罗尔斯顿菌(Ralstonia sp.) T6对三氯吡啶醇污染土壤的修复及能完全矿化毒死蜱工程菌株的构建

    Remediation of 3,5,6-Trichloro-2-Pyridinol Polluted Soil With Ralstonia sp. T6 and Construction of a Chlorpyrifos-Mineralizing Engineered Strain

    • 摘要: 以毒死蜱降解中间代谢产物3,5,6-三氯-2-吡啶醇(TCP)高效降解菌罗尔斯顿菌(Ralstonia sp. )T6 为材料,研究其在土壤中对TCP 的降解特性。结果表明,温度、接菌量和初始底物浓度对TCP 的降解都有影响,T6 菌株降解TCP 的最适温度为30 ℃,当土壤含菌量〔以菌落形成单位(CFU)计〕小于10×108 kg-1 时,降解率随着含菌量的增加而提高,当含菌量超过10×10 kg-1 时,降解率不再提高。降解率随着TCP 初始浓度的增加而降低, 当TCP 初始浓度为50~100 mg·kg-1时,6 d 内可将50 mg·kg-1 TCP 降解80%。利用基因工程手段,将来源于寡养单胞菌(Stenotrophomonas sp. )DSP-1 的甲基对硫磷水解酶基因(mpd)插入菌株T6 基因组16S rRNA 基因中,成功构建一株可彻底矿化毒死蜱的重组工程菌株T6-mpd。生长试验结果表明罗尔斯顿菌T6-mpd 和T6 的生长特性基本一致。对T6-mpd 菌株降解毒死蜱的特性研究结果表明,在LB 培养基中,T6-mpd 对毒死蜱的水解效率与DSP-1 基本一致,但在基础盐(MSM)培养基中,T6-mpd 在60 h 内对50 mg·L-1 毒死蜱的降解率仅为36%,显著低于DSP-1。模拟土壤原位修复试验结果表明,在含菌量为10 kg-1条件下,T6-mpd 在2 d 内可将50 mg·kg-1毒死蜱降解64%。认为T6-mpd 菌株在毒死蜱残留污染环境修复中具有潜在的应用前景。

       

      Abstract: TCP is a kind of metabolic product of degradation of chlorpyrifos. Ralstonia sp. T6, a TCP (3,5,6-trichloro-2-pyridinol) degrading strain of bacteria, was used in an experiment to explore its TCP degrading characteristics in soil. Results show that T6 degraded 80% of 50 mg·kg-1 TCP in 6 days. Temperature, inoculation rate and initial concentration of the substrate were factors affecting its degrading efficiency. The optimum temperature for T6 degrading TCP was 30℃; in soils lower than 10×108 CFU/kg in bacteria content, TCP biodegradation rate rose with rising bacteria content, while in soils higher than that, the rate did not rise, but fall instead with rising bacteria content. A methyl parathion hydrolase gene (mpd) from Stenotrophomonas sp. DSP-1 was inserted into 16S rDNA of Ralstonia sp. T6 by conjugation and a genetically engineered T6-mpd that is supposed to be able to completely mineralize chlorpyrifos was obtained. Culturing tests show that T6-mpd and Ralstonia sp. T6 displayed similar growth characteristics. Chlorpyrifos degrading tests show that in LB medium, the chlorpyrifos hydrolysis efficiency of T6-mpd was basically the same as that of Stenotrophomonas sp. DSP-1, but in inorganic salt medium, T6-mpd‘s 50 mg·L-1 chlorpyrifos hydrolysis rate in 60 h reached only 36%, significantly lower than DSP-1’s. Simulated in-situ soil remediation shows that T6-mpd, 10×108 CFU·kg-1 in content, could degrade 64% of 50 mg·kg-1 chlorpyrifos. It is, therefore, believed that Strain T6-mpd is a promising tool for bioremediation of chlorpyrifos-contaminated environment.

       

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