acetamiprid And Study On Its Metabolic Pathway

Acetamiprid is an insecticide belonging to the neonicotinoids, first launched by Nippon Soda in 1995 in Japan. It is a systemic and contact insecticide, which is widely used, both in agriculture and domestically, against numerous varieties of insects. This extraordinary spectrum of efficacy, together with full exploitation of the nAChR, plant systemicity, long-lasting effect and versatile uses make acetamiprid a substitute for organophosphates, methylcarbamates and pyrethroids. With continuous research in the insecticide, its toxicity has become more and more obvious. Acetamiprid increases sensitivity to antennal stimulation by sucrose solutions at doses of 1μg/bee and impaired long-term retention of olfactory learning at the dose of 0.1μg/bee. Also acetamiprid is genotoxic and cytotoxic to human peripheral lymphocytes in vitro. So it's necessary to grasp the treatment of acetamiprid residues in the environment to reduce its harm to the environment. As for the present metabolic studies of acetamiprid, more researches are reported in plants, animals and the environment, with quite less in its microbial metabolism. This research's purpose is to screen microorganisms which can degrade acetamiprid to nontoxic substances to the environment. Also study the metabolites of acetamiprid, explore microbial metabolic pathways of it.This study established an experimental method to isolate highly efficient degradation bacteria of acetamiprid, identified the efficient degradation strain, and researched the degradation activity of this bacterium. Degradation product and molecular structure of it has been identified, also the degradation conditions of acetamiprid by this strain were optimized.We established a method using thin layer chromatography(TLC) as qualitative detection and HPLC as quantitative detection of acetamiprid. TLC can be used to analysis acetamiprid concentration very quickly in the initial experiments of isolating bacteria. HPLC can be used to measure the microbial degradation ability of the isolated bacterium.For strain's identification, experiments were designed in the aspects of colony morphology in the medium, microscopic morphology, 16S rDNA gene sequence analysis, whole-cell fatty acid analysis. At last we identified the strain as a Gram-negative bacterium, with polar flagella, short rod shape, 1μm long, 0.5μm wide, a strain of Stenotrophomonas sp.Gas chromatography-mass spectrometry (GC-MS) was used to study the degradation product of acetamiprid. With nuclear magnetic resonance analysis results, we identified that molecular weight of the degradation product is 156 Da, with a molecular formula Cl-C₅H₃N-CH₂-NH -CH₃, which is identical to acetamiprid degradation product IM-1-4 in honeybee and soil.At last we optimized the degradation conditions of acetamiprid in the aspects of N source nutrients, acetamiprid concentration, temperature, medium pH and rotation speed. By considering the growth of bacterium and the degradation ability of acetamiprid, we use the formula of 1 g/L yeast extract as a source of N nutrient, 1 g/L acetamiprid as substrate, 30oC, pH 7 and 200 r/min as the best degradation condition.