| Astaxanthin has powerful antioxidantive activities and the function of scavenging Free Radicals. It has wide applications in the aquaculture, food, medicine etc. At present, Haematococcus pluvialis is regarded as the best biological source of astaxanthin. In this thesis, original strain was treatd repeatedly with UV. After screening, a mutant named ZW-Ⅱ-6 was selected. The mutant can be fit for mixotrophic culture and has high biomass and high production of astaxanthin by the culture medium added with organic carbon source. Compare to the original strain , the cell dry weight of ZW-Ⅱ-6 was increased from 0.1877 g/L to 0.3176 g/L, enhancing about 69.2%,and the production of astaxanthin was increased from 2.0835 mg/L to 5.2706mg/L, about 1.405 times higher than the original strain. Subculture showed that the genetic stability of ZW-Ⅱ-6 is good.The biomass of Haematococcus pluvialis was determined by cell dry weight. The optimal medium and suitable culture conditions for the high-biomass of Haematococcus pluvialis were studied. Mixotrophic culture added with sodium acetate and glucose, and results showed that the medium added with glucose was better. So glucose was chosen as the carbon source of mutant ZW-Ⅱ-6. On the base of single factor tests , the orthogonal test was designed. Results showed that the optimal culture medium for ZW-Ⅱ-6 is decided as: glucose 3g/L, KNO3 0.5g/L,KH2PO4 0.03 g/L,MgSO4·7H2O 0.2 g/L,CaCl2 0.08 g/L. Studied the conditions such as light intensity,temperature,initial pH, and got the suitable culture conditions is that the light intensity 2500lx, temperature 22℃and initial pH 8. Under the optimal culture medium and suitable conditions for 8 days, the biomass of Haematococcus pluvialis was 1.1274g/L.The effects of light intensity and concentration of organic carbon source on the growth of vegetative cell of Haematococcus pluvialis was investigated. The growth of Haematococcus pluvialis was affected by light intensity and carbon source concentration. The optimal light intensity is approximately 2500lx, and the optimal carbon source is glucose for ZW-Ⅱ-6. Effects of light intensity and organic carbon source concentration on the growth were concomitant. When the light intensity was 0 or 500lx, the glucose concentration supporting the highest cell dry weight was 5 g/L. When the light intensity rose to 2500lx, the glucose concentration supporting the highest cell dry weight reduced to 3g/L. By the CCD experiment (Center Combination Design), the highest cell dry weight was 1.2375g/L, when the light intensity was 2259lx and the glucose concentration was 3.1113g/L.The production of astaxanthin under mixotrophic culture was investigated and astaxanthin content was determined by DMSO extraction. Light intensity and glucose concentration have reciprocal influence on the production of astaxanthin in Haematococcus pluvialis. When the light intensity was 0lx, the production of astaxanthin under mixotrophic culture was lower than autotrophic culture and when light intensity rose from 0lx to 2500lx, the glucose concentration supporting the highest production of astaxanthin reduced from 5g/L to 3g/L. By the CCD experiment, the highest production of astaxanthin was 41.06mg/L, when the light intensity was 2605.66lx and the glucose concentration was 3.1616 g/L. It shows that the production of astaxanthin is related with the biomass of Haematococcus pluvialis.By the preliminary studies on the cell transformation of Haematococcus pluvialis for astaxanthin accumulation, the optimal transformation conditions were: KNO3 0.01g/L, sodium acetate 1.5 g/L, transformation temperature 30℃, transformation pH 10, transformation time 10days, transformation light intensity >7000lx. The concomitant effects of the transformation conditions need further research. |
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