Screen, Identification And Tentative Cultivation Of Tropical Freshwater Microalgae For Lipid Production

The Twelfth Five-Year Plan in China has emphasised the development of liquid fuels from non-food biomass and encouraged the industrialization of microalgal biofuels. Hainan is a tropical island in South-China with hot and humid climate, vast water area and abundant microalgal germplasm resources, which has considerable potential for biofuel production. In this thesis, there are three goals, e.g. to collect the fresh-water microalgae and build the germplasm resources database, to set up a system for evaluation of microalgal strains for biodiesel production, to study the effects of nitrogen sources, concentration of phosphate, concentration of iron on growth and lipid content of microalgal strains and obtain salinity tolerance by gradually acclimation.Fifity fresh-water samples were collected from Haikou and surrounding areas. And three hundred microalgal strains were isolated by traditional methods such as agar plates and micropipette. Preliminary screening was done using Nile Red stain and observation by fluorescent inverted microscope, then thirty strains were selected as candidates for second screening. Rescreening was done using a self-designing system, and seven strains were charactered as potential strains for biodiesel production. Species identification was performed using morphological and molecular methods.three of these cultures were identified as Scenedesmus, one was Coelastrum, one was Chlorella, one was Mychonastes and one was Chlamydomonas. The fatty acids were analyzed using GC-MS.The results showed that major fatty acid composition of seven strains of microalgae were C16and C18. The total of palmitic (C16:0), stearic (C18:0), palmitoleic (C16:1), oleic (C18:1) acid comprised43.52～77.56%in these strains, which means that they were suitable isolate for the production of good quality biodiesel.Two strains e.g. Coelastrum. reticulatum and Scenedesmus. ecornis were used as starting materials to evaluate the effects of nitrogen sources, concentration of phosphate, concentration of iron on growth and lipid content. Results showed that:for nitrogen experiment of C. reticulatum and S.ecornis, higher biomass (0.72±0.08g/L and0.80±0.03g/L) and higher lipid content (38.35±0.96%and30.25±4.43%) were obtained when using NaNO3and NH4Cl as nitrogen sources.respectively. Advanced research revealed that under nitrogen starvation condition (about as10% concentration of NaNO3in BG11medium), the growth condition of C. reticulatum was less affected, but the lipid content was as high as40.64%, and the lipid yield was as high as386.03mg/L. For phosphate experiment, lipid accumulation was enhanced at phosphate starvation condition, while high(>10mg/L) or low (microscale) concentration of phosphate blocked the growth of C. reticulatum. What’s more, growth and lipid accumulation were both enhanced in S. ecornis at phosphate concentration of5mg/L. For iron experiment, growth and lipid accumulation of C. reticulatum were enhanced at relatively high iron concentration (0.03mmol/L), while growth and lipid accumulation of S. ecornis were enhanced at relatively low iron concentration (0.01mmol/L).C. reticulatum and S. ecornis were also cultivated in medium of gradually increasing salinity to acclimatise to rigorous saline water. Results showed that the biomass of two strains rose from1.42±0.12g/L and0.84±0.04g/L to2.89±0.15g/L and2.24±0.14g/L, respectively, which means that the increase of salinity stimulated microalgal cells division. At the preliminary stage of acclimation, their lipid contents were decreased sharply, e.g. from38.67±2.84%to24.17±2.01%and from24.25±0.26%to15.91±0.74%. But the biomass and lipid contents increased next with the progress of acclimation, and finally they are all acclimatise to rigorous saline water (salinity25%ow/v) with almost the same lipid contents of the non-domesticated.Above all, screen, identification, tentative cultivation and salinity accumulation of tropical freshwater microalgae of Hainan for lipid production, which was meaningful for the production of biodiesel using tropical freshwater microalgae, was developed in this study.