Research On The Culture Techniques And Accumulation Of Lipids In Marine Oil-rich Microalgae

With the rapid socio-economic development, the demand of human on fossilfuels is increasing. The excessive use of crude oil results in sharp drop of the oilreserves and serious environmental damage such as air pollution, greenhouse effectand oil spills. Owing to the advantages of diversity of feedstock, good combustionproperties, low environmental pollution and renewable ability, biodiesel isconsidered to be an ideal alternative energy of fossil fuels. Marine microalgae isbelieved to be a promising feedstock for biodiesel production for its high lipidscontent, as well as its cultivation without occupation of arable land and freshwater.However, how to obtain a large number of microalgae biomass by large-scalecultivation and increase the output of lipids effectively are the key issues forexploitation of microalgae biodiesel.In this study, a determinate method of lipids content in microalgae wasestablished and improved based on Nile Red staining. Compare to the tranditionalextraction method by organic reagent, it was more rapid. As quantitative detectionmethod of lipids content, it could be used in the optimization experiments formicroalgae culture conditions. Axenic purification of four marine microalgaespecies （Isochrysis galbana CCMM5001, Chlorella sp. CV, Chlorella sp. C95andChlorella sp. C146） was carried out by using several common antibiotics. Followingby heterotrophic cultivation, one species Chlorella sp. CV showed goodheterotrophic character with the lipids content increasing from12.8%to40.1%. Thisspecie was considered to be heterotrophic engineering oil-rich microalgae andoptimization culture conditions were studied. The results showed that the highestbiomass, lipid content and productivity were, and respectively.Aiming to lipids-riched marine microalgal Isochrysis galbana CCMM5001, theinfluence of different culture conditions on its growth and lipids accumulation in columnar light bioreactor were studied. The results indicated that low Nconcentration and temperature were suitable for lipids accumulation，but the lipidscontent reduced if the illumination intensity was too high or too low. The optimumconditions for growth and lipids accumulation were:1323and441μmol·L^-1for Nconcentration,148.0and92.5μmol·m-2·s-1for illumination intensity,25and15℃fortemperature respectively. The optimized culture condition and a two-stagecultivation were carried out to increase both the lipids content and productivity of63.3%and22mg·L^-1·d^-1.The correlation between the lipids content and the level of ACC geneexpression of Isochrysis galbana CCMM5001was investigated. The results showedthat the level of ACC gene expression and the lipids content both would increaseunder carbon dioxide enrichment, nitrogen deprivation and low temperature cultureconditions. This implied that the high level of ACC gene expression could lead tolipids accumulation in Isochrysis galbana CCMM5001.In this dissertation, the lipids determination method, large-scale cultivationtechnology, lipids accumulation conditions and genetic basis of lipids sythesis ofmarine microalgae were studied. It will provide the pre-experimental basis andnecessary theory evidence for development and utilization of marine microalgaebiodiesel.