半连续稀释异养-贫营养两段法培育富油小球藻及调控机制

The improvement of microalgal biomass and intracellular lipid accumulation is a big challenge in the field of microalgal energy, which correlates directly with the commercial development of microalgal biodiesel. A two-stage cultivation model has been proposed to promote the increase in Chlorella protothecoides biomass and accumulation of intracellular lipid by our preliminary results. However, the fundamental mechanism underlying this promotion effect is not clear yet. In this proposal, the semi-continuous dilution heterotrophic-N deprivation two-step cultivation model will be employed to culture rich-oil Chlorella protothecoides. And the heterotrophic high-density Chlorella cells and nitrogen deprived rich-oil Chlorella cells will be adopted as control group, the two-stage cultured rich-oil Chlorella cells will be adopted as the experimental group to carry out study. The integrated modern biological means based on proteomics, Gene Ontology,KEGG (Kyoto encyclopedia of genes and genomes) metabolic pathway, in vivo confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) will be employed to comprehensively and systematically analysize the physiological and biochemical changes and differentially expressed proteins in Chlorella cells. The aims of this proposal is to screen metabolic regulatory factors related to lipid accumulation (i.e. the regulated enzymes related to synthesis and accumulation of lipid) and reveal the regulated mechanism of lipid accumulation in response to this two-step cultivation model. This proposal will provide foundations for manual adjustment of lipid accumulation of microalgae and large-scale development of microalgal biodiesel, and also provide a reference for other microalgae species lipid metabolism research.

如何提高微藻细胞生物量并有效富集胞内油脂一直是微藻能源领域的一大难题，也是微藻生物柴油实现产业化的关键。我们前期研究结果表明，两段培养法可以促使小球藻（Chlorella protothecoides）生物量的提高及胞内油脂的大量积累，这种现象产生的原因尚不明确。本项目采用半连续稀释异养-贫营养两段法模式培育小球藻，拟以异养高密度藻细胞和缺氮积脂细胞为对照组，两段法培育积脂藻细胞为实验组，应用蛋白组学、GO富集、KEGG代谢通路、活体共聚焦显微（CLSM）、流式细胞术（FCM）等现代生物学集成研究手段，全面系统地分析不同培养条件、不同培养阶段小球藻的生理生化变化及其蛋白质组的表达差异，甄选与小球藻油脂富集有关的代谢调控因子（即油脂合成积累途径的调控酶），揭示调控因子响应两段培育富集油脂的调控机制，为人工干预微藻油脂积累、推进微藻生物柴油规模化发展提供基础，同时也为其它藻类代谢研究提供参考。

如何提高微藻细胞生物量并有效富集胞内油脂一直是微藻能源领域的一大难题，也是微藻生物柴油实现低成本产业化的关键技术之一。本项目开展了以两段法模式（半连续稀释异养-贫营养）培育小球藻提高其油脂产率的研究，并对两段法模式对促进小球藻生物量提高及胞内油脂大量积累的调控机制进行了探索，取得的重要研究结果如下：.针对两段法模式中异养培养底物高成本瓶颈问题，提出以浓缩马铃薯淀粉水解物、蔗渣水解液作为碳源补给的半连续稀释异养，经氮饥饿诱导后使得小球藻细胞油脂终产率提高近10倍，其胞内脂肪酸组分C16:0、C16:2Δ9,12、C18:1Δ9、C18:2Δ9,12是制备生物柴油的理想原料；将抽提油脂经转酯化后获得的微藻生物柴油燃料性能（如密度、黏度、闪点、冷凝点、热值等）完全符合EN 14214、ASTM 6751生物柴油质量标准。研究成果为人工干预微藻油脂积累及利用生物质废弃物低成本开发微藻生物柴油提供新思路。.借鉴其他物种脂质代谢理论基础，通过生物集成分析手段，甄选出果糖-1,6-二磷酸醛缩酶（FBA）、核酮糖羧化酶（RuBisCO）、酰基载体蛋白（ACP）、苹果酸酶（ME）等与油脂富集相关的关键调控酶；另新发现催化脂肪酸β-氧化的脂肪酰CoA脱氢酶ACD、氮饥饿信号调控因子-硝酸还原酶NR也是小球藻油脂产率提高的潜在调控因子，并推测出关键调控酶调控的脂质合成积累路径，揭示了半连续稀释异养-贫营养两段法模式在小球藻积累油脂过程中的正调控机制。研究成果为构建高产油脂“工程藻株”及推进微藻生物柴油规模化进程提供理论依据。