【作者】 郭宁；

【导师】 刘新星；

【作者基本信息】 中南大学 ， 微生物学， 2010， 硕士

【摘要】 磁小体是一种理想的新型磁性纳米材料,可以广泛应用在磁性材料、生物医学工程、环境保护等各领域。磁小体一般在趋磁细菌胞内排列成磁链,因此趋磁细菌得到了趋磁性,基于这种特性,可以使用磁分离方法分离纯化趋磁细菌。本课题组首次发现氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)胞内可以合成磁性颗粒。但是类似氧化亚铁硫杆菌这种磁性细菌的趋磁性非常微弱,分离强磁性菌有一定的困难。本文对磁性细菌的磁分离进行了研究,设计开发了一种有效的磁性细菌分离仪,并应用分离仪分离得到磁敏感氧化亚铁硫杆菌,还探索了其培养条件和批量培养。本文在磁分离理论计算的基础上设计开发出一种磁性细菌分离仪,经过特殊设计的磁极头可以产生利于磁分离的梯度分布强磁场。利用氧化亚铁硫杆菌为样品进行了磁分离实验：分离得到的强磁菌平均每个细胞含有4颗磁性颗粒,84.21%的细胞含有磁性颗粒,多数细胞含有2-5颗,而弱磁菌平均每个细胞含有2颗磁性颗粒,65.79%的细胞含有磁性颗粒,多数细胞只含有1颗；在人工磁场中,强磁菌的趋磁性明显强于弱磁菌。结果表明磁分离仪可以有效地分离磁性细菌。磁分离仪的最优操作条件是：电磁铁通2A电流,以单位时间流量884mL/h,分离浓度108ml-1的细胞悬液。本文应用磁性细菌分离仪多次分离磁敏感氧化亚铁硫杆菌,得到了趋磁性比较强的菌株,然后对得到的菌株的生长影响因素进行了研究。通过正交实验得到磁敏感氧化亚铁硫杆菌的最佳培养条件为：温度T=35℃；250mL摇瓶培养时,摇床转速为150rpm；初始pH=2.5；9K培养基初始FeSO4浓度为44.7g/L。本文通过补加Fe2+的方式,实现了磁敏感氧化亚铁硫杆菌的实验室规模(2L)批量培养,初始培养体积为1L,终体积为2L,最佳条件为发酵40h时,一次补入浓度5%的补料,最终菌体浓度比不补料对照提高了60.69%；相同条件一次补入9K培养基全料,菌浓度相比只补入Fe2+提高了6.01%,比不补料提高了70.34%。原子力显微镜观察表明发酵得到的菌体有明显的磁信号,即胞内含有磁性颗粒。更多还原

【Abstract】 The magnetosome is an ideal kind of bio-nano-magnetic material. Magnetotactic bacteria could migrate along the magnetic field lines, which was due to that they could synthesize intra-cellular magnetosomes. So the magnetotactic bacteria could be separated easily by magnetic separation. The research in this group discovered the fact that Acidithiobacillus ferrooxidans could synthesize intra-cellular magnetic particles. But the magnetotaxis of Acidithiobacillus ferrooxidans was so weak that strong magnetic cells could hardly be separated from cells suspension. In this paper we researched the magnetic separation of magnetic bacteria. Based on the magnetic separation theory we designed and developed a kind of magnetic bacteria separator. As the sample Acidithiobacillus ferrooxidans was separated by this magnetic separator, and the optimal operation condition was studied. A strain of strong magnetic Acidithiobacillus ferrooxidans was isolated by magnetic separation, and the culture condition of it was researched including the influence of magnetic field and optimal condition of maximum culture density. In addition, magnetic Acidithiobacillus ferrooxidans could be batch cultured in the laboratory first time by fed-batch fermentation.We developed a kind of magnetic bacteria separator, which could create high gradient magnetic field. Acidithiobacillus ferrooxidans was investigated as an example. Strong magnetic and weak magnetic cells were separated and collected. On average, the number of the magnetic particles presented in the strong magnetic cells was more than that of the weak magnetic cells.84.21% of the strong magnetic cells contained 4-10 magnetic particles. By comparison only 65.79% of the weak magnetic cells contained 1-3 magnetic particles. Moreover, semisolid-plate magnetophoresis showed that the magnetotaxis of strong magnetic cells was stronger than the weak magnetic cells. These results suggest that the magnetic separator could be used to isolate the magnetic bacteria. When the magnetic flux density of outlet a and b reached 1220mT and 900mT and the suspension flow with the cells density of 108 ml-1 was controlled by a applying a rate of 884mL/h, the separation efficiency achieved maximum in the orthogonal experiments.We separated Acidithiobacillus ferrooxidans using the magnetic separator and optimized the culture conditions of Acidithiobacillus ferrooxidans by orthogonal experiments. We found that Acidithiobacillus ferrooxidans grew best at initial pH value of 2.0,35℃and 150rpm when added 44.7g/L FeSO4 into 200mL cultured medium.We batch cultured Acidithiobacillus ferrooxidans in the laboratory by fed-batch fermentation. In the shake-flask fermentation, the final volume of liquid was 2 L. After 40 h in the fermentation Fe2+(5%) was added in the fermentation liquid. The max cell density could reach 2.33×107 mL-1. Compared with control experiments it increased by 60.69%. At the same condition, the completion 9K culture medium was added in the fermentation liquid. The max cell density could reach 2.47×107 mL-1. Compared with control experiments it increased by 70.34%. And atom force microscopy and magnetic force microscopy showed that cells in the fermentation were magnetic.更多还原