葉黃素為普遍存於植物、微藻與其他光合生物中的一種類胡蘿蔔素。在植物中，葉黃素扮演著光合輔助色素的角色。文獻指出，適量葉黃素可有效延緩老化、預防心血管，並降低罹患眼疾、癌症與慢性疾病之風險。由於人體無法自行合成葉黃素，故需經食物補充。目前葉黃素之主要來源為金盞花，但近年來，微藻成為葉黃素之替代來源之一。由於微藻具高生長速率且不受季節採收之限制，且相較於金盞花，微藻之生長較為快速，且具有較高之葉黃素含量，而其葉黃素主要以非酯化之小分子存在，因此以微藻作為葉黃素生產料源有其優勢。
本研究之目標為建立微藻葉黃素之生產技術平台，起初篩選數株能利用醋酸鈉生長之本土微藻，如Chlorella sp., Scenedesmus abundans GH-D11, Scenedesmus Obliquus AS-6-1, 與Chlorella sorokiniana HCH-2，進行混營培養，並初步鑑定其葉黃素生產能力。實驗結果顯示，本土微藻Chlorella sp. 具最佳之生物量產率及葉黃素生產效率(1.10 mg/L與2.79 mg/L/d)。為進一步提升葉黃素生產效能，本研究以系統性策略進行培養基成分之優化。首先，進行探討不同培養基(Basal medium, modified Bold Basal medium, modified Bristol's medium, and BG-11 medium)對於葉黃素生產效率之影響。而結果顯示，本土微藻Chlorella sp. 培養於BG-11培養基中，可獲得最高之葉黃素生產效率(3.39 mg/L/d)，其培養基組成成本亦為最低。由於先前結果顯示，於批次培養的過程中，本土微藻Chlorella sp. 之葉黃素含量及產率與培養基內之醋酸鈉(有機碳源)及硝酸鈉(氮源)之消耗密切相關，且其對於生物量生產及收成時機亦具顯著之影響。因此，本研究以反應曲面實驗設計法(RSM)針對碳氮源組成進行最適化。而RSM模擬結果顯示，於添加4.88 g/L醋酸鈉及1.83 g/L硝酸鈉的條件下，可獲最佳葉黃素產率為 3.86 mg/L/d。此結果與三重複驗證實驗結果十分相符。再者，為進一步進行培養基微量元素組成之優化，兩水準部分因子實驗被用以全面性檢測具顯著性之影響因子，並以陡升法探尋中央合成實驗設計之中心點，以針對微量金屬進行反應曲面模型之建構。實驗結果顯示，於添加51 mg/L二水合氯化鈣及218 mg/L氯化鈉，其葉黃素產率可進一步提升至4.10 mg/L/d。此外，光照強度對於本土微藻Chlorella sp. 之葉黃素生產無顯著性之影響，亦無顯著之光抑制生長效應，因此有利於未來戶外大規模化培養。
最後，以上述所得之最適化條件進行半批次操作(semi-batch operation)及兩階段半批次操作策略(semi-batch integrated with two-stage)以提升其生物量產率及葉黃素產率。實驗結果顯示，半批次操作於培養基取代率為80% 情況下，生物量產率及葉黃素生產速率分別可提升至1.55 g/L/d與5.51 mg/L/d。而於兩階段半批次操作策略中，於培養基取代率為80%時可得到其最大生物量產率及葉黃素生產效率，分別為1.98 g/L/d與7.62 mg/L/d，相較於批次條件分別提升約87 %與86 %。

Lutein is a kind of carotenoids widely existing in plants, microalgae and other photosynthetic organisms. In addition to acting as photosynthetic pigments, uptaking lutein can also effectively prevent aging, retinal diseases, cardiovascular diseases and chronic diseases. Compared to conventional lutein production source (i.e., marigold petals), microalgae have emerged as a promising lutein producer due to their high growth rate, no limitation of seasonal harvesting and most importantly, high lutein content in microalgal biomass, high lutein productivity, and the structure of lutein in microalgae being the free form. Therefore, using microalgae as the lutein production sources has some advantages over marigold.
This study was to establish the platform technology on lutein production from the indigenous microalgal strains. First, several indigenous microalgal strains isolated from aquatic environments of Taiwan, such as Chlorella sp., Scenedesmus abundans GH-D11, Scenedesmus obliquus AS-6-1, and Chlorella sorokiniana HCH-2, were examined for their capabilities to accumulate lutein under mixotrophic cultivation. The results show that the indigenous microalga Chlorella sp. had the highest biomass and lutein productivity (1.10 mg/L/d and 2.79 mg/L/d, respectively). In order to further enhance the performance of lutein productivity of Chlorella sp., a series of systematic strategies were applied. First, four kinds of broth media (Basal, MBM, MBBM, and BG-11) were used to examine the microalgae cell growth and lutein content of Chlorella sp. The results show that BG-11 medium was more suitable for lutein production (2.96 mg/L/d). Furthermore, since our previous work showed the performance of lutein production with Chlorella sp. was markedly dependent on the concentration of sodium acetate and sodium nitrate, which also further influenced the biomass productivity and harvest timing. Therefore, the response surface methodology (RSM) experimental design was used to optimize the composition of carbon and nitrogen source. The simulated results based on RSM analysis predicted a maximal lutein productivity of 3.86 mg/L/d when adding 4.88 g/L of sodium acetate and 1.83 g/L of sodium nitrate. This predicted value was confirmed with experiments conducted based on the optimized conditions. The lutein productivity obtained from the confirmation experiments was 3.87 mg/L/d, which is quite similar to the predicted one. Finally, in order to investigate the influence of trace elements in the medium on lutein productivity and avoid neglecting the main and interaction effects among those ions, the two-level fractional factorial experimental design was applied to find out the significant factors. Afterwards, the steepest ascent method was used to locate the center for central composite designs. Lastly, the response surface methodology was employed for optimizing the trace composition. The RSM analysis demonstrated a little improvement on lutein production, as the productivity further increased to 4.10 mg/L/d with the optimal trace element composition of 51 mg/L calcium chloride dihydrate and 218 mg/L sodium chloride. The investigation on the effect of light intensity shows that light intensity did not have significant effect on the performance of lutein production. Also, there was also no light inhibition on the cell growth. Hence, this strain may be feasible for outdoor cultivation.
Next, the operation modes were studied (including semi-batch and semi-batch integrated with two-stage system) using optimal medium composition as mentioned above to improve the biomass productivity and lutein productivity. The results of semi-batch system show that the maximal biomass productivity and maximal lutein productivity were 1.55 g/L/d and 5.51 mg/L/d, respectively, at 80% medium replacement ratio. The results of semi-batch integrated with two-stage operation strategy demonstrated a maximal biomass productivity and maximal lutein productivity of 1.98 g/L/d and 7.62 mg/L/d, respectively, also occurred at a medium replacement ratio of 80% ratio. Compared with the optimal condition of batch cultivation, the lutein productivity was enhanced by 87% and to 86%, respectively, for semi-batch operation and semi-batch integrated with two-stage system.

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