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| 产油单针藻LB50和微芒藻XJ-2的培养及NaCl诱导的油脂积累研究 | |
| 其他题名 | Cultivation and lipid accumulation by NaCl induction of oleaginous microalgae Monoraphidium dybowski |
| 杨海剑 | |
| 出版年 | 2015 |
| 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 | 在能源微藻产业链的上游，高含油量的生物质的培养是最为关键的限制瓶颈。为了在室外条件下稳定的获得较高的生物量和油脂含量，本文主要以单针藻LB50和微芒藻XJ-2为研究对象，结合室内外封闭式和开放式反应器的培养、不同浓度和不同时期的NaCl诱导，探索了油脂积累的效率及其机理，主要结果如下： 初选的6株产油绿藻(Micractinium sp. XJ-2，Westella botryoides XJ-4，Monoraphidium dybowskii XJ-2，Monoraphidium sp. 1，Monoraphidium dybowskii LB50，Monoraphidium sp. LB59)经室内外培养发现，虽然它们的脂肪酸组分都具有作为生物柴油的潜力，但生长和油脂含量存在较大差异，依据油脂产率，筛选M. dybowskii LB50和Micractinium sp. XJ-2为后期重点研究藻株。 然后对2株筛选出的微藻(M. dybowskii LB50和Micractinium sp. XJ-2)的氮素类型进行了优化，发现尿素完全可以作为它们室外大规模培养的氮源，适宜浓度均为0.25 g L-1。但M. dybowskii LB50需在0.1 M NaHCO3下才能更好生长。而且在该营养条件下，M. dybowskii LB50在室外1400 L柱状反应器培中的油脂含量能稳定达到30%，油脂产率达24.41 m3 ha-1 y-1。 为了进一步提高油脂产率，对不同生长阶段的M. dybowskii LB50用不同浓度的NaCl进行诱导，以找到最适的时间节点。研究发现在不同的生长阶段，适合的NaCl浓度诱导，都可使M. dybowskii LB50油脂的积累量和油脂品质显著改善。最佳的时期是在指数生长的后期，此时加入20 g L-1 NaCl (1.4 mg/107cells)可以显著提高M．dybowskii LB50的脂质含量(40%)和脂质生产力(分别增加10%和30%)。 在室外较大规模(140 L)下，以确定时间节点加入NaCl后发现，在20 g L-1 NaCl诱导下增加的油脂就是中性脂，而且只需诱导24 h，M. dybowskii LB50和 Micractinium sp. XJ-2的中性脂分别增加了54%和69% (占干重的15.80%和12.99%)，且中性脂的面积生产率分别达到了3.43 g m-2 d-1和2.59 g m-2 d-1。通过生命周期评估中经济和能量的分析，我们发现这种光合自养两步法(NaCl诱导)是经济可行的。特别在增加油脂产率、工艺节能、经济上都具较明显应用前景。 代谢组学分析进一步发现，不同浓度NaCl诱导的油脂积累虽有共性，但有明显不同。共性是大量的糖降解提供了碳源，能生成丙酮酸或乙酰乙酸的氨基酸降解也提供碳源，膜脂降解转化积累TAG。不同的是：20 g L-1 NaCl诱导积累TAG主要是靠光合固定的CO2提供碳源，横向物质转化较少，糖脂转化TAG 为18%左右；40-60 g L-1是靠胞内大分子物质(淀粉)降解，能产生丙酮酸和乙酰乙酸的氨基酸(赖氨酸、亮氨酸、甘氨酸、苏氨酸)降解提供了合成TAG的碳源和糖脂(DGDG、MGDG)以及DGTS的转化成TAG。60 g L-1 NaCl诱导时渗透调节物(甘油、可溶性糖)大量积累，TAG虽有升高，但总脂没有升高。 跑道池培养中进一步发现，M. dybowskii LB50比Micractinium sp. XJ-2更具有应用潜力。即在5 m2跑道池中M. dybowskii LB50比Micractinium sp. XJ-2的油脂生产率显著高(分别为2.61±0.1 g m-2 d-1和2.28±0.06 g m-2 d-1)。且M. dybowskii LB50在200 m2跑道池半连续培养中的油脂产率可高达5.43 g m-2 d-1 (18.51 m3 ha-1 y-1)，半连续的培养中，M. dybowskii LB50和Micractinium sp. XJ-2不仅都能增加油脂产率，并且每批都可进行3轮半连续培养。针对M. dybowskii LB50跑道池半连续培养工艺的数据，经生命周期评价和技术经济分析发现，生产1 Kg M. dybowskii LB50的粗油，需消耗18.7 MJ 能量(200 m2)，可以固定0.8 Kg CO2，释放5.8 Kg O2，成本在4 USD。与现有培养工艺相比，半连续培养在能耗、固定CO2、经济上均占巨大优势，加之M. dybowskii LB50在耐盐环境下有更大应用潜力，因而，该工艺与M. dybowskii LB50结合将在干旱区及盐碱地有非常大的应用优势。 |
| 英文摘要 | To achieve the sustainable industrial production of bioenergy from microalgae, the crucial technological bottleneck of providing sufficient oleaginous feedstock must be addressed. This study focused on cultivation and lipid accumulation of Monoraphidium dybowskii LB50 and Micractinium sp. XJ-2, developed lipid induction methods in closed photobioreactor and open raceway ponds using different concentrations of NaCl induction in photoautotrophic two-step cultivation, to explore the efficient cultivation strategy and the mechanism of TAG accumulation through NaCl induction. The main results are provided follows: Six preliminary selected microalgae (Micractinium sp. XJ-2，Westella botryoides XJ-4，Monoraphidium dybowskii XJ-2，Monoraphidium sp. 1，Monoraphidium dybowskii LB50，Monoraphidium sp. LB59) were different in growth and lipid contents. But the pattern of the six strains outdoors was similar to that indoors. Finally, Micractinium sp. XJ-2 and M. dybowskii LB50 were further studied in the subsequent trials. The investigation of medium optimization found that the urea can be used as nitrogen source for cultivation M. dybowskii LB50 and Micractinium sp. XJ-2 outdoors, and the concentration was 0.25 g L-1. But for cultivation of M. dybowskii LB50 outdoors, the 0.1 M NaHCO3 must be added in the medium. Further, during a two month period cultivation under a large scale of 1400 L outdoors, the average lipid content and productiovity were about 30% and 24.41 m3 ha-1 y-1, respectively. In order to further improve oil productivity, the technology of NaCl induction in photoautotrophic two-step cultivation was widely used, but the time node and concentration range of the added NaCl should be determined in photoautotrophic two-step cultivation. We found lipid content and quality were improved in all growth phases with NaCl induction. the