【作者】 王光云；

【导师】 吴涓；

【作者基本信息】 安徽大学 ， 微生物学， 2012， 硕士

【摘要】 针对近年来微囊藻毒素(MCs)的全球性污染问题,本研究以蓝藻爆发较严重和频繁的巢湖为研究对象,对其进行了较为详细的研究。本文以MCs为唯一碳氮源,采用富集驯化培养的办法,从巢湖蓝藻爆发的底泥中分离筛选MCs降解菌,同时对筛选出的MCs降解菌进行鉴定,主要通过形态观察、生理生化实验以及16S rDNA序列分析来确定其分类学地位,为巢湖的藻毒素污染的微生物降解治理提供有效地菌源。本研究从巢湖底泥中分离纯化出一株MCs降解菌株M6,其16S rDNA的长度为1408bp,经鉴定该降解菌株M6为蜡状芽孢杆菌(Bacillus cereus)。为了进一步优化该降解菌株M6的降解效果,本研究以MCs为唯一的碳源和氮源,分别从不同的外加碳源和氮源,pH值,接种量,MCs的初始浓度等方面,考察这些因素对降解菌株M6降解MC-LR效果的影响,并初步确定该降解反应的最适环境条件。结果表明外加碳源甘油可大幅促进降解菌株M6的生长及其对MC-LR的降解效率,降解率可达到66.2%；外加氮源硝酸铵可以促进降解菌株M6对MC-LR的降解效率；菌株在pH值为7.0-8.0时,菌株的生长状况良好,并且对MC-LR的降解效率都比较高,当培养基中的pH值为8.0时,MC-LR的降解效率达到最大,可达到62.0%；当接种量介于1%-10%时,菌株的生长量及MC-LR的降解效率均随接种量的增大而增大,降解效率最高可达51.3%；当MC-LR初始浓度在1.95-15.6mg/L之间时,MC-LR的降解效率随着MC-LR的初始浓度的增大而逐渐增大,当初始浓度为15.6mg/L时,MC-LR的降解效率最高为59.2%。本研究还对降解菌株M6降解MC-LR的降解机理进行了初步的探究,对降解菌株M6降解MC-LR进行了定位研究,考察MC-LR诱导前后的降解菌M6进行了SDS-PAGE全细胞电泳,确定经MC-LR诱导前后胞内蛋白质的变化,并且从不同pH值,接种量,MCs的初始浓度等方面,考察这些因素对降解菌株M6胞内粗酶液降解MC-LR的影响。结果表明,降解菌M6降解MC-LR的活性物质位于细胞内,属于胞内酶,SDS-PAGE全细胞电泳发现至少有三种酶参与了MC-LR的降解过程,而且实验表明这三种酶为降解菌本身的组织酶而不是诱导酶。当反应体系中胞内粗酶液浓度达到404.9mgL,MC-LR的初始浓度为10mg/L,pH为8.0,时,MC-LR的降解率达到最高,16h降解率可达98.7%。同时本研究还在不同条件下对降解菌株M6进行培养,提取粗酶液并降解一定量的MC-LR,以初步了解环境因素对产酶量多少的影响,发现当在外加碳源为甘油、氮源为硝酸铵,pH为8,接种量为10%,MC-LR初始浓度为15.6mg/L的诱导条件下,菌株M6的胞内粗提液对MC-LR有较大的降解效率,说明在此条件下降解MC-LR的酶表达量较多。更多还原

【Abstract】 For the global pollution problem of microcystins (MCs) in recent years, the research choose Chaohu Lake where blue-green algae is more serious and frequent as research object and carry out some detailed studies. In this paper, MCs degradation bacterium is separated and identificated in the way of enrichment training based on MCs as the only carbon source. Taxonomy status of MCs degradation bacterium is determined by the morphology, physiological biochemistry experiment and16S rDNA sequence analysis to provide effective bacterium source for the cyanobacteria toxin microbial degradation of pollution control in the chaohu lake. The result indicats that the MCs degradation bacterium named M6with length of1408bp shows similarity of99%to candle shape Bacillus.In order to optimize the degradation effect of the MCs degradation bacterium, this study research the efect to degradation effect of some facts such as carbon, nitrogen source, pH value, quantity and the initial concentration of the MCs based on the MCs as the only carbon, nitrogen source. It also determines the optimal environment degradation of the reaction of degradation and the maximum efficiency. The result indicates that plus carbon source (glycerin) can largely promote the growth of the M6degrading bacteria and the degradation of efficiency and the degradation rate of MC-LR, up to66.2%. Plus plus nitrogen source (ammonium) can promote the degradation efficiency of MC-LR. When the pH value is7.0-8.0, the growth of the strains and the degradation of efficiency of MC-LR is higher. When the pH value is8.0, the degradation efficiency reachs the maximum value, up to62.0%. When inoculation amount ranges from1%-10%, the degradation rate of MC-LR grows as the degradation rate increases and the degradation of highest efficiency strains is51.3%. When initial concentration of MC-LR is between1.95to15.6, the degradation rate of MC-LR increses. When the initial concentration of MC-LR is15.6mg/L, MC-LR degradation rate is up to59.2%.This study also researches degradation mechanism of M6and positions the degradation bacterium M6degradation MC-LR. At the same time, it carry out the SDS-PAGE whole cell electrophoresis on M6before and after being inducted by MC-LR to be sure the change of the cell protein. It also investigates efect of environmental factors such as pH value, quantity and the initial concentration of the MCs on the inside the cell thick liquid enzyme degradation effect. The result shows that degradation bacterium M6degradation MC-LR of the active material is located in cells, belonging to the intracellular enzyme, based on SDS-PAGE whole cell electrophoresis, we find that at three enzymes at least joins in the MC-LR of degradation process, and these three enzymes is degradation bacterium itself, not the organization of the enzyme inducible enzyme. When the cell in thick liquid enzyme concentration reached404.9mg/L, MC-LR of initial concentration of10mg/L and pH values8.0in the system, MC-LR degradation rate reaches the highest, up to98.7%after16h. At the same time the research inducts degradation bacterium M6in different conditions, thick liquid extraction enzyme degradation and a certain amount of MC-LR preliminary understanding the environment factors on the enzyme production quantity of how much influence, we find that when have carbon source glycerin, ammonium nitrate nitrogen source, pH values8, inoculated quantity is10%and initial concentration of MC-LR is15.6mg/L, intracellular coarse extraction liquid of strains M6have larger degradation of efficiency to MC-LR. It indicates that drop solution-LR enzyme expression of MC is more in this condition.更多还原