哈尔滨产乙醇杆菌木糖产氢工程菌构建及秸秆高效产氢工艺研究

China has taken the leading position worldwide in fundamental theory and engineering technology for biological hydrogen production. The reconstruction of hydrogen-producing bacteria based on the metabolic engineering theory has been the key to further stimulate technological development and cut costs of biological hydrogen production. Straws are cheap, high-available and reliable feedstocks for biohydrogen production. However, xylose is the main composition of straws. Thus, the efficient utilization of xylose is a cutting-edge topic to improve energy recovery from straws and further save the cost of hydrogen production, which meets the strategic demands of scientific and technological innovation ecological environment protection of the 13th national five-year in China. This project will optimize the genetic manipulation tools for Ethanoligenens harbinense, screen the suitable xylose metabolism genes, and then construct the xylose metabolic pathway in Ethanoligenens harbinense. This project will obtain the engineering strain with high performance for hydrogen production from xylose. The operation parameters influencing the cell growth and xylose metabolism will investigated and optimised, and metabolites repressor effect on engineering bacteria will be revealed. The genomics and transcriptomics will be applied to investigate the molecular mechanism of the mixed sugar utilization mechanisms and establish the metabolism model of the engineered strain. Finally, hydrogen production process from straw hydrolysate will be developed with the engineered E. harbinense, which will break through the bottleneck of straw utilization. This project will enable efficient resource and energy recovery from agricultural waste, minimize hydrogen production costs and provide technical support for large-scale hydrogen production, which will make positive contributions to China's energy strategic transition and ecological environment protection.

我国在生物制氢基础理论和工程技术领域已经处于国际领先地位，基于代谢工程理论的产氢细菌改造成为进一步提升生物制氢技术水平和降低制氢成本的关键。秸秆能够为生物制氢提供廉价、丰富、可靠的原料，而其主要成分木糖的高效利用是提高秸秆利用效率、降低制氢成本的前沿课题，符合国家“十三五”科技创新规划和生态环境保护的战略需求。本项目拟通过代谢工程技术手段，优化遗传操作工具，筛选合适的木糖代谢基因，在哈尔滨产乙醇杆菌中构建木糖代谢途径，获得高效木糖产氢工程菌株；调控影响工程菌生长和代谢条件，优化木糖工程菌产氢效率，解析工程菌代谢物阻遏效应，利用基因组、转录组等手段解析生物学机制，建立混合糖高效代谢模型；构建木糖工程菌利用秸秆水解液的发酵产氢工艺，有效解决秸秆利用的瓶颈问题，实现农业废弃物高效资源化能源化，最大限度地降低制氢成本，为氢能大规模生产提供技术支撑，为我国能源战略转型和生态环境保护做出积极贡献。

生物制氢能够有效保障能源安全，减少环境污染。实现对秸秆的主要成分木糖的高效利用，对秸秆能源化和降低制氢成本具有重要的推动作用。本项目构建了高效产氢工程菌，开发了生物炭及银杏叶等发酵制氢添加剂，证实了利用木糖产氢菌进行秸秆发酵产氢的可行性，为推动秸秆生物制氢的产业化应用做出了重要贡献。.本项目揭示了哈尔滨产乙醇杆菌的5个活跃的氢化酶和3种产氢途径，并利用其歧化氢化酶构建了高效产氢工程菌，产氢速率和氢气产量分别提高101.96%和10.57%。证明了生物炭的添加对乙醇型发酵产氢具有显著的促进作用，产氢量和产氢速率分别提升118.4%和220.3%，并从pH缓冲、ORP变化、细胞固定化和矿物质元素方面系统分析了生物炭的影响机制；解析了银杏叶对糖蜜乙醇型发酵产氢的影响，氢气产率最高可达到1.58 mol/mol，采用比较转录组学的手段在分子层面揭示了银杏叶对发酵产氢的促进机制。.分离了能够利用木糖发酵产氢的新菌种，其利用木糖和葡萄糖的平均比产氢量分别为2.32 mol-氢气/mol-xylose和2.76 mol-氢气/mol-glucose，平均比产氢速率分别为10.07 mmol-氢气/g-cdw/h和9.47 mmol-氢气/g-cdw/h，产氢能力居于现有产氢菌的前列；分析了木糖产氢菌利用木糖和葡萄糖生长产氢动力学及基质代谢途径，推测其是通过木糖异构酶直接将木糖转化为木酮糖进入磷酸戊糖途径进行利用。.解析了木糖产氢菌利用秸秆的发酵产氢能力，分步酶解发酵和同步酶解发酵获得的最大比产氢量分别为71 ml/g-秸秆和110 ml/g-秸秆，展现出优良的秸秆发酵产氢特性；构建了高效低温碱尿预处理体系，最佳产氢效率达到213.06 mL/g-底物，比未处理秸秆产量提升1.48倍，同时证明了其在寒区冬季室外实施的可行性；构建了秸秆发酵产氢烷一体化反应系统，总氢烷效率达到544.23 mL/g-TS，优于目前绝大多数氢烷系统的效率，为实现秸秆生物质高效利用提供了新思路。