基于显微拉曼光谱技术的活体微藻油脂含量快速无损检测研究

The non-destructive detection on lipid in microalgae is critical to the cultivation of oil-producing microalgae and also very important to extend the application of using microalgae as new energy and in other fields such as food area. In this project, the main lipid components such as saturated fatty acids, unsaturated fatty acids in chlorella and grape algae were investigated using confocal micro-Raman spectroscopy technology, lipid components and its accumulation procedure will be analyzed in a quantitative and nondestructive approach. Firstly the Raman characteristic bands will be identified according to the Raman scattering rationale and the Raman spectroscopy of standard lipid, then with the aid of chemometrics the obvious and specific Raman bands will be selected as quantitative analysis peaks, and combined with internal standard methods the main lipid components for microalgae can be detected in a quantitative way. In addition, the dynamic accumulation process of lipid in microalgae was also monitored and captured for analysis. In this project, the response behavior of microalgae will also be investigated with different nutrition so as to find out the relationship between nutrition needs and development of lipid in microalgae. The study will try to reveal the key procedure and control methodology in microalgae cultivation and guide the cultivation of high lipid productivity microalgae so as to extend the microalgae application to energy and industry fields.

产油微藻培养过程中油脂含量的快速无损检测是推广微藻作为新能源、食品等领域应用的基础和关键。项目以探究产油微藻培养过程中油脂积累的规律、油脂成分（饱和脂肪酸、不饱和脂肪酸等）的检测机理和快速获取方法为主线，首先对活体微藻细胞的共聚焦显微拉曼光谱进行测试条件的优化，探明微藻油脂主要成分的拉曼光谱响应机理，实现饱和脂肪酸、不饱和脂肪酸的定性检测；然后采用光谱预处理及波段优化方法结合谱峰比值对不同培养液营养成分下微藻油脂积累的规律及时滞特性进行研究，同时结合特征峰内标法实现油脂主要成分（饱和脂肪酸、不饱和脂肪酸等）的定量检测，最后实现油脂主要成分分布、不饱和度等的动态监测。该项目的研究成果将为活体微藻的培养、产油率及过程控制提供理论基础和信息支持。

产油微藻培养过程中油脂含量的快速无损检测是推广微藻作为新能源、食品等领域应用的基础和关键。项目以探究产油微藻培养过程中油脂积累的规律、油脂成分（饱和脂肪酸、不饱和脂肪酸等）的检测机理和快速获取方法为主线。具体研究内容主要分为：探明微藻油脂主要成分的拉曼光谱响应机理，实现饱和脂肪酸、不饱和脂肪酸的定性检测；采用光谱预处理结合特征峰内标法实现油脂主要成分不饱和脂肪酸等的定量检测；实现油脂主要成分分布、不饱和度等的动态监测。研究表明：通过选择四种波段（全波段建模、色素、油脂以及混合特征）建立PCA-LDA模型可对氮胁迫培养时间不同的斜生栅藻细胞进行有效判别，将重量法测得的脂肪酸含量与拉曼光谱法测得的油脂特征峰（1445 cm-1）的积分拉曼强度进行相关性分析，两者存在显著相关性（R2=0.83）。结果显示拉曼光谱技术应用于对氮胁迫天数不同的藻细胞的鉴别模型和对藻细胞内油脂含量进行定性定量分析具有可行性。. 项目执行后期，在前期研究的基础上，将共聚焦显微拉曼技术采集的单点微藻细胞信号用于建立细胞内油脂含量预测模型和面扫描信号用于绘制油脂分布伪彩色图。分析绘制的油脂分布伪彩图，可以直观的反应出细胞核位置以及油脂积聚的位置。为了直观体现拉曼光谱对斜生栅藻胞内物质的检测能力，对藻细胞进行拉曼面扫描采集，并对数据进行可视化处理。藻细胞拉曼光谱中1266 cm-1与1656 cm-1处的峰分别对应顺式=C-H平面变形与顺式C=C拉伸，两个峰主要代表脂肪链不饱和程度。另外1302 cm-1与1445cm-1处的峰分别对应CH2旋转与CH2卷曲，代表脂肪酸饱和碳链。建立饱和脂肪酸（1445 cm-1）和不饱和脂肪酸（1266 cm-1）的拉曼成像图，藻细胞拉曼成像图中，细胞核区域无明显拉曼响应，形成一个近圆形低值区域，结果与NR镜检结果一致。. 本项目的主要技术具有较大的原始创新性，项目研究成果将为活体微藻的培养、产油率及过程控制提供信息支持、检测方法和途径，并推动我国新能源—微藻产油的产业发展。