淨零排放是目前一個全球性的挑戰，主旨為透過減輕大氣中的二氧化碳水平，減少溫室氣體排放，同時利用碳捕獲技術來達到淨零碳排的目的。通過微藻捕捉二氧化碳的獨特能力，用於進行光合作用，提供了一個新的淨零碳排方案。微藻可以透過吸收光能，有效地將二氧化碳轉化為生物質，並提供可持續、再生的能源和高價值的化學品。本文深入探討以二種藻種：藍綠藻及萊茵衣藻作為二氧化碳減排的主要對象，開發高效生物質生產、廢水培養和環境修復方面的潛力，也利用基因工程技術突破二氧化碳同化能力及耐熱性等。最終分析生產之油脂、蛋白質、葉黃素等高價值化合物的產量。
基於微藻的二氧化碳減排戰略，目前仍需要解決的問題包括選擇具有高二氧化碳吸收率和生物質產率的藻種、優化培養系統以實現最大效率、快速生長的培養策略、開發具有成本效益之收獲和加工方法。所以我們由培養策略開始，優化營養成分、光照條件、二氧化碳濃度、溫度和鹽度，以提高微藻的生物量和化學品產量。其中，藍綠藻PCC10605利用N1A2培養基，在2%的二氧化碳，100 μmol/m2/s的光照強度和16:8的光照/黑暗時間培養，可以獲得最高的生物量1.17 g/L。Synechococcus sp. PCC7002 可利用Tris-HCl萃取溶液直接獲得藻藍蛋白（C-PC），而Cyanobacterium aponinum PCC10605則由磷酸萃取溶液以及破細胞的方法取得藻藍蛋白。其中PCC10605的C-PC最高的產量為0.652 g-CPC/g-DCW。此外，PCC10605藍綠藻具有廢水處理的潛力，生物量達到1.23 g/L，其C-PC對鉛金屬離子的吸附可達84.1 mg/g-CPC，並證明C-PC具有抗氧化和抗菌等活性，表明PCC10605強大的應用價值。
研究的第二部分，選擇基因組序列已被完全解密的萊茵衣藻進行基因工程改造。分別在萊茵衣藻CC400過表達三個獨立基因，包含吡哆醇激酶（pdxY）、碳酸酐酶（CA）和伴侶蛋白（GroELS）基因；當中PdxY用於提高生物量生產，CA用於增強二氧化碳同化能力，而GroELS用於提高微藻在高溫的耐熱性。由整合pdxY基因在CC400的 PY9藻株中，評估在不同培養系統，即一般燒瓶、雙層光反應器（TLR）和氣舉式光生物反應器（PBR）的生物量產量。當PY9菌株在PBR以1%二氧化碳進行培養時，生物量累積明顯增強，達到1.442 g/L，這是野生型藻株生物量的兩倍。由基因轉錄分析得知PY9菌株的RuBisCo和pdxY的表達水平上升，但碳酸酐酶（CA）下調。於此，利用混營培養時，pdxY基因成功提高了CC400藻株的二氧化碳利用率，達到-1.183 g-CO2/g-DCW，是野生株的2.68倍。
為了提升衣藻整體生物量的局限性，採用基因工程整合來自Mesorhizobium loti（MlCA）和Sulfurihydrogenibium yellowstonense（SyCA）的異源碳酸酐酶（CA），獲得pCHM及pCHS藻株。CA在微藻的碳濃縮機制（CCM）中非常重要，它將大氣中的二氧化碳轉化為碳酸氫鹽，對於生長和細胞密度至關重要。由不同的培養裝置中，得知以Tris acetate phosphate（TAP）混營培養到利用BG11m的自養條件，CA基因都能提高生物量、葉黃素和脂質的產量。在5%二氧化碳的PBR培養時，pCHS藻株的生物量達到了3.65 g/L、21.32 mg/L的葉黃素和672 mg/L的脂質。在混養和自養條件下，二氧化碳同化率在混營培養為2.748 g/g-DCW和自養的2.792 g/g-DCW；當中生物量的累積與CA活性呈正相關，參與葉黃素和脂質生物合成代謝途徑的關鍵基因的表達水平明顯上調。這些發現強調了基因工程和利用異源CA酶來提高CCU，更大幅地提高微藻的生產潛力。
最後以CRISPRi技術抑制CC400代謝中的磷酸烯醇丙酮酸羧化酶（PEPC1）基因，改變碳流量至油脂及蛋白質累積。進一步將GroELS基因整合在CC400取得PGi藻株，在35oC時7天時，PGi的生物量可達到2.56 g/L的最高產量。經由所有優化條件，最高的脂質為871 mg/L和葉黃素產量達22.3 mg/L。此外，PGi在35oC的混營培養下具有出色的二氧化碳同化能力，達到1.087 g/g-DCW。
微藻能夠有效地捕獲二氧化碳並將其轉化為生物質，重點在於選擇合適藻株、設計最佳化培養、善用其優勢成份；另一方面可由基因改造提昇其能力，使其突破生長速度及化學品產量。透過對微藻深入的研究和應用層面，在二氧化碳減排、生物燃料、動物飼料、營養品和其他有價值的產品將一定可以提供實現淨零碳排的新契機。

Net-zero emissions is now a global challenge that aims to reduce greenhouse gas emissions by reducing atmospheric CO2 levels while integrating carbon capture technologies to achieve net-zero carbon emissions. Meanwhile, microalgae offer a promising solution through their unique ability to capture and utilize CO2 for photosynthesis. By harnessing the power of sunlight, microalgae can efficiently convert CO2 into biomass, providing a sustainable and renewable source of energy and valuable products. In this thesis, we explored the potential of two microalgae species, blue-green microalgae and Chlamydomonas reinhardtii, as the main targets for CO2 mitigation, to develop sustainable biomass production and environmental remediation. Another approach is to apply genetic engineering techniques to break through the CO2 fixation capacity and heat tolerance. The final analysis is performed to produce high-value compounds such as oils, proteins and lutein.
