| Fossil energy crisis and global greenhouse effects are two major challenges of the21st century. Microalgae as an alternative and renewable feedstock for biodiesel production, has drawn wide attention. However, there are still many challenges for the commercial utilization of biodiesel from microalgae as the algal-oil price remained rather higher than that of fossil fuels. Particularly, the further improvements of microalgal lipid production, as well as the reduction of lipid extraction costs are two problematic areas for the algal biofuel production technology. In this study, two different aspects of genetic engineering strategy and high CO2concentration domestication strategy were used for the enhancement of microalgal lipid production with microalgae Chlorella sp., and a new membrane-based lipid extraction method was also proposed for the reduction of extraction costs. The main research of the thesis included the following five aspects:1. The core metabolic network and lipid accumulation network of Chlorella sp. under autotrophic condition was constructed and analyzed. The reliability of the metabolic network model was proved by the partially explanation of the environmental pressures impact on metabolic flux distribution and the competition between starch and TAG synthesis pathways. Combination of metabolic network analysis with CASOP algorithm analysis showed that fatty acid synthesis and Kennedy pathway, especially the DGAT enzyme, was of great importance for the TAG accumulation.2. Separation, purification and identification of Chlorella sp. were carried out. Antibiotic combination treatment and streak plate method were used to remove bacteria and mold, respectively, and the sterile cultivation system was established. The comparison of three genomic DNA extraction methods showed that the plant genomic DNA extraction kit was esay operated and short time consuming, and high DNA purity and DNA yield could be obtained together with the improved SDS-CTAB method. PCR amplification of rbcL gene indicated that the Chlorella sp. was further belonged to Chlorella vulgaris species, and the phylogenetic analysis was accomplished.3. A diacylglycerol acyltransferase (DGAT) gene from Saccharomyces cerevisiae S288c was constructed to pBI121vector, and the Agrobacterium-mediated transformation of DGAT in Chlorella vulgaris was established with tris acetate phosphate (TAP) as the co-cultivation medium and G418as the selection antibiotic. The results showed that the transformation frequency was127-148per106cells, and the colony PCR could be adopted to monitor the stability of DGAT transformation in Chlorella vulgaris when6%w/v Chelex-100solution was used as the DNA extraction buffer for10min of the incubation time. Although this transformation was still found to be unstable, the colony PCR did show its application in screening of the large scale genetically-engineered transformants besides that in the general amplification of genomic DNA fragments.4. The high CO2concentration domestication and lipid enrichment were carried out in the airlift photobioreactor, and three methods of gravimetric determination, FT-IR spectroscopy and Nile Red staining were evaluated for algal lipid content analysis. The gravimetric determination was preferable for large-scale cultivation with low-frequency monitoring, while FT-IR and Nile Red were suitable for general laboratory cultivation with medium-frequency monitoring, in particularly Nile Red was appropriate for small samples when high-frequency screening was required. Moreover, the FT-IR method can simultaneously analyze the carbohydrate and protein contents, while the Nile Red staining can locate the lipid droplets. The biomass concentration reached1.37g L-1with the lipid concentration and lipid content of589mg L-1and47.2%after domestication, respectively, and the CO2fixation rate reached the maximum of106.9mg L-1h-1under1.0%CO2condition.5. Microalgal lipid extraction with membrane based technology was established. High pressure homogenization was suitable for cell fragmentation with efficiency of91.7%after manipulation three times. PVDF (small one) hollow fiber membrane material was suitable for membrane based extraction, with the static contact angle of inner and outer surfaces of75.0°and77.1°, respectively. The inner surface was flat mesh structure, and the outer surface was rough and porous structure which had a large number of10-100nm circular holes, combining finger-like structures at two sides of the cross-section with microporous sponge-like structures in the middle. Ethyl acetate was suitable organic extractant and the extraction efficiency was56.2%. The operating conditions were optimized with aqueous phase flow of40mL min-1(0.28cm s-1) and organic phase flow of21mL min-1(2.79cm s-1). and the maximum extraction efficiency reached71.3%. As for the algal liquds concentrated with2.5times,5times.15times and30times, the extraction efficiency was65.2%.57.4%.45.9%and36.1%, respectively. |
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