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| 光强影响产油微藻油脂积累的机制及其在规模化培养中的应用 | |
| 其他题名 | On lipid accumulation in oleaginuous microalgae affected by light intensity and its application in scale up cultivation |
| 何桥宁 | |
| 出版年 | 2017 |
| 学位类型 | 博士 |
| 导师 | 胡春香 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 限制微藻生物质能源发展的最大瓶颈是规模化培养中低油脂产率所带来的高成本。强光能有效提高油脂产率，是既清洁又可持续的环境因素。因此，本文从高产油藻株的筛选，不同光强下藻株生长生理、生化组分变化，高光强下转录组、代谢组分析及规模化培养的应用等方面，围绕光强对微藻油脂积累展开了探究。主要结果如下： 从沙漠土壤结皮筛选到2株优良产油藻株Chlorella sp. L1和M. dybowskii Y2，室内的生理生化分析发现，高光强 (HL，400 mmol photons m-2s-1)虽然使它们的ROS含量上升，光合活性降低，叶绿体降解， 蛋白和碳水化合物含量下降，但脂质含量普遍升高，细胞体积整体变大，而且极性脂含量均下降，中性脂(NL)含量升高，产率分别达51.36 mg L-1d-1和 49.71 mg L-1d-1。室外波动光强下，高波动光强也显著利于油脂积累和NL的大幅提高，只是来源于膜脂重塑的TAG比例较低(11%-20%)，大部分来自脂肪酸(FA)的从头合成或其它物质的转化。 转录组学(RNA-Seq)进一步分析发现，1) HL下M.dybowskii Y2的光合作用表达明显下调，但氧化磷酸化、ATP转运酶上调弥补了ATP生成的减少，TCA循环(sucB、CS、MDH)的上调表达提供了更多还原力。2) 参与C3固定、 糖酵解、戊糖磷酸和淀粉代谢的基因 第1d 显著上调，之后下降(如PFK、TPI、PGM、ENO、GAPDH、及淀粉和蔗糖合成酶等)；而糖异生途径中的PCK和PPDK，及戊糖磷酸途径中RPE、G6PD、rpiA、PGM均明显上调，这为FA的生物合成提供了更多的中间 代谢物及能量。3) 氮代谢中谷氨酸脱氢酶转录水平剧烈上调，将谷氨酸氧化成α-酮戊二酸并释放，为TCA循环提供碳骨架；说明氮代谢通过增强TCA循环向碳代谢转化。4) FA合成相关的Accase、ACSL、FASN、FAB和TAG积累的PAP、DGAT和PDAT等基因均显著上调表达，这说明从头合成和膜脂转化是TAG大量积累的主要来源。首次在产油微藻中探究并揭示了强光影响油脂和TAG积累机制。鉴于盐诱导与光调控的互补性，本研究利用HL与盐(20 g L-1 NaCl)胁迫结合的两步诱导方法，使M. dybowskii XJ-15的油含量提高到50.37%，产率达76.72 mg L-1d-1，油脂品质均显著提高。代谢组学分析进一步发现，氨基酸、胺类、蛋白均被分解重新利用，碳水化合物在后期也被分解，海藻糖、多元醇等参与渗透平衡的调节；TCA循环中间代谢产物的明显下降，意味着它们是FA合成中乙酰CoA的主要来源；而C16:0和C18:1比例的显著升高、及C18:2和C18:3比例的显著下降，证明膜脂的重塑和FA从头合成促进了TAG的大量积累。在光强调控的规模化应用中，温度常伴随作用；本研究首先探究了温度对M. dybowskii Y2油脂积累的影响。发现15 oC和35 oC条件均能促进油脂积累，35 oC下油脂产率最高；油脂组成方面，15 oC促进极性脂积累，而35 oC促进NL积累。然后将HL与15oC、25oC和35 oC联合诱导油脂积累；结果显示各组均能促进NL积累，中性脂产率都超过40 mg L-1 d-1 ，且HL与 35 oC下的产率最高。 最后，将多株候选产油微藻在强光下进行了规模化培养，发现柱状反应器(140 L)中，Chlorella sp. L1和M.dyboswkii Y2批量培养的油脂产率分别为9.06 和8.05 g m-2d-1 ；平板生物反应器下，可多次半连续培养，且使油脂产率分别达16.22和13.17 g m-2d-1(15 L)，24.31 和16.36 g m-2d-1 (96 L)；开放池(200 m2 ORP)中，半连续培养也较批量培养更高(前者为5.15 vs 4.06 g m-2d-1；后者为 5.35 vs 3.00 g m-2d-1)；高光强与盐诱导下(5 m2 ORP)，使M. dybowskii XJ-151的油脂产率(3.74 g m-2d-1)提高了27.21%。然后，基于两年200 m2 ORP的培养数据，对Chlorella sp. L1和M. dybowskii Y2的生产工艺做了生命周期评价和经济成本分析。 |
| 英文摘要 | The commercial application of microalgal biodiesel is impeded by low lipid productivity and high cost in scale-up cultivation. High light intensity (HL) is a clean and sustainable environmental factor and can promote lipid productivity efficiently. Therefore, this study investigate the isolation of potential oleaginous microalgae, the physiological and biochemical changes under different light intensities, transcriptomic and metabolomic analysis of strains in HL, and the application of microalgae cultivation outdoors. The main results are provided as follows: Chlorella sp. L1 and Monoraphidium dybowskii Y2 obtained from biological soil crusts were selected for its high lipid accumulation potential. Analysis of their indoors physiological changes indicated that chlorophyll degraded, Fv/Fm decreased, ROS scavenging enzyme increased, protein and carbohydrate content decreased under HL(400 mmol photon m-2s-1), but more carbon allocated into lipid accumulation as well as most of intracellular space was occupied by lipid bodies. Moreover, membrane lipid reduced dramatic to format neutral lipid (NL). The NL productivity (NLP) in Chlorella sp. L1 (51.36 mg L-1d-1) and M. dybowskii Y2 (49.71 mg L-1d-1) were greatly promoted. Outdoors application in photobioreactors with high fluctuating intensity (HFI) also found the accumulation of lipid and NL. But the increased NL come from membrane lipid remodeling accounted for 11% to 20%, the remaining from de novo fatty acid synthesis and other materials transformation.The transcriptome results (RNA-Seq) with the KEGG pathways analysis indicated that 1) Photosynthesis related genes were down-regulated, but genes in oxidative phosphorylation and transporting ATPase were up-regulated to make up the loss of ATP, TCA cycle (sucB, CS, MDH) upregulated to provided more NAD(P)H for fatty acid synthesis. 