臺灣博碩士論文加值系統

依照人類利用能源的速度來看，化石燃料即將耗盡，故發展替代性能源是刻不容緩的事，而氫氣是一種具發展潛力之替代能源，故許多國家將氫氣生產納入政策當中；利用生物方式產生氫氣是一種不會污染環境且不需另外消耗能源之氫氣生產方式，具有產氫能力之微生物有許多種，其中藍綠菌由於可利用光能產生氫氣，同時消耗二氧化碳減少溫室效應等優點，適合做為研究生物產氫之材料。
本研究將藍綠菌株CH1、CH2、CH3培養於好氧光照之培養槽內，使菌株大量生長後，進行生物產氫批次實驗，探討不同生長期之細胞、不同種類及濃度之碳源添加，氮源有無、厭氧或好氧，及光照或黑暗等條件下之生物產氫能力。
結果顯示，對數生長期之菌株CH1於未添加碳源與氮源之最大氫氣累積量為4 mL H2 / 100 mL Head Space，而對數生長後期之菌株CH3在添加4% 二氧化碳培養條件下之最大氫氣累積量，於66小時為8 mL H2 / 100 mL Head Space。
於添加不同濃度碳源之批次實驗結果顯示，菌株CH1與CH3培養於添加各種濃度醋酸鈉之氫氣累積量皆小於1 mL H2 / 100 mL Head Space。而添加1500 mg/L葡萄糖後菌株CH3於150小時後開始產生氫氣，最大氫氣累積量達23.1 mL H2 / 100 mL Head Space；添加2000 mg/L果糖之菌株CH3最大氫氣累積量達到88 mL H2 / 100 mL Head Space。
添加固定濃度碳源進行生物產氫批次實驗發現，（1）菌株CH1於添加2000 mg/L果糖之基質利用率最高，且氫氣累積量可達79 mL H2 / 100 mL Head Space，而添加蔗糖之氫氣累積量達32 mL H2 / 100 mL Head Space，但添加半乳糖、鼠李糖、蔗糖、乳糖之基質利用率皆小於10%，且不會產氫。（2）菌株CH3於添加2000 mg/L果糖時，基質利用率高達97.5%，且氫氣累積量達79 mL H2 / 100 mL Head Space，添加蔗糖之基質利用率高於60%，氫氣累積量達55 mL H2 / 100 mL Head Space，添加葡萄糖之基質利用率亦高達90%，而氫氣累積量為27 mL H2 / 100 mL Head Space，添加半乳糖、鼠李糖、蔗糖、乳糖之基質利用率為40% 至60%，氫氣累積量皆小於10 mL H2 / 100 mL Head Space。（3）菌株CH2不具產氫能力。
在對數生長期添加2000 mg/L果糖之生物產氫批次實驗發現，菌株CH1於添加2000 mg/L果糖後，氫氣累積量最高達到25.1 mL H2 / 100 mL Head Space，細胞生質量增加量為759 mg/L。菌株CH3於添加2000 mg/L果糖後，氫氣累積量最高達到27.6 mL H2 / 100 mL Head Space，細胞生質量增加量為522 mg/L；菌株CH2則不具產氫能力。
在同時添加2000 mg/L果糖與660 mg/L氨氮後，菌株CH1與CH3皆不會氣體，菌株CH1與CH3之果糖與氨氮含量下降至一定濃度後不再下降，有機酸含量亦較未添加氨氮者低。
在好氧條件下，添加2000 mg/L果糖後菌株CH1與CH3之基質利用率高達98%以上，且碳源在120小時內即利用完全，不會產生氫氣。
培養至對數生長後期之菌株CH1與CH3，在黑暗下之氫氣累積量約為8 mL H2 / 100 mL Head Space，較光照下對數生長後期之菌株其氫氣累積量為低。
綜合結果發現，於厭氧光照下，對數生長後期之菌株CH1與菌株CH3，在添加2000 mg/L果糖且不添加氮源之條件下，生物產氫能力較其他條件為強；菌株CH2則不具生物產氫能力。

Hydrogen evolution by cyanobacteria is a potential way of biohydrogen production for the future. Nitrogen-fixing cyanobacteria are photosynthetic organism with simple need, using CO2 and N2 as source of carbon and nitrogen, and sunlight as their source of energy. Cyanobacteria are able to evolve H2 in light catalysed by nitrogenase activity, with water serving as the primary electron donor.
