低温木质纤维素降解菌降解机制及对秸秆还田土壤的影响

Straw is one of the most abundant biomass resources on earth. Inefficient treatment of straw, such as burning, not only wastes straw resources, but also causes pollution. Returning straw to the soil can improve resource utilization. However, the problems of low temperature and a drying climate in northern China have limited the rapid degradation of straw returning to soil and have become the bottleneck for straw resource utilization. The research objectives of this project are as follows: 1) to screen highly efficient lignocellulose-degrading microorganisms from soil under a decomposing straw pile at low temperature; 2) to determine the best enzyme production conditions and degrading enzyme properties; 3) to analyze the lignocellulose-degrading products by psychrotrophic degrading bacteria with FTIR, GC, GC-MS, and NMR; 4) to investigate the biodegradation pathway; 5) to analyze the dynamics of expression quantities for related enzymes by qRT-PCR; 6) to sequence the transcriptomes of degrading bacteria using RNA-Seq technology, analyzing gene function, genetic differences and metabolic pathways in the process of lignocellulose degradation and exploring lignocellulose-degrading mechanisms by degrading bacteria at low temperature; and 7) to prepare a mixed psychrotrophic straw-degrading bacterial agent and evaluate its soil environmental release through microbial community structure, microbial biomass, soil organic matter and the change of nutrient content, effect on succeeding crop. The biodegradation mechanism of the main ingredients of straw at low temperatures is expected to be cleared through this study and improve the efficiency of returning degraded straw to soil, and the study is expected to offer a solution strategy for straw resource utilization technological bottleneck of returning degraded straw to soil at low temperatures.

秸秆是地球上最丰富的生物质资源之一，目前由于燃烧等不合理处理方式，不仅造成秸秆资源浪费，同时引起环境污染。秸秆降解还田可实现秸秆资源化利用，但由于我国北方地区存在气温低、干燥等问题限制还田秸秆快速降解，成为秸秆资源化利用瓶颈问题。本课题从腐烂秸秆堆底部土壤分离低温高效木质纤维素降解菌，研究其最佳产酶条件及酶学特性。采用FTIR、GC、GC-MS和NMR分析低温降解菌对木质纤维素降解产物，推测降解途径；采用qRT-PCR法分析相关酶表达量动态变化，采用RNA-Seq对菌株进行转录组测序，分析其降解木质纤维素过程中相关基因功能、基因差异及代谢通路，探讨降解机制；制备秸秆低温复合菌剂，从土壤微生物群落结构、有机质及养分含量变化等方面探讨木质纤维素低温降解菌对土壤环境释放安全性。预期通过本研究明确低温条件下微生物对秸秆主要成分降解机理，提高秸秆降解还田效率，为秸秆资源化利用瓶颈问题提供解决对策。

秸秆作为一种富含有机质和营养元素的农业废弃物，在改善土壤质量方面具有潜在能力。然而，严寒地区的低温环境抑制秸秆腐解，造成一系列环境问题。本项目利用耐冷微生物加速秸秆在低温条件下分解，以提高严寒地区秸秆还田效率。结果表明，通过富集筛选法获得了多株耐冷木质纤维素降解菌，采用单因素、响应面分析法对降解菌的生长以及产酶条件进行了优化以达到强化降解木质纤维素的效果；利用GC-MS和FTIR并结合相关文献，对木质纤维素降解产物进行鉴定，并分别推测出木质素、纤维素和半纤维素的降解途径；全基因组和转录组测序分析结果表明，耐冷木质素降解菌 Arthrobacter sp. C2中的脱色过氧化物酶 (DyP)、耐冷半纤维降解菌 Raoultella sp. HC6中的β-木糖苷酶（XynB）和耐冷纤维素降解菌 Aspergillus sp. YC2中的纤维素酶（CMC）分别是木质素、半纤维素和纤维素分解代谢过程中的关键降解酶，这些酶的作用位点、方式与推测的木质纤维素降解途径一致；RT-qPCR结果表明，dyp、xynb和cmc基因分别是菌株C2、HC6和YC2中木质纤维素降解过程关键基因，该结果与前期研究推断相吻合；对DyP酶、XynB酶和CMC酶进行酶学特性分析，确定了其最佳反应条件；联合耐冷木质纤维素降解菌制备了以生物炭为载体的低温高效秸秆降解菌剂，通过低温秸秆降解复合菌剂配施秸秆还田试验发现，施用该菌剂能显著改善土壤肥力，提高土壤细菌群落丰富度和多样性；盆栽试验结果表明，施用低温秸秆降解菌剂处理组小麦的株高、根长和鲜重均显著增加，并且该组SOD酶活和SP含量均升高。本项目揭示了耐冷木质纤维素降解菌对木质纤维素的降解途径与代谢机制，复配低温高效降解菌制备低温秸秆降解菌剂强化了低温下秸秆降解转化效果，为提高严寒地区秸秆还田效率提供理论参考和技术支撑。