多重荧光标记结合拉曼光谱原位研究多粘芽胞杆菌降解秸秆的生物膜特征

Characterization of Biofilm Formed by Paenibacillus polymyxa SYX-1 During the Degradation of Rice Straw Using Multiple Fluorescence Labeling Combined with Raman Spectroscopy

利用多粘芽胞杆菌(Paenibacillus polymyxaSYX-1)快速降解稻草秸秆,用多重荧光标记技术进行标记,共聚焦激光显微镜观察(CLSM)并结合激光拉曼光谱(Raman),原位研究纤维素降解过程中形成的生物膜特征,进一步探究植物纤维的酶解机制。共聚焦激光扫描跟踪结果表明:在0～13d的降解过程中,SYX-1菌株定殖的稻草秸秆表面物质成分呈复杂化趋势,表明在秸秆表面有良好的生物膜形成;拉曼光谱结果显示SYX-1菌株对稻草秸秆的降解主要发生在拉曼强度1580～1590cm-1处,证实了相关秸秆纤维素降解酶主要以β-折叠构象和无规卷曲构象共存。多重荧光标记技术和拉曼光谱手段相结合,可以作为深入研究纤维素降解过程中生物膜的形成特征及探究植物纤维酶解机制的有效手段。

In this study, Paenibacillus polymyxa SYX-I was used to rapidly degrade rice straw. The formed biofilm was characterized by the combination of confocal laser scanning microscopy （CLSM） and laser Raman spectroscopy. The distribution patterns of proteins, α-polysac- charides, cellulose, total cells, and dead cells in rice straw were simultaneously visualized using CLSM after labeling by fluoresceinisothio- cyanate （FITC）, concanavalin A（Con A）, calcofluor white （CW）, STYO 63, and SYTOX blue, respectively. The results demonstrated that SYX-1 was colonized on the surface of straw and formed a good biofilm during the degradation process from 0 to 13 days. A lignocellulose structure was observed obviously in~ rice straw at 0 day but obscured after cultivation of 13 days, supporting the degradation of rice straw by SYX-1. The laser Raman spectra demonstrated that the degradation of rice straw mainly occurred at the bands of 1580 cm-~ and 1590 cm-＇, which is belonged to the stretch vibration modes of amide groups. Therefore, the combination of multiple fluorescence labeling-CLSM obser- vation and laser Raman spectroscopy could be used as a suitable tool to characterize the architecture and function of biofilm. Knowledge on the architecture and function of biofilm contributes to our understanding of the degradation and utilization of lignocellulose biomass and pro- vides novel information for engineering applications and scientific research.