木质纤维素水解残渣厌氧消化过程中微生物群落的组成与功能分析

Lignin is a byproduct that can be recovered from Unhydrolyzed Solids (UHS) derived from lignocellulosic biorefineries and upgraded to produce value-added products, improving the economics of the whole biorefinery process. However, the carbohydrates covalently bound to lignin in UHS need to be removed before the utilization of lignin. The applicant developed a biological method of anaerobic digestion previously, which can selectively decompose of the recalcitrant carbohydrates while preserving lignin intact for downstream valorization. However, the composition of microbial community and the mechanism of complete polysaccharide hydrolysis in UHS are not clear. Based on the previous work, anaerobic digestion of UHS generated from enzymatic hydrolysis of AFEX pretreated corn stover will be investigated in this study. The applicant are proposing to study the change of microbial community structure, carbohydrate-degrading enzyme profiling and carbohydrate linkages during AD by using the technologies of 16S-DNA high-throughput sequencing, metagenomic analysis, quantitative proteomic analysis and glycome analysis. This knowledge will provide understanding of the key microorganisms and enzymes that are required for efficient carbohydrate decomposition, as well as the relationship between microbial community/key enzyme expression and individual carbohydrate linkage disruption. This work will provide fundamental insight about anaerobic degradation mechanism of recalcitrant carbohydrates in lignocellulosic biomass and contribute to the future application of anaerobic microorganisms in improving lignin properties for further valorization.

从木质纤维素生物炼制水解残渣中回收木质素并转化为高值产品可以降低木质纤维素生物炼制的成本，但首先需要将水解残渣中与木质素共价结合的糖类去除。厌氧微生物能选择性地分解上述难降解性糖类且保留木质素的完整性，但这些微生物的组成及分解难降解性糖类的作用机理尚不清楚。在前期工作的基础上，本项目拟采用16S-rDNA测序、宏基因组、定量蛋白组和糖组分析等技术，研究氨纤维爆破处理后的玉米秸秆水解残渣厌氧消化过程中微生物群落结构、糖类降解酶表达谱和糖类连接键的动态变化，通过识别断裂的糖间连接键，探明作用于难降解性糖类分解的关键微生物和糖类降解酶表达及其与单个糖间连接键断裂的关系，为进一步解析木质纤维素中难降解性糖类的厌氧降解机理及推动厌氧微生物在高品质木质素制备领域的应用奠定基础。

如何有效地去除秸秆水解残渣中的糖类和灰分等杂质是木质素高值利用的关键。本研究利用厌氧消化的生物方法从秸秆水解残渣中富集木质素，再利用溶剂萃取的方法从消化残渣中纯化木质素。. 系统地研究了不同温度和接种比例条件下，氨纤维爆破预处理秸秆水解残渣中的聚糖含量变化、聚糖降解率以及产气性能，阐明了水解残渣中难降解性糖类的厌氧降解规律，获得了优化的水解残渣厌氧降解条件。. 采用16S-rDNA测序技术分析了秸秆水解残渣厌氧消化过程的细菌和古菌群落结构的演变规律，获得了不同发酵阶段的优势菌群组成；采用宏基因组测序技术解析水解残渣厌氧消化过程关键时间点的细菌群落结构及功能基因组成，明确了参与糖类降解的主要细菌以及与糖类降解相关的功能基因。. 通过优化条件下厌氧消化过程固体残渣的组成变化，明确了厌氧消化对水解残渣中木质素的富集程度；通过核磁共振、凝胶色谱、热重分析等表征，阐明了厌氧消化对水解残渣中木质素分子量大小、热稳定性以及主要官能团和化学连接键的影响规律。. 利用不同有机溶剂从厌氧消化残渣中萃取木质素，获得了不同溶剂条件下的木质素质量得率和纯度；通过凝胶色谱、热重分析、核磁共振等手段表征了木质素的分子量大小、热稳定性和结构，获得了不同萃取木质素的品质和应用潜力。