數十年來海洋觀測及研究表明，大氣中的二氧化碳濃度不斷地增加，海洋持續吸收大氣中的二氧化碳的過程正在改變海水的化學反應，這個過程被稱為大洋酸化，同時造成一系列的環境改變，影響著許多海洋生物的生長。前人研究也指出在早期地球處於環境較還原狀態時，已有固氮微生物進行固氮作用及光合作用，讓海洋及大氣產生了改變，持續影響著地球環境至今。
為探討大洋酸化對固氮藍藻生長的影響，本研究從台灣東部黑潮海域附近採集副熱帶西北太平洋固氮藍藻，主要研究內容分為五個部分：(1) 固氮藍藻篩選及培養；(2) 培養環境中添加Fe3+/Fe2+，對藻類生長之影響；(3) 添加抗壞血酸，對藻類生長之影響；(4) 添加抗壞血酸及不同價態鐵，對藻體細胞利用碳、磷、鎂及其他微量元素之影響；(5) 探討海水酸化及無氧、有氧的環境下，對固氮藍藻生長速率之影響。
本研究在固氮藍藻篩選及培養的實驗過程中，通過吸收光譜分析結果，發現在額外添加磷的情形下，大於20微米組分中，非固氮藻類死亡後，固氮藻在培養第五週開始大量繁殖，觀察到藻類明顯生長。此結果研究表明，副熱帶西北太平洋的海水通過20微米孔徑的濾膜過濾後，於額外添加磷的條件下培養可以篩選出固氮藍藻，也證明副熱帶西北太平洋海水中固氮藻存在於大於20微米的顆粒組分中，而小於20微米組分可能主要含有細菌、病毒及其他非固氮藻。
另外利用吸收光譜儀、ICP-OES及TOC分析固氮藍藻在添加Fe3+/Fe2+及有無添加抗壞血酸條件下培養的各項結果表明，於本研究中使用無菌海水作為基本配方的培養液，不額外添加鐵進行培養，固氮藍藻仍可生長，且在培養環境中，隨二價鐵濃度增加，固氮藍藻生長速率有增快的趨勢，而添加抗壞血酸不利於固氮藻使用碳、氮、磷..等元素，也減少細胞外的元素吸附，同時減緩固氮藻的生長速率，使藻體細胞延緩老化，延長固氮藻的生長週期。
本研究採取曝入空氣、氮氣、二氧化碳，三種氣體不同組合的方式，使固氮藻在海水酸化及無氧、有氧的條件培養下，探討不同環境對固氮藍藻生長速率之影響，結果表明在海水酸化及有氧的環境下，固氮藻生長速率較高，無氧環境下固氮藻生長緩慢，由此證明酸化的環境及含氧的條件，有利於副熱帶西北太平洋海水中的固氮藻生長。

SUMMARY
This study collected subtropical Northwest Pacific cyanobacteria from Kuroshio waters near the east coast of Taiwan to Fugang (22°47’N,121°11’E). Under controlled conditions in the laboratory, I explore the impact of ocean acidification on the cyanobacteria growth, and their rate of nitrogen and carbon fixation. The study is divided into five parts: (1) develop a method for selection and culture of nitrogen-fixational cyanobacteria, (2) access the growth of nitrogen-fixational cyanobacteria under different Fe3+/Fe2+ concentrations, (3) access growth of nitrogen-fixational affected by ascorbic acid, (4) access the uptake of elements (P, Mg, Mn, Cu, Zn, Co…) by cyanobacteria under the different cultural conditions which adding ascorbic acid and iron in different valence state. (5) access the growth rate of nitrogen-fixational cyanobacteria affected by CO2 acidification under oxygenated and non-oxygenated conditions. The purpose, methods, major results, and conclusions are summarized as follows.
Keyword：ocean acidification, carbon dioxide, nitrogen fixation cyanobacteria, iron(II)

