里氏木霉促进秸秆发酵废物形成类腐殖物质特异性的研究

The utilization of waste straw is an urgent need for the sustainable development of Agriculture. It is an effective way to increase soil organic matter content by straw carbon sequestration. Humic substance (HS) is the main body of soil organic matter. Currently, the mechanism of the specific and structural formation of HS is a hot topic of debate and research. Cellulase production from straw waste by Trichoderma reesei has been widely applied. However, there are few reports about the conversion of fermentation residues into humic substances. Solid fermentation will be performed in this project with Trichoderma reesei and rice, corn and rape straw respectively. Elemental analysis, thermogravimetric analysis, infrared spectroscopy, nuclear magnetic resonance will be used to explore the dynamic changes of straw degradation, humic substances structure and the mechanism of formation and transformation of humic like components. In order to enhance the effect of carbon sequestration, the optimum parameters will be tested and given as a result. At the same time, this project also tries to analyze the formation mechanism of HS specificity using biotechnology of high throughput metabarcoding DNA which is useful for uncovering the diversity of a large array of microorganisms, and the relationship between soil microbial community structure succession and chemical structure changes of HS is studied as well. The project will provide more scientific basis for the illustration of HS formation , which has important guiding significance for the effective utilization of waste straw in agriculture.

废弃秸秆的有效利用是农业可持续发展的迫切需求。通过秸秆固碳增加土壤有机质含量是一种有效手段，其中构成土壤有机质主体腐殖物质（HS）的特异性和结构性形成的机理是国内外争论和研究的热点。利用里氏木霉发酵秸秆废弃物生产纤维素酶应用广泛，而对发酵剩余物转化为类腐殖物质的机制还不清楚。本课题将里氏木霉分别与水稻、玉米、油菜秸秆进行固态发酵，运用元素分析、热重分析、红外光谱、核磁共振等技术手段，从秸秆降解的动态变化规律、形成的秸秆类腐殖质各组分的动态变化规律、以及秸秆类腐殖物质的结构特征变化规律等进行深入研究，探讨类腐殖物质的形成转化规律，形成更多稳定的腐殖物质的最佳条件。同时运用DNA metabarcoding技术，从生物多样性角度分析HS形成的特异性，研究土壤微生物群落结构演替与HS化学结构变化之间的关系。该项目为HS的形成研究提供更多科学依据，对废弃秸秆在农业的有效利用具有重要指导意义。

本课题从类腐殖物质的转化数量、形成的类胡敏酸组成和结构特点以及秸秆发酵前后的微生物多样性三方面入手，重点研究了里氏木霉（T.reesei）对不同秸秆分解转化为类腐殖物质的效率和机理。研究表明：添加里氏木霉后，玉米秸秆形成的类胡敏酸和PQ值较对照组分别增加了17.5%和8.9%，而类富里酸和类胡敏素分别降低了5.54%和7.38%。说明里氏木霉在短期内能促进秸秆转化为类腐殖物质。部分富里酸和胡敏素降解或转化为胡敏酸，最高降解率和最高酶活（纤维素酶活、木聚糖酶活、β-葡萄糖苷酶活）性都出现在发酵第四天。不同秸秆因为含有的木质纤维素含量不同，即使在完全烘干的情况下，粉碎度也存在明显差异,玉米秸秆较但发酵后秸秆的粉碎度明显增加，均达到90%以上，较不发酵秸秆增加约20%，同时，类胡敏酸的缩合度、氧化度、芳香化度以及热稳定性都增强，说明里氏木霉产生纤维素酶，促进秸秆废物向稳定的类胡敏酸转化。类胡敏酸的H/C值近似为2.0，高于大多数土壤，说明秸秆发酵产生的类胡敏酸与土壤胡敏酸的前体物质并不相同，发酵过程加速了胡敏酸的氧化及芳香化，进而形成具有特异性的更为复杂的分子结构。通过16S rRNA序列测定与分析，对比发酵前后秸秆的细菌多样性，发现以发酵25天的玉米秸秆胡敏酸提取液为唯一碳源获得的细菌多样性高于未发酵秸秆，其中食烷菌属（Alcanivorax）与假单胞菌属（Pseudomonas）获得比率分别占21.7%和13.5%，为腐殖质形成的优势种群。未发酵秸秆中交替单胞菌属（Alternomona）和食烷菌属（Alcanivorax）为优势属，分别占29.6%和13.5%。本课题揭示了促进秸秆废物向稳定的类胡敏酸转化的分子机制和腐殖物质特异性形成的生物学基础，为里氏木霉在农业土壤修复方面的作用提供了理论指导。