阿特拉津降解菌AG1、ADG1和SA1的分离鉴定及其降解特性的研究
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摘要
污染环境的微生物修复具有其它修复方法不可比拟的优势，如何充分开发利用参与环境净化的微生物资源已成为一项重要研究课题。阿特拉津是国内外使用时间最长，使用量最大的除草剂之一。因其长时间大范围使用，造成大面积的土壤、地表水、地下水等环境的污染。本论文的研究目的是使用一种不同于常规富集分离培养的降解菌分离方法，分离阿特拉津污染土壤中的原位优势降解菌，并在国内外阿特拉津的微生物降解研究的基础上对这些降解菌的降解特性、降解基因到生物修复进行较为系统的研究。
一、阿特拉津降解菌的分离与鉴定
不经过富集培养，直接将长期受阿特拉津污染的土壤稀释涂布加有土壤浸出液和阿特拉津的平板，分别从三个采自不同地区的污染土壤S1、S2和S3中各分离了一株阿特拉津高效降解菌株AG1、ADG1和一个混合降解菌AH，通过反复的液体培养和划线分离从AH中分离到一株纯的降解菌SA1。AG1、ADG1和SA1均可快速有效地降解阿特拉津。16S rDNA核甘酸序列分析结合培养和生理生化特征，AG1与ADG1被鉴定为Arthrobacter spp.，SA1被鉴定为Pseudomonas sp.。
二、降解菌生长特性和降解特性的研究
AG1的最适碳源为麦芽糖、蔗糖和甘露醇，最适氮源为有机氮；最适碳氮比6／1(摩尔比)；最适生长温度为25℃-30℃；最适生长pH值范围7-10；静息细胞在25℃-42℃时降解效果较好，降解率受pH值影响很小；能以西玛津、特丁津、莠灭净、西草净、扑草净为唯一氮源生长；AG1以阿特拉津为唯一碳氮源生长时，降解终产物为氰尿酸，降解4000mg／L的阿特拉津也不会产生反馈抑制现象。
ADG1以葡萄糖、麦芽糖、蔗糖、淀粉、甘露醇、柠檬酸钠为碳源，有机氮、硝态氮、铵态氮和脲为氮源时生长都很好；最适碳氮比6／1(摩尔比)；最适生长温度为25℃-37℃；在pH值4-10范围内生长都能很好地生长；静息细胞在25℃-42℃时降解效果较好，降解率受pH值影响很小；ADG1降解谱与AG1相同；ADG1以阿特拉津为唯一碳氮源生长时，降解终产物为氰尿酸。
SA1最适碳源为葡萄糖、麦芽糖和柠檬酸钠，最适氮源为硝态氮和铵态氮；所测
Bioremediation has advantages other pollution remediation methods absent. It has been an important research item for us to exploit and utilize microbial resource to remove environmental pollutions. In recent years, a large amount of unexploited microbial resources were detected by culture-independent microbiological molecular technology, which give us a hint that there are also many pollutant-degrading microbial resources untouched now. Atrazine has been widely used for a long time in the world, which lead to the pollution of a large area of soil, ground water and underground water. In this dissertation we tried to isolate degrading bacteria by the way different from enrichment isolation usually employed, to isolate dominant in situ atrazine-degrading bacteria from polluted soil, and studied their characteristics, degrading genes and applied them to remove atrazine in simulated pollution soil on the base of others' research achievements.
1. Isolation and identification of high efficient atrazine-degrading bacteria
High efficient atrazine-degrading bacteria AG1, ADG1 and a consortium AH were isolated without enrichment by diluting and plating long time atrazine-contaminated soil samples(S1, S2, S3), which was collected from different area, directly onto plates containing soil extracts and atrazine. By repeated alternation of liquid cultivating and plate streaking for a long time, a pure atrazine-mineralizing bacterium strain SA1 was isolated from AH. AG1, ADG1 and SA1 all could degrade more than 500 mg ·L-1 of atrazine. Combined their 16S rDNA sequences analysis with physiobiochemical characteristics, AG1 and ADG1 were identified as Arthrobacter spp., and SA1 was Pseudomonas sp..
