Title Page
Abstract
Contents
1. Introduction 13
2. Literature review 17
2.1. Microalgae 17
2.1.1. Characteristics and global market of microalgae 17
2.1.2. Recent values of microalgae 18
2.1.3. Autotrophic cultivation of microalgae 20
2.1.4. Heterotrophic cultivation of microalgae 21
2.2. Phycocyanin 24
2.2.1. Characteristics and structure of phycocyanin 24
2.2.2. Biosynthesis of phycocyanin 24
2.2.3. Applications of phycocyanin 25
2.3. Galdieria sulphuraria 27
2.3.1. General characteristics of Galdieria sulphuraria 27
2.3.2. Heterotrophic cultivation of G. sulphuraria 28
2.3.3. Heterotrophic cultivation of G. sulphuraria for PC production 30
3. Materials and methods 32
3.1. Materials 32
3.2. Methods 34
3.2.1. Autotrophic culture of G. sulphuraria 074 34
3.2.2. Separation of strains from G. sulphuraria 074 by pigmentation 34
3.2.3. Heterotrophic cultivation of G. sulphuraria 35
3.2.4. Measurement of growth parameters 37
3.2.5. Measurement of pigments 39
3.2.6. Genomic analysis of G. sulphuraria 41
3.2.7. Measurement of antioxidant contents and activities 43
3.2.8. Statistical analysis 46
4. Results and discussion 47
4.1. Separation of G. sulphuraria 074 strains by pigmentation 47
4.1.1. Separation of G. sulphuraria 074 into G. sulphuraria 074W1 and G. sulphuraria 074G1 47
4.1.2. Spectral characteristics of pigments from the culture of G. sulphuraria 074G1 49
4.2. Optimization of culture conditions for heterotrophic cultivation 51
4.2.1. Selection of basal medium for heterotrophic cultivation 51
4.2.2. Determination of initial inoculation concentration 53
4.2.3. Determination of carbon:nitrogen ratio 56
4.3. Second separation of G. sulphuraria 074G1 strains by pigmentation 59
4.3.1. Separation of G. sulphuraria 074G2 from G. sulphuraria 074G1 59
4.3.2. Morphology of G. sulphuraria 074G2 61
4.4. Heterotrophic cultivation of G. sulphuraria 074G2 for PC production 62
4.4.1. Effects of glucose and ammonium sulfate on algal growth 62
4.4.2. Effects of glucose and ammonium sulfate on PC production 64
4.4.3. Comparison of algal growth and PC production of G. sulphuraria 074G2 with other separated strains 66
4.5. Characteristics of the pigments produced by separated strains from G. sulphuraria 074 69
4.5.1. Color properties of the cultures and freeze-dried cells of separated strains 69
4.6. PCR analysis and Sanger sequencing of genes related to phycocyanin systhesis 72
4.7. Antioxidant substances and activities of cell extracts from G. sulphuraria 074 strains 76
4.7.1. Antioxidant substances of algal extracts of G. sulphuraria 074W1, G. sulphuraria 074G1 and G. sulphuraria 074G2 76
4.7.2. Antioxidant activities of cell extracts from G. sulphuraria 074 strains 78
5. Conclusions 81
6. 국문요약 83
7. References 86
Table 1. The composition of the autotrophic and heterotrophic growth media 33
Table 2. Sequence of the primers used for PCR 42
Table 3. Sequence of the primers used for Sanger sequencing 43
Table 4. Color properties of freeze-dried cell powders of G. sulphuraria 074W1, G. sulphuraria 074G1 and G. sulphuraria 074G2 70
Table 5. BLAST sequence analysis with cpcA and cpcB of G. sulphuraria 074W1, G. sulphuraria 074G1 and G. sulphuraria 074G2 75
Figure 1. Colony colors of G. sulphuraria 074 (A and B), G. sulphuraria 074W1 (C) and G. sulphuraria 074G1 (D) cultivated heterotrophically on solid medium. 48
Figure 2. Absorption spectra of cell culture (A) and culture extract (B) of heterotrophically cultivated G. sulphuraria 074G1. 50
Figure 3. Absorbance and phycocyanin concentrations of heterotrophically cultivated G. sulphuraria 074G1. 52
Figure 4. Effects of initial inoculation concentration of G. sulphuraria 0741 on PC production during heterotrophic cultivation. 55
Figure 5. Effect of C:N ratio on PC production of heterotrophically cultivated G. sulphuraria 074G1. 58
Figure 6. Colony colors of G. sulphuraria 074G1-G2 mixtures (A and B), G. sulphuraria 074G1 (C) and G. sulphuraria 074G2 (D) cultivated heterotrophically on solid medium. 60
Figure 7. Microscopic images of G. sulphuraria 074G2 that were cultivated autotrophically (A) and heterotrophically in the presence of glucose (B). 61
Figure 8. Effects of glucose and ammonium sulfate on heterotrophic cultivation of G. sulphuraria 074G2. 63
Figure 9. Effects of glucose and ammonium sulfate on phycocyanin production of heterotrophically cultivated G. sulphuraria 074G2. 65
Figure 10. Algal growth and PC production of G. sulphuraria 074 strains during heterotrophic cultivations. 68
Figure 11. The colors of the culture broth and freeze-dried cell powders of G. sulphuraria 074W1 (A and D, respectively), G. sulphuraria 074G1 (B and E,... 70
Figure 12. The specific PC (A), total chlorophyll (B) and carotenoid (C) contents of the freeze-dried cell powders of G. sulphuraria 074W1, G. sulphuraria 074G1 and G. sulphuraria 074G2. 71
Figure 13. Agarose gel electrophoresis analysis of PCR products of cpcA and cpcB genes extracted from G. sulphuraria 074W1, G. sulphuraria 074G1 and... 74
Figure 14. Total phenolic (A) and total flavonoid (B) contents of algal extracts of G. sulphuraria 074W1, G. sulphuraria 074G1 and G. sulphuraria 074G2. 77
Figure 15. 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity (A) and ferric-reducing antioxidant power (FRAP) values (B) algal extracts of G. sulphuraria 074W1, G. sulphuraria 074G1 and G. sulphuraria 074G2. 80