Isolation, Identification And Fatty Acid Composition Of Microalgaes From The South China Sea

With the rapid development of the global economy, energy and environmental crisis become more and more serious. Microalgae has been recognized as a raw material for biodiesel production because of its rapid growth speed, high photosynthetic efficiency and its ability to accumulate significant amounts of lipids. However, these organisms have great diversity, more than30000species have been investigated. How to filter out high quality algae species that suit for industrial production from this huge system has become the basis of other research directions. Because of the distribution of four seasons in China, we must find out the appropriate species of algae to adapt to different growth temperatures, to achieve stability in microalgae biodiesel feedstock supply. The South China Sea has a high biodiversity that are almost warm-water species. The algae samples collected from the South China Sea are purified, in order to get a microalgae with high lipid content, rapid growth speed and good tolerance to high temperature, to achieve industrial production of microalgae biodiesel.In chapter one, twelve different kinds of microalgae had been isolated from the samples from the South China Sea by micropipette separation method. Pure algaes were expanding cultivated to extract total DNA using the plant gene group DNA extraction reagents box. PCR technology was used to amplify the18S rDNA. The result of amplification was tested. Homology analysis was performed using BLAST in NCBI. Twelve different kinds of microalgae had been identified using the molecular biology method, combining with observed under optical microscope, scan electric mirror and transmission electric mirror. Twelve strains of algaes contained5kinds of Chlorophytas,6kinds of Bacillariophyceaes and1kind of Chrysophyta. The homologies of the twelve different kinds of microalgae all reached95%. Combining molecular and morphological identification methods greatly increased the accuracy of identification of microalgae.In chapter two, the biomass, total fat content and fatty acid composition of12microalgaes isolated from the South China Sea were analyzed. Growth curves indicated that all the microalgaes growed well. The total fat content were determined through methanol-chloroform (2:1) gravimetric method. The results showed that Cyclotella meneghiniana had the highest oil content(39.95%), and the second largest biomass(2.8535g/L). Nannochloropsis Salina had the largest biomass(4.3969g/L), and its oil content was23.46%. Two kinds of microalgaes owned both rapid growth and high oil content, and they were both high quality algae species for the production of microalgae biodiesel. The lipids extracted from the microalgae were tested by GC after esterification reaction. The long-chain fatty acids content were very high in all the12kinds of microalgaes, and they were all above80%. Results showed that the fatty acid composition among different kinds of categories varied greatly. The main fatty acids in Chrysophyta were C18:1, C14:0, C18:3and C16:0, and the DHA content was8.19%. Chlorophyta contains characteristics fatty acids such as C16:0, C16:1and C18:1. Other types of fatty acid in Chlorophyta had low level and were decentralized. The specific features of Bacillariophyceae were high in contents of C14:0, C16:0, C16:1and C22:4. The distribution of fatty acids in Bacillariophyceae are more concentrated. All the6kinds of Bacillariophyceaes had a small amount of DHA except Amphiprora paludosa.In chapter three, the effects of temperature, illumination intensity and nutrient concentrations on the growth and lipid accumulation of Cyclotella meneghiniana were studied by single factor experiments. A fast determination method of lipid content in microalgae using BODIPY was built. The result indicated that on the10day after vaccination, Cyclotella meneghiniana reaches a plateau. Cyclotella meneghiniana had a short growth cycle, and was suitable for industrialized production of microalgae biodiesel. The microalgae biomass and lipid content could be the two key factors that measure a energy microalgae. Taking the two aspects into consideration, the optimal culture conditions on lipid produced by Cyclotella meneghiniana were:the culture temperature was25℃, the illumination intensity was2500lux, the nitrate concentration was75g/L, the phosphate concentration was120g/L, the silicate concentration was15g/L,and the iron concentration was12.6g/L. Optimizing the culture conditions is an important way to improve the biomass and lipid accumulation of microalgae.