INTRODUCTION
Carbon dioxide (CO2) is the major greenhouse gas that results from human activities and causes global warming and climate change. And ocean acidification is the ongoing decrease in the pH of the Earth's oceans, caused by the uptake of CO2 from the atmosphere. The change impact to atmosphere, ocean and biosphere. Recently, many studies about N2-fixation cyanobacteria which fixing carbon and nitrogen into oceans. And many study indicate that increasing CO2 will lead to enhance N2-fixation, C-fixation photosynthesis and growth rates in nitrogen fixation cyanobacteria. So we want to investigate the effect of ocean acidification on growth of N2-fixational cyanobacteria from the subtropical NW Pacific.
The study is divided into five parts: (1) develop a method for selection and culture of nitrogen-fixational cyanobacteria, (2) access the growth of nitrogen-fixational cyanobacteria under different Fe3+/Fe2+ concentrations, (3) access growth of nitrogen-fixational cyanobacteria affected by ascorbic acid, (4) access the uptake of elements (P, Mg, Mn, Cu, Zn, Co…) by cyanobacteria under the different cultural conditions which adding ascorbic acid and iron in different valence state. (5) access the growth rate of nitrogen-fixational cyanobacteria affected by CO2 acidification under oxygenated and non-oxygenated conditions.
MATERIALS AND METHODS
Seawater was picked up from seacoast in Taitung (22°47’N, 121°11’E). In first part of experiment, unsterilized seawater was incubate with N and P, while the other bottle incubating with P as control. In addition, the siftings of seawater sorted by 20, 2.7, 0.7 and 0.45 µm filter paper were added into culture medium separately and the absorbance values of medium were observed.
In second part of experiment, Fe3+/Fe2+ in different concentrations was added into culture medium and the growth rate of N2-fixational cyanobacteria was observed with or without ascorbic acid. After three months culture, I collected the dry algae to assay element concentrations with ICP-OES and carbon concentration with TOC.
Last part of experiment, I prepared three bottles filled with culture medium that contained N2-fixational cyanobacteria. I pumped air into medium of first bottle, N2 and CO2 into medium of second bottle (non-oxygenated), besides, air and CO2 into third bottle (acidification). I incubated those culture and observed absorbance values of medium.
RESULTS AND DISCUSSION
In unfiltered seawater which both nitrogen and phosphorus are added, a non-nitrogen-fixing cyanobacteria has rapid growth ; in unfiltered seawater which without the addition of nitrogen, at first most of algae grow very slowly or even die but nitrogen-fixation cyanobacteria begin reproducing quickly one month later.The results shows that without the addition of nitrogen, the siftings of seawater sorted by 20, 2.7, 0.7 and 0.45 µm filter paper exhibit no significant growth of cyanobacteria within four weeks. However, after 5 weeks of continuous culture, the growth of cyanobacteria was observed obviously in the siftings greater than 20µm. My result proved nitrogen-fixational cyanobacteria can be collected by this method.
In my experiment, nitrogen-fixational cyanobacteria still grow under no add additional iron condition. The growth rate of nitrogen-fixation cyanobacteria had trend to faster, when I increased the concentration of divalent iron. And the growth rate of nitrogen-fixational cyanobacteria deacreased with ascorbic acid.
Under acidification condition, nitrogen-fixational cyanobacteria grew fast. The growth rate of nitrogen-fixational cyanobacteria incubated under oxygenated environment higher than non-oxygenated environment.
CONCLUSION
Nutrient restriction and particle selection is a method to collect nitrogen-fixational cyanobacteria. Moreover, in the subtropical seawaters of the Northwest Pacific, nitrogen- fixational cyanobacteria present in greater than 20 microns fraction.
In the experiment of adding iron to culture nitrogen-fixing cyanobacteria, the growth rate of cyanobacteria increased in higher iron(II) concentration. However, ascorbic acid inhibits the growth rate and also slow down cell metabolism of nitrogen-fixing cyanobacteria. Furthermore, ascorbic acid reduce cyanobacteria to use C, N, P and trace metal elements.
Ocean acidification lead to increasing growth rate of N2-fixational cyanobacteria from the subtropical NW Pacific. Oxygen is an advantageous condition for the growth of the N2-fixational cyanobacteria from the subtropical NW Pacific.

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