摘要
为了应对日益严峻的能源和环境问题,生物柴油越来越受到各国研究者的关注。微藻油脂的主要成分为甘油三酸酯,是一种理想的生物柴油原料。由于微藻具有不争耕地与淡水资源,不影响粮食安全,生长迅速且不受季节影响等优势而倍受青睐。然而由于成本的居高不下,微藻生物柴油一直没能进行大规模商业化生产。在制备微藻生物柴油过程中,富油微藻的培养是最为基础的一个环节,选育含油量高、生物量产量大、培养成本低的富油微藻是目前研究的热点问题。本文选择一株富油微藻,球等鞭金藻(Isochrysis galbana)CCMM5001,对其异养生长的条件和可行性,粗脂含量的快速测定方法以及生长过程中环境因子的影响等作了一系列研究。
首先研究了四种不同碳源葡萄糖、果糖、乙酸钠和甘油对球等鞭金藻CCMM5001异养生长的影响。结果表明,球等鞭金藻CCMM5001对碳源具有选择性,果糖对其异养生长促进效果最好,其次为葡萄糖,再者乙酸钠,甘油最差。果糖在50mM浓度时促进异养生长的效果好于5mM浓度时;葡萄糖、乙酸钠和甘油则相反。
综合考虑异养效果和经济因素,研究不同浓度的葡萄糖、光照强度以及通气水平对球等鞭金藻CCMM5001异养生长的影响。结果表明,球等鞭金藻CCMM5001利用葡萄糖异养生长的最佳浓度为2g/L,低于或高于2g/L时,最高细胞密度均有所下降;生长能力正常光照下高于弱光条件,非通气培养高于通气培养,其异养生长能力低于光合自养生长能力。
分析了粗脂含量和尼罗红染色荧光强度的关系。球等鞭金藻CCMM5001细胞中累积的脂类物质为中性脂,被尼罗红染色后发金黄色荧光,在480nm激发波长时,脂的发射峰为575nm,其荧光强度与总脂含量线性相关。根据线性相关曲线,建立了利用尼罗红染色配合荧光分光光度计快速测定球等鞭金藻CCMM5001细胞粗脂含量的方法。
分析了球等鞭金藻CCMM5001细胞密度与TD-700叶绿素荧光仪测定的活体荧光值之间的关系,确定两者的线性相关曲线。建立通过测定活体荧光值快速测定细胞密度的方法,以表示其生长情况。
设计不同的温度、盐度、光照强度和初始pH梯度,并以温度和盐度、光照强度和初始pH分别作为双因子进行正交试验。结果表明,球等鞭金藻CCMM5001生长的适宜温度为18~36℃,最适温度为24℃;适宜盐度为12~38,最适盐度为24;适光范围为2000lux~9000lux,饱和光强为3500lux;适宜初始pH范围4.8~8.8,最适pH范围6.8~7.8。在温度24℃,盐度24,光照强度3500lux,初始pH7.8的条件下培养,第15天可以得到最高细胞密度值7.24×10~6 cells/ml,为基础培养条件下细胞密度的1.08倍,培养周期缩短14天。其中,温度和盐度的双因子正交试验进一步表明,球等鞭金藻CCMM5001对高温高盐条件有一定耐受力,随着温度上升,高盐组的生长逐渐占优势。在温度为36℃,盐度为38时,最高细胞密度可达到3.90×10~6 cells/ml,高于相同温度下其他盐度组。
Biodiesel has received much attention by global researchers recently for dealing with the energy crisis and environment problems which become worse each day. The major components of lipids in microalgae are triglycerides, ideal materials in the prodution of biodiesel. Microalgae are more considerable because of several advantages: the cultivation of microalgae can reduce the competition for arable land and fresh water, has no effection on the food safety, and microalgae grow rapidly in every season in the year. However, the cost of biodiesel from microalgae is still too high for the sweeping production in commercial field. The culture of oil-rich microalgae is the basal step during biodiesel production, selecting microalgae with rich oil content, high biomass and low culture cost is a main problem recently.
The present study investigate the oil-rich Isochrysis galbana CCMM5001. The heterotrophic growth characteristics, the fast measurement of lipid content and the effect of some environmental factors are investigated.
Effcets of four different carbonaceous glucose, fructose, acetic natrium, glycerol on heterotrophic growth of Isochrysis galbana CCMM5001 are studied. The results show that the utilization of carbonaceous in Isochrysis galbana CCMM5001 is selective. Fructose is the best carbonaceous for heterotrophic growth, secondly glucose, then acetic natrium, glycerol is the worst. The heterotrophic productivity is better with fructose at 50mM concentration than 5mM, which opposite with other three carbonaceous.
Consider with the heterotrophic productivity and commercial factor, effects of a variety of glucose concentration, light intensity and aeration level on heterotrophic growth of Isochrysis galbana CCMM5001 are studied. The results show that the optimal glucose concentration for heterotrophic Isochrysis galbana CCMM5001 is 2g/L. The maximum cells concentration reduces at both higher and lower glucose concentration value. The productivity is better under normal intensity of light than low intensity, non-aeration than aeration, and photoautotrophic than heterotrophic.
The relation between lipid content and fluorescent intensity from Nile Red is investigated. The lipid content in Isochrysis galbana CCMM5001 cells is neutral, which can make the fluorescence from Nile Red become golden. The emission maximum of lipid is at 575nm wavelength, while the excitation wavelength is 480nm. The fluorescent intensity is linear relative with lipid content. The method that determining the lipid content in Isochrysis galbana CCMM5001 quickly by fluorescent measurement of Nile Red is found on the linear equation.
The relation between the cells concentration of Isochrysis galbana CCMM5001 and fluorescent intensity determined by TD-700 fluorometer is investigated, and the linear equation is found out. A method that determining the cells concentration by fluorescent measurement using TD-700 fluorometer is found, in order to describe the growth of the microalgae.
Effects of temperature, salinity, light intensity and pH on Isochrysis galbana CCMM5001 growth are studied when cultivated under a variety salinity with different temperature conditions and a variety pH with different lingt intensity. The results show that the temperature ranges for Isochrysis galbana CCMM5001 growth is 18~36℃, the optimal temperature is 24℃; the salinity ranges for growth is 12~38, the optimal salinity is 24; the light intensity ranges for growth is 2000lux~9000lux, the saturated light intensity is 3500lux; the pH ranges for growth is 6.8~7.8, the optimal pH range is 5.8~8.8. The maximum biomass concentration is 7.24×10~6 cells/ml when cultivated under the condition temperature 24℃, salinity 24, light intensity 3500lux, PH 7.8, which is 1.08 times compared with normal condition. Meanwhile, the investigation about effects of both temperature conditions and salinity show that Isochrysis galbana CCMM5001 can grow under high level of both temperature and salinity. The productivity under high salinity level becomes better while the temperature increased. The maximum biomass concentration can reach 3.90×10~6 cells/ml when cultivated under the condition temperature 36℃, salinity 38, much more than that cultivated under the same temperature with other variety of salinity.
引文
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