| With the rapid development of biomass energy, microalgal biofuel has become one ofthe hot spots. Chlorella protothecoides is a species of microalgae, which is easy to grow,with high content of lipid and valuable metabolites. Therefore, it has been one of the mainmicroalge species being widely resaerched. However, it has been proven that there is anincompatibility for the microalgae between biomass accumulation and high lipid content.When cultivated in rich medium, C. protothecoides rapidly accumulates biomass but withrelatively low content of lipid, while under the stress of nitrogen starvation, or strongradiation, it accumulates biomass very slowly but with high content of lipid. This problemseriously obstructs the large-scale application of C. protothecoides. Thus, in this study, thefermentation conditions for more biomass accumulation and higher lipid content wereoptimized respectively. Then, a novel cultivation strategy, named "heterotrophy-stress"two-step cultivation, was employed to attain the both goals at the same time. In addition,C. protothecoides cells contain a lot of high-valued products. So, a preliminary study wasalso conducted on the extraction, purification and analysis of monosaccharide compositionof the microalgal polysaccharide from the biomass residue after lipid extration. The mainwork and results were as follows:1. To improve biomass production, the medium of C. protothecoide was optimized bysingle factorial experiments and orthogonal experiments. The obtained optimal cultureconditions for maximum biomass were: glucose17.5g/L, NaNO31.25g/L, MgSO40.12g/L, and K2HPO40.72g/L. At the same time, the optimal culture conditions for the largestlipid content were also optimized. They were: NaNO30g/L, FeCl36H2O0.0015g/L, andMgSO40.040g/L.2. In500mL flask, with the "heterotrophy-stress" two step cultivation strategy,6.17g/L dry-weight of C. protothecoides biomass was achieved, which was close to the level（6.25g/L） of traditional heterotrophic cultivation, but lipid content increased from15.40%to34.81%,2.26fold of that of the traditional heterotrophic cultivation.. Furthermore, a3-L fermenter experiment showed a consistent pattern. Under traditional heterotrophiccondition, the biomass reached14.10g/L dry-weight and lipid content attained17.16%. While after light and CO2being introduced in the two-step cultivation, the accumulatedbiomass was13.20g/L dry-weight and lipid content reached40.15%,2.34fold of that ofthe traditional heterotrophic cultivation. In comparison to traditional heterotrophiccultivation, the content level of pigments, including chlorophyll and β-carotene, oftwo-step cultivation has both been decreased, and the fatty acid profiles become evensimplier, mainly C18:1（59.41%） and C16:0（26.63%）, which was a better choice forbiodiesel feedstock.3. The process was optimized for extraction of microalgal polysaccharide from the residueafter lipid extraction using single factorial experiments and orthogonal experiments. Theoptimal conditions were: temperature90°C, soaking time2h, solid-liquid ratio1:30, andrepeated3times. Under the optimal conditions, the extraction efficiency was20.15%.Compared with traditional heterotrophic cultivation, the microalgal polysaccharide contentof the two-step strategy was increased from9.57%to18.06%in500mL flask. While in3-L fermenter, the algal polysaccharide content was increased from10.16%to24.74%,exhibiting the similar pattern. The monosaccharide composition of the microalgalpolysaccharide was measured with FR-IR and GC-MS, and the major monosaccharideswere glucose, mannose and galactose. |
