基于磷过量补给提高氮限制异养培养普通小球藻的油脂产量

发布时间：2018-08-06 13:35

【摘要】：生物柴油是一种可替代传统化石燃料的可再生能源。微藻具有生长周期短、含油量高、不占用耕地等优点,是制备生物柴油的最佳原料,因此引起越来越多的关注。提高油脂产量是降低生物柴油成本的关键,可通过提高微藻生物量和胞内油脂含量两个角度寻求解决方案。氮饥饿是最常用的刺激微藻细胞累积油脂的手段,然而氮饥饿条件下会影响微藻细胞生长,引起生物量降低,从而导致油脂产量增加不明显甚至降低。本文针对这一问题,探究了微藻培养过程中,同时提高生物量和油脂含量的新型微藻培养方案。(1)利用氮限制磷过量条件异养培养微藻。在氮磷浓度正常条件(对照组)培养时,生物量为4.73 g/L,氮限制条件下,生物量仅有2.34 g/L,在氮限制条件下,增加磷浓度至45 mg/L,生物量可达到4.53 g/L,与对照组接近。过量的磷以聚磷的形式储存在细胞内,可为微藻细胞生长和油脂合成提供必要的物质和能量。通过分析生长相关基因表达发现,油菜素类固醇的合成、碳和氨基酸的代谢在微藻细胞生长过程中具有重要作用。氮限制磷过量培养条件下,微藻细胞的油脂含量可达42.3%,油脂产率为310.0 mg/(L·d),分别为氮磷正常培养条件下的2倍和1.32倍。油脂组分分析得出C16和C18的脂肪酸比例高达95%以上,并且,饱和脂肪酸和单不饱和脂肪酸的比例均高于对照组。因此,氮限制磷过量策略培养微藻不仅可以提高生物量、油脂含量,而且可以提升生物柴油的品质。(2)氮限制磷过量培养条件下磷的投加方式对微藻生长和油脂合成的影响。氮限制磷过量培养条件下,增加磷的投加次数对微藻的生长有一定抑制作用,分2次投加的最大生物量为3.95 g/L,而分8次投加的生物量为3.53 g/L。增加磷的投加次数可提高微藻细胞的内油脂含量,分8次投加的油脂含量可达到41.5%,与2次投加相比提高了1.2倍。通过分析与油脂合成相关基因表达发现,编码ACCase的基因Unigene7398,编码DGAT的基因CL2735.Contig2、CL73.Contig8、CL12.Contig1、Unigene7590、CL2735.Contig1、Unigene1920,增加磷的投加次数有利于提高油脂合成基因的表达水平。在培养周期末期,不同磷的投加方式对微藻细胞内磷的贮存形态没有显著影响,过量的磷主要以正磷酸单酯的形式存在。此外,分多次投加磷有利于细胞内快速累积以C16:0为主的饱和脂肪酸,而投加次数少有利于C18:0和C18:1两种脂肪酸的累积。因此,增加磷的投加次数对油脂产量的增加没有明显的促进作用,但可以提高细胞内的油脂含量。
[Abstract]:Biodiesel is a renewable energy source that can replace traditional fossil fuels. Microalgae have the advantages of short growth cycle, high oil content and no occupation of cultivated land. It is the best raw material for the preparation of biodiesel, so more and more attention has been paid to microalgae. Increasing the oil production is the key to reduce the cost of biodiesel. The solution can be found by increasing the biomass of microalgae and the content of intracellular oil. Nitrogen starvation is the most commonly used method to stimulate the accumulation of oil in microalgae cells. However, nitrogen starvation can affect the growth of microalgae cells and lead to the decrease of biomass, which leads to the increase of oil production and even the decrease of oil production. In order to solve this problem, a new microalgae culture scheme, which can increase the biomass and oil content simultaneously, was studied. (1) the heterotrophic microalgae were cultured under the condition of nitrogen limiting phosphorus excess. Under normal nitrogen and phosphorus concentration (control group), the biomass was 4.73 g / L, the biomass was only 2.34 g / L under nitrogen limitation, and the biomass reached 4.53 g 路L ~ (-1) by increasing phosphorus concentration to 45 mg / L under nitrogen limitation. Excessive phosphorus is stored in cells in the form of phosphorous, which can provide essential material and energy for microalgae cell growth and oil synthesis. By analyzing the expression of growth-related genes, it is found that the synthesis of rapesin steroids, the metabolism of carbon and amino acids play an important role in the growth of microalgae cells. Under the condition of nitrogen limiting phosphorus excess culture, the oil content of microalgae cells reached 42.3%, and the oil yield of 310.0 mg/ (L d), was 2 times and 1.32 times higher than that of normal culture condition, respectively. The fatty acid ratio of C16 and C18 was over 95%, and the ratio of saturated fatty acid to monounsaturated fatty acid was higher than that of control group. Therefore, the cultivation of microalgae not only increased the biomass and oil content, but also improved the quality of biodiesel. (2) the effect of phosphorus addition on the growth and lipid synthesis of microalgae under the condition of nitrogen limiting phosphorus excess. Under the condition of nitrogen limiting phosphorus excess culture, the growth of microalgae was inhibited by increasing the times of phosphorus addition. The maximum biomass of microalgae was 3.95 g / L in two times and 3.53 g / L in 8 times. The content of oil in microalgae cells could be increased by increasing the number of times of adding phosphorus. The oil content of microalgae cells added in 8 times could reach 41.5 times, which was 1.2 times higher than that of two times. By analyzing the expression of genes related to oil synthesis, it was found that Unigene7398, a gene encoding ACCase, and CL773. Contig8, a gene encoding DGAT, CL12. Contig1: Unigene7590, CL2735. Contig1 + Unigene1920, increased the number of phosphorus dosages to improve the expression level of oil synthesis genes. At the end of the culture cycle, different phosphorus dosages had no significant effect on the storage form of phosphorus in microalgae cells, and the excess phosphorus existed mainly in the form of orthophosphate monophosphate. In addition, multiple dosages of phosphorus were beneficial to the rapid accumulation of saturated fatty acids, mainly C16: 0, and less times to the accumulation of C 18: 0 and C 18: 1 fatty acids. Therefore, increasing the number of phosphorus dosages had no significant effect on the increase of oil yield, but could increase the oil content in cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q945;TE667

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