Based on the CO2 capture and utilization strategies of microalgae, there are still issues that need to be addressed, including the selection of microalgal species with high CO2 uptake and biomass yield, optimization of the culture system to achieve maximum efficiency, fast-growing culture strategy, and development of cost-effective harvesting and processing methods. Therefore, we started with culture strategies to optimize nutrient composition, light conditions, CO2 concentration, temperature and salinity to increase the biomass and chemical production of microalgae. Among them, the highest biomass of 1.17 g/L could be attained from the blue-green algae PCC10605 using N1A2 medium, incubated at 2% CO2, 100 μmol/m2/s light intensity and 16:8 light-dark period. Synechococcus sp. PCC7002 can be directly used to extract C-phycocyanin (C-PC) with Tris-HCl buffer, while Cyanobacterium aponinum PCC10605 need to be extracted by phosphate buffer and homogenization. The highest yield of C-PC for PCC10605 was 0.652 g-CPC/g-DCW. In addition, PCC10605 have potential for wastewater treatment with a biomass of 1.23 g/L. The extracted C-PC can adsorb 84.1 mg/g-CPC of lead metal ions and the antioxidant and antibacterial activities of C-PC are demonstrated, indicating the convincing applicability of PCC10605.
Another approach, Chlamydomonas reinhardtii was selected for genetic engineering host, whose genome sequence has been fully decoded. In our study, we investigated the overexpression of three genes, namely pyridoxal kinase (pdxY), carbonic anhydrase (CA) and chaperone protein (GroELS) genes. Among them, pdxY gene is used to enhance biomass production, CA gene was utilized to enhance CO2 assimilation capability, and GroELS gene is for increasing heat tolerance of microalgae. We first focused on integrating the pdxY gene, resulting in the creation of the PY9 strain of microalgae. The biomass production of PY9 strain is evaluated in altered cultivation systems, i.e., flasks, Two-layer Photo-Reactor (TLR) and Photo-Bioreactor (PBR). Remarkably, when the PY9 strain was cultured in the PBR with 1% CO2, we observed a significant enhancement in biomass accumulation, reaching up to 1.442 g/L, which was approximately twice the biomass yield of the wild-type strain. Furthermore, our investigation revealed an interesting observation regarding the gene expression profiles in the PY9 strain. We observed a downregulation of carbonic anhydrase (CA) at transcriptional levels and higher expression levels of RuBisCo and pdxY. Thus, the pdxY gene successfully improved CO2 assimilation capacity of CC400 genetic strain, which was -1.183 g-CO2/g-DCW, which is 2.68 times higher than that of the wild-type strain. To understand the limitations of the overall biomass production of microalgae, we investigated the potential of enhancing carbon dioxide capture and utilization (CCU) in CC400 by introducing heterologous carbonic anhydrase (CA) enzymes from Mesorhizobium loti (MlCA) and Sulfurihydrogenibium yellowstonense (SyCA) through genetic engineering approaches, namely pCHM and pCHS. CA is important in carbon-concentrating mechanism (CCM) of microalgae by converting atmospheric CO2 into bicarbonate, which is essential for optimal growth and cell density. In our study, we employed different culture devices to explore the impact of these genetically modified CC400 strains on CCU. Notably, the engineered microalgae demonstrated the ability to transition from mixotrophic (TAP) to autotrophic (BG11m) conditions, resulting in improved biomass, lutein, and lipid production. Specifically, when cultured in a PBR supplemented with 5% CO2, pCHS strain reached a biomass of 3.65 g/L, with lutein and lipid concentrations reaching 21.32 mg/L and 672 mg/L, respectively. Moreover, the CO2 assimilation rate was measured at 2.748 g-CO2/g-DCW and 2.792 g-CO2/g-DCW under mixotrophic and autotrophic conditions, respectively. Importantly, the biomass accumulation positively correlated with CA activity. Additionally, the expression levels of key genes involved in the metabolic pathways of lutein and lipid biosynthesis were significantly upregulated. These findings highlight the potential of genetic engineering and the utilization of heterologous CA enzymes to enhance CCU and maximize the productivity of microalgae.
Finally, CRISPRi system is also applied to suppress phosphoenolpyruvate carboxylase (PEPC1) gene in CC400 metabolism, altering carbon flux to lipid and lutein accumulations. Further integration of the GroELS gene in CC400 resulted in a PGi strain with a maximum biomass production of 2.56 g/L at 35oC for 7 days. Moreover, PGi has excellent CO2 assimilation capacity of 1.087 g-CO2/g-DCW under mixotrophic condition.
In conclusion, microalgae are effective in capturing CO2 and converting it into biomass. The focus is on selecting the suitable strain of microalgae, designing the optimal cultivation, and making the best use of its advantageous features. While genetic engineering can enhance its ability to break through the growth rate and chemical yield. Through in-depth research and application of microalgae, new opportunities for CO2 emission reduction, biofuels, animal feeds, nutritional products and other valuable products will surely be offered to achieve net-zero carbon emission in the future.

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