毒死蜱降解菌株Sphingopyxis terrae R17的分离鉴定及其luxAB基因标记研究
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摘要
1.采用摇瓶富集法和土壤富集法,对农药厂排污沟污泥和土壤进行驯化培养,从中分离得到24株菌,其中有6株菌对毒死蜱的降解效果较好,命名为E30、R17、P5、B1、Z5、T15,在48h对10 mg-L~(-1)毒死蜱的降解效率分别达55.47%、32.27%、58.15%。34.76%、27.10%、42.01%。
2.采用三亲本结合法将发光酶基因luxAB成功导入R17菌株得到标记菌株LuxR17,在暗室里可以观察到明显的荧光现象,连续转代20次均无质粒丢失现象。
3.对R17菌株进行生理生化和16S rDNA序列同源性分析,发现该菌株能以毒死蜱为唯一碳源进行生长,鉴定为Sphingopyxis terrae。
4. R17、LuxR17菌株的最适生长温度为35℃、最适pH为7~8。在此条件下R17培养28h后到达稳定期,菌落浓度达9.18×10~8 cfu-mL~(-1)。LuxR17菌株培养32h达稳定期,菌落浓度达1.04×10~9 cfu-mL~(-1)。
5. R17、LuxR17菌株在不同时间对10 mg-L~(-1)毒死蜱的降解特性研究结果表明,在1d、2d、3d和4d内,当R17菌株的接种量为9.18×10~7 cfu-mL~(-1)时,毒死蜱降解率分别达到18.59%、31.23%、36.55%和47.69%;LuxR17菌株的接种量为1.04×10~8 cfu-mL~(-1)时,毒死蜱的降解效率分别为17.72%、24.14%、35.06%和48.48%。
6. R17、LuxR17菌株对不同浓度毒死蜱的降解特性研究结果表明,对1 mg-L~(-1)、5 mg-L~(-1)和10 mg-L~(-1)的毒死蜱降解2d,R17对毒死蜱的降解率分别达到50.21%、43.46%和31.23%;LuxR17菌株对毒死蜱的降解效率分别为41.64%、35.20%和24.14%。
7. LuxR17菌株在自然土壤中和灭菌土壤中的存活状况研究结果表明,LuxR17菌株在土壤中的变化总的来说呈下降趋势,但在不同处理土壤中其下降的速度有所不同;在灭菌土壤中LuxR17的活菌数要比自然土壤中的多,在加有毒死蜱的土壤中LuxR17的活菌数要高于未加毒死蜱的土壤。
8.毒死蜱在不同处理土壤中的降解动态研究结果表明,自然土壤中毒死蜱的降解速率较灭菌土壤中的快,在加LuxR17菌液的土壤中毒死蜱的降解速率要显著高于未加LuxR17菌液的土壤。将LuxR17加入含20 mg-L~(-1)毒死蜱的灭菌土壤和自然土壤中降解3、6、9、12d后,灭菌土壤中毒死蜱的降解率分别达到42.37%、70.92%、80.04%和94.70%,自然土壤中毒死蜱的降解率分别达到55.92%、72.27%、89.13%和98.31%。
1. 24 strains were isolated from the sludge and soil of pesicide factory through shaking culture and soil enrichment method, among whcih six strain have better degrading efficiency of chlorpyrifos that are named as E30, R17, P5, B1, Z5, T15and the degrading rate were55.47%、32.27%、58.15%。34.76%、27.10%、42.01% respectively in 2d.
2. By the method of tri-parental mating, the marker gene luxAB was successfully transferred into R17 strain, and a marked strain, LuxR17, was successfully obtained. The plasmid with luxAB did not lose after 20 transfers, and the strain was genetically stable.
3. Strain R17 was identified preliminarily as Sphingopyxis terrae based on its physiological and biochemical characters and its 16 S rDNA homologue sequence analysis.
