| With the increasing depletion of fossil fuels and continuous deterioration of ecologicalenvironment, many countries are paying efforts to seek for renewable and environmentallyfriendly fuels to replace fossil fuels. Oleaginous microalgae could quickly grow uponphotosynthesis, accumulate oil through assimilating CO2, and gain high oil content and areayield. Therefore，it is considered as a good raw material of biofuels, which is also the firstchoice for energy conservation, emissions reduction and production of renewable clean energy.In this paper, Coccomyxa subellipsoidea C-169was used as the research object, which hasthin walls and completed genome sequencing information. A uniform and stablehigh-throughput culture system was constructed, and combined with cytomic analysistechnologies platform and Nile Red fluorescence staining technology, cells growth and oilaccumulation condition of Coccomyxa subellipsoidea C-169were quickly optimizated. It is ofgreat importance to fully master basic biological information of Coccomyxa subellipsoideaC-169and further study the mechanism of producing oil. The main results were as follows:1. Coccomyxa subellipsoidea C-169were grown autotrophically in Basal and BBMculture media, respecyively. The effects of2%CO2supplement or air on cell specific growthrate, biomass dry weight, content and yield of total fatty acids, neutral lipids content wereinvestigated in this study. Compared with the results from aeration with air,2%CO2supplement could significantly increase the cell specific growth rate by2.6times in Basalmedium and2.3times in BBM medium, respectively. After13days culture, the maximalbiomass concentration was obtained at4.67g/L by2%CO2supplement in Basal medium,which was significantly higher than other groups (p <0.05). The highest content of total fattyacids reached37.53%DW by2%CO2supplement in BBM medium, and neutral lipidsreferred as fluorescence intensity was significantly higher than other groups (p＜0.05). Theseresults indicated that2%CO2supplement in Basal medium could promote cells to producehigher biomass, and that2%CO2supplement in BBM medium could increase the yield offatty acids and neutral lipids in this microalgae.2. Based on cytomic analysis technologies platform, such as flow cytometer,fluorescence microplate reader, confocal laser scanning microscope, high-throughputevaluation system on determining neutral lipids of Coccomyxa subellipsoidea C-169wereestablished. The effects of cell concentration, Nile Red dye concentration, different treatment,solvent concentration, staining temperature, staining time, vibration time on neutral lipidscontent referred as average fluorescence intensity of Coccomyxa subellipsoidea C-169were investigated and optimized in this study. The results indicated that106/mL is the best cellconcentration, and that Nile Red dye concentration between0.05and0.2μg/mL is suitable.The addition of15%~30%(v/v) glycerol could significantly improve the permeability of cellmembrane. Staining temperature between25to40℃was found no significant difference onthe fluorescence intensity (p＞0.05). When the staining time was longer than10min andvibration time longer than30s, there was no significant increase on fluorescence intensity ofneutral lipids. In conclusion, systematically optimization could significantly improve theeffect of microalgae fluorescent staining.3. Throughput cultivation system of Coccomyxa subellipsoidea C-169were established,and the uniformity and stability of the system was perfect. Combined with cytomics analysistechnologies, varieties of factors (kind and concentration of nitrogen, concentration ofNH4HCO3and phosphate, initial pH value, NaCl concentration, etc) on cell specific growthrate, biomass dry weight and oil accumulation capacity were investigated in this study.Results indicated that nitrogen-rich cultivation was significantly beneficial to cell growth andbiomass accumulation of Coccomyxa subellipsoidea C-169, whereas nitrogen-deficientcultivation was in favour of neutral lipids content accumulation. When NH4HCO3concentration was0.23g/L, cells biomass accumulation and metabolism of lipid synthesiswere significantly enhanced, and increasing or decreasing of NH4HCO3concentration wouldinhibit cell growth and oil accumulation. Phosphate demand of Coccomyxa subellipsoideaC-169was very little, therefore reducing phosphate concentration did not significantly affectthe biomass and neutral lipids content. The initial pH value of4~6was in favor of the cellgrowth and oil accumulation. In0~15‰salinity range Coccomyxa subellipsoidea C-169grewwell and there was no significant difference (p>0.05), while cell growth was significantlysuppressed in higher salinity concertration of15‰, and biomass dry weight and growth ratewas significantly lower than the control group (p<0.05). Neutral lipids referred asfluorescence intensity at10‰salinity cultivation was significantly higher than all the othergroups (p<0.05). |
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