海水小球藻缺氮光合产氢的分子基础及调控机制

Hydrogen photoproduction from microalgae is considered to be an ideal source of clean and renewable energy. Hydrogen photoproduction by marine algae might be an effective method for scaling-up bio-hydrogen production, because it has the advantage of saving freshwater resources. In our previous study, a marine Chlorella pyrenoidosa was found to produce hydrogen efficiently using natural seawater medium under nitrogen deprivation condition, which made it possible to produce hydrogen at relatively low cost. However, the molecular basis and regulation mechanism of its hydrogen photoproduction are still unclear. In this project, genes, proteins and metabolic pathways associated with hydrogen production of C. pyrenoidosa will be screened and analyzed through transcriptomic, proteomic and metabonomic approaches. In order to clarify the relationships between hydrogen photoproduction and other metabolic pathways (e.g. respiratory pathways), the changes in activities of photosynthetic, mitorespiratory and chlororespiratory pathways etc. will be analyzed during the hydrogen producing process after adding specific inhibitors and uncouplers. Meanwhile, the expression levels in key genes, proteins and the contents in key metabolites of these metabolic pathways will also be determined in the presence of specific inhibitors and uncouplers. Based on above analysis, the molecular basis and regulation mechanism of hydrogen photoproduction in nitrogen-deprived marine C. pyrenoidosa will be elucidated. The result of this project could help us to understand the mechanism of hydrogen photoproduction in marine green alga more deeply, and could provide theoretical supports in artificial regulation or genetic engineering methods for improving the hydrogen photoproduction efficiency of marine green algae.

微藻产氢作为生产可再生洁净能源的理想途径备受关注，海水微藻光合产氢因可直接利用海水（节省宝贵的淡水资源）而更具潜力。我们前期分离到一株海水小球藻，发现该藻在天然海水培养基中仅通过简单地缺氮处理即可高效产氢，具有其它海水微藻不可比拟的优势，但其产氢机制亟待挖掘。本项目拟采用转录组、蛋白质组及代谢组技术从分子与代谢等层面对其产氢过程进行综合解析，筛选产氢可能的相关基因及代谢调控途径。通过施加不同干扰剂后分析光合作用、线粒体呼吸、叶绿体呼吸等生理生化过程，结合对关键基因、蛋白表达水平及代谢产物含量的测定分析验证各代谢途径在其光合产氢中的功能。通过上述研究阐明海水小球藻缺氮光合产氢的分子基础及调控机制，丰富海水绿藻光合产氢理论，并为通过人工调控或遗传改良提高其产氢效率提供指导。

绿藻光合产氢是一种非常有吸引力的生产可再生清洁能源的方式。我们前期研究发现，海水小球藻IOAC707S在缺氮的自然海水培养基中可以产生氢气，但其产氢机制尚不完全清楚，其产氢量也有待进一步提高。在本项目研究中，我们建立了一种在低氮的天然海水培养基中实现海水小球藻长期光合产氢的新方法。此外，从生理、生化、转录组学、蛋白质组学和代谢组学等方面探讨了海水小球藻在缺氮条件下光合产氢的分子机制。. 研究发现，于低氮培养基中预培养的海水小球藻，在氮源耗尽后进入缺氮状态，培养144小时后开始产生氢气。气相中的初始氧气浓度对氢气的产生有显著影响。与单纯低氮相比，低氮缺磷双重胁迫下海水小球藻的产氢量减少了85%。经优化培养后，海水小球藻在缺氮天然海水培养基中的产氢量可达到186 mL/L，不仅比之前的研究结果提高了6倍，而且比野生型莱茵衣藻在缺硫条件下的产氢量高出了20～60%。在缺氮条件下，海水小球藻IOAC707S的光系统II光化学活性和光合放氧速率降低，呼吸速率增加，有利于厌氧的建立及氢气的产生。在培养基中加入3-（3,4-二氯苯基）-1,1-二甲基脲（DCMU）可使总产氢量减少85%，说明水是氢酶的主要电子供体。在缺氮条件下，淀粉对海水小球藻产氢的贡献不大。与缺硫条件下莱茵衣藻产氢不同，醋酸在海水小球藻光合产氢过程中不仅参与厌氧环境的建立，而且作为外源电子供体起着重要作用。转录组学、蛋白质组学和代谢组学分析表明，在海水小球藻产氢过程中，光合活性并不是简单地下降，而是经历了一个复杂的调节，为光合产氢做准备；糖酵解、戊糖磷酸途径、三羧酸循环、卡尔文循环、光呼吸和线粒体呼吸等途径在海水小球藻光合产氢过程中均起到重要作用。. 上述研究结果有助于更深入地了解海洋绿藻的产氢机理，并为通过人工调控或基因工程方法提高海水小球藻的产氢效率提供理论依据。

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