双极膜电渗析辅助乙醇型发酵产氢及其代谢调控机制研究

As increasingly tensive energy supply and serious environment pollution, there is an urgent need to develop a clean and renewable energy source for sustainable development. Dark fermentative biological hydrogen production is promising for its industrializing potential. However there are some technical barriers in fermentation process, for example, hydrogen molar yield is still low, product feedback inhibitory action is severe and raw material utilization is incomplete. This research will take E.Harbinese B49, a representative strain of ethanol-type fermentation as research object, and demonstrate a comparative study of inhibition effects of added ethanol and acetate acid on cell growth and hydrogen production by batch test. Quantify the products inhibition strength, then identify the key inhibitory product. Based on bipolar membrane electrodialysis technology, develop a product separation unit for the key inhibitor removing, and build an integrated fermentative hydrogen production and product separation process. Investigate the operating conditions and hydrogen production features of the integrated process, through the comparative study with traditional fermentative hydrogen production. Conduct research on hydrogen produce under different operation models and different culture conditions respectively, thus obtain the optimized operation parameters of the integrated process. Perform metabolic flux analysis on the hydrogen production tests above, according to the intracellular flux distribution, determine the key metabolic regulatory nodes, and reveal the relationship between product metabolism and hydrogen elevation. The results of this research will illustrate internal control mechanism of promoting hydrogen production through extracellular products control, and provide technical support and logical proofs for optimizing control of bio-hydrogen production process.

面对日益紧张的能源供应和严峻的环境污染，研发清洁的可再生能源成为可持续发展的迫切需求。暗发酵生物制氢具有工业化发展潜力，但目前存在氢气摩尔产量低、产物抑制影响大和底物利用不完全等技术壁垒。本课题以乙醇型发酵代表菌株E.Harbinese B49 为研究对象，通过外源投加产物的静态实验，比较主要液相产物乙醇和乙酸对细胞生长和产氢的抑制影响，通过量化抑制强度，识别关键抑制产物；基于双极膜电渗析技术开发去除关键抑制物的产物分离单元，构建产氢发酵与产物分离耦合制氢工艺，对比传统发酵工艺的产氢过程，提出耦合工艺的运行条件和产氢特性；分别进行不同运行模式和不同培养条件下的产氢研究，优化耦合工艺运行参数；对上述产氢过程进行代谢通量分析，根据代谢流分布变化，确定关键代谢调控点位，揭示产物代谢与产氢代谢的关系，研究结果将阐释控制胞外产物促进产氢的内在调控机制，为生物制氢工艺的优化调控提供技术支持和理论依据。

研发清洁的可再生能源是实现可持续发展的必由之路。暗发酵生物制氢具有能源生产和降解污染双重效益。本研究以提高氢气摩尔产量、解除产物抑制影响和提高底物利用率为目标，以乙醇型发酵代表菌株E.Harbinese B49 为研究对象，考察主要液相产物乙醇和乙酸对细胞生长和产氢的抑制影响，通过量化比较产物抑制强度，识别出乙酸为关键抑制产物。根据主要代谢反应中间代谢产物的物量平衡构建代谢通量模型，从胞内代谢的角度分析不同浓度抑制产物对产氢量的影响，并获得关键点位G6P属于挠性节点，PYR属于刚性节点，乙酰辅酶A为弱刚性节点。在此基础上，提出了基于双极膜电渗析技术的高效分离乙酸的产物分离耦合产氢发酵工艺，开展连续分离、连续分离连续补料产氢研究。对不同运行模式的代谢通量分析表明产氢差异的根本原因在于葡萄糖代谢过程中的丙酮酸至乙酰辅酶A途径中的主要产氢前体Fdred的生成量的不同，连续补料连续分离模式提高了E. harbinese B49代谢葡萄糖的过程中主要的产氢前体Fdred的生成量，从而得到了较高的产氢效率，研究揭示产物代谢与产氢代谢的关系，阐释控制胞外产物促进产氢的内在调控机制，同时验证了本研究的E. harbinese B49代谢通量分析方法是可行的。