[农业]海洋微藻论文：三种海洋产能微藻规模化养殖及采收技术的初步研究.doc

海洋微藻论文：三种海洋产能微藻规模化养殖及采收技术的初步研究【中文摘要】在化石能源日益衰竭及其燃烧带来环境污染问题日益严重的严峻形势下,寻求可再生能源受到普遍关注,其中生物质能源以其较高的可持续性和清洁无污染的特点成为全球关注的焦点。微藻生物质能源是一种以开发藻类中丰富的油脂制备生物柴油的生物质能源,是一种具有巨大开发潜力的优质生物质能源,但原料供给不足和生产成本高昂始终是制约微藻生物柴油产业发展的主要原因。微藻的生物质产量和油脂含量是影响生产成本的关键因素,已有大量研究表明,微藻生物量和油脂含量受到环境条件和营养盐成分调控。为优化培养条件,获得高生物量,并提高微藻油脂产量,本研究以三种常见海洋微藻盐藻（ Dunaliella salina ）、湛江等边金藻（ Isochrysis zhanjiangensis）和微绿球藻（Nannochloropsis oculata）为研究对象,开展了室内培养条件优化、室外中试试验和采收技术等方面的研究,通过研究,初步建立了海洋产能微藻开发利用技术线路。（1）本研究采用索氏提取法,以乙醚为溶剂,在6080的温度条件下,对7种海洋微藻细胞内粗脂肪含量进行测定,并对提取方法做了优化。优化的方法是将样品预先在乙醚溶剂中浸泡一定时间,使部分油脂在乙醚溶剂中浸出,然后再按索氏方法进行脂肪抽提。研究表明,优化后的索氏提取法具有提取效率高、提取成本低和提取完全等优点,并且该方法缩短了抽提时间,从而降低了乙醚等有机萃取剂因挥发而造成的污染。（2）通过两种不同充气方式培养3种海洋微藻,研究了CO2对微藻生长和脂肪积累的影响。试验设组纯充空气组,组充CO2与空气混合气体组,结果表明,三种海洋微藻均能在充气条件下迅速进入指数生长期,平均生长率均在1以上,组较组生长和脂肪积累显著（P0.05）。生长指数末期,三种微藻的最高密度分别达到3.33106、9.20106和1.75107个/mL,充CO2和空气混合气体组细胞密度分别是纯充空气组的1.28、6.23和3.09倍。湛江等边金藻的脂肪含量最高,在充CO2和空气混合气体组其脂肪含量较纯充空气组翻倍增加,达到40%,提高了122.8%,而盐藻和微绿球藻分别提高了69.5%和49.6%。（3）采用单因素试验研究了盐度对盐藻生物量和脂肪积累的影响,结果表明盐度变化对盐藻生物量和脂肪积累均有显著的影响。在本实验条件下,盐度在20100的范围内,盐藻均能正常生长,且在盐度40时,能够获得最高生物量和最高的脂肪含量,分别达到1.19 g/L和33.84%。（4）通过添加硅酸钠培养湛江等边金藻,研究得出,硅酸钠不是影响湛江等边金藻油脂生产力的主要原因,但是,当藻液硅酸钠浓度为10 mg/L时,藻液生长率和生物量最高;而当藻液硅酸钠浓度为5 mg/L时能够收获最大的脂肪含量63.93%和产油生产力71.056 mg/Ld;研究表明,添加硅酸钠浓度在510 mg/L的范围能够提高湛江等边金藻油脂生产力。（5）自行设计竖立薄膜袋培养海洋微藻,研究发现,接种后第二天两种试验微藻均能进入指数生长期,并且维持指数生长期时间长。8 d后湛江等边金藻最大密度2.99106个/mL,计算最大生长率为0.626;10 d后微绿球藻密度达到8.50106个/mL,计算生长率为0.565,而且10 d后仍有升长的趋势,一直维持至第15 d。研究结果表明,利用竖直薄膜袋室外培养海洋微藻不仅具有受光面积大、成功率高、污染机会小、操作简单等优点,而且设备材料成本低、占地面积小、薄膜袋不容易破损和方便采收等优点。（6）本研究利用两种化学试剂分别对两种微藻进行絮凝采收试验,结果表明,两种絮凝剂对两种海洋微藻的絮凝效果明显。进一步研究表明,0.3 g/L的硫酸铝对湛江等边金藻絮凝作用明显,1000 mL藻液20 min后发生絮凝作用,80 min沉淀度可达70%以上;0.2 g/L的硫酸锌对微绿球藻絮凝效果极为显著,1000 mL藻液10 min内沉淀度在50%以上,其最大絮凝度超过90%。【英文摘要】Due to the severely situation of the shortages of fossil fuels and the production of greenhouse gas carbon dioxide on their combustion, finding the renewable energy is receiving considerable attention. Because of the cleaner-burning and renewable fuel, biodiesel can be a focus of the bio-energy. Microalgae biomass energy is to find high efficient microalgae resource for the preparation of biodiesel, and it has great exploitation and utilizing potential. However the raw materials supply and cost constraints are always important factors in development of biodiesel industry. Microalgae biomass productivity and its lipid content are the key factors of the biodiesel cost. Many investigations indicated that lipid content and the dry matter yield of microalgae could be influenced by environmental conditions. In the present experiment, the cultural conditions for optimizing lipids productivity in Dunaliella Salina, Isochrysis zhanjiangensis and Nannochloropsis oculata were studied, and a technical route of microalga oil production was built.（1） In the present study, the crude lipid of seven species of marine microalgae were extracted by ethyl ether using soxhlet extraction method, under a temperature of approximately 6080. The soxhlet extraction method was improved by dipping the microalgae in ethyl ether for some time before leaching. The results show that the improved soxhlet extraction was more efficient, lower cost and more thoroughly extraction. Whats more the pollution caused by ethyl ether was reduced due to a shorter extraction time.