【作者】 胡江；

【导师】 李顺鹏；

【作者基本信息】 南京农业大学 ， 微生物学， 2004， 博士

【摘要】 阿特拉津，也称莠去津，是在世界范围內广泛使用的一种三嗪类除草剂，在国内外杂草防除上占有重要的地位。阿特拉津虽然是一种低毒除草剂，但在土壤中具有比较长的持留性，其半衰期长，使用量大，因此在国外很多自然水体中都检测到了阿特拉津及其降解产物，并且很多浓度都远远超过美国环保局规定的安全浓度，造成严重的环境污染，随着除草剂在我国的推广使用，阿特拉津给我国的环境也带来了很大的影响，多次发生严重污染事故。阿特拉津的长残留性还有一个非常严重的后果，即对后茬作物的药害问题。有研究表明，当土壤中含量大于0.1mg／kg时，即影响小麦的萌发与生长，而对于极其敏感的作物水稻来说，河水中阿特拉津对水稻苗期的安全灌溉浓度为0.01mg／L，而对水稻的致死浓度是0.1mg/L。在阿特拉津污染修复的过程中，通常使用的两种生物处理的方案：生物刺激和生物原位修复。对生物刺激来说，如果要成功，土著微生物必须已经具备阿特拉津代谢的能力，它们的代谢活性可以通过对土壤补充适当的营养物质而得到增强。在生物原位修复中，土著微生物不具有阿特拉津降解能力而必须通过外源微生物的投加来获得这一能力。一系列的实验表明采用生物原位的方法来消除土壤中阿特拉津的污染，比投加营养物质进行生物刺激的方法有效，但是实际使用的效果依菌株不同也有很大差异，因此选择一个合适的菌株进行生物修复也显得至关重要。本实验室以减少农业环境中阿特拉津残留为主要目的，另外针对一些突发的污染事件非常需要生物处理方法来减少污染这一需求，对阿特拉津高效降解菌株进行了分析和研究。 本实验室采用驯化富集的方式，从能够稳定降解阿特拉津的混合菌群中，分离得到一株能够在阿特拉津固体培养基上产生水解圈的革兰氏阳性细菌，命名为BTAH1。该菌株能够以阿特拉津为唯一碳氮源进行生长，在126小时內将培养基中1000mg／L的阿特拉津降解至9.05mg／L，降解率达99.01％，属于降解性状良好的阿特拉津降解菌株。该菌株为盐酸硫胺的营养缺陷型，可以利用常见的碳氮源进行生长，但是高浓度的蛋白胨对其生长有强烈的抑制作用；最适生长温度为30℃左右，最适DH为7.5左右，通气量对菌株的生长影响不大。通过生理生化和16S rDNA同源性分析，初步将该菌株鉴定为微小杆菌属(Exiguobacterium sp．)细菌，该属细菌降解阿特拉津尚属首次报道。进一步实验比较了菌株BTAH1与微小杆菌属(Exiguobacterium sp.)的四个模式菌株(Exiguobacterium aurantiacum、Exiguobacterium antarcticum、南京农业人学博卜论文:除草剂阿特拉津降解菌的分离鉴定、降解特性及初步应用研究Ex心uobacterium unda和Ex妙obaeterium aee如zicum)在形态学，生理学和遗传学方面的相似性和差异。结果表明，BTAHI在菌体大小，菌落形态等形态学特征上与四个菌株有较大差异，尤其在鞭毛这一形态学特征上，BTAHI不具有其他菌株都具有的周生鞭毛这一共有特征;从糖类发酵实验来看，BTAHI与Exiguobacteriuma:‘厂anriacum、Exiguobaczerium antarcricum、ExigUobaczerium unda和Exiguobacleriun7ace川icun，四个模式菌株也明显不同，具有较多的独特发酵类型;165 rDNA同源性表明，BTAHI与Exiguobacreriuo aurantiaeom最为接近，同源性为98.1%，与其他三个菌株Exiguobacrerium anrarcricum、Exiguobacrerium unda和Exiguobaereriumacel)，lic um的同源性分别为94.0%、93.8%和94.5%;但是DNA一DNA液相杂交表明，BTAHI与四个模式菌株的DNA同源性都较低，与的同源性分别为26.25%、24.67%、48.11%和51 .22%，而一般来说，当DNA一DNA液相杂交值低于70%时，就可以认为两个菌株不是同一个种。根据形态学，生理学及遗传学上的差异，尤其是DNA一DAN液相杂交的结果，我们初步认定菌株BTAHI为微小杆菌属中的一个新种。 菌株BTAHI可以利用阿特拉津为唯一碳氮源进行生长，但是在培养基中添加碳源不利于菌体对阿特拉津的降解.而外源氮源的添加则会抑制菌体的生长，因为降解过程中该菌株会以NH3的形式释放阿特拉津化学结构中所有的五个氮原子，同时使溶液的pH值大幅上升(在磷酸缓冲体系中，变化幅度约为1一1.5).液体培养状态下，温度对BTAHI的降解影响较大，在25一30℃时，该菌株可以很好地降解溶液中的阿特拉津;pH值对BTAHI的降解影响相对较小，在 pH7一9时，该菌株都可以很好地降解溶液中的阿特拉津。BTAHI在降解阿特拉津的过程中，首先水解阿特拉津结构上的氯离子，同时产生羚基阿特拉津，与大多数革兰氏阳性阿特拉津降解菌株不同，BTAHI不会在溶液中累积氰尿酸，可以完全矿化阿特拉津为CO:和NH3。菌株BTAHI的降解谱比较窄，只能降解氛取代的三嗓类除草剂，不降解甲硫基取代等其他结构的三漆类除草剂，可能具有与革兰氏阴性细菌类似的降解基因和途径。采用PCR扩增和Southem杂交等方法研究了阿特拉津降解菌株BTAHI阿特拉津降解基因的保守性，结果表明从该菌株中扩增出的三个阿特拉津降解基因(阿特拉津氛水解酶基因a创，羚基阿特拉津脱乙胺基水解酶基因atzB和N-异丙基氛尿酸氨异丙氛基水解酶基因a七C)的保守片段与已报更多还原

