碳源浓度影响微生物PHB合成代谢的单细胞拉曼光谱分析  

Raman Spectral Profiles of PHB Synthesis by Cupriavidus necator H16 at Different Fructose Levels

作　　者：覃赵军[1,2] 彭立新[3] 竺利波[3] 宋水山[4] 刘军贤[1] 王桂文[3] 

机构地区：[1]广西师范大学物理科学与技术学院,广西桂林541004 [2]广西大学行健文理学院,广西南宁530004 [3]广西科学院生物物理实验室,广西南宁530007 [4]河北省科学院生物研究所,河北石家庄050051

出　　处：《中国激光》2015年第3期324-332,共9页Chinese Journal of Lasers

基　　金：广西壮族自治区自然科学基金(2012GXNSFCA053001,2013GXNSFAA019043)

摘　　要：应用激光镊子拉曼光谱实时监测杀虫贪铜菌(Cupriavidus necator)H16菌株在特定氮源、不同果糖浓度下生物塑料聚β-羟基丁酸(PHB)的合成过程及其生化动态,以期获知碳源浓度对PHB合成的影响机制。结果显示:1)2.0%果糖浓度(质量分数)下的PHB产量获得最佳发酵效果;2)发酵启动及发酵前期胞内物质的合成速度基本相同,但果糖浓度影响对数生长期的长短;3)782、1574、1656和1732 cm-1等峰信号强度变化动态显示,高碳源浓度下核酸物质的拉曼信号长时间维持在较高的水平,这可能是影响细胞合成PHB的重要因素;4)单个细胞在1732 cm-1峰处的平均强度与发酵液PHB总含量线性相关,可用于定量监测发酵过程中PHB总量动态。发现了C.necator H16菌株在不同碳源浓度下PHB合成代谢过程中胞内主要生物大分子的变化动态,为PHB发酵提供全新的光谱学信息,也为基于单细胞拉曼光谱快速分选高产优良菌株提供实验依据。Laser tweezers Raman spectroscopy technology is used to explore the accumulation of biodegradable plastic poly-β-hydroxybutyric acid（PHB） in Cupriavidus necator H16 at single-cell level. With ammonium sulfate（2 g/L） as nitrogen source, the fermentation of PHB is performed with 0%, 0.5%, 1.0%, 2.0% or 3.0% of fructose（mass fraction） and Raman spectra of single cell are acquired at different time of incubation respectively. Major results are as follows. 1） The fermentation performs ideal PHB product yield at 20 g/L of fructose. 2） At different fructose levels, C. necator H16 cells take almost the same steps to synthesize bio-macromolecule in the initial and early stages of fermentation, while higher fructose level extends the log phase of growth. 3） The dynamics of Raman intensity from peaks 782, 1574, 1656 and 1732 cm–1, which derive from RNA, DNA, proteins and PHB respectively,reveals that the cells show stronger Raman intensity of nucleic acid for longer time under higher level of fructose,and this may be of important factor affecting the accumulation of PHB. 4） The average intensity of single cell at peak1732 cm- 1shows linear relationship with yield of PHB and can quantitatively follow the PHB production. This observation uncovers the kinetics of major bio-macromolecule of C. necator H16 during the PHB accumulation at different fructose levels, provides novel spectral information for the PHB fermentation. This work shows the potential of laser tweezers Raman spectroscopy for the quick screening of high-yield PHB strains.

关 键 词：光谱学 拉曼光谱 单细胞分析 聚Β-羟基丁酸 

分 类 号：O657.37[理学—分析化学]