Trichosporon dermatis油脂含量和油脂组成细胞荧光分析方法与机理研究

Microbial oil production by fermentation of oleaginous yeasts on lignocellulosic hydrolysates can offer low-cost, great available, and renewable lipid feedstock for biodiesel production. This technology still needs great amount of research with various factors and levels to satisfy the requirement of industrial application, but the complex and trivial analytical method of lipid content and lipid composition reduce the efficiency of the research on this technology, thus it is important to explore new kind of analytical method. In this study, new oleaginous yeast Trichosporon dermatis CH007 which can utilize lignocellulosic hydrolysates efficiently for fermentation is chosen as research object. By revealing the relationship between fluorescent intensity of fluorescent dyes and lipid content / lipid composition, and the laws of fluorescent dyes’ entering into yeast cells, fluorescent stability, etc., the cell fluorescent analytical mechanism of cellular lipid content and lipid composition can be elucidated, this can offer theoretical basis for the establishing its analytical method. In addition, fluorescent dyes whose fluorescent intensity related significantly to cellular lipid content / lipid composition will be screened, then the optimal cell fluorescent analytical method will be obtained by response surface analysis, and the adaptability, accuracy and stability of this method will be evaluated in the actual fermentation. Finally, efficient, accurate and convenient cell fluorescent analytical method for lipid content and lipid composition measurement will be built, and this will increase the research efficiency of lipid fermentation technology effectively.

酵母利用木质纤维素水解液发酵产微生物油脂可为生物柴油的制备提供廉价、来源广泛、可再生的油脂原料。该技术仍需大量多因素、多水平的研究以满足工业应用要求，但目前微生物油脂含量和油脂组成复杂繁琐的分析方法降低了研究的效率，因此开发新型的分析方法十分重要。本项目以可高效利用木质纤维素水解液发酵的新型油脂酵母Trichosporon dermatis CH007为研究对象，通过揭示荧光染料荧光强度与油脂含量、油脂组成的关系及染料进入酵母细胞和荧光稳定性等规律，阐明细胞油脂含量和油脂组成的细胞荧光分析机理，为建立其分析方法提供理论基础；筛选获得荧光强度与细胞油脂含量、油脂组成具有显著相关性的荧光染料，然后使用响应面分析获得最优的细胞荧光分析方法，并在实际发酵中评价该方法的适应性、准确性和稳定性，最终建立高效、准确、简便的油脂含量和油脂组成的细胞荧光分析方法，有效提高油脂发酵技术的研究效率。

油脂酵母利用木质纤维素水解液等廉价原料发酵制备微生物油脂是生物质化工的研究热点之一。当前微生物油脂的定量定性分析方法较为复杂繁琐，一定程度上降低了酵母油脂发酵的研究效率。本项目以油脂酵母Trichosporon dermatis CH007为主要研究对象，以建立高效、准确、简便的酵母油脂细胞荧光分析方法为主要目标，从胞内外两个角度研究了荧光染料与油脂的特异性结合现象，阐明了T. dermatis油脂细胞荧光分析机理；系统对比了常用油脂荧光探针Nile Red与BODIPY 505/515用于T. dermatis油脂细胞荧光分析的效果，筛选出较优的荧光探针；系统考察了悬浮液种类、预处理温度、细胞浓度、染色剂浓度、染色时间等关键因素对酵母油脂细胞荧光分析方法的影响规律，建立了准确性、适应性与稳定性高的T. dermatis油脂细胞荧光分析方法，为其它油脂酵母建立相似的方法及建立同时适用于多种油脂酵母分析的方法提供了方法学依据；构建了基于表面活性剂（SDS）与酸活化凹凸棒石（H-ATP）的Nile Red溶解体系，比较了丙酮、SDS、SDS+H-ATP等Nile Red负载溶液的内部结合情况、光学性质、水溶液中稳定性及细胞荧光分析效果，为进一步改进T. dermatis油脂细胞荧光分析方法奠定了基础。