裂解多糖单加氧酶在粪污秸秆异位发酵床微生物降解木质纤维素中的作用研究

Efficient degradation of lignocellulose is a key process during the fermentation of manure and straws, and it can affect the capacity-increase of soil carbon pool after applying organic fertilizer.The degradation due to the lignocellulosic-degradation enzymes such as lytic polysaccharide monooxygenase (LPMO), which has synergistic effect with cellulase on efficiently degrade lignocellulose. However, the function of LPMO in lignocellulosic degradation of complex ecosystems like ectopic fermentation system is not clear but important for contributing to the improvement of effectiveness of waste composting. This study investigated the microbial succession of microorganisms and specific functional microorganisms during the ectopic fermentation process through high-throughput sequencing. Furthermore, the diversity of LPMO-encoding genes was analyzed. Real-time PCR and Real-time RT-PCR methods were utilized for accurate quantification of gene copy numbers and gene expression level of LPMO genes, respectively. In order to illustrate the role of LPMO in cellulose degradation, the cellulases and LPMO activities and the degradation products were evaluated through structural equation modeling and canonical correlation analysis. The influence factors of ectopic fermentation system were also investigated. The above results will not only contribute to improve the microbial mechanisms of lignocellulose degradation in ectopic fermentation system, but also explore the possible modes of action of LPMO in complex environment and provide theoretical guidance for resource utilization of wastes.

木质纤维素的高效降解影响着粪污秸秆的发酵腐熟，进而影响还田后土壤碳库增容。裂解多糖单加氧酶（LPMO）由于可以高效降解木质纤维素而广受关注。然而LPMO在异位发酵床等复杂生境木质纤维素降解中的作用却不清晰，严重阻碍了粪污秸秆高效利用。本研究拟从粪污秸秆异位发酵床木质纤维素降解出发，通过高通量测序手段研究其中微生物群落及产LPMO功能微生物的组成及演替，在此基础上解析其中LPMO基因的多样性，并结合real-time PCR、real-time RT-PCR技术对LPMO基因数量及转录水平进行定量研究，同时对纤维素酶系的酶活、LPMO的氧化活性及代谢产物进行鉴定与分析，通过结构方程模型和相关分析，解析LPMO及其功能微生物在木质纤维素降解中的效应机制及影响因素。研究结果不仅可揭示异位发酵床中木质纤维素降解的微生物机制，还解析了功能LPMO的潜在作用模式，最终为废弃物的资源化利用提供理论依据。

木质纤维素的高效降解影响着粪污秸秆的发酵腐熟，裂解多糖单加氧酶（LPMO）由于可以高效降解木质纤维素而广受关注。本研究以粪污秸秆异位发酵床为对象，从木质纤维素降解出发，通过高通量测序手段研究其中微生物群落及LPMO在木质纤维素降解中的作用，添加秸秆可增强木质纤维素分解酶活性，增加木质纤维素分解微生物和编码CAZymes（碳水化合物活性酶）基因的丰度。冗余分析表明，木质纤维素降解与鞘状杆菌、假单胞菌、芽孢杆菌和放线菌含量呈显著正相关。利用宏基因组学分析发现发酵床进程中含有AA9、AA10，其中AA10含量较高。此外，还讨论了LPMO 与纤维素酶的共表达情况，以及辅助氧化还原酶（AA3、AA4、AA7）为LPMO 提供驱动力。荧光定量PCR测定木质纤维素降解不同阶段LPMO基因的相对丰度，发现AA10是在腐熟阶段显著提高，且秸秆的添加会显著提高AA9和AA10的丰度，这与宏基因组的分析结果相同，进一步证明AA10是促进发酵床中木质纤维素降解的主要LPMO，同时秸秆的添加更有利于其表达。同时结构方程模型进一步验证了发酵床中微生物功能多样性的增加是木质纤维素降解的主要驱动力，这是受添加秸秆后温度升高的影响。该研究为农业废弃物发酵中木质纤维素降解研究奠定基础，也为农业废弃物的循环利用提供了支撑。