Degradation Of Synthetic Pyrethroids Residues By Photosynthetic Bacteria

Synthetic pyrethroids (SPs), which have exellent biological pesticide acivity, are a kind of chemical pesticides. It is one of main type of pesticides residue in agricultural products because of long-time and vast quantities using.A polyclonal antibody (PAb) specific for fenpropathrin was prepared. Hapten fenpropathric acid (FA) was coupled with carrier protein bovine serum albumin (BSA) to form an antigen FA-BSA via 1-ethyl-3-(3-dimethyl aminopropyl) and curborimide hydrochloride (EDC). The conjugates used to immunize New Zealand white rabbits were identified with UV scanning spectrum and the couple rate of the antigen was 8.8:1. After the antisera were obtained and purified using 50% saturated ammonium sulfate method, the specificity of PAb was detected by IC-ELISA (indirect complete enzyme-linked immunosorbent assay). It showed that the optimal concentration of coating antigen and PAb against fenpropathrin prove to be 1.0μg·mL-1 and 1:12800, respectively. The limited detectable concentration was 8.5μg·L-1. The cross-reaction rate between PAb and other SPs was low, except deltamethrin. The results showed that the anti-fenpropathrin PAb had a high specificity and was good enough for immunoassay of fenpropahtrin residue, and the IC-ELISA was efficient for screening degrading microorganism of SPs residues.SPs residues degrading anaerobic microbial community JZ-1 were domesticated by selective culture. Degrading experiments showed the optimum degrading conditions were pH 7,30℃. Community JZ-1 degraded fenpaprothren, cyfluthrin and cypermethrin with a concentration of 100 mg·L-1 up to 53.27%,33.36%,41.39% in 15 days in optimum conditions, respectively. The nearly complete 16S rDNA were amplified from community JZ-1 using bacterial university primers 27F and 1492R, the restriction analysis was performed following separate digestion with enzyme Rsa I, revealed that the coverage (C) of the 16S rDNA library of community JZ-1 was 93.54%, means better representative of community JZ-1; Shannon-Wiener index (H) was 1.20, means plenty of degradation bacterial resources in community JZ-1. The representative 16S DNA sequences of six clusters were sequenced, and shown that the major clusters were Rhodopseudomonas and Porphyromonadaceae bacterium, respectively.On the basis of domesticated SPs residues degrading anaerobic microbial community JZ-1, photosynthetic bacterial strains, which could degrade SPs residues were isolated by IC-ELISA and then by single spore separating and pure culturing. Isolated strains (named as PSB07-6, PSB07-15, PSB07-19, PSB07-21) were identified as members of genus Rhodopseudomonas based on its morphology, physiology and homology and phylogenetic analysis of 16S rDNA gene sequence. The degrading velocity of isolated strains PSB07-6, PSB07-15, PSB07-19, PSB07-21 were 1.02 mg·d-1,1.48 mg·d-1,1.13 mg·d-1,2.94 mg·d-1, respectively, cultured in 35℃,3000 Lx.Photosynthetic bacterial strain PSB07-15 with better fenpropathrin residue degrading efficency was selected for the degradation characterristics test. The results showed that isolated strain PSB07-15 could grow in PSB media with fenpropathrin up to 600 mg·L-1. The optimal conditions of strain PSB07-15 degrading fenpropathrin were 20-30℃, pH 6.5-7.5,2000-3000 Lx. The degradation velocity of fenpropathrin residue of strain PSB07-15 was 1.87 mg-d"1 in optimal degradation conditions. Strain PSB07-15 degraded fenpropathrin residue in co-metabolic way. The degrading-enzyme(s) of strain PSB 07-15 was endoenzyme.The pH valve monitoring in the process of degrading fenpropathrin residue by isolated strain PSB07-15 showed that there are complex varied pH value, decreasing in 1-3d and 9-15d, raising rapid in 5-9d. Gas chromatography with mass selective detector (GC-MS) identified m-phenoxyphenyl-acetonitril as the stable metabolic product and from this a biodegradation pathway of hydrolasis of fenpropathrin residue is proposed. The results of biological characteristics showed that strain PSB07-15 could produce IAA (indole-3-acetic acid) (1.81±0.09 mg·L-1) and possess higher activity of ACC deaminase (0.35±0.02 U·mg-1) in 24h, and the efficiency of IAA production (1.02±0.05 mg·L-1) and ACC deaminase activity (0.12±0.01 U·mg-1) of strain PSB07-15 could inhibited by fenpropathrin residue with concentration of 100 mg·L-1. Strain PSB07-15 could increase root length of maize significantly.Fenpropathirn residue bioremediation efficiency by isolated strain PSB07-15 in hydroponic cucumber system was evaluated in lab, for actual application of bioremediation by photosynthetic bacterium. The results showed, strain PSB07-15 degraded 47.63% fenpropathrin residue up to a concentration of 100 mg·L-1 in cucumber nutrient fluid, and degraded 59.73% fenpropathirn residue in cucumber within 30d. Strain PSB07-15 could enhance significantly root-length increment and biomass increment; however, enhance root activity value and root system catalase activity value not significantly of cucumber.Strain PSB07-21 was selected for cloning and analysis of degrading-enzyme gene. The optimum conditions of degrading SPs residues were at 35℃, pH 7 and 3000 Lx. PSB07-21 could degrade fenpropathrin, cypermethrin and bipthenthrin by 66.63%,43.25% and 50.18% in a concentration of 600 mg·L-1 at day 15, respectively. We cloned a putative gene which was 326 bp long with 37.0% identical to 2OG-Fe(Ⅱ) oxygenase gene. When compensating low concentration Fe(Ⅱ) in PSB medium with synthetic pyrethroids, degradation efficiency of strain PSB07-21 was enhanced.This study contributes to screen and apply SPs residues degrading organisms for SPs residues biodegradation. And it is theorial references for degrading enzyme gene cloning, constructing efficiencial degrading engeneering bacteria.