水域沤竹体系中菌群结构多样性和酶活力分布表征及沤竹机理研究

The theories and methods of environmental microbiology, molecular biology, enzymology and materialogy have been employed in this project to investigate the dynamic chanages of microorganism community structure and diversity as well as enzyme activity distribution in natural aquatic bamboo retting system and to analyze the bamboo degradation model during retting and thus to reveal the retting mechanism. The microorganism community is analyzed based on the culture-dependent technique with the target to obtain and identify the culturable strains and analyze their diversity and enzyme-prodution capacity. Based on culture-independent technique, metagenome from bamboo retting system was extracted and bacterial community structure and diversity were analyzed and compared by PCR-SSCP technique. Dominant bands corresponding to the different bacterial communities were excised from SSCP gels and the DNA was recovered and sequenced，providing the information of uncultured bacteria and the bacteria can not be obtained through traditional cultivations.The dynamic change of enzyme-complex structure druing retting was demonstrated through enzyme activity distrubution analysis. The bamboo degradation model was constructed based on the investigation of bamboo chemical components and structure change. Based on the analysis of the correlation between structure and diversity change of microbial community and enzyme-complex , as well as the relationship between enzyme adsorption/penetration campacity on bamboo and bamboo degradation mode, the degumming model of retting related to 'microbial community - Enzymes - Bamboo' was established. Such model is further applied in high-speed and eco-friendly bamboo retting system construction as well as bast retting industry.

本项目将环境微生物学、分子生物学、酶学及纺织材料学等方法进行有机结合，研究天然水域沤竹体系中微生物群体结构多样性及酶活力分布动态变化规律，分析沤制过程中竹子的降解模式，揭示沤竹机制。在微生物群体分析中，基于可培养方法，进行菌株筛选鉴定、产酶分析及多样性研究；基于非培养方法，提取沤竹体系宏基因组，通过PCR-SSCP技术对沤竹过程中细菌群体结构和多样性进行分析和比较，通过对SSCP指纹图谱中的主要条带DNA的回收测序，获取不可培养的以及传统培养所不易得到的微生物的信息。通过酶活力分布研究，揭示沤竹过程中酶系结构动态变化规律；通过竹子的成分分析及结构变化规律研究，建立沤制过程中竹子的降解模型。基于微生物菌群结构及多样性变化与体系中酶系动态变化的关系，以及酶系中酶在竹上的吸附渗透力度与竹降解模式的关系，建立"菌群-酶系-竹"的沤竹脱胶模型，并将其应用在高效清洁沤竹体系构建及韧皮纤维沤制工序中。

竹纤维在纺织中的应用符合“绿色、生态、环保”的消费理念和回归自然的需求。竹纤维生物脱胶的研究尚处在起步阶段，缺乏针对竹子特定原料结构及组分特性的菌株或酶系的发掘及对竹体系所需酶复配体系的本质探索。本项目立足于天然水域沤竹体系分析，通过纯培养技术及非培养技术揭示沤制体系中微生物群体多样性及结构进化。结合水体酶活力监控、水质分析及沤制过程竹材的结构演变，阐明“菌群-酶系-竹”的沤制模型，建立竹子酶法高效清洁处理体系，为发展竹类资源的生物加工技术奠定基础。研究表明，天然沤制工艺可以获得性能稳定的竹束纤维。沤竹过程中水体溶解氧含量持续下降，pH呈“U”型变化；总氮、氨氮、总磷含量呈“N”型变化且变化趋势相同；总有机物含量持续上升，水质逐步恶化, 但沤竹液有良好的可生化性。沤制前期木质素降解酶系起主要作用，后期木聚糖酶占主导地位。木质素降解酶系组成为Lip-Mnp-Laccase，其与纤维素酶有一定的协同作用。沤液中存在木质素、半纤维素和部分纤维素，其含糖量变化规律与酶系活力变化规律相匹配。沤制过程中物种多样性呈现一定变化规律。可筛选的优势菌株占总菌属的比例较少，例如在对芳香族化合物有极广泛代谢能力的鞘氨醇单胞菌无法通过可培技术获得。沤制中后期出现的芽胞杆菌属和链霉菌属，分别具有较强的果胶酶和木聚糖酶活性。基于沤竹体系分析，构建酶法高效清洁竹处理体系。单一酶和复配酶对竹子作用效果有限，可通过超声波等预处理方法，增强酶对底物的可及度，进而增强酶作用效果。预处理萃取液中的部分物质，可以作为酶催化的底物，合成绿色功能聚合物，增强竹类资源的综合利用度。最后，在本项目的支持下，对纤维基材及面料的功能改性方面进行了拓展延伸，为开辟竹类高品质、高附加值产品奠定基础。本项目在国内外核心期刊发表论文36篇（录用3篇），其中SCI收录15篇；授权国家发明专利2件，申请国家发明专利5件。本项目实施中培养研究生9名，其中3人已答辩。