碩士 / 元智大學 / 生物科技與工程研究所 / 103 / Microalgae biotechnology has been widely used in various fields such as healthy food and biofuels. High-density fermentation tanks have been applied to culture microalgae to produce docosahexaenoic acid (DHA). Cells require sufficient oxygen to sustain the growth, and oxygen is a limiting factor in high-density culture fermenter. Injection of pure oxygen into a large tank is more efficient than air, but it is easy to make tank exploding, thereby high safety facilities are required. Hemoglobin from Vitreoscilla (VGB) is an oxygen carrier, which has been applied to increase cell density of aerobic organisms in a bioreactor. In this study, VGB gene was fused under a constitutive promoter, and then it was electroporated into two microalgae, Chlamydomonas reinhardtii and a high DHA strain of Aurantiochytrium limacium SR21. At least three independent transgenic microalgal cell lines were obtained and confirmed by genomic PCR and RT-PCR. Cell growth was faster in VGB containing transgenic microalgae than wild-type or vector only transgenic control cell lines under low oxygen condition. Cryopreservation technology is applied for a long-term storage of the original cell lines. However, the intracellular ice crystals formed during the freezing process damage the cell membrane and cause cell death. In this study, various croprotectants were applied to increase the survival rate of transgenic microalgae.
Saussurea involucrata is a traditional herbal medicine that contains plant substances to treat illness. Syringin is a S. involucrata metobolite with anti-hypersensitivity and anti-inflammatory on autoimmune disease. Due to human over-exploitation of S. involucrate which naturally grows in 4000 to 4500 meter-high mountain, making an insufficient supply of wild S. Involucrata with high price. By plant tissue culture, S. involucrata plants are able to regenerate from undifferenticated callus. In this study, we established an efficient system to get the regenerated S. involucrata plants from leaf disks. In addition, fast growth of S. involucrata suspension cells were set up. Suspension cells under different culture conditions were oven-dried and then analyzed by high performance liquid chromatography (HPLC) to detect compounds contained in cell extract. With hormone combinations, rapid growth of suspension cells producing high Syringin could be achieved under either light or dark conditions. Bioreactor was then applied to scale up suspension cell cultured S. involucrata. The cells were dried by either oven-drying process or freeze-dry, and then analyzed by HPLC to detect Syringin. The result showed that the freeze-dry method had five folds higher Syringin than the oven dry method. In this study, the microalgae used cryoprotectant and four cryoprotectants were aplied to establish snow lotus suspension cell cryopreservation system by liquid nitrogen. The highest survival rate after cells recovered from cryopreservation was 83.3%.
| Identifer | oai:union.ndltd.org:TW/103YZU05111115 |
| Creators | Ying-Kai Chu, 褚英凱 |
| Contributors | Li-Fen Huang, 黃麗芬 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 79 |
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