Since the optimal operating conditions for the CO2 uptake and H2 production are different, a two-stage system can be effectively employed to separate these two phases. In this study, Anabaena sp. CH1, CH2, and CH3 has been isolated and purified from paddy soils of middle Taiwan and the physiology of nitrogen metabolism of these two species has been studied, ether. These cyanobacterium were incubated in 4% CO2 and 96% air with 40 Wm2 intensity of light in the 150 mL volume reactors. After cultured a pried of time, cyanobacterium was translated into a 60 mL volume batch reactor proceeded a series of biohdyrogen batch experiments such as different kinds and concentrates of carbon source, log or later growth phase, with or without 660 mg/L ammonium, anaerobic or aerobic, light or dark, and different species of cyanobacterium.
When 2000mg/L carbohydrates, such as fructose, glucose, galactose, rhamnose, lactose, and sucrose, been used as carbon source, it indicated that fructose is one of the most helpful inducer to product hydrogen.
Cumulative H2 production was higher when cyanobacterium growing in later phase then in log phase. And there was no H2 production when incubated in 660 mg/L ammonium, nether in the dark.
Over all, it was found that when added 2000 fructose under argon as gas phase with 5 Wm2 intensity of light and growth in later phase, cyanobacterium CH3 produce a maximum cumulative hydrogen pruduction as 80 mL H2 / 100 mL Head Space without oxygen co production.

目 錄
摘要 I
Abastrate III
目錄 V
表目錄 X
圖目錄 XI
第1章 前言 1
第2章 文獻回顧 2
2-1 研究源起 2
2-1-1 能源危機 2
2-1-2 氫能源之優點 2
2-1-3 氫能源利用 3
2-1-4 氫氣生產方式 3
2-2 生物產氫 4
2-2-1 具有產生氫氣能力之微生物種類 4
2-2-2 藍綠菌產氫之優點 5
2-3 藍綠菌之分類、光合作用與固氮作用 7
2-3-2 藍綠菌之光合作用 8
2-3-3 藍綠菌之固氮作用 8
2-4 藍綠菌之生物產氫作用 9
2-4-1 藍綠菌之生物產氫機制 9
2-4-2 藍綠菌產氫之酵素系統 12
2-4-3 藍綠菌產生氫氣之影響因子 14
2-5 菌株CH1、CH2、CH3之研究 16
第3章 研究架構 18
第4章 材料與方法 19
4-1 菌種來源、純化與保存方式 19
4-1-1 菌種來源 19
4-1-2 菌種純化 19
4-1-3 菌種保存/純化固體培養基 20
4-1-4 菌種保存/增殖液體培養基 21
4-2 菌種培養 23
