乙醇预处理对芦竹细胞壁的影响及荧光可视化分析

Effects of ethanol pretreatment on the cell wall of Arundo donax and fluorescence visualization analysis

为探究NaOH-乙醇预处理过程中NaOH浓度变化对芦竹纤维表面结构、细胞壁区域化学成分以及酶解效果的影响,采用NaOH质量浓度分别为2.5,5.0,10.0 g/L的NaOH-乙醇溶液和10.0 g/L的NaOH水溶液于90℃水浴条件下,分别对芦竹粉末和切片预处理2 h。结果表明:当碱质量浓度为10.0 g/L时,NaOH-乙醇预处理后木质素和木聚糖脱除率达到最大值,分别为47.11%和35.12%,芦竹酶解葡萄糖得率和木糖得率达到49.41%和77.61%,分别是未处理样品的6.2倍和7.4倍。场发射扫描电镜观察显示,NaOH-乙醇预处理后,芦竹纤维细胞壁表面微纤丝暴露。预处理过程的荧光显微镜跟踪观察表明,木质素的脱除均由薄壁细胞开始,逐渐向与之靠近的厚壁纤维过渡,最后到维管束内部的厚壁纤维,细胞角隅的木质素相对较难脱除,预处理后仍显示较明显的木质素信号;NaOH-乙醇溶液预处理后的切片整体木质素自发荧光现象减弱,细胞壁中木质素相对浓度下降,对酶解葡萄糖和木糖得率的提高都起到促进作用。

Using enzymes,such as cellulases,the hydrolysis of lignocellulosic biomass has been considered as one of the eco-friendly methods to produce bioethanol in bioconversion.However,for the enzymatic hydrolysis,the biomass materials should be pretreated to improve hydrolysis efficiency.This is because of the intrinsic recalcitrance of cell walls in the biomass materials,which is caused by the complexity of chemical composition,multi-laminated cell-wall structure,diversity of cell types and cell combinations.Thus,an efficient pretreatment is an essential step for increasing enzymatic digestibility of the biomass.Arundo donax,the typical monocot family Gramineae,is widely cultivated in China,which has the characteristics of the high biomass yield and fast-growing for cultivation and offers great potential for bioethanol production.In this study,A.donax was pretreated by NaOH-ethanol solution.Both sections and powder of A.donax were pretreated by 2.5 g/L,5.0 g/L and 10.0 g/L NaOH-ethanol solutions,and 10.0 g/L NaOH solution at a 90℃water bath and kept for 2 h.After the pretreatment,using the field emission scanning electron microscope(FE-SEM),X-ray diffraction(XRD),fluorescence light microscope(FLM)and high-performance liquid chromatography(HPLC),the surface morphology of the fibers,the topochemical changes in the cell walls and the enzymatic hydrolysis efficiency were investigated.The 10.0 g/L NaOH-ethanol was demonstrated to be the optimal condition for the pretreatment and evidenced by the maximum yield of glucose and xylose,which was 6.2 times and 7.4 times of the yields using the un-treated A.donax.The results of the wet-chemical analysis and XRD examination indicated that changes of crystallinity were affected by the removal of the amorphous cellulose,hemicellulose and lignin in the cell walls together with the swelling of cellulose fibrils.FE-SEM images showed that,after the 10.0 g/L NaOH-ethanol pretreatment,more separated microfibers were exposed on the surface of the cell walls,which could increase the accessibility of the enzyme.By tracing the pretreatment process,the autofluorescence images showed that the removal of lignin among different cell types began at the parenchyma,then the sclerenchyma fibers located at the outer regions of vascular bundles and followed by the inner parts.The fluorescence signal still occurred in the cell corner after the pretreatment,indicating that the lignin in this region was relatively difficult to remove.After the NaOH-ethanol pretreatment,the whole autofluorescence from the sections was reduced,which suggested that the effect of the NaOH-ethanol pretreatment was more homogeneous compared with the NaOH pretreatment.Thus,the NaOH-ethanol pretreatment could be an effective method to gain fermentable sugars from A.donax.