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

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10×10^8 kg－1时，降解率随着含菌量的增加而提高，当含菌量超过10×10^8 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10^8 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。

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 10×10^8 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, 10×10^8 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.