4-2-1 菌種培養槽架設情形 23
4-2-2 菌種培養槽生長曲線測定 24
4-3 批次實驗 26
4-3-1 批次實驗進行之環境條件與流程 26
4-3-1-1 批次實驗進行之環境條件 26
4-3-1-2 批次實驗流程 27
4-3-2 厭氧光照下，不同生長期菌株CH1與CH3未添加碳氮源生物產氫批次實驗 30
4-3-3 厭氧光照下，不同生長期菌株CH1與CH3於氣相中添加4% 二氧化碳之生物產氫批次實驗 30
4-3-4 厭氧光照下，對數生長後期菌株CH1與CH3添加不同濃度醋酸鹽及醣類之生物產氫批次實驗 30
4-3-5 厭氧光照下，對數生長後期菌株CH1、CH2、CH3添加單糖及雙糖之生物產氫批次實驗 30
4-3-6 厭氧光照，對數生長期菌株CH1、CH2、CH3之生物產氫批次實驗 31
4-3-7 厭氧光照下，對數生長後期菌株CH1與CH3添加果糖及氨氮之生物產氫批次實驗 31
4-3-8 好氧光照下，對數生長後期菌株CH1與CH3添加果糖之生物產氫批次實驗 31
4-3-9 厭氧黑暗下，對數生長後期菌株CH1與CH3添加果糖之生物產氫批次實驗 31
4-4 分析方法 33
4-4-1 細胞濃度 33
4-4-2 葉綠素含量 33
4-4-3 pH值分析 34
4-4-4 細胞生質量 34
4-4-5 氣體含量分析 34
4-4-6 化學需氧量-COD值分析 36
4-4-7 果糖含量分析 37
4-4-8 氨氮含量分析 38
4-4-9 有機酸分析 39
4-4-10 異形細胞頻率分析 40
4-4-11 葡萄糖含量分析 41
4-5 實驗用水及鹼洗液 41
第5章 結果與討論 42
5-1 菌種於培養槽內生長曲線測定 42
5-2 厭氧光照下不同生長期菌株CH1與CH3未添加碳氮源與於氣相中添加4% 二氧化碳之生物產氫批次實驗 45
5-2-1 不同生長期菌株CH1與CH3未添加碳氮源之產氫批次實驗 45
5-2-2 不同生長期菌株CH1與CH3於氣相中添加4% 二氧化碳之生物產氫批次實驗 46
5-3 厭氧光照下，對數生長後期菌株CH1與CH3添加不同濃度醋酸鹽及醣類之生物產氫批次實驗 48
5-3-1 菌株CH1與CH3添加不同濃度醋酸鈉之生物產氫批次實驗 49
5-3-2 菌株CH1與CH3添加不同濃度葡萄糖之生物產氫批次實驗 51
5-3-3 菌株CH1與CH3添加不同濃度果糖之生物產氫批次實驗 53
5-4 厭氧光照下，對數生長後期菌株CH1、CH2、CH3添加單糖之生物產氫批次實驗 55
5-4-1 菌株CH1、CH2、CH3添加2000 mg/L果糖之生物產氫批次實驗 55
5-4-2 菌株CH3添加2000 mg/L葡萄糖之生物產氫批次實驗 64
5-4-3 菌株CH1與菌株CH3添加2000 mg/L半乳糖生物產氫批次實驗 67
5-4-4 菌株CH1與菌株CH3添加2000 mg/L鼠李糖生物產氫批次實驗 70
5-5 厭氧光照下，對數生長後期之菌株CH1與CH3添加雙糖生物產氫批次實驗 73
5-5-1 菌株CH1與CH3添加2000 mg/L乳糖之生物產氫批次實驗 73
5-5-2 菌株CH1與CH3添加2000 mg/L蔗糖之生物產氫批次實驗 76
5-6 厭氧光照下，對數生長後期菌株CH1與CH3添加不同碳源之生物產氫能力比較 79
5-7 厭氧光照下，對數生長期菌株CH1、CH2、CH3添加果糖之生物產氫批次實驗 84
5-7-1 厭氧光照下，對數生長期菌株CH1、菌株CH2、菌株CH3添加2000 mg/L果糖之生物產氫批次實驗 84
5-7-2 厭氧光照下，對數生長期與對數生長後期菌株CH1、CH2、CH3添加果糖之生物產氫能力比較 88
5-8 厭氧光照下，對數生長後期菌株CH1與CH3添加果糖及氨氮之生物產氫批次實驗 90
5-8-1 厭氧光照下，對數生長後期菌株CH1與CH3只添加碳源與同時添加碳源氮源之生物產氫能力比較。 90
5-9 好氧光照下，對數生長後期菌株CH1與CH3添加果糖之生物產氫批次實驗 95
5-9-1 厭氧與好氧光照下，對數生長後期菌株CH1與CH3添加果糖之生物產氫能力比較 95
5-10 厭氧黑暗下，對數生長後期菌株CH1與CH3添加果糖之生物產氫批次實驗 100
5-10-1 厭氧光照與黑暗下，對數生長後期菌株CH1與CH3添加果糖之生物產氫能力比較 100
第6章 結論與建議 102
6-1 結論 102
6-2 建議 103
參考文獻 104

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