2) Carbon fixation, most synthetase in starch and sucose, glycolysis pathway genes (PFK, PK, TPI, PGM, ENO, GAPDH) were greatly up regulated in 1d HL induction, but decreased in following days. Gluconeogenesis pathway (PCK, PPDK) and pentose phosphorylation (RPE, G6PD, rpiA, PGM) were upregulated indicating the provision of carbon intermediates and reducing power. 3) The levels of glutamate dehydrogenase in nitrogen metabolism increased dramatic, providing more carbon skeleton for TCA cycle. 4) FA synthesis genes of ACSL, FASN, FAB, G3PDH and key genes in TAG accumulation pathway (PAP, DGAT and PDAT) were significantly up-regulated. The results verified the role of de novo FA synthesis and membrane lipid remodeling in TAG accumulation. For the first time, these work revealed the metabolic pathways changes and TAG accumulation mechanism.Third, the study explored the compensation effects of HL with NaCl induction in M. dybowskii XJ-151. The lipid content increased to 50.37% with high lipid productivity (LP) of 76.72 mg L-1d-1, and biodiesel quality were greatly improved. Metabolic profiling revealed that amino acids, amines and proteins were remobilized through catabolism, and carbohydrate were catabolized to provide C sources for lipid synthesis at the last stage. Trehalose and polyols responded to stress environment might participate in defending osmotic fluctuations by removing the excess reducing equivalents and balancing the redox status, their abundance continuously increased. TCA cycle intermediates generally decreased due to the provision of acetyl-CoA for FA biosynthesis. C16:0 and C18:1 significantly increased, while C18:2 and C18:3 markedly decreased. Membrane lipid remodeling and de novo lipid biosynthesis all contributed to TAG accumulation. Considering that temperature changes always accompanied with the HL outdoors, the study also explored the effects of temperature in M. dybowskii Y2. Both of 15 oC and 35 oC can promote lipid content, but more polar lipid were found in 15 oC and NL found in 35 oC. The results of HL combined with different temperatures (15 oC, 25 oC, 35 oC) indicated that NL can be greatly enhanced (NLP all exceed 40 mg L-1 d-1) in all groups, especially under HL combined with 35 oC.At last, several oleaginous strains cultivated outdoors under natural high light intensity. In columnar photobioreactors (140 L), the LP in Chlorella sp. L1 and M. dybowskii Y2 with batch culture was 9.06 and 8.05 g m-2d-1. In flat bioreactors, Chlorella sp. L1 and M. dybowskii Y2 can cultured with several times of semi-continuous, and their LP promoted to 16.22, 13.17 g m-2d-1(15 L), and 24.31, 16.36 g m-2d-1(96 L). In open raceway pond (200 m2 ORP), higher lipid productivities (4.06 versus 5.15g m-2d-1 for Chlorella sp. L1, 3.00 versus 5.35 g m-2d-1 for M. dybowskii Y2) were achieved under semi-continuous mode. Combination effect of HL with NaCl were verified 5 m2 ORP, the LP of 3.74 g m-2d-1 promoted for 27.21%. Then, based on the cultivation data in 200 m2ORP, the LCA (life cycle assessment) and financial costs analysis were carried out. |
| 中文关键词 | 高光强 ; 转录组学 ; 代谢组学 ; 油脂产率 ; 脂质重塑 |
| 英文关键词 | high light intensity transcriptomic metabonolomic lipid productivity lipid remodeling |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 水生生物学 |
| 来源机构 | 中国科学院水生生物研究所 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/287917 |
| 推荐引用方式 GB/T 7714 | 何桥宁. 光强影响产油微藻油脂积累的机制及其在规模化培养中的应用[D]. 中国科学院大学,2017. |
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