【作者】 王能飞；

【导师】 袁红莉；

【作者基本信息】 中国农业大学 ， 微生物学， 2004， 硕士

【摘要】 微生物在治理镉污染中有着广阔的应用前景，其中微生物吸附剂的研究已成为目前研究的热点之一。本文对一株可在高镉浓度(2000mg／L)下存活的酵母菌Y11吸附镉的特性作了详细的研究，并对其吸附镉的机制进行初步探讨，为实现微生物的镉污染治理奠定了基础。 根据形态学观察和葡萄糖发酵实验，脲酶实验以及重氮基蓝B实验结果，结合《酵母菌的特征与鉴定手册》，确定Y11为一株红酵母(Rhodotorula sp．)。 生长曲线结果表明菌体液体培养7小时进入对数期，18小时左右进入稳定期，因此初步确定菌体培养时间为20小时。在此基础上，研究了吸附时间、菌体加入量及pH对Y11吸附效果的影响。结果表明菌体对镉的吸附在7～8分钟达到峰值，30分钟基本平衡。在镉浓度为48.1mg／L的溶液中，菌体加入量为6g／L时，镉的去除率最大，达到95.6％，菌体中镉含量为干重的9‰。pH对吸附效率有决定性的影响，随着pH的升高，菌体吸附镉的能力增强，但低pH条件有利于镉的解吸。 Y11菌体具有重复吸附镉的能力。经五次吸附循环，其对50.7mg／L含镉溶液中镉的去除能力保持在90.87％～93.23％之间。将菌体用明胶—海藻酸钠固定后吸附镉，最大去除率达到77.91％。 研究了不同处理的菌体吸附的镉在细胞内的分布情况，结果发现在菌体培养过程中添加浓度为100mg／L镉时，菌体吸附的镉有3.27％进入细胞内；但是用培养好的菌体，冻干后吸附镉，菌体吸附的镉只有1％可进入原生质体，其余99％都分布在细胞壁周围。 探讨了Y11菌产金属硫蛋白(MT)的情况，结果表明镉诱导能明显促进Y11菌MT的产量，菌体经诱导后的MT产量达到638.8μg／g，是非诱导菌体的85倍，远大于报道的酵母菌中MT的产量。采用玻璃珠破碎细胞，经煮沸除去不耐热蛋白，再通过乙醇沉淀得到MT粗品。依次经过Sephadex G-50凝胶过滤，DEAE-52离子交换，再通过Sephadex G-25凝胶过滤脱盐纯化MT，经SDS-PAGE检测MT周围没有其它杂蛋白，分子量为6500 Da。更多还原

【Abstract】 Environmental cadmium pollution is of increasing concern. Microorganisms are deemed to have great potential in treating cadmium pollution. Biosorption has emerged as one of the most promising process because of its high efficiency and favorable economics. A Yeast strain, isolated and designated as Y11 in our lab, could survive 2000mg/L of cadmium. Its characteristics and mechanism of cadmium removal were studied in this paper.According to its morphological characteristics and the ability of glucose fermentation, urease production, Fast Blue BB Salt(DBB) experimental 1 was identified as a Rhodotorula sp.Growth curve of Y11 showed the logarithmic phase began from 7 hr and stationary phase began from about 18 hr. 20hr was consided as the optimal culture time for Y11. The effect of adsorption time, the amount of biomass, and the pH on the Cd2+-adsorption by Y11 were studied. The results showed that the adsorption peak value is about 7 min and equilibrium time was about 30 min. When the initial cadmium concentrations is 48.1mg/L, with the optimum biomass quantum of 6g/L, the best removal ratio was 95.6% and Cd content of biomass reached 9‰. pH play a key effect on adsorption. Absorption of Cd2+ effectively enhanced with increasing pH. While low pH had good effect on Cd2+ desorption from biomass.Y11 had good repetitious ability of cadmium adsorption. With initial Cd2+ concentrations of 48.1mg/L in solution, after 5 absorption and desorption cycles, the cadmium absorptivity kept 90.87%~93.23%. Immobilized Yl 1 cell with gelatin-alginate can removal 77.91% Cd2+.The distribution of accumulated Cd2+ in different disposed Y11 is different too. 3.29% of the total Cd2+ can entered cytoplasm during growth culture with 100mg/L of Cd2+ in medium. But only 1% entered cytoplasm and 99% existed around cell wall when used the lyophilized biomass to adsorb cadmium.Metallothionein (MT) was found in Y11, and Cd could induce a high output of MT. When Y11 was cultured with 100mg/L cadmium, the MT content was 638.8ug/g biomass, which is 85 times of non-induced Y11 (7.51ug/g) and by far more than other Yeast strains reported to this day. After breaking up Y11 cell by glass beadings, seething solution to remove heat-sensitive proteins, MT was deposited by alcohol. The purification procedure involved gel filtration on Sephadex G-50, ion exchange chromatography on DEAE-52, and gel filtration on Sephadex G-25 and discontinuous polyacrylamide gel electrophoresis. It exhibited a clear MT stripe with molecular weight of 6500 Da in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.更多还原