【摘要】：生物柴油是一種可替代傳統(tǒng)化石燃料的可再生能源。微藻具有生長周期短、含油量高、不占用耕地等優(yōu)點(diǎn),是制備生物柴油的最佳原料,因此引起越來越多的關(guān)注。提高油脂產(chǎn)量是降低生物柴油成本的關(guān)鍵,可通過提高微藻生物量和胞內(nèi)油脂含量兩個角度尋求解決方案。氮饑餓是最常用的刺激微藻細(xì)胞累積油脂的手段,然而氮饑餓條件下會影響微藻細(xì)胞生長,引起生物量降低,從而導(dǎo)致油脂產(chǎn)量增加不明顯甚至降低。本文針對這一問題,探究了微藻培養(yǎng)過程中,同時提高生物量和油脂含量的新型微藻培養(yǎng)方案。(1)利用氮限制磷過量條件異養(yǎng)培養(yǎng)微藻。在氮磷濃度正常條件(對照組)培養(yǎng)時,生物量為4.73 g/L,氮限制條件下,生物量僅有2.34 g/L,在氮限制條件下,增加磷濃度至45 mg/L,生物量可達(dá)到4.53 g/L,與對照組接近。過量的磷以聚磷的形式儲存在細(xì)胞內(nèi),可為微藻細(xì)胞生長和油脂合成提供必要的物質(zhì)和能量。通過分析生長相關(guān)基因表達(dá)發(fā)現(xiàn),油菜素類固醇的合成、碳和氨基酸的代謝在微藻細(xì)胞生長過程中具有重要作用。氮限制磷過量培養(yǎng)條件下,微藻細(xì)胞的油脂含量可達(dá)42.3%,油脂產(chǎn)率為310.0 mg/(L·d),分別為氮磷正常培養(yǎng)條件下的2倍和1.32倍。油脂組分分析得出C16和C18的脂肪酸比例高達(dá)95%以上,并且,飽和脂肪酸和單不飽和脂肪酸的比例均高于對照組。因此,氮限制磷過量策略培養(yǎng)微藻不僅可以提高生物量、油脂含量,而且可以提升生物柴油的品質(zhì)。(2)氮限制磷過量培養(yǎng)條件下磷的投加方式對微藻生長和油脂合成的影響。氮限制磷過量培養(yǎng)條件下,增加磷的投加次數(shù)對微藻的生長有一定抑制作用,分2次投加的最大生物量為3.95 g/L,而分8次投加的生物量為3.53 g/L。增加磷的投加次數(shù)可提高微藻細(xì)胞的內(nèi)油脂含量,分8次投加的油脂含量可達(dá)到41.5%,與2次投加相比提高了1.2倍。通過分析與油脂合成相關(guān)基因表達(dá)發(fā)現(xiàn),編碼ACCase的基因Unigene7398,編碼DGAT的基因CL2735.Contig2、CL73.Contig8、CL12.Contig1、Unigene7590、CL2735.Contig1、Unigene1920,增加磷的投加次數(shù)有利于提高油脂合成基因的表達(dá)水平。在培養(yǎng)周期末期,不同磷的投加方式對微藻細(xì)胞內(nèi)磷的貯存形態(tài)沒有顯著影響,過量的磷主要以正磷酸單酯的形式存在。此外,分多次投加磷有利于細(xì)胞內(nèi)快速累積以C16:0為主的飽和脂肪酸,而投加次數(shù)少有利于C18:0和C18:1兩種脂肪酸的累積。因此,增加磷的投加次數(shù)對油脂產(chǎn)量的增加沒有明顯的促進(jìn)作用,但可以提高細(xì)胞內(nèi)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q945;TE667

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