2. Studies of growth and degradation characteristics
The optimal carbon sources for AG1 were maltose, sucrose and mannitaol, and the optimal nitrogen source was organic nitrogen, and the most optimal C/N (mol atom) was 6/1. It grew well under conditions of 25℃-30℃ and pH7-10. Its resting cellsdegraded atrazine well at 25℃-42℃, and their degrading efficiency was not affected greatly by pH. AG1. Besides atrazine, AG1 could also grow on simazine, terbuthylazine, ametryn, simetryn and prometryn as sole nitrogen sources. AG1 could grow on atrazine as
一、阿特拉津降解菌的分离与鉴定
不经过富集培养，直接将长期受阿特拉津污染的土壤稀释涂布加有土壤浸出液和阿特拉津的平板，分别从三个采自不同地区的污染土壤S1、S2和S3中各分离了一株阿特拉津高效降解菌株AG1、ADG1和一个混合降解菌AH，通过反复的液体培养和划线分离从AH中分离到一株纯的降解菌SA1。AG1、ADG1和SA1均可快速有效地降解阿特拉津。16S rDNA核甘酸序列分析结合培养和生理生化特征，AG1与ADG1被鉴定为Arthrobacter spp.，SA1被鉴定为Pseudomonas sp.。
二、降解菌生长特性和降解特性的研究
AG1的最适碳源为麦芽糖、蔗糖和甘露醇，最适氮源为有机氮；最适碳氮比6／1(摩尔比)；最适生长温度为25℃-30℃；最适生长pH值范围7-10；静息细胞在25℃-42℃时降解效果较好，降解率受pH值影响很小；能以西玛津、特丁津、莠灭净、西草净、扑草净为唯一氮源生长；AG1以阿特拉津为唯一碳氮源生长时，降解终产物为氰尿酸，降解4000mg／L的阿特拉津也不会产生反馈抑制现象。
ADG1以葡萄糖、麦芽糖、蔗糖、淀粉、甘露醇、柠檬酸钠为碳源，有机氮、硝态氮、铵态氮和脲为氮源时生长都很好；最适碳氮比6／1(摩尔比)；最适生长温度为25℃-37℃；在pH值4-10范围内生长都能很好地生长；静息细胞在25℃-42℃时降解效果较好，降解率受pH值影响很小；ADG1降解谱与AG1相同；ADG1以阿特拉津为唯一碳氮源生长时，降解终产物为氰尿酸。
SA1最适碳源为葡萄糖、麦芽糖和柠檬酸钠，最适氮源为硝态氮和铵态氮；所测
Bioremediation has advantages other pollution remediation methods absent. It has been an important research item for us to exploit and utilize microbial resource to remove environmental pollutions. In recent years, a large amount of unexploited microbial resources were detected by culture-independent microbiological molecular technology, which give us a hint that there are also many pollutant-degrading microbial resources untouched now. Atrazine has been widely used for a long time in the world, which lead to the pollution of a large area of soil, ground water and underground water. In this dissertation we tried to isolate degrading bacteria by the way different from enrichment isolation usually employed, to isolate dominant in situ atrazine-degrading bacteria from polluted soil, and studied their characteristics, degrading genes and applied them to remove atrazine in simulated pollution soil on the base of others' research achievements.
1. Isolation and identification of high efficient atrazine-degrading bacteria
High efficient atrazine-degrading bacteria AG1, ADG1 and a consortium AH were isolated without enrichment by diluting and plating long time atrazine-contaminated soil samples(S1, S2, S3), which was collected from different area, directly onto plates containing soil extracts and atrazine. By repeated alternation of liquid cultivating and plate streaking for a long time, a pure atrazine-mineralizing bacterium strain SA1 was isolated from AH. AG1, ADG1 and SA1 all could degrade more than 500 mg ·L-1 of atrazine. Combined their 16S rDNA sequences analysis with physiobiochemical characteristics, AG1 and ADG1 were identified as Arthrobacter spp., and SA1 was Pseudomonas sp..
2. Studies of growth and degradation characteristics
The optimal carbon sources for AG1 were maltose, sucrose and mannitaol, and the optimal nitrogen source was organic nitrogen, and the most optimal C/N (mol atom) was 6/1. It grew well under conditions of 25℃-30℃ and pH7-10. Its resting cellsdegraded atrazine well at 25℃-42℃, and their degrading efficiency was not affected greatly by pH. AG1. Besides atrazine, AG1 could also grow on simazine, terbuthylazine, ametryn, simetryn and prometryn as sole nitrogen sources. AG1 could grow on atrazine as
引文
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