盐生杜氏藻对盐度变化和渗透胁迫下的细胞反应及其中性脂肪的积累
发布时间：2019-01-08 19:10
【摘要】：杜氏盐藻是一种绿色微藻,能够在盐度极其广泛的环境下生长并积累含有类胡萝卜素的中性脂肪球,因此可以用来生产生物柴油和色素。杜氏盐藻是迄今为止研究发现的最为耐盐的真核生物,适合开放式的大规模培养,具有广泛的和潜在的工业应用前景。杜氏盐藻的另外一个重要的生物学特性是其无刚性细胞壁,有助于从其细胞中提取中性脂肪或类胡萝卜素并进行生物炼制。这种藻类在细胞受到低盐渗透胁迫或高盐渗透胁迫时能够迅速改变其体积和形状。为了进一步开发和利用杜氏盐藻生产生物柴油的实际应用和研究盐度对细胞生长和脂肪产生的影响,本论文通过改变培养基中氯化钠的浓度分析了盐度对于杜氏藻细胞的形态、生长以及中性脂质积累情况的短期和长期影响。研究结果发现:(1)在不同程度的盐渗透胁迫下,细胞的大小与形态会发生明显的变化。低盐渗透胁迫下的细胞会迅速增大,而高盐渗透胁迫下的细胞会迅速变小。这种现象说明了杜氏藻的特殊膜结构能够使其在不同盐度的环境中使细胞迅速膨胀或收缩而避免细胞破裂,反映了其细胞内离子浓度的变化。(2)盐浓度对细胞生长速度和细胞最大生长量也有影响:盐浓度越低,杜氏盐藻的生长速率越快,细胞最大生长量越高;反之,盐浓度越高,细胞生长速度越慢,细胞最大生长量越低。(3)将杜氏藻细胞从含有9%NaCl培养基中转移到含有15%NaCl培养基中的细胞所积累的中性脂质含量最高。然而在这种盐胁迫条件下,杜氏藻生长缓慢,细胞分裂能力下降,但渗透胁迫并未对细胞造成致命性的伤害,细胞能自动调节体积以适应这种变化。将杜氏藻从浓度为3%的NaCl培养基中转移到浓度为9%的NaCl培养基后能迅速恢复生长并未能积累中性脂质,说明细胞在该浓度下的生长并未受到抑制。因此,杜氏盐藻细胞的中性脂质的积累应该是建立在细胞生长受到抑制的基础之上而获得的。(4)在含有3%-15%NaCl浓度范围内分批培养的杜氏盐藻的结果都显示:营养元素的限制是减缓细胞分裂和抑制细胞生长的另一个重要因素,所有分批培养的细胞在进入了生长停滞期时都开始积累中性储存脂质。综上所述,杜氏盐藻只有在高盐度渗透胁迫下,短期内会引起细胞中中性脂质的积累,且抑制细胞的分裂并造成细胞生长缓慢。尽管在高盐度下生长的杜氏藻会积累中性脂质,但同时其缓慢的生长速度的也会影响细胞中中性脂肪的总产量。因此,工业化培养杜氏藻时建议在低盐浓度条件下进行藻体细胞的培养,等细胞生长进入到指数生长期后再将其转移到高盐浓度中,这样既可获得大量的藻细胞,又能收获大量的中性脂肪。然而,这种培养方式周期较长,因此在探究利用微藻生产生物柴油领域的应用前景时,最有待解决的问题是如何同时实现细胞生长量和单细胞脂质产率的最大化。
[Abstract]:Dunaliella Salina is a green microalgae, which can grow and accumulate carotenoid neutral fat spheres in a very wide range of salinity, so it can be used to produce biodiesel and pigment. Dunaliella Salina is the most salt-tolerant eukaryote found so far. It is suitable for large-scale open culture and has wide and potential industrial application prospects. Another important biological characteristic of Dunaliella Salina is its non-rigid cell wall, which helps to extract neutral fat or carotenoid from its cells and biorefine them. The algae can change its size and shape rapidly under low salt osmotic stress or high salt osmotic stress. In order to further develop and utilize the application of Dunaliella Salina to produce biodiesel and to study the effect of salinity on cell growth and fat production, the morphology of Dunaliella sp. Cells was analyzed by changing the concentration of sodium chloride in culture medium. Short-and long-term effects of growth and neutral lipid accumulation. The results showed that: (1) under different degree of salt osmotic stress, the size and morphology of cells changed obviously. The cells under low salt osmotic stress will increase rapidly, while those under high salt osmotic stress will rapidly become smaller. This phenomenon shows that the special membrane structure of Dunaliella can cause the cells to expand or contract rapidly in different salinity environment to avoid cell rupture. (2) Salt concentration also affects the cell growth rate and the maximum cell growth: the lower the salt concentration, the faster the growth rate of Dunaliella Salina and the higher the cell growth; On the other hand, the higher the salt concentration, the slower the cell growth rate and the lower the maximum cell growth. (3) the higher the salt concentration, the higher the accumulation of neutral lipid in the cells transferred from the culture medium containing 9%NaCl to the medium containing 15%NaCl. However, under the condition of salt stress, the growth of Dunaliella was slow and the ability of cell division was decreased. However, osmotic stress did not cause fatal damage to the cells, and the cells could automatically adjust their volume to adapt to this change. When the algae was transferred from 3% NaCl medium to 9% NaCl medium, the growth of Dunaliella spp recovered rapidly and the neutral lipid was not accumulated, which indicated that the growth of the cells was not inhibited at this concentration. therefore The accumulation of neutral lipid in Dunaliella Salina cells should be based on the inhibition of cell growth. (4) the results of batch culture of Dunaliella Salina in the concentration range of 3%-15%NaCl showed that Nutrient restriction is another important factor in slowing cell division and inhibiting cell growth. All the cells cultured in batches began to accumulate neutral storage of lipid at the stage of growth stasis. In conclusion, only under high salinity osmotic stress, Dunaliella Salina can induce the accumulation of neutrophil lipid, inhibit cell division and slow cell growth. Although the growth of Dunaliella spp under high salinity will accumulate neutral lipid, but its slow growth rate will also affect the total production of cellular neutral fat. Therefore, it is suggested that the algae somatic cells should be cultured in low salt concentration, so that the cells grow into exponential growth period and then transfer to high salt concentration, so that a large number of algal cells can be obtained. And it can harvest a lot of neutral fat. However, this culture method has a long period of time, so the most important problem to be solved in exploring the application prospect of microalgae in biodiesel production is how to maximize both cell growth and single cell lipid yield.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q945.78