（2） In the present experiment, the effect of CO2 on the growth and lipid content three marine microalgae in two kinds of aerobic culture medium, groupaeration of pure air and groupaeration of mixture with and CO2 for experimental were studied. The results indicated that the logarithmic phase of growth of the three marine microalgae was reached quickly, with the special growth higher than 1, but the growth and lipid content in experimental groupwere significantly lower than that in experimental group. At the end of logarithmic phase, the maximum densities of the three marine microalgae were 3.33106, 9.20106 and 1.75107 cell/mL, respectively. The densities were more than 1.28, 6.23 and 3.09 times in the experimental groupas that in the experimental groupof the three marine microalgae. In addition, the lipid content of Isochrysis zhanjiangensis was the highest, and its content was significantly higher in groupthan that in group, which uped to 40% and adding to 122.8%, while the lipid contents of Dunaliella Salina and Nannochloropsis oculata rose to 69.5% and 49.6% respectively. （3） The influences of salinity on the biomass and the total lipid of D. Salina were studied by one-factor experimental design. The results showed that there were remarkably effects of different salinity on the biomass and lipid of D. Salina. The microalgae could grow normally in the scope salinity of 20 to 100 , in addition, the salinity of 40 made the highest biomass and the highest lipid content, they got to 1.19 g/L and percentage at 33.84, respectively.（4） Added different sodium silicate concentrations to culture I. zhanjiangensis. The result showed that thought the added sodium silicate was not the key role of the lipid productivity of I. zhanjiangensis, the maximum specific growth rate and biomass were in the treatment group with a sodium silicate concentration of 10 mg/L. Whereas, the highest lipid content and lipid productivity were observed in the treatment group with a sodium silicate of 5 mg/L, which were 63.93% （dry weight basis） and 71.056 mg/Ld, respectively. According to the results of the present experiment, the sodium silicate concentration in the culture medium of I. zhanjiangensis for oil production should be 510 mg/L.（5） Standing polyethylene film bags were used to culture marine microalgae. The two marine microalgae grew to log phase directly without lag phase at the second day. The highest density and specific growth rate of I. zhanjiangensis could high to 2.99106 cell/mL and 0.626, respectively at the 8th day;Whereas, Nannochloropsis oculata could high to 8.50106 cell/mL and 0.565, respectively in the 10th day, and it seen continue to rose. Results invented that standing polyethylene film bags were flexible enough for good condition for the algae culture and prevent the film bags from damage. It was cheap, occupied small space and easy to harvest.（6） The harvest of I. zhanjiangensis and N. oculata scale culture were studied. The results indicated that flocculants has significant effects on the two marine microalgae were significant. The condensational degree of aluminium sulfate on I. zhanjiangensis was higher than 70% in 80 minutes, and the condensational degree of the zinc sulfate on N. oculata was higher than 50% within 10 minutes.【关键词】海洋微藻 生物燃料 微藻培养 索氏提取 絮凝【英文关键词】marine microalgae biodiesel microalgae culture soxhlet extraction flocculation【目录】三种海洋产能微藻规模化养殖及采收技术的初步研究摘要4-7ABSTRACT7-10引言14-29第一章 海洋微藻粗脂肪含量测定及其方法优化研究29-351.1 材料和方法29-311.1.1 实验材料291.1.2 主要设备291.1.3 微藻采收29-301.1.4 提取准备工作301.1.5 索氏提取301.1.6 脂肪测定30-311.2 结果31-321.2.1 提取时间311.2.2 微藻粗脂肪含量31-321.3 讨论32-341.4 小结34-35第二章 二氧化碳对微藻生长和脂肪含量的影响35-442.1 材料与方法35-372.1.1 材料352.1.2 培养用水和营养盐352.1.3 试验设计35-362.1.4 生长测定362.1.5 油脂含量的测定362.1.6 数据分析36-372.2 结果37-422.2.1 三种微藻细胞密度与吸光值的线性关系372.2.2 培养过程中pH 的变化37-382.2.3 充气对微藻生长的影响38-422.2.4 充气对微藻脂肪含量的影响422.3 讨论42-432.4 小结43-44第三章 盐度变化对盐藻生物量和总脂含量的影响44-503.1 材料与方法44-453.1.1 材料443.1.2 方法步骤44-453.1.2.1 培养用水和营养盐44-453.1.2.2 实验设计453.1.2.3 生长测定453.1.2.4 藻生物量的采收与测定453.1.2.5 微藻粗脂肪百分含量的测定453.2 结果与分析45-483.2.1 生物量与吸光值的线性关系45-463.2.2 盐度对盐