【作者】 李自达；

【导师】 刘军贤； 王桂文；

【作者基本信息】 广西师范大学 ， 理论物理， 2011， 硕士

【摘要】 随着化石能源的日益枯竭和环境问题的日趋严重,开发洁净可再生资源已成了紧迫的课题。一些微生物在合适的生长环境下能在细胞内部合成或者代谢转化出乙醇、油脂,或者有重要经济价值的代谢产物,如类胡萝卜素等,其中微生物油脂能够替代化石能源来制取生物柴油,类胡萝卜素可以作为食物色素添加剂及药物添加剂,而乙醇则可以提纯后直接做为燃料,可见这些产物均具有比较高的经济价值。因此,本文在前人研究的基础上,通过结合激光镊子拉曼光谱系统,尝试进一步探索研究生物质能源微生物发酵过程中,探测发酵产物、底物以及细胞内部的物质动态变化过程,为实际发酵生产提供理论依据和重要参数参考,同时结合拉曼光谱和光学操控便利,探索发展分选微生物的新方法。主要开展了以下研究工作：1.微生物乙醇发酵中,实验室研究和工业生产的必检项目是发酵液乙醇含量的测定。为此应用拉曼光谱在倒置显微镜上建立一种用96孔板高通量快速检测发酵液乙醇含量的新方法,通过最小二乘法拟合标准乙醇溶液与内标物的拉曼信号比值,得到回归方程,根据回归方程计算乙醇溶液和发酵液中的乙醇含量,与气相色谱法的检测结果进行对比。用本方法检测了以木薯淀粉为发酵基质的菌株筛选、三角瓶发酵和500升发酵罐发酵过程的乙醇含量,t-测验表明,与气相色谱法结果有很好的重现性(p=0.05),准确度高。并在此基础上,对比利用一元线性回归法、多元线性回归法、主成分回归法和偏最小二乘法建立定量分析模型,探索同时定量检测乙醇和葡萄糖的快捷方法。结果表明基于拉曼光谱技术定量方法,能满足实验和生产对乙醇和葡萄糖检测精度的要求。2.采用拉曼光谱在分子水平上对单个活细胞的结构变化,物质转换和生物活性进行研究。基于拉曼光谱,探索实验室500 L中式发酵罐木薯淀粉浓醪发酵乙醇过程中,酿酒酵母细胞内生物大分子的生化信息,以及发酵底物和细胞代谢产物的变化。分别追踪发酵液光谱中乙醇和葡萄糖的特征峰880 cm-1和1127 cm-1谱带的变化,与气相色谱法和DNS比色法检测结果比较发现,其变化曲线基本吻合,表明特征峰面积与物质的含量存在对应关系。此外,采用多种分析方法从拉曼光谱数据中提取细胞在生长发育过程中的演变信息。结果表明,应用激光镊子拉曼光谱技术,探索微生物发酵研究的新方法,为优化发酵参数,指导乙醇发酵生产调控提供借鉴。3.为了探求建立准确快速分选酵母细胞的方法,尝试利用拉曼光谱与激光镊子技术,从细胞群体中分选出目标酵母。收集各酵母细胞的拉曼光谱,经去背景、17点S-G平滑滤波、多项式拟合基线校正和矢量归一化等数据预处理,结合主成分分析,从酵母细胞的拉曼光谱中提取胞内物质的主要特征信息,根据物质构成的差异来识别目标酵母,进而建立分选模型。激光镊子俘获单个酵母细胞并收集其拉曼光谱,实时判别细胞的归属,并根据判别结果收集细胞。将收集的酵母细胞经菌落培养和苏丹黑B进行染色等方法进行验证,结果显示,分选出的细胞均含有目标产物。这一方法在实现分选所选目标酵母的同时,也为利用激光镊子拉曼光谱分选其它单细胞提供了借鉴方法。更多还原

【Abstract】 Along with the depletion of fossil fuels and the environmental problem is becoming increasingly serious, the development of clean and renewable resources has become a pressing issue. There are some microorganisms can be used to synthesize or convert some special material if cultured in the right condition, such as the microbial oil which can substitute for fossil fuels to produce biodiesel, the carotenoid can be used as food and drug additives, and ethanol can be used as fuel after purification, This means that these products all have relatively high economic value. This work, combined with the previous research, and the laser tweezers Raman spectroscopy system was used, trying to further explore the dynamic development of the products and the substrates of fermentation liquor and the material of intracellular during the process of fermentation, then we can provide a theoretical and an important reference parameters for the ferment industrial production, At the same time, we trying to carve from a selected group of yeast cells with the production capacity of some special products of the target cells. For that reason, the main research was carried out the following:1. This work is an attempt to study about fermentation detection of ethanol which is important to the laboratory research and industrial production. A high-throughput method used in our study is based on Raman Spectroscopy in the inverted microscope and 96-well plates. Base on the Raman signal ratio of standard ethanol solution and the internal, I establish a regression equation by the least squares fitting. And then the equation was used to calculate the content of ethanol, it was stem from the strains with cassava starch as a fermentation substrate, flask fermentation and the 500 liters of ethanol fermentation tank separately. At the same time the gas chromatography was used to detect the foregoing item. The t-test indicate that the results of Raman and gas chromatography have good reproducibility (p=0.05), this means that Raman test has a good accuracy. On the basis of Raman spectroscopy and 96-well plate, the linear regression, multiple linear regression, principal component regression and partial least squares (PLS) were employed to measure the concentration of ethanol and glucose aqueous mixtures simultaneously. A group of standard concentration of ethanol and glucose mixtures are was detected and fermentation broth of cassava starch was used to validate. The results show that this method could be applied to the real-time monitoring of ethanol fermentation.2. The structure, transformation, and bioactivity of single living cells at the molecular level have been studied by Raman spectroscopy. In this study, I explore the biochemical information about their biological macromolecules and the metabolic product from the Raman spectra of Saccharomyces cerevisiae, which was cultured by cassava starch with the 500 L fermentor. At first, in order to measure the ethanol and glucose concentration of the fermentation broth, we track the 880 cm-1 and 1127 cm-1 spectra band, and the results were compared with that of gas chromatography and the DNS colorimetry. In addition, several approaches such as the study of mean spectra, the difference spectra, the usage of histogram of characteristic absorption peak area and the application of principal component analysis were applied. These methods were applied on a data set containing Raman spectra of yeast in three stages of its growth, aiming to extract information about the evolution of cell components during growth. The result show that the laser tweezers Raman spectroscopy approach has the potential to open a new frontier to study microorganisms, which were used to optimize the fermentation parameters, and direct practical production of ethanol.3. Extensive research has been carried out in an effort to screen the microorganisms. Here, Raman Spectroscopy and laser tweezers were used to sort yeast from mixed yeast cells. The preprocessing of subtracted background,17 points S-G smoothing filter, polynomial fitting baseline correction and vector normalization were performed and the main features information of intracellular substances from the Raman Spectroscopy of yeast cells were extracted by combining principal component analysis. Basing on the distinguished composition of the traget yeast and the other yeast, a sorting model was established. The test yeast cell in optical trapping was distinguished real-time by the model referring to its Raman spectra. The cells distinguished as target yeast was collected by means of optical manipulation. The sorted traget yeast cells were verified by microbial culture and Sudan black B test etc. The result illustrates that Raman spectroscopy combining with optical manipulation is an effective technique for sorting yeast and other economic microorganisms.更多还原