4. Under the optimal growth condition, 35℃and pH 7-8, Strain R17 could reach 9.18×10~8 cfu-mL~(-1) after 28h and LuxR17 strain reach 1.04×10~9 cfu-mL~(-1) after 32h.
5. The chlorpyrifos degradation characteristics of R17 strain and LuxR17 strain were studied at different times under the initial concentration of 10 mg-L~(-1). The results showed that the degradation rate of chlorpyrifos by strain R17 with the inoculation of 9.18×10~7 cfu-mL~(-1) were18.59%, 31.23%, 36.55% and 47.69%, and that by LuxR17 with the inoculation of 1.04×10~8 cfu-mL~(-1) were 17.72%, 24.14%, 35.06% and 48.48% respectively in 1d, 2d, 3d and 4d.
6. The chlorpyrifos degradation characteristics of R17 strain and LuxR17 strain were studied with different initial chlorpyrifos. The results showed that with initial concentration of 1 mg-L~(-1), 5 mg-L~(-1), 10 mg-L~(-1), the chlorpyrifos degradation rate by R17 strain reached 50.21%, 43.46% and 31.23% and that by LuxR17 strain reached 41.64%, 35.20% and 24.14% after 2d respectively.
7. Experiments were conducted to investigate the distribution of LuxR17 strain in different soil. The results showed that the trends of LuxR17 strain in different treatments of soils were generally declined, but the dicline speeds were different in different treatments of soils. The cell number of LuxR17 strain in sterilized soil was higher than that in non-sterilized soils and the cell number in soil with chlorpyrifos was higher than that in soil without chlorpyrifos.
8. Biodegradation kinetics of chlorpyrifos in different treatments of soil was studied. The results showed that the degradation rate of chlorpyrifos in no-sterilized soil was higher than that in sterilized soils and the degradation rate in soil with LuxR17 strain was obviously higher than that in soil without LuxR17strain. With initial concentration of 20 mg-L~(-1) chlorpyrifos, the degradation rates of chlorpyrifos by LuxR17 strain were 55.92%, 72.27%, 89.13% and 98.31% in no-sterilized soil and were 42.37%, 70.92%, 80.04% and 94.70% in sterilized soil respectively in 3d, 6d, 9d and 12d.
2.采用三亲本结合法将发光酶基因luxAB成功导入R17菌株得到标记菌株LuxR17,在暗室里可以观察到明显的荧光现象,连续转代20次均无质粒丢失现象。
3.对R17菌株进行生理生化和16S rDNA序列同源性分析,发现该菌株能以毒死蜱为唯一碳源进行生长,鉴定为Sphingopyxis terrae。
4. R17、LuxR17菌株的最适生长温度为35℃、最适pH为7~8。在此条件下R17培养28h后到达稳定期,菌落浓度达9.18×10~8 cfu-mL~(-1)。LuxR17菌株培养32h达稳定期,菌落浓度达1.04×10~9 cfu-mL~(-1)。
5. R17、LuxR17菌株在不同时间对10 mg-L~(-1)毒死蜱的降解特性研究结果表明,在1d、2d、3d和4d内,当R17菌株的接种量为9.18×10~7 cfu-mL~(-1)时,毒死蜱降解率分别达到18.59%、31.23%、36.55%和47.69%;LuxR17菌株的接种量为1.04×10~8 cfu-mL~(-1)时,毒死蜱的降解效率分别为17.72%、24.14%、35.06%和48.48%。
6. R17、LuxR17菌株对不同浓度毒死蜱的降解特性研究结果表明,对1 mg-L~(-1)、5 mg-L~(-1)和10 mg-L~(-1)的毒死蜱降解2d,R17对毒死蜱的降解率分别达到50.21%、43.46%和31.23%;LuxR17菌株对毒死蜱的降解效率分别为41.64%、35.20%和24.14%。
7. LuxR17菌株在自然土壤中和灭菌土壤中的存活状况研究结果表明,LuxR17菌株在土壤中的变化总的来说呈下降趋势,但在不同处理土壤中其下降的速度有所不同;在灭菌土壤中LuxR17的活菌数要比自然土壤中的多,在加有毒死蜱的土壤中LuxR17的活菌数要高于未加毒死蜱的土壤。
8.毒死蜱在不同处理土壤中的降解动态研究结果表明,自然土壤中毒死蜱的降解速率较灭菌土壤中的快,在加LuxR17菌液的土壤中毒死蜱的降解速率要显著高于未加LuxR17菌液的土壤。将LuxR17加入含20 mg-L~(-1)毒死蜱的灭菌土壤和自然土壤中降解3、6、9、12d后,灭菌土壤中毒死蜱的降解率分别达到42.37%、70.92%、80.04%和94.70%,自然土壤中毒死蜱的降解率分别达到55.92%、72.27%、89.13%和98.31%。
