草除灵高效降解菌的分离鉴定、降解特性及降解途径的研究
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摘要
从农药厂污水处理池的活性污泥中分离得到一株能以草除灵为唯一碳源生长的菌株。根据其表型特征、生理生化特性,结合菌株的16S rRNA基因序列同源性分析,将此菌株初步鉴定为Methyloversatilis sp.,命名为cd-1。
菌株cd-1液体培养时呈絮状生长,在温度为30℃、pH 8.0的R2A培养基中生长最好；利用有机氮源和有机碳源生长较好；菌株对通气量没有特殊要求；该菌株不耐盐,NaCl浓度对菌株生长影响很大,NaCl浓度在0g/L～5g/L时生长最好；菌株cd-1对氯霉素和氨苄青霉素有抗性,对供试的其他抗生素均敏感。
菌株cd-1在温度为30℃、pH为8.0时降解草除灵最好；接种量为2%时,菌株在草除灵浓度为100mg·L-1的无机盐液体培养基培养48h后,草除灵残留浓度仅为0.85mg·L-1,降解率达到99%；接种量由0.5%提高到8%,可以迅速加快草除灵的降解；当草除灵浓度为25 mg·L-1时,16h时草除灵残留浓度仅为1.44 mg·L-1,草除灵浓度提高到400 mg·L-1时,48h时草除灵浓度仍高达284.56mg·L-1,说明高浓度会对菌株产生毒害作用,抑制菌株对草除灵的降解；菌株利用有机氮源降解草除灵最快；通气量对菌株降解草除灵没有影响；加入蛋白胨、酵母粉和LB溶液可以提高菌株对草除灵的降解率。在土壤中接种降解菌cd-1可以加快草除灵的降解,土著微生物与外源投加的降解菌以协同作用的方式降解草除灵,提高了土壤中草除灵的降解速率。
通过MS/MS和GC-MS联合检测,研究了草除灵在高效降解菌株cd-1作用下的降解途径,总共检测到了3个新的代谢产物。通过质谱图分析及代谢产物累积转换关系,鉴定3个代谢产物分别为benazolin、7-chloro-3-Methylbenzo[d]thiazol-2(3H)-one和2-chloro-6-(methyle-neamino)benzenethiol。初步确定代谢途径,先由benazolin-ethyl转化为benazolin,接着由benazolin转化为7-chloro-3-methylbenzo[d]thiazol-2(3H)-one,最终转化为2-chloro-6-(methyle-neamino)benzenethiol。
为了便于菌株的实际应用,还对菌株的固定化进行了研究。采用海藻酸钠包埋法固定高效降解菌,结果表明海藻酸钠浓度为3%,CaCl2浓度为3%,固化时间为12h时,制得的固定化小球机械强度最好,对草除灵的降解效果也最好。固定化细菌的降解速度比游离态细菌的降解速度慢,但是固定化细菌抗pH和温度变化能力比游离态细菌强,对pH和温度的忍受范围更广。固定化降解菌在合适的重复使用次数内有较高的稳定性和反应活性,可用于固定化微生物反应器的构建。
A highly efficient benazolin-ethyl degrading strain was isolated from pesticide wastewater treatment pool,this strain could use benazolin-ethyl as the sole carbon source for growth.It was identified as Methyloversatilis sp. according to its physiological and biochemical properties, and 16S rRNA gene sequence analysis,and the strain was designated as cd-1.
The optimum growth condition was temperature 30℃and pH 8.0 in R2A culture medium. In liquid cultures, cells of stain cd-1 tended to clump together, and forming white flakes, and using organic carbon source and organic nitrogen source grew best; Ventilation had on effect on the growth of the strain; Concentration of NaCl had great impact on the growth of stain cd-1, when the concentration of NaCl was 0～5g/L,the strain grows best; Strain cd-1 was resistant to chloramphenicol and ampicillin.
