| The demand for energy is increasing with the rapid development of the global economy. However, the reserves of fossil fuel will be depleted in the future decades, and combustion of fossil fuel leads to global warming and many other environment problems. Therefore, developing clean and renewable energy has become an important part of worldwide energy policy. Biofuels have many advantages, such as having high calorific value, properties are similar to fossil diesel thus can be directly applied to internal combustion engine. Biodiesel production does not compete for land with food and is friendly to environment. Dunaliella salina is the first microalgae to be commercially produced. It is easy to grow, contains high content of valuable metabolites, and has greater potential than other oil resources. Therefore, in this study, D. salina was selected as the model microalgae. First, simultaneous assay method was applied to measure lipid, total carotenoids, pigments and carbohydrates in D. salina. Then response surface methodology(RSM) was applied to optimize the culture conditions. Varieties of single factorial experiments were exploded to induce lipid and total carotenoids accumulation. Finally, proper strategies were selected and combined together to maximize the lipid and total carotenoids accumulation, and carbohydrate and chlorophyll contents were also measured. The main results are summarized below:1. Lipid of D. salina exhibited a sensitive peak in the ultraviolet region at 260 nm, and total carotenoids' maximum absorbance appeared at 430, 450 and 480 nm. The standard curves of lipid and total carotenoids were plotted. The simultaneous assay was optimized, 30 seconds of vortex time was enough for complex reaction and estimation of lipids should be done within 3h after reaction began.2. Response surface methodology(RSM) was employed to optimize the culture medium for D. salina. The optimized cultivation conditions for lipid production were Na Si O3 4.3362 mg/L, Tris base 223 ?L per 200 m L medium and Na NO3 71.50 mg/L.3. Single factorial experiments were conducted to improve lipid and other high-valued products accumulation of D. salina.(1) Adding 80 g/L Na C l could significantly improve the lipid(0.134 mg/m L), total carotenoids(1.55 ?g/m L) accumulation and growth(600.5 mg/L) of D. salina. They were 46.9%?3.9% and 11.1% higher than the control(Lipid content 0.091 mg/m L, total carotenoids 1.50 ?g/m L, weight 540.8 mg/L), respectively.(2) The effects of p H were different between lipid and total carotenoids accumulation. The optimal p H for D. salina to accumulate lipid(0.122 mg/m L) was p H7.5, which was 4.7% higher than the control(p H7, lipid 0.116 mg/m L), total carotenoids(0.588 ?g/m L) content reached most at p H7, which was higher than other groups, growth(753.1 mg/L) achieve the best at p H8, which was 42.4% higher than the control(p H7, weight 532 g/m L).(3) Adding ferric citrate in the medium significantly improved lipid, total carotenoids accumulation and growth of D. salina. Lipid(0.112 mg/m L) content was 14.3% higher than the control(Fe C l3, lipid 0.098 mg/m L), total carotenoids(1.915 ?g/m L) was 10.6% higher than the control(Fe C l3, total carotenoids 1.732 ?g/m L) and the growth(533.7 mg/L) was 28.4% higher than the control(Fe Cl3, weight 415.9 mg/m L).(4) Culture method was optimized to accumulate more lipid, D. salina was inoculated in 100 m L medium, then added 50 m L fresh medium every 7 days, ultimately achieved 200 m L. The lipid(0.096 mg/m L) content of D.salina was 37.1% higher than the control(Batch, lipid 0.070 mg/m L), total carotenoids(2.10 ? g/m L) content was 28.1% higher than the control(Batch, total carotenoids 1.64 ? g/m L), and growth(554.5 mg/L) was 43.3% higher than the control(Batch, weight 386.9 mg/L).(5) The appropriate amount of glucose(10 mg/L) could stimulate lipid(0.127 mg/m L) accumulation and growth(1226.4 mg/L) of D. salina, each was 35.1% and 73.3% higher than the control(0 g/L glucose, lipid 0.094 mg/m L, weight 707.5 mg/L). Total carotenoids(0.765 ?g/m L) content of D. salina attained the highest when the glucose concentration was at 15 mg/L, which was 156.8% higher than the control(0 g/L glucose, Total carotenoids 0.298 ?g/m L).(6) The lipid(0.1153 mg/m L) and total carotenoids(0.676 ?g/m L) content attained the highest when the concentration of zinc was at 100 mg/L, which was 11.3% and 121.6% higher than the control(lipid 0.104 mg/m L, total carotenoids 0.305 ? g/m L), respectively. Whereas the weight(603.1 mg/L) of D. salina was decreased after adding 100 mg/L zinc in the culturing base compared with the control(weight 709 mg/L).(7) N itrogen deficient culture base could significantly induce lipid(0.123 mg/m L) accumulation, which was 75.7% higher than the control(lipid 0.072 mg/m L), the total carotenoids content(2.672 ?g/m L) and weight(720.1 mg/L) of D. salina cultured in phosphorus deficient culture base were 19.2% and 36.9% higher than the control(Total carotenoids 2.242 ?g/m L, weight 526.9 mg/L).4. Appropriate strategies were selected and combined together to maximize the lipid and total carotenoids accumulation. Group H had the highest weight(687.7 mg/L), which was 43.1% higher than the control(480.5 mg/L). The weight of group F(612.6 mg/L) and N(630.7 mg/L) were second only to the group H, they were 27.5% and 31.2% higher than the control, respectively. Group F(0.149 mg/m L) and N(0.147 mg/m L) had the highest lipid content, they were 79.8% and 76.5% higher than the control(lipid 0.083 mg/m L), respectively. Group F and N had no significant difference between lipid contents, however, the total carotenoids of group N(1.827 ?g/m L) was higher than group F(1.978 ?g/m L), and the difference was significant. Therefore, the best strategy was group N : combination of adding zinc ions 100 mg/L in the culture base, applying fractional addition method to culture D. salina, and employing nutrient deficiency of nitrogen. |
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