1. 24 strains were isolated from the sludge and soil of pesicide factory through shaking culture and soil enrichment method, among whcih six strain have better degrading efficiency of chlorpyrifos that are named as E30, R17, P5, B1, Z5, T15and the degrading rate were55.47%、32.27%、58.15%。34.76%、27.10%、42.01% respectively in 2d.
2. By the method of tri-parental mating, the marker gene luxAB was successfully transferred into R17 strain, and a marked strain, LuxR17, was successfully obtained. The plasmid with luxAB did not lose after 20 transfers, and the strain was genetically stable.
3. Strain R17 was identified preliminarily as Sphingopyxis terrae based on its physiological and biochemical characters and its 16 S rDNA homologue sequence analysis.
4. Under the optimal growth condition, 35℃and pH 7-8, Strain R17 could reach 9.18×10~8 cfu-mL~(-1) after 28h and LuxR17 strain reach 1.04×10~9 cfu-mL~(-1) after 32h.
5. The chlorpyrifos degradation characteristics of R17 strain and LuxR17 strain were studied at different times under the initial concentration of 10 mg-L~(-1). The results showed that the degradation rate of chlorpyrifos by strain R17 with the inoculation of 9.18×10~7 cfu-mL~(-1) were18.59%, 31.23%, 36.55% and 47.69%, and that by LuxR17 with the inoculation of 1.04×10~8 cfu-mL~(-1) were 17.72%, 24.14%, 35.06% and 48.48% respectively in 1d, 2d, 3d and 4d.
6. The chlorpyrifos degradation characteristics of R17 strain and LuxR17 strain were studied with different initial chlorpyrifos. The results showed that with initial concentration of 1 mg-L~(-1), 5 mg-L~(-1), 10 mg-L~(-1), the chlorpyrifos degradation rate by R17 strain reached 50.21%, 43.46% and 31.23% and that by LuxR17 strain reached 41.64%, 35.20% and 24.14% after 2d respectively.
7. Experiments were conducted to investigate the distribution of LuxR17 strain in different soil. The results showed that the trends of LuxR17 strain in different treatments of soils were generally declined, but the dicline speeds were different in different treatments of soils. The cell number of LuxR17 strain in sterilized soil was higher than that in non-sterilized soils and the cell number in soil with chlorpyrifos was higher than that in soil without chlorpyrifos.
8. Biodegradation kinetics of chlorpyrifos in different treatments of soil was studied. The results showed that the degradation rate of chlorpyrifos in no-sterilized soil was higher than that in sterilized soils and the degradation rate in soil with LuxR17 strain was obviously higher than that in soil without LuxR17strain. With initial concentration of 20 mg-L~(-1) chlorpyrifos, the degradation rates of chlorpyrifos by LuxR17 strain were 55.92%, 72.27%, 89.13% and 98.31% in no-sterilized soil and were 42.37%, 70.92%, 80.04% and 94.70% in sterilized soil respectively in 3d, 6d, 9d and 12d.
引文
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