臺灣博碩士論文加值系統

生物產氫是微生物分解有機物的過程中產生氫氣的代謝途徑，包含生物光解、光醱酵及暗醱酵，其中以暗醱酵產氫最具發展效益，於厭氧環境進行糖解發酵等代謝產生氫氣與二氧化碳，過程中也會產生具有價值的醇類與揮發性有機酸。然而，生物產氫受限於微生物作用故近期學者開始研究添加奈米材料以提升微生物產氫效率。奈米碳管(carbon nanotube, CNT)是種特殊的碳材料，具有高比表面積、良好導電性及對機械、化學與熱抗性高等特性，故有作為生物催化的潛能。

Clostridium pasteurianum是暗醱酵菌中常見的研究菌種，為革蘭氏陽性短桿狀菌，生長為絕對厭氧的環境下且會產生內生胞子。本研究選用Clostridium pasteurianum(CH5)純菌株，與經修飾改良的多壁奈米碳管(MWCNT-COOH)進行共培養暗醱酵產氫研究。分別添加不同濃度0、200、600、800、1000 mg/L之MWCNT-COOH與CH5進行共培養，分析生物累積產氣量、氣體組成比、Volatile Fatty Acids(VFAs)、葡萄糖利用率等，找尋最佳的共培養添加濃度。並經由胞子染色法觀察，其CH5於不同環境濃度中之胞子產生情形，再依最佳濃度進行分生檢測、產氫酵素活性分析，最後探討當將MWCNT-COOH與CH5進行共固定化顆粒的可行性。

從實驗結果得知，添加濃度為1000 mg/L可得到最大生物產氣量175.4 mL，且高於控制組8%。最高氫氣產量則為添加200與1000 mg/L時約為66 mL。產氫量(Hydrogen production yield, HPY)的表現也以添加200與1000 mg/L時表現較佳分別為 1.06與1.07 mol H2/mol glucose。而添加200 mg/L時，其產氫率(Hydrogen production rate