【Abstract】 Through a long term enrichment, a Gram positive bacterium was isolated from a stable atrazine degrading consortium, named BTAH1, which could form a clearing zone on atrazine agar. This strain could use atrazine as sole carbon and nitrogen source, and degrade 1.000 mg/L atrazine within 126 hr completely. It could grow on most common carbon and nitrogen source, but it was an auxotroph strain of thiamine, and was strongly inhibited by high concentration of peptone. The optimum temperature and pH for BTAH1 growth was 30 C and 7.5, respectively. However, the bacterium was alkalophilic and could grow at a wide pH range, i.e., pH 6.5-11.5. Phylogenetic tree based on 16SrDNA sequence data showed that BTAH1 was closed to Exiguobacterium sp.We studied The differences in morphology, physiology, and genetics between BTAH1 and four type strains of Exiguobacterium sp., Exiguobacterium aurantiacum DSMZ 6208. Exiguobacterium antarcticum DSMZ 14480, Exiguobacterium unda DSMZ 14481, and Exiguobacterium acetylicum DSMZ 20416. The results showed that the bacterium size, clone shape, and glycolytic profile of BTAH1 were very different from those of four type strains. Especially. BTAH1 had no flagellum, but all four type strains had peritrichous flagella. 16S rDNA sequence analysis revealed 98.1% similarity between the 16S rDNA sequence of BTAH1 and Exiguobacterium aurantiacum DSMZ 6208, 94.0% between BTAH1 and Exiguobacterium antarcticum DSMZ 14480, 93.8% between BTAH1 and Exiguobacterium unda DSMZ 14481, and 94.5% between BTAH1 and Exiguobacterium acetylicum DSMZ 20416. DNA-DNA reassociation values indicated that BTAH1 was in a new genomic cluster of Exiguobacterium sp., and the DNA of BTAH1 and strain 6208 showed only 26.25% similarity, BTAH1 and strain 14480 24.67%, BTAH1 and strain 14481 48.11%, BTAH1 and strain 20416 51.22%, respectively. Based on genomic destinctiveness and the clear difference in chemotaxonomy, BTAH1 is proposed to be a strain in new specie of Exiguobacterium.Strain BTAH1 could grow in liquid medium with atrazine as sole carbon and nitrogensource, but extra carbon source in the medium would repress the degradation of atrazine by strain BTAHl. When BTAHl degraded atrazine, it could release all five nitrogen atoms from the chemical structure of atrazine as NH3, which led to a great increasing of solution pH. Therefore extra nitrogen source in the medium would greatly inhibit the growth of strain because of the high pH in solution. In lab conditions, strain BTAH1 could degrade atrazine very efficiently at 25-30C and at pH 7-9. During the course of atrazine degradation by BTAHl, it hydrolyzed the chlorine atom in the structure at first, and produced hydroxyatrazine. Furthermore, strain BTAHl did not cumulate cyanuric acid in the medium, which differed from most Gram positive atrazine degrading strains. Substrate range experiments showed that strain BTAHl could use chlorine-substituted s-trizine herbicide, but not use methyltyio-substitude and other structure s-trizine herbicide. The narrow substrate specificity of BTAHl suggested that strain BTAHl might has the same degradation pathway as Pseudomonas sp. strain ADP, but not most Gram positive strains.PCR amplification and Southern blotting were used to study the conservation of atrazine degrading genes in strain BTAHl. Results showed that the conserved fragments sequences of three atrazine degrading genes {atzA, atzB, and atzC) in strain BTAH1 shared 99% identity with those in strain ADP, which indicated that BTAHl had the same degrading pathway as strain ADP, and was rare in Gram positive strains. Strain BTAHl had two big plasmids, named pBTAHll and pBTAH12. The location experiment by Southern blotting showed that the first degrading gene, atzA, was located on chromosome DNA, and the other two on plasmid pBTAHl 1.To confirm the resource of atrazine degrading genes in strain BTAHl, an 8 kb PstI fragment was cloned into E. coli by clone hybridization, which contain atzB gene of BTAHl. Sequencing analysis of the fragment showed that it was 8581 bp, had 5 ORFs, and atzB更多还原