共培养菌群强化木质纤维素厌氧消化及其形成机制研究

Rumen microorganisms can play an important role in lignocelluloses degradation to methane as an effective approach for biomass utilization. During the investigation, a series of co-cultural consortia with specific functions are isolated from rumen microorganisms by diluted culture technique, under combined selective pressure of characteristic metabolites and metabolic inhibitors. Function and interaction of different co-cultural consortia are further elaborated by characteristic metabolites analysis during the process of lignocelluloses degradation, which can give guidance for shaping the consortial relations of mutualism, cross-feeding and syntrophy purposely. Moreover, formation mechanism about co-cultural consortia is studied from the multi-level including microbial community structure, functional gene transcription, energy metabolism and quorum-sensing etc. The potential for consortial synergy action is defined during the degradation process. Finally, functions of electron transfer and quorum-sensing signal on effectively regulating co-cultural consortial structure and function during consortial formation are investigated for strengthening methane production from lignocellulosic materials. This study could provide theoretical and practical experience for co-cultural consortia research, and present new methods and theoretical foundation for highly efficient production of biomass energy from lignocelluloses.

木质纤维素物质的生物降解是其资源化利用的重要途径，瘤胃微生物对降解木质纤维素产甲烷有重要作用。在特征代谢物、代谢抑制剂复合选择性压力下采用连续稀释培养技术，从瘤胃微生物中分离出一系列具有特定功能的共培养菌群；通过研究特征代谢物的降解过程，揭示不同结构共培养菌群在木质纤维素降解过程中的功能和相互作用，并有目的地塑造互利、互养、互营三种菌群关系；在微生物菌群结构、功能基因转录、能量代谢、群体感应信号等多维水平上研究菌群关系的形成机制，明确功能菌群协同作用在木质纤维素降解产甲烷过程中的效能；通过考察电子传递和群体感应信号在菌群关系形成中的作用，有效调控共培养菌群的结构和功能，最终强化木质纤维素产甲烷过程。本研究可为菌群关系研究提供理论和实践经验，为高效利用木质纤维素产生生物质能源提供技术手段。

瘤胃微生物对降解木质纤维素产甲烷有重要作用，本项目通过厌氧污泥和瘤胃微生物构建共培养菌群，在批次和半连续模式下研究秸秆厌氧消化的过程强化；借助于分子生物学，物质形态分布特征和三维荧光光谱-平行因子分析等技术手段，阐明共培养菌群降解木质纤维素的微生物学机制。主要研究内容和结果如下：. 半连续条件下，有机负荷为1.5，3.5和7g/(L·d)时，共培养体系比单瘤胃体系降解秸秆的甲烷量分别提高1.79，2.07和2.26倍；滤纸酶和羧甲基纤维素酶酶活呈增加趋势，高于对照组。厌氧消化后，Methanobrevibacter成为优势菌属。共培养水解菌群结构变化明显，Clostridium仍是优势菌属，Ruminococcus提高显著。来源于瘤胃的Bacteroides，Fibrobacter和Acetivibrio从体系中消失。共培养体系中Ruminococcus与Methanobrevibacter功能菌群的协作实现了水解和产甲烷活性的提高。. 瘤胃菌群和厌氧污泥在接种比1:1，1:2，2:1，1:0和0:1（VS比）条件下，研究稻秸的消化特性。共培养体系的甲烷产率提高了约三倍，在1:1时最高甲烷产率为273.64mL/g VS。共培养显著提高了木聚糖酶和纤维素酶活性，是稻秸高效处理的关键原因之一。厌氧消化后，固相中纤维素水解菌主要来自梭菌目（Clostridiales）。水解菌和嗜乙酸型甲烷菌共存于稻秸残渣上。液相中甲烷丝菌属与互营杆菌属或消化肠状菌属形成互营氧化菌群减少丙酸积累，加快了厌氧消化的启动时间。. 在高固态半连续消化中，容积产气率最高达到1.04L/(L·d)。当OLR为2.26g/(L·d)时，甲烷含量稳定在54.39%左右，甲烷产率均值为280.90mL/gVS；卧式反应装置中纤维素酶均值达到2632.18U/mL，有利于纤维素的降解。PARAFAC模型确定荧光组分数为3，辅酶F420和NADH荧光强度可以反映产甲烷活性和消化效率的变化。当OLR提高到2.47g/(L·d)时，嗜氢型Methanobacteriales数量下降到1.04×106 拷贝数/gTS，嗜乙酸型Methanosarcinales数量增加到9.44×106拷贝数/gTS，产甲烷途径发生了适应性变化。