[Abstract]:Dunaliella Salina is a green microalgae, which can grow and accumulate carotenoid neutral fat spheres in a very wide range of salinity, so it can be used to produce biodiesel and pigment. Dunaliella Salina is the most salt-tolerant eukaryote found so far. It is suitable for large-scale open culture and has wide and potential industrial application prospects. Another important biological characteristic of Dunaliella Salina is its non-rigid cell wall, which helps to extract neutral fat or carotenoid from its cells and biorefine them. The algae can change its size and shape rapidly under low salt osmotic stress or high salt osmotic stress. In order to further develop and utilize the application of Dunaliella Salina to produce biodiesel and to study the effect of salinity on cell growth and fat production, the morphology of Dunaliella sp. Cells was analyzed by changing the concentration of sodium chloride in culture medium. Short-and long-term effects of growth and neutral lipid accumulation. The results showed that: (1) under different degree of salt osmotic stress, the size and morphology of cells changed obviously. The cells under low salt osmotic stress will increase rapidly, while those under high salt osmotic stress will rapidly become smaller. This phenomenon shows that the special membrane structure of Dunaliella can cause the cells to expand or contract rapidly in different salinity environment to avoid cell rupture. (2) Salt concentration also affects the cell growth rate and the maximum cell growth: the lower the salt concentration, the faster the growth rate of Dunaliella Salina and the higher the cell growth; On the other hand, the higher the salt concentration, the slower the cell growth rate and the lower the maximum cell growth. (3) the higher the salt concentration, the higher the accumulation of neutral lipid in the cells transferred from the culture medium containing 9%NaCl to the medium containing 15%NaCl. However, under the condition of salt stress, the growth of Dunaliella was slow and the ability of cell division was decreased. However, osmotic stress did not cause fatal damage to the cells, and the cells could automatically adjust their volume to adapt to this change. When the algae was transferred from 3% NaCl medium to 9% NaCl medium, the growth of Dunaliella spp recovered rapidly and the neutral lipid was not accumulated, which indicated that the growth of the cells was not inhibited at this concentration. therefore The accumulation of neutral lipid in Dunaliella Salina cells should be based on the inhibition of cell growth. (4) the results of batch culture of Dunaliella Salina in the concentration range of 3%-15%NaCl showed that Nutrient restriction is another important factor in slowing cell division and inhibiting cell growth. All the cells cultured in batches began to accumulate neutral storage of lipid at the stage of growth stasis. In conclusion, only under high salinity osmotic stress, Dunaliella Salina can induce the accumulation of neutrophil lipid, inhibit cell division and slow cell growth. Although the growth of Dunaliella spp under high salinity will accumulate neutral lipid, but its slow growth rate will also affect the total production of cellular neutral fat. Therefore, it is suggested that the algae somatic cells should be cultured in low salt concentration, so that the cells grow into exponential growth period and then transfer to high salt concentration, so that a large number of algal cells can be obtained. And it can harvest a lot of neutral fat. However, this culture method has a long period of time, so the most important problem to be solved in exploring the application prospect of microalgae in biodiesel production is how to maximize both cell growth and single cell lipid yield.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q945.78
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