不同形态氮盐对铜绿假单胞菌SD-1种群稳定性的影响及机制研究
发布时间：2018-11-01 19:13
【摘要】：铜绿假单胞菌SD-1是一株高效杂环化合物降解菌株,可通过生物强化的方式在污染物治理中发挥重要作用。已有研究提示,强化菌株的稳定性对于生物强化系统稳定运行具有重要意义。虽然有大量报道关注了强化菌株在生态系统中的种间竞争,但目前尚未有研究涉及强化菌株的种内生态稳定性。鉴于此,本论文以铜绿假单胞菌SD-1作为试验菌株,通过连续传代的方式,探究了废水处理系统中不同形态氮盐对菌株种群稳定性的影响;对比野生株和群体感应欺骗株在不同形态氮盐中的生长差异,从代谢角度解析了不同形态氮盐对菌株种群稳定影响的群体感应机制;此外,通过对比不同形态氮盐条件下分离的进化合作者群体感应效应差异,进一步从进化角度探究了不同形态氮盐对菌株种群稳定影响的群体感应机制。该研究对于丰富群体感应理论、拓展群体感应理论在生物强化系统中的应用、优化生物强化系统的运行策略具有重要意义。所获得的主要结果如下:(1)长期进化过程中,部分组别菌群密度下降了 1-2个数量级,出现种群崩溃现象。菌株在外加铵盐,硝酸盐、亚硝酸盐和不外加氮盐的对照组别中崩溃的机率分别为0%,33.3%,66.6%和11.1%。稳定组别中,欺骗子比例范围为0%-53%,崩溃组别中欺骗子比例为51%-81%。崩溃组别中,蛋白酶含量显著下降。不同形态的外加氮盐对菌株SD-1种群稳定性产生显著影响,外加亚硝酸盐和硝酸盐组别中种群更容易崩溃。(2)对不同培养条件下菌株SD-1氮源利用情况进行对比,显示氨氮、硝氮的变化趋势相同,但亚硝氮的变化趋势存在差异。在硝酸盐和亚硝酸盐培养基中,菌株SD-1公共物质分泌要较其他组别中高2-3倍。在外加硝酸盐的葡萄糖培养基中,野生株和欺骗子利用氨氮的能力相同;但在外加硝酸盐的葡萄糖培养基中,欺骗子前12 h比合作者生长更快;而外加亚硝酸盐的葡萄糖培养基中,欺骗子的生长则始终高于合作者。硝酸盐和亚硝酸盐使合作者菌株肩负了更多的公共物质代谢负担,同时使得欺骗子菌株获得了较之合作者更高的生存优势,即群体感应介导的代谢差异是外加亚硝酸盐和硝酸盐条件下种群更容易崩溃的根本原因。(3)从长期进化试验中筛选了 24株进化合作者,其群体感应均发生了显著变化。虽然进化菌株与初始合作者菌株的生长无显著差异,但是所有进化菌株的30C12-HSL,C4VHSL和蛋白酶产量不同程度地低于初始合作者菌株,变得更加"谨慎";而所有进化菌株的绿脓素合成不同程度地高于初始合作者菌株,稳定维持机制被加强。合作行为"谨慎"进化以及维稳能力提升是菌群在长期进化中维持稳定的重要原因。
[Abstract]:Pseudomonas aeruginosa SD-1 (Pseudomonas aeruginosa) is a high efficient heterocyclic compound degrading strain which can play an important role in pollutant treatment through biointensification. It has been suggested that the stability of the enhanced strain is of great significance for the stable operation of the biological strengthening system. Although there have been a lot of reports on the inter-species competition of the enhanced strains in the ecosystem, there are no studies on the intraspecific ecological stability of the enhanced strains. In this paper, using Pseudomonas aeruginosa SD-1 as the experimental strain, the effects of different nitrogen salts in wastewater treatment system on the population stability of the strains were studied by means of continuous passage. By comparing the growth differences of wild plants and colony sensing deceptive strains in different forms of nitrogen salts, the effects of different forms of nitrogen salts on the population stability of strains were analyzed from the point of view of metabolism. In addition, by comparing the population induction effects of evolutionary collaborators with different nitrogen salts, the population sensing mechanism of different forms of nitrogen salts on the population stability of strains was further explored from the perspective of evolution. This study is of great significance to enrich the theory of group induction, expand the application of group induction theory in biological reinforcement system, and optimize the operation strategy of biological reinforcement system. The main results are as follows: (1) during the long-term evolution, the density of some groups of bacteria decreased by 1-2 orders of magnitude, resulting in the phenomenon of population collapse. The probability of the strain collapsing in the control group with added ammonium salt, nitrate, nitrite and no nitrogen salt was 66.6% and 11.1%, respectively. In the stable group, the deceptive sub-ratio ranges from 0 to 53, and in the collapse group the deceptive sub-ratio is 51-81. In the collapse group, the protease content decreased significantly. The population stability of strain SD-1 was significantly affected by different forms of nitrogen salts, and the population in the groups of nitrite and nitrate was more likely to collapse. (2) the nitrogen source utilization of strain SD-1 was compared under different culture conditions. The results showed that the change trend of ammonia nitrogen and nitrite nitrogen was the same, but the change trend of nitrite nitrogen was different. In nitrate and nitrite medium, the secretion of common substance of the strain SD-1 was 2-3 times higher than that of other groups. In glucose medium supplemented with nitrate, wild plant and deceiver had the same ability to utilize ammonia nitrogen, but in glucose medium with nitrate added, deceiver grew faster than collaborator 12 hours before the addition of nitrate. In glucose medium supplemented with nitrite, the growth of deceivers was always higher than that of collaborators. Nitrate and nitrite make the co-strains bear more burden of common substance metabolism, and make the deceptive strains gain higher survival advantages than the collaborators. That is, the metabolic difference mediated by population induction is the root cause of population collapsing more easily under the conditions of nitrite and nitrate. (3) 24 evolutionary collaborators were selected from the long-term evolutionary experiment, and their population induction changed significantly. Although there was no significant difference between the growth of the evolutionary strain and the initial co-strain, the production of 30C12-HSLC4VHSL and protease of all the evolutionary strains was lower than that of the initial co-strain, and it became more "cautious". The pyocyanin synthesis of all the evolutionary strains was higher than that of the initial co-strains to some extent, and the stability and maintenance mechanism was strengthened. The evolution of cooperative behavior and the improvement of stability are the important reasons for the stability of the microflora in the long-term evolution.
