Degradation Characteristics And Pathway Of Cypermethrin And 3-Phenoxybenzoic Acid By Aspergillus Niger YAT1

Pyrethroid insecticides (PY) has been widely used and also is the most promising one. However, its degradation is difficult under natural conditions and can accumulate in the environment and remain in agricultural produces for a long time, thus resulting in severe damages to environment and human healthy. Moreover, most pyrethroid pesticides can be degraded into an intermediate product 3-phenoxybenzoic acid (3-PBA). This product can not only cause secondary pollution to agricultural produces but also retard the biomineralization of its parent pesticide. Microbial degradation is an efficient and green method to degradate various pesticides and their intermediates. It has become a hotspot on C Y residues-degrading.Based on the current incomplete researches on the source of the CY-degrading strain, single degrading pathway of CY and risk of CY residues degraded by microorganism, the uneven distribution or dispersion of pyrethroid pesticides and poor repeatability in detected C Y concentrations caused by the adsorption or absorption of microbial cells in microbial degradation systems, a method for simultaneous detection of CY and 3-PBA in microbial degradation was established to screen beneficial fungi in traditional fermented food, which has the ability to degrade CY and 3-PBA. The degradation character and the pathway of CY by strain was studied and proposed to provide reference for the further research and practical application of degrading strain.The method for simultaneous detection of CY and 3-PBA in microbial degradation systems A pretreatment method for high-performance liquid chromatography (HPLC) to simultaneously determine CY and 3-PBA concentration in the systems was established, including sampling and extraction. Total sampling was suitable for mold systems. CY and 3-PBA could be fully extracted by ultrasonic treatment with acetonitrile under ultrasonic condition. The concentration of CY and 3-phenoxybenzoic acid (3-PBA) in microbial degradation systems were determined by HPLC-UV. CY and 3-PBA were separated by Gemini 100A C18 column (5.0μm,150 mm×4.60 mm, i.d.) at wavelength of 210 nm,1.0 mL/min and column temperature of 25℃, with acetonitrile and phosphoric acid solution (pH=2.5) as the mobile phase in gradient elution. The calibration curve of CY and 3-PBA standards obtained under the given chromatographic conditions showed a good linearity relationship at CY and 3-PBA concentrations range from 0.50 to 50.0 mg/L. The simultaneous determination of CY and 3-PBA in the pretreated sample by HPLC showed a high precision (CY/RSD= 1.23% and 3-PBA/RSD= 2.08%, n=5) and a good stability over a period of five days (CY/RSD=1.57% and 3-PBA/RSD=1.50%, n=5). The recoveries of CY and 3-PBA in microbial degradation systems at three different spiked levels ranged from 98.38% to 105.80%(CY/RSD=1.49%-3.93%, n=5) and 99.59% to 101.20%(3-PBA /RSD=0.58%-3.64%, n=5).Screening of cypermethrin-degradative fungi and its characterization to cypermethrin and 3-phenoxybenzoate acid.Two cypermethrin-degrading fungi strains were isolated from vinegar grains and brick tea, and only the strain of YAT1 have further degradation of 3-PBA ability. The YAT1 could degrade 41.32% of cypermethrin in the PD medium (50mg/L) in 120 h. Combination of morphological and the ITS sequence analysis(GenBank Accession No. JQ3660700), YAT1 was identified as Aspergillus niger.The cypermethrin degradating characteristic research of Aspergillus niger YAT1 shows that it degradation of cypermethrin was carried out with the cometabolism way in the presence of the suitable carbon source, and the degradation of cypermethrin could be described by a first-order kinetics model. In addition, the t1/2 of cypermethrin were 3.573-11.748d when the concentration of CY、temperature、and pH was limited. The temperature and pH had less effect on the t1/2, but the concentration of CY could prolong the t1/2. The Aspergillus niger YATl have different degree of degradation to deltamethrin, fenvalerate, cyfluthrin, and permethrin. The 3-PBA degradating characteristic research of Aspergillus niger YAT1 shows that the strain YAT1 could degraded all of the 3-PBA after 22h incubation in the PD medium with 100mg/L 3-PBA, and the degradation could be described by a first-order kinetics model.The t1/2 of 3-PBA were 5.635-12.160h when the concentration of 3-PBA、temperature、and pH was limited, far less than the ti/2 in natural environment. The pH had less effect on the t1/2, but the temperature and concentration of 3-PBA could prolong the t1/2.Pathway for degrading of cypermethrin by Aspergillus niger YAT1 According to an HPLC method for the detection of characteristic peak, LC-MS confirmation of intermediate degradation product, related detection of enzymatic activity and utilization of assistant analysis on model substrate, inferred the pathway of 3-PBA for degrading of cypermethrin, and the patheway for more degrading of 3-PBA: effect of esterase on making cypermethrin hydrolysis of permethrin acid and a-cyano-3-Phenoxybenzalcohol; the latter spontaneous oxidized to 3-Phenoxybenzaldehyde,and further oxidized to 3-Phenoxybenzoic Acid; effect of dioxygenase on making 3-Phenoxybenzoic Acid oxidized to phenol and protocatechuic acid; or effect of hydroylase on making 3-Phenoxybenzoic Acid oxidized to 5-hydroxyl-3-Phenoxybenzoic Acid,5-hydroxyl-3-Phenoxybenzoic Acid bond cleavage to phenol and gallic acid. Phenol degrades into catechol through ortho electrophilic substitution reaction by hydroxylation oxygenase. Catechol, protocatechuate acid and gallic acid were oxidated to straight-chain olefin acid by dioxygenase. Straight-chain olefin acid was finally oxidated to carbon dioxide.The results from this study can not only provide method on CY detection, but also perfect the biodegradation degradation pathway of CY. In addition, this study provides strains resources and basic data on applying the biodegradation of CY to treat environmental pollution and to eliminate or reduce pesticides residue in agricultural produces, thus possessing important theoretical significance and practical value.