【作者】 高影影；

【导师】 王长海；

【作者基本信息】 南京农业大学 ， 应用海洋生物学， 2013， 博士

【摘要】 随着全球经济的快速发展,不可再生的石化能源被过度开发,导致世界范围内能源呈现日益短缺的状态。开发可再生、环境友好的生物柴油替代能源已成为目前能源研究的重要课题,受到了各国研究者的广泛关注。微藻生长速度快、生长周期短、油脂含量高、环境友好、不占用耕地等诸多优势使其成为开发生物柴油的重要原料来源。由于微藻生产成本居高不下,微藻生物柴油至今尚未实现大规模商业化生产。可见,降低生产成本成为微藻生物柴油生产中亟待解决的重点和难点。而选育生物量产率大、油脂含量高、培养成本低的富油微藻是降低微藻生物柴油生产成本的有效途径之一。本文对取自青岛汇泉湾天然海水中的海洋微藻进行了分离纯化和形态鉴定,并分析了其生长和油脂积累特性；对分离出的油脂产率较高的藻与实验室原有藻种进行了生长和油脂积累分析,发现我们分离到的海洋小球藻NJ101的生长速度快,油脂产率高,脂肪酸组成适于生产生物柴油。于是,选择了海洋小球藻进行下一步的实验,探讨了一系列营养元素对其生长和油脂积累的影响。最后,通过营养缺乏增加了海洋微藻的油脂含量。主要研究结果如下：(1)分离纯化出两株海洋微藻,经鉴定,一株绿藻为海洋小球藻(Chlorella sp. NJ101),另一株硅藻为小新月菱形藻(Nitzschia closterium f. minutissima NJ112)。通过对分离到的海洋小球藻与其它九种实验室原有海洋微藻的研究,发现海洋小球藻、牟氏角毛藻和杜氏盐藻是生长速度、生物量产率和油脂产率最大的三株海洋微藻(即富油微藻),其中海洋小球藻和牟氏角毛藻的饱和脂肪酸和单不饱和脂肪酸的含量占总脂肪的80%以上,杜氏盐藻的饱和脂肪酸和单不饱和脂肪酸的含量更是高达90%以上。尤其是所分离的海洋小球藻生长最快、生物量产率与油脂产率最高,且其脂肪酸组成符合生物柴油生产的欧洲标准,是生产生物柴油的良好原料。(2)在海洋小球藻的培养基中加入NaHCO3、Na2CO3和葡萄糖作为碳源时,降低了海洋小球藻的生物量产率和油脂产率。海洋小球藻以NaNO3为氮源时生物量产率和油脂产率最大,NaNO3比NH4Cl和CO(NH2)2更适合作为海洋小球藻的氮源来生产生物柴油。NaNO3和NaH2PO4对海洋小球藻生物量产率和油脂产率有很大的影响。实验发现：当NaNO3浓度在2.2～8.8×10-3mol·L-1范围内时,对海洋小球藻的油脂产率影响最大；当NaH2PO4浓度在1.8×10-5mol·L-1～1.8×10-4mol·L-1范围内时,对海洋小球藻的油脂产率影响最大。不同的Na2SiO4浓度对海洋小球藻的生物量产率和油脂产率的影响均不显著。MgSO4的添加与否及浓度大小对海洋小球藻的生长和油脂积累的影响均不显著。在海洋小球藻的培养基中加入CaCl时,海洋小球藻的生物量产率不显著的增加,而油脂产率在适当浓度时显著增加。(3)培养基中添加FeCl3时,海洋小球藻的油脂产率明显高于无铁培养基中的,但是添加的浓度对海洋小球藻的油脂产率没有显著的影响。在实验设置的梯度范围内,CuSO4、Na2MoO4、ZnSO4、CoCl2和MnCl2的添加与否及浓度大小对海洋小球藻的生长和油脂积累的影响均不显著。(4)正交实验获得的使海洋小球藻的油脂产率最大的营养盐浓度为NaNO3-N8.8×10-4mol·L-1; NaH2PO4-P7.2×10-5mol·L-1; FeCl3-Fe0.5×10-5mol·L-1.在所有的实验组中,海洋小球藻的饱和脂肪酸和单不饱和脂肪酸的含量都接近或超过其细胞干重的90%,亚麻酸(C18：3)含量均小于总脂含量的10%,满足生物柴油生产的欧洲标准EN14214。(5)全营养缺乏对牟氏角毛藻、杜氏盐藻和海洋小球藻的生物量的降低最多,其次分别是缺氮、缺磷和缺铁。对于牟氏角毛藻和海洋小球藻而言,缺氮是最大的生理压力,其次分别是全营养缺乏、缺磷和缺铁；对于杜氏盐藻而言,全营养缺乏是最大的生理压力,其次分别是缺氮、缺磷和缺铁。缺氮条件下,牟氏角毛藻的油脂含量最高,达到了细胞干重的46%。全营养缺乏条件下,杜氏盐藻和海洋小球藻的油脂含量最高,分别达到细胞干重的54%和64%。即通过营养缺乏提高油脂含量后,我们分离到的海洋小球藻Chlorella sp.NJ101的油脂含量达到了最大。总之,本文从天然海水中筛选出一株富油微藻,经鉴定为海洋小球藻；探讨了不同营养条件对海洋小球藻的生长和油脂积累的影响；并利用营养缺乏增加了海洋小球藻的油脂含量。为进一步开发微藻生物能源、提高微藻的油脂含量及早日实现微藻生物柴油的产业化提供了理论依据和实验基础。更多还原

