氯嘧磺隆高效降解菌的分离筛选及其生物学特性研究
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摘要
氯嘧磺隆属磺酰脲类除草剂,其具有超高效、广谱、低毒和高选择性等特点,已在我国广泛使用,但其残留期长,易形成农田环境污染,对后茬敏感作物有严重的药害,是农业生产的一个重要问题。本文针对氯嘧磺隆残留的农田污染问题,研究其微生物降解技术,采用摇瓶富集驯化方法分离筛选高效降解菌株,应用形态、生理生化和分子方法对新菌株进行了分类地位鉴定,研究了该菌的主要生物学特性和降解酶粗酶液的特性,并采用盆栽实验法对该株降解菌的效果进行了初步评价。本研究对有效控制农田农药污染,保障农产品安全生产均具有重要的意义。具体研究结果如下:
应用摇瓶富集驯化培养方法,从河北、北京、天津等地的农药厂排污口淤泥中分离到1株氯嘧磺隆高效降解菌株E-1。菌株在含10 mg/L氯嘧磺隆的液体无机盐培养基中,反应72h,其降解效率可以达到94.74%。
运用形态、生理生化及分子方法对该菌株进行了鉴定。鉴定结果显示,该菌株的16SrDNA基因序列与巨大芽孢杆菌Bacillus megaterium的相似性达到100%,结合菌株的其他特征,综合判定该株菌为巨大芽孢杆菌。
研究明确了E-1菌株的生长特性和营养特性。E-1菌株在牛肉膏蛋白胨培养基上生长迅速,pH5～9均能生长,pH10时不生长,最适pH为6.0;菌株在15～45℃均能生长,50℃不能生长,最适生长温度为30℃;菌株生长对氧气的需求要量不高,装瓶量40 mL/250mL至200mL/250mL均能良好生长;菌株的最适碳氮源分别为葡萄糖和酵母粉。
研究明确,该菌株的降解酶位于细胞内,为诱导型酶。该菌的粗酶液在pH为7.0的磷酸缓冲液中,温度30℃时,对氯嘧磺隆的降解率可达到52.73%(蛋白含量4.747mg/ml,反应30min);底物氯嘧磺隆的浓度对粗酶的活性有抑制作用,酶活性随着氯嘧磺隆浓度的增加而降低,底物氯嘧磺隆浓度从5mg/L上升到50mg/L,酶的降解效率从52.79%降低到27.41%。
生测结果显示,施用降解菌对氯嘧磺隆药污染具有显著的修复作用,施用降解菌可保证敏感作物的出苗率及较正常的株高。在本实验条件下,当土壤中氯嘧磺隆浓度为14μg/kg时,降解菌的修复作用最明显,不加菌处理的玉米株高被抑制率为58.35%,加入降解菌液后其株高被抑制率可降低到了15.66%,如果提高施菌量,其修复效果可能更佳。
Chlorimuron-theyl has become one of the worst pollutants in soil because of extensive using in the world. As a result, effects of degrading bacteria on chlorimuron-theyl and its bio-degradation has attracted increasing attention. In present research, degrading bacteria were isolated and identified, and its degradation effects were analyzed.
One highly effective degrading bacterium of chlorimuron-ethyl named E-1. The E-1 was isolated from soil which has long term applied with chlorimuron-ethyl through enrichment culture method. The E-1 used chlorimuron-theyl as sole nitrogen source. The degradation percentage of chlorimuron-ethyl was 94.74% after cultured 72h in liquid mineral medium containing 10 mg/L chlorimuron-ethyl. Based on the morphological appearances, physiological biochemical characters and 16SrDNA sequence analyse, the E-1 was 100% homologous with Bacillus megaterium. The E-1 was identified as Bacillus megaterium.
The E-1 grew fast when the pH value of 5.0～9.0 in beef peptone medium, the E-1 grew best when the pH was 6.0 and stopped grow when the pH value of 10.0. The E-1 grew fast when the temperature was 15～45℃, the E-1 grew best when the temperature was 30℃and stopped grow when the temperature was 50℃. The appropriate aeration was 40～200mL/250mL. The optimum carbon and nitrogen was glucose and yeast powder, respectively.
The degrading-enzymes produced by E-1 and located in cells, a kind of inducible enzyme. Temperature and pH were key factors affecting degrading activity of enzymes. The enzymes showed the highest activity at 30℃and pH 7.0 in the Na_2HPO_4-NaH_2PO_4 buffer. The degradation percentage of chlorimuron-ethyl was 52.73% ,when the enzyme content was 4.747 mg/ml andreaction time was 30 min in degradation experiment of chlorimuron-ethyl. The degrading-enzymes were much more stable at pH 6.5～7.0 and 4℃. While the chlorimuron-ethyl initial concentration raised, the degrading activity of enzymes declined; the enzymes degrading activity declined along with the enzymes concentration at the same chlorimuron-ethyl concentration.