【学位授予单位】：浙江工商大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172
[Abstract]:Pseudomonas aeruginosa SD-1 (Pseudomonas aeruginosa) is a high efficient heterocyclic compound degrading strain which can play an important role in pollutant treatment through biointensification. It has been suggested that the stability of the enhanced strain is of great significance for the stable operation of the biological strengthening system. Although there have been a lot of reports on the inter-species competition of the enhanced strains in the ecosystem, there are no studies on the intraspecific ecological stability of the enhanced strains. In this paper, using Pseudomonas aeruginosa SD-1 as the experimental strain, the effects of different nitrogen salts in wastewater treatment system on the population stability of the strains were studied by means of continuous passage. By comparing the growth differences of wild plants and colony sensing deceptive strains in different forms of nitrogen salts, the effects of different forms of nitrogen salts on the population stability of strains were analyzed from the point of view of metabolism. In addition, by comparing the population induction effects of evolutionary collaborators with different nitrogen salts, the population sensing mechanism of different forms of nitrogen salts on the population stability of strains was further explored from the perspective of evolution. This study is of great significance to enrich the theory of group induction, expand the application of group induction theory in biological reinforcement system, and optimize the operation strategy of biological reinforcement system. The main results are as follows: (1) during the long-term evolution, the density of some groups of bacteria decreased by 1-2 orders of magnitude, resulting in the phenomenon of population collapse. The probability of the strain collapsing in the control group with added ammonium salt, nitrate, nitrite and no nitrogen salt was 66.6% and 11.1%, respectively. In the stable group, the deceptive sub-ratio ranges from 0 to 53, and in the collapse group the deceptive sub-ratio is 51-81. In the collapse group, the protease content decreased significantly. The population stability of strain SD-1 was significantly affected by different forms of nitrogen salts, and the population in the groups of nitrite and nitrate was more likely to collapse. (2) the nitrogen source utilization of strain SD-1 was compared under different culture conditions. The results showed that the change trend of ammonia nitrogen and nitrite nitrogen was the same, but the change trend of nitrite nitrogen was different. In nitrate and nitrite medium, the secretion of common substance of the strain SD-1 was 2-3 times higher than that of other groups. In glucose medium supplemented with nitrate, wild plant and deceiver had the same ability to utilize ammonia nitrogen, but in glucose medium with nitrate added, deceiver grew faster than collaborator 12 hours before the addition of nitrate. In glucose medium supplemented with nitrite, the growth of deceivers was always higher than that of collaborators. Nitrate and nitrite make the co-strains bear more burden of common substance metabolism, and make the deceptive strains gain higher survival advantages than the collaborators. That is, the metabolic difference mediated by population induction is the root cause of population collapsing more easily under the conditions of nitrite and nitrate. (3) 24 evolutionary collaborators were selected from the long-term evolutionary experiment, and their population induction changed significantly. Although there was no significant difference between the growth of the evolutionary strain and the initial co-strain, the production of 30C12-HSLC4VHSL and protease of all the evolutionary strains was lower than that of the initial co-strain, and it became more "cautious". The pyocyanin synthesis of all the evolutionary strains was higher than that of the initial co-strains to some extent, and the stability and maintenance mechanism was strengthened. The evolution of cooperative behavior and the improvement of stability are the important reasons for the stability of the microflora in the long-term evolution.
【学位授予单位】：浙江工商大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172
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