| Recent soaring oil prices and diminishing world oil reserves, coupled with rise of greenhouse gases and the predicted climate change threat, have generated renewed interest in using algae as an alternative and renewable feedstock for energy production. However, algal-oil price remained rather higher than that of fossil fuels, and the harvesting and extraction of fuel precursors from microalgal cultures was one of the most problematic areas for the algal biofuel production technology. In this study, a novel method, milking microalgal oil in situ based on membrane techonology for simultaneous production and selective extraction of algal lipid in an aqueous-organic biphasic system was proposed. The main research of the thesis included:1. Nannochloropsis sp. and Botryococcus braunii FACHB-357 with high lipid content were screened from 6 blue-green algal species. To establish the rapid method for algal lipid content analysis, gravimetric determination, Nile red dye and FT-IR were evaluated according to their accuracy and convenience. Among these three methods, FT-IR could be used to semi-quantitatively and simultaneously analyze lipid, protein and carbonhydrate content of algae biomass. Nile red fluorescence determination was a rapid method to characterize algal neutral lipid content by spectrofluorometer and locate lipid body in algal cell by using a fluorescence microscope.2. Due to the slow growth rate of B. braunii FACHB-357, the culture medium BG11 was optimized by the response surface method (RSM) to enhance the algal lipid and biomass systhesis. Nutrients of N, P and Fe were screened as the key impact factors with the PBD analysis. The experimental results show that the higher lipid productivity could be obtained under the lower nitrate concentrations (50 mg L-1) and ammonium ferric citrate concentration (6 mg L-1). N-starvation was applied to enhance lipid accumulation both in Nannochloropsis sp. and B. braunii FACHB-357. The results of Nile red analysis and transmission electron microscope (TEM) photos showed that algal lipid production could be greatly enhanced by N-starvation treatment. After one week, the lipid contents of Nannochloropsis sp. and B. braunii FACHB-357 increased to 55% and 39%, respectively. The average lipid productivity of Nannochloropsis sp. and B. braunii FACHB-357 were 25.5 mg L-1 d-1 and 7.4 mg L"1 d-1, respectively.3. To extract the microalgal lipid in situ, biocompatible solvents were screened for lipid milking of Nannochloropsis sp. and B. braunii FACHB-357 in an aqueous-organic system. The effects of organic solvent on the microalgal growth, lipid extractability, dehydrogenases activity and cell membrane integrity were investigated by using a UV-visible spectrophotometer, FT-IR spectroscopy, 2,3,5-triphenyltetrazolium chloride (TTC) and Evans Blue stain method, respectively. The results showed that alkane solvents with log P>5.5 were biocompatible while the hydrophilic solvents with log P<5.5 were toxic to Nannochloropsis sp. and B. braunii FACHB-357 due to deactivating dehydrogenase and reducing cell membrane integrity. When 10% (v/v) hexadecane was used to establish the biphasic system, the total lipid production of Nannochloropsis sp. was increased by 28.9% compared to the control. The screened biocompatible solvent hexadecane enhanced not only the algal growth but also the lipid accumulation, showing an effective way to facilitate the process for in situ lipid milking from Nannochloropsis sp. On the other hand, for B. braunii FACHB-357,10%(v/v) tetradecane was choosen to eastablish the aqueous-organic biphasic system due to the better biocompatibility, lipid extracbility and solvent recovery rate (10.16%). The cell ultra-structure and cell membrane permeability of B. braunii FACHB-357 were investigated to understand the mechanism of lipid extraction within the biphasic system. The results showed that biocompatible solvent of tetradecane could induce algal lipid accumulation, enable the cell membrane more active and the cell wall much looser. The exocytosis process was observed to be one of the mechanisms for lipid cross-membrane extraction in the presence of organic solvent.4. B. braunii FACHB-357 could excrete and store lipid in the outter cell wall which was revealed by the transmission electron microscope (TEM) and fluorescence microscope photos. However, lipid was accumulated only in Nannochloropsis sp. cells, and the cell wall of this alga was thicker than 0.3μm. Lipid of B. braunii FACHB-357 and Nannochloropsis sp. was good raw material to produce biodiesel because of high content of C16～C18 fatty acids. Based on membrane-dispersion technologies, the steps of algal harvesting and lipid extraction were integrated to allow in situ lipid milking by tetradecane from B. braunii FACHB-357 and hexadecane from Nannochloropsis sp. The experimental results showed that the lipid extractability of B. braunii FACHB-357 was enhanced to 50.15% with a membrane module compared to the control (38.05%). But only 7% lipid of Nannochloropsis sp. was extracted by organic solvent. In addition, above 90% cells of B. braunii FACHB-357 and Nannochloropsis sp. were still active during lipid in situ extraction.
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