LuxAB Labelling Of Chlorpyrifos-degrading Strain X1of Cupriavidus Taiwanensis And Its Fate In Soils

Chlorpyrifos is one of the most widely used insecticides in the world. It could contaminatethe environment and harm the health of human with numerous agricultural crop and urban pest control uses. There were many studies conducted on the microbial remediation of chlorpyrifos, and most of them focused on laboratory connditions by pure culture, few studies on biodegradation of chlorpyrifos in the soil environment. In this paper, plasmid pTR102that contained luxAB genes was successfully introduced into chlorpyrifos-degrading strain Cupriavidus taiwanensis X1by electroporation. Then we studied the XI strain and Lux-X1, including their physiological, degradation of chlorpyrifos, the main factors which influence survival condition and chlorpyrifos-degrading efficiency of Lux-X1in three soils. The main results are as follows.The gradient plate method was used to investigate the minimum inhibitory concentration (MIC) of km and tet to X1strain, and the MIC were17.1μg/mL and2.4μg/mL respectively.The main factors which influences the transformation efficiency of X1strain were optimized, and the optimal conditions for electroporation were as follows:the electric field strength was10kv/cm, the electric time was4ms and the concentration of plasmid was100ng/μL. By the method of electroporation, gene luxAB was successfully introduced in X1strain, and a marked strain, Lux-X1, was successfully obtained. The luxAB gene can express in Lux-X1, after continously transfering for15generations, and the characteristic of luminescence was still stable.The optimal growth condition of strain XI and strain Lux-X1were determined separately. The optimal growth temperature and pH was37℃and pH7.0, but the growth of Lux-X1was more sensitive to pH than XI, when the pH was below6or above8; Lux-X1grew a little slowly. The growth curve of X1and Lux-X1showed the comparable growth tendency.The chlorpyrifos degradation characteristics of strain X1and strain Lux-X1were studied. With initial chlorpyrifos concentration of200mg/L, the degrading rate of Lux-X1at2h,6h,10h, were53.04%,3.89%,93.13%respectively, while the degradation rate of X1at2h,6h,10h, were52%,88.63%,97.02%respectively.The survival conditions of Lux-X1in shajiang black soil, yellow cinnamon soil and red soil were studied. The results showed that Lux-X1stably colonized in the three kinds soils after30d. The colonization of Lux-X1was better in shajiang black soil and poor in red soil. The number of Lux-X1in the soil rose at day2, and then decreased slowly in shajiang black soil while decreased from the beginning both in yellow cinnamon soil and red soil. Comparely the colonization of Lux-X1was quite poor in red soil with the population from2.0×l07at day1decreased to6.8×104CFU/g dry soil at day10.The500mg/L chlorpyrifos added to three soils was degraded by Lux-X1inoculated in soils after16d, and the order of degradation rate in soils, reflecting degradation effect of Lux-X1were as following:shajiang black soil> yellow cinnamon soil> red soil, the degradation rate constant of chlorpyrifos in the three soils were0.3185/d,0.2206/d and0.1732/d respectively, and the half life were2.1765d,3.1423d and4.0023d respectively.