Results showed that the strain degraded benazolin-ethyl best at 30℃and pH 8.0.When the inoculation rate was 2% and the concentration of benazolin-ethyl was 100mg·L-1, the residue concentration of benazolin-ethyl was only 0.85mg·L-1 in salts liquid medium after 48h by the strain cd-1, the degradation of benazolin-ethyl could reach 99%; Improving inoculation rate from 0.5% to 8% speeded up the degradation of benazolin-ethyl; When the concentration of benazolin-ethyl was 25mg·L-1, the residue concentration was only 1.44mg·L-1 after 16h by the strain cd-1, and when the concentration of benazolin-ethyl was up to 400mg·L-1, the residue concentration was still 284.56mg·L-1 after 48h, showing that increasing concentration of benazolin-ethyl produced toxic effects on the strain,inhibited the degradation of benazolin-ethyl;The strain was easy to use organic nitrogen, and degraded benazolin-ethyl the fastest; Ventilation had no effect on the degradation of benazolin-ethyl;Added peptone, yeast extract and LB solution was beneficial to the degradation of benazolin-ethyl.Degradation in soil inoculated with bacteria cd-1 could accelerate the degradation of benazolin-ethyl,because the indigenous microbiology and the exogenous degradation bacteria degraded benazolin-ethyl in a synergy way, and improved the soil degradation rate of benazolin-ethyl.
The degradation pathway of benazolin-ethyl in the effect of the benazolin-ethyl degrading strain cd-1 cd-1 had been learned, and three new metabolites had been detected by MS/MS and GC-MS combined detection. Benazolin、7-chloro-3-Methylbenzo[d] thiazol-2(3H)-one and 2-chloro-6-(methyle-neamino)benzenethiol were successfully detected and identified based on mass spectrum analysis and transformation between metabolite accumulation.A biochemical degradation pathway of benazolin-ethyl was proposed, benazolin-ethyl first changed into benazolin,and then benazolin changed into 7-chloro-3-methylbenzo[d]thiazol-2(3H)-one,last changed into 2-chloro -6-(methyle-neamino) benzenethiol。
In order to facilitate the practical application of the strain, the immobilization of the strain was also studied. Used sodium alginate embedding the benazolin-ethyl degrading strain cd-1,results showed that mechanical strength of immobilized beads best and the degradation of benazolin-ethyl effect was best when the concentration of sodium alginate was 3%, the concentration of CaCl2 was 3% and immobilized time was 12h.The degradation rate of immobilized bacteria was slower than the free bacteria, but immobilized bacteria could tolerance pH and temperature in a wider range.Immobilized degrading bacteria could keep high stability and reactivity in a suitable number of reuse, and coula be used for immobilized reactor building.
菌株cd-1液体培养时呈絮状生长,在温度为30℃、pH 8.0的R2A培养基中生长最好；利用有机氮源和有机碳源生长较好；菌株对通气量没有特殊要求；该菌株不耐盐,NaCl浓度对菌株生长影响很大,NaCl浓度在0g/L～5g/L时生长最好；菌株cd-1对氯霉素和氨苄青霉素有抗性,对供试的其他抗生素均敏感。
菌株cd-1在温度为30℃、pH为8.0时降解草除灵最好；接种量为2%时,菌株在草除灵浓度为100mg·L-1的无机盐液体培养基培养48h后,草除灵残留浓度仅为0.85mg·L-1,降解率达到99%；接种量由0.5%提高到8%,可以迅速加快草除灵的降解；当草除灵浓度为25 mg·L-1时,16h时草除灵残留浓度仅为1.44 mg·L-1,草除灵浓度提高到400 mg·L-1时,48h时草除灵浓度仍高达284.56mg·L-1,说明高浓度会对菌株产生毒害作用,抑制菌株对草除灵的降解；菌株利用有机氮源降解草除灵最快；通气量对菌株降解草除灵没有影响；加入蛋白胨、酵母粉和LB溶液可以提高菌株对草除灵的降解率。在土壤中接种降解菌cd-1可以加快草除灵的降解,土著微生物与外源投加的降解菌以协同作用的方式降解草除灵,提高了土壤中草除灵的降解速率。
通过MS/MS和GC-MS联合检测,研究了草除灵在高效降解菌株cd-1作用下的降解途径,总共检测到了3个新的代谢产物。通过质谱图分析及代谢产物累积转换关系,鉴定3个代谢产物分别为benazolin、7-chloro-3-Methylbenzo[d]thiazol-2(3H)-one和2-chloro-6-(methyle-neamino)benzenethiol。初步确定代谢途径,先由benazolin-ethyl转化为benazolin,接着由benazolin转化为7-chloro-3-methylbenzo[d]thiazol-2(3H)-one,最终转化为2-chloro-6-(methyle-neamino)benzenethiol。
为了便于菌株的实际应用,还对菌株的固定化进行了研究。采用海藻酸钠包埋法固定高效降解菌,结果表明海藻酸钠浓度为3%,CaCl2浓度为3%,固化时间为12h时,制得的固定化小球机械强度最好,对草除灵的降解效果也最好。固定化细菌的降解速度比游离态细菌的降解速度慢,但是固定化细菌抗pH和温度变化能力比游离态细菌强,对pH和温度的忍受范围更广。固定化降解菌在合适的重复使用次数内有较高的稳定性和反应活性,可用于固定化微生物反应器的构建。
A highly efficient benazolin-ethyl degrading strain was isolated from pesticide wastewater treatment pool,this strain could use benazolin-ethyl as the sole carbon source for growth.It was identified as Methyloversatilis sp. according to its physiological and biochemical properties, and 16S rRNA gene sequence analysis,and the strain was designated as cd-1.
The optimum growth condition was temperature 30℃and pH 8.0 in R2A culture medium. In liquid cultures, cells of stain cd-1 tended to clump together, and forming white flakes, and using organic carbon source and organic nitrogen source grew best; Ventilation had on effect on the growth of the strain; Concentration of NaCl had great impact on the growth of stain cd-1, when the concentration of NaCl was 0～5g/L,the strain grows best; Strain cd-1 was resistant to chloramphenicol and ampicillin.
Results showed that the strain degraded benazolin-ethyl best at 30℃and pH 8.0.When the inoculation rate was 2% and the concentration of benazolin-ethyl was 100mg·L-1, the residue concentration of benazolin-ethyl was only 0.85mg·L-1 in salts liquid medium after 48h by the strain cd-1, the degradation of benazolin-ethyl could reach 99%; Improving inoculation rate from 0.5% to 8% speeded up the degradation of benazolin-ethyl; When the concentration of benazolin-ethyl was 25mg·L-1, the residue concentration was only 1.44mg·L-1 after 16h by the strain cd-1, and when the concentration of benazolin-ethyl was up to 400mg·L-1, the residue concentration was still 284.56mg·L-1 after 48h, showing that increasing concentration of benazolin-ethyl produced toxic effects on the strain,inhibited the degradation of benazolin-ethyl;The strain was easy to use organic nitrogen, and degraded benazolin-ethyl the fastest; Ventilation had no effect on the degradation of benazolin-ethyl;Added peptone, yeast extract and LB solution was beneficial to the degradation of benazolin-ethyl.Degradation in soil inoculated with bacteria cd-1 could accelerate the degradation of benazolin-ethyl,because the indigenous microbiology and the exogenous degradation bacteria degraded benazolin-ethyl in a synergy way, and improved the soil degradation rate of benazolin-ethyl.
The degradation pathway of benazolin-ethyl in the effect of the benazolin-ethyl degrading strain cd-1 cd-1 had been learned, and three new metabolites had been detected by MS/MS and GC-MS combined detection. Benazolin、7-chloro-3-Methylbenzo[d] thiazol-2(3H)-one and 2-chloro-6-(methyle-neamino)benzenethiol were successfully detected and identified based on mass spectrum analysis and transformation between metabolite accumulation.A biochemical degradation pathway of benazolin-ethyl was proposed, benazolin-ethyl first changed into benazolin,and then benazolin changed into 7-chloro-3-methylbenzo[d]thiazol-2(3H)-one,last changed into 2-chloro -6-(methyle-neamino) benzenethiol。
In order to facilitate the practical application of the strain, the immobilization of the strain was also studied. Used sodium alginate embedding the benazolin-ethyl degrading strain cd-1,results showed that mechanical strength of immobilized beads best and the degradation of benazolin-ethyl effect was best when the concentration of sodium alginate was 3%, the concentration of CaCl2 was 3% and immobilized time was 12h.The degradation rate of immobilized bacteria was slower than the free bacteria, but immobilized bacteria could tolerance pH and temperature in a wider range.Immobilized degrading bacteria could keep high stability and reactivity in a suitable number of reuse, and coula be used for immobilized reactor building.
引文
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