【Abstract】 With the rapid development of the global economy, non-renewable fossil fuels are overexploited, leading to a worldwide increasingly shortage of energy. Biodiesel is a renewable and ecologically friendly energy resource, and the development of it becomes increasingly important and is attractive to the researchers of the world.Microalgae are of particular interest as a most promising source of biomass for biodiesel production due to their rapid growth rate, short growth cycle, high lipid content, strong adaptability to environment and easy to cultivation and so on. However, the production of biodiesel from microalgae is still too expensive to meet the market requirements. To solve this problem, it is essential to identify suitable strains of microalgae for mass cultivation and improve the lipid content of them.In this study, we isolated and purified marine microalgae from natural seawater and identified them by their morphology. Oil-rich marine microalgea was screened from microalgal species of our laboratory and the marine microalgae we isolated. The lipid productivity of Chlorella sp. NJ101was the highest, so the effects of nutrient conditions on the growth and lipid accumulation of Chlorella sp. were explored. Nutrient deprivation was used to enhance the lipid content of marine microalgae. The main results are as follows:(1) Two marine microalgae were isolated and purified form natural seawater. One of them was green alga which was identified as Chlorella sp. NJ101and the other was diatom which was identified as Nitzschia closterium f. minutissima NJ112. Three marine microalgae with faster growth, higher biomass productivity and higher lipid productivity were screened out from the ten strains; they are Chlorella sp. NJ101, Chaetoceros muelleri and Dunqliella salina. The saturated and monounsaturated fatty acids content of Chlorella sp. NJ101and Chaetoceros muelleri were more than80%and that of Dunaliella salina even more than90%. The growth rate of Chlorella sp. NJ101was the faster and the biomass productivity and the lipid productivity of Chlorella sp. NJ101was the highest and the fatty acid composition of Chlorella sp. NJ101meets the requirement of EN14214, so, Chlorella sp. NJ101was a promising biodiesel feedstock.(2) When NaHCO3, Na2CO3and glucose were added to the medium as carbon source, the biomass productivity and lipid productivity of Chlorella sp. NJ101was reduced. The highest biomass productivity and lipid productivity of Chlorella sp. NJ101was obtained when NaNO3was used as nitrogen source. So, when Chlorella sp. NJ101was used to produce biodiesel, NaNO3was more suitable to use as the nitrogen source than NH4CI and CO(NH2)2. NaNO3and NaH2PO4have a great influence on the biomass productivity and lipid productivity of Chlorella sp. NJ101. The most significant effect on the lipid productivity of Chlorella sp. NJ101was obtained when the concentrations of NaNO3were between2.2-8.8×10-3mol·L-1or the concentration of NaH2PO4were between1.8×10-5mol·L-1-1.8x10-4mol·L-1. Different concentration of Na2SiO4didn’t have significant effect on the biomass productivity and lipid productivity of Chlorella sp. NJ101. None of the addition or the concentration of MgSO4had significant effect on the growth and lipid productivity of Chlorella sp. NJ101. When CaCl was supplemented to the medium of Chlorella sp. NJ101, the biomass productivity was increased though not significant, and the lipid productivity was significantly increased with suitable CaCl concentration.(3) The addition of FeCl3significantly increased the lipid productivity of Chlorella sp. NJ101, but the concentration of FeCl3didn’t have significant effect on the lipid productivity of Chlorella sp. NJ101. None of the addition or the concentration of CuSO4, Na2Mo04, ZnSO4, CoCl2and MnCl2had significant effect on the growth and lipid productivity of Chlorella sp. NJ101.(4) The optimum nutrients concentration with which the lipid productivity of Chlorella sp. NJ101was highest was obtained form the orthogonal experiment:NaNO3-N8.8×10-4mol·L-1; NaH2PO4-P7.2×10-5mol·L-1; FeCl3-Fe0.5×10-5mol·L-1. And the fatty acid composition of Chlorella sp. NJ101in all the trials meets the requirement of EN14214.(5) Complete nutrition deprivation resulted in the largest reduction of C. muelleri, D. salina and Chlorella sp. NJ101growth, followed by deprivation of nitrogen, phosphate and iron from the medium. Deprivation of nitrogen gave the greatest physiological stress to C. muelleri and Chlorella sp. NJ101, followed closely by deprivation of complete nutrient, phosphate and iron. Complete nutrition deprivation gave the greatest physiological stress to D. salina, followed closely by deprivation of nitrogen, phosphate and iron. The highest lipid content for C. muelleri was achieved by nitrogen deprivation and the lipid content were46%of dry cell weight. The highest lipid content for D. salina and Chlorella sp. NJ101was achieved by complete nutrient deprivation, and the lipid content were54%and64%of dry cell weight, respectively. It means that Chlorella sp. NJ101isolated form seawater has the highest lipid content when the nutrient deprivation was used.In short, one microalge with high lipid productivity was screened form natural seawater; it is Chlorella sp. NJ101. The effects of different nutrient conditions on the growth and lipid accumulation of Chlorella sp. NJ101were studied. Nutrient deprivation was used to enhance the lipid content of marine micoalgae with high lipid productivity. This article provides a theoretical and experimental basis to the further development of microalge biofuel and the enhancement of microalgal lipid content and will speed up the commercialization of microalgae biodiesel.更多还原