Bioassay experiment showed that the seeding and height of sensitive crop of degrading bacteria treatment were compatible with the control, on the whole, it considered that E-1 could recover the contamination of chlorimuron-ethyl. The recover effects of E-1 were the most prominent when the chlorimuron-ethyl concentration was 14μg/kg in soil, which the corns’hight inhibition rate of degrading bacteria treatment was 15.66% lower than that of the chlorimuron-ethyl control. The recover ability of E-1 might be even better when the introduction of degrading bacteria was increased.
应用摇瓶富集驯化培养方法,从河北、北京、天津等地的农药厂排污口淤泥中分离到1株氯嘧磺隆高效降解菌株E-1。菌株在含10 mg/L氯嘧磺隆的液体无机盐培养基中,反应72h,其降解效率可以达到94.74%。
运用形态、生理生化及分子方法对该菌株进行了鉴定。鉴定结果显示,该菌株的16SrDNA基因序列与巨大芽孢杆菌Bacillus megaterium的相似性达到100%,结合菌株的其他特征,综合判定该株菌为巨大芽孢杆菌。
研究明确了E-1菌株的生长特性和营养特性。E-1菌株在牛肉膏蛋白胨培养基上生长迅速,pH5～9均能生长,pH10时不生长,最适pH为6.0;菌株在15～45℃均能生长,50℃不能生长,最适生长温度为30℃;菌株生长对氧气的需求要量不高,装瓶量40 mL/250mL至200mL/250mL均能良好生长;菌株的最适碳氮源分别为葡萄糖和酵母粉。
研究明确,该菌株的降解酶位于细胞内,为诱导型酶。该菌的粗酶液在pH为7.0的磷酸缓冲液中,温度30℃时,对氯嘧磺隆的降解率可达到52.73%(蛋白含量4.747mg/ml,反应30min);底物氯嘧磺隆的浓度对粗酶的活性有抑制作用,酶活性随着氯嘧磺隆浓度的增加而降低,底物氯嘧磺隆浓度从5mg/L上升到50mg/L,酶的降解效率从52.79%降低到27.41%。
生测结果显示,施用降解菌对氯嘧磺隆药污染具有显著的修复作用,施用降解菌可保证敏感作物的出苗率及较正常的株高。在本实验条件下,当土壤中氯嘧磺隆浓度为14μg/kg时,降解菌的修复作用最明显,不加菌处理的玉米株高被抑制率为58.35%,加入降解菌液后其株高被抑制率可降低到了15.66%,如果提高施菌量,其修复效果可能更佳。
Chlorimuron-theyl has become one of the worst pollutants in soil because of extensive using in the world. As a result, effects of degrading bacteria on chlorimuron-theyl and its bio-degradation has attracted increasing attention. In present research, degrading bacteria were isolated and identified, and its degradation effects were analyzed.
One highly effective degrading bacterium of chlorimuron-ethyl named E-1. The E-1 was isolated from soil which has long term applied with chlorimuron-ethyl through enrichment culture method. The E-1 used chlorimuron-theyl as sole nitrogen source. The degradation percentage of chlorimuron-ethyl was 94.74% after cultured 72h in liquid mineral medium containing 10 mg/L chlorimuron-ethyl. Based on the morphological appearances, physiological biochemical characters and 16SrDNA sequence analyse, the E-1 was 100% homologous with Bacillus megaterium. The E-1 was identified as Bacillus megaterium.
The E-1 grew fast when the pH value of 5.0～9.0 in beef peptone medium, the E-1 grew best when the pH was 6.0 and stopped grow when the pH value of 10.0. The E-1 grew fast when the temperature was 15～45℃, the E-1 grew best when the temperature was 30℃and stopped grow when the temperature was 50℃. The appropriate aeration was 40～200mL/250mL. The optimum carbon and nitrogen was glucose and yeast powder, respectively.
The degrading-enzymes produced by E-1 and located in cells, a kind of inducible enzyme. Temperature and pH were key factors affecting degrading activity of enzymes. The enzymes showed the highest activity at 30℃and pH 7.0 in the Na_2HPO_4-NaH_2PO_4 buffer. The degradation percentage of chlorimuron-ethyl was 52.73% ,when the enzyme content was 4.747 mg/ml andreaction time was 30 min in degradation experiment of chlorimuron-ethyl. The degrading-enzymes were much more stable at pH 6.5～7.0 and 4℃. While the chlorimuron-ethyl initial concentration raised, the degrading activity of enzymes declined; the enzymes degrading activity declined along with the enzymes concentration at the same chlorimuron-ethyl concentration.
Bioassay experiment showed that the seeding and height of sensitive crop of degrading bacteria treatment were compatible with the control, on the whole, it considered that E-1 could recover the contamination of chlorimuron-ethyl. The recover effects of E-1 were the most prominent when the chlorimuron-ethyl concentration was 14μg/kg in soil, which the corns’hight inhibition rate of degrading bacteria treatment was 15.66% lower than that of the chlorimuron-ethyl control. The recover ability of E-1 might be even better when the introduction of degrading bacteria was increased.
引文
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