optimal time node was the late-exponential growth phase(LEGP). Lipid content and productivity can be significantly improved (increased by 10% and 30%) by 20 g L-1 NaCl (1.4 mg/107cells) induction at LEGP, and the effect was also been tested in a large-scalecolumn photobioreactor (140 L). After determine the time node, we made a further discussion on neutral lipid accumulation by NaCl induction in a large-scale column photobioreactor (140 L) outdoors. In the outdoor the increment of lipid was neutral lipids by 20 g L-1 NaCl-induced and the induction period was only 24 hours, shorten the cultivation time. The neutral lipids of M. dybowskii LB50 and Micractinium sp. XJ-2 increased by 54% and 69% (15.80% and 12.99% of dry weight), respectively. Thus, the areal neutral lipid productivity (140 L) of M. dybowskii LB50 and Micractinium sp. XJ-2 reached 3.43 and 2.59 g m-2 d-1, respectively. Through the life cycle assessment, economy and energy analysis, we found that this technology of NaCl induction in photoautotrophic two-step cultivation was economically feasible. Therefore, from the perspective of productivity, the energy-saved and economical in the process of industrial production of bioenergy from microalgae, the strategy of NaCl induction was very promising. It is urgent to further explore the mechanism of TAG accumulation by NaCl induction. The mechanism of different concentrations NaCl induction had commonness and characteristics. Commonality is that a lot of sugar degradation provided carbon for TAG biosynthesis, the amino acidsdegradation also provided carbon, and membrane lipids transformed into TAG. Specifically, TAG accumulation through 20 g L-1 NaCl-inducted mainly depended on carbon through photosynthetic CO2 fixation, less substances transversed conversion into TAG, the neutral lipid (mainly TAG) accumulation approximately 18% came from glycolipids (GL). But TAG accumulation through 40-60 g L-1 NaCl-inducted was mainly on intracellular substances (starch, sugar) degradation to provide carbon for TAG biosynthesis, the amino acids (Lys, Leu, Gly, Thr) degradation also provided carbon to synthesize TAG, and the glycolipids (MGDG, DGDG) phospholipids and DGTS transformed into TAG. TAG increased through 60 g L-1 NaCl-inducted, but total lipid did not accumulation, mainly because some osmotic regulation substances accumulated by osmotic pressure. At the same time, M. dybowskii LB50 and Micractinium sp. XJ-2 also cultivated in raceway ponds outdoors to determine the more widely use. We found M. dybowskii LB50 had more potential than Micractinium sp. XJ-2. The lipid productivity of M. dybowskii LB50 reached 5.43 g m-2 d-1 (the annual yield was 18.51 m3 ha-1 y-1, which had slightly lower than in column column photobioreactor cultivation). The lipid productivity of M. dybowskii LB50 and Micractinium sp. XJ-2 had been increased by the semi-continuous cultivation, what’s more, each batch can be carried out three times with semi-continuous culture. Further to study on the date that M. dybowskii LB50 cultured in 200 m2 raceway ponds with semi-continuous mode through the LCA and TEA. Production for 1 Kg crude bio-oil of M. dybowskii LB50 need energy consumption 18.7 MJ (200 m2), could fix 0.8Kg CO2-eq, and release 5.8 Kg O2. And the total costs of production for 1 Kg crude bio-oil is4$. The semi-continuous culture has a huge advantage, not only in energy consumption, CO2fixation, but also in economy. What’s more, alkali-resistant oleaginous M. dybowskii LB50 has even more potential under saline-alkaline condition. Thus, the combination of technology and M. dybowskii LB50 will have great application advantages in arid and saline land. |
| 中文关键词 | Monoraphidium dybowskii LB50 ; Micractinium sp. XJ-2 ; NaCl诱导 ; 油脂生产率 ; TAG |
| 英文关键词 | Monoraphidium dybowskii LB50 Micractinium sp. XJ-2 NaCl induction lipid production TAG |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 环境科学 |
| 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/2XILL650/287580 |
| 推荐引用方式 GB/T 7714 | 杨海剑. 产油单针藻LB50和微芒藻XJ-2的培养及NaCl诱导的油脂积累研究[D]. 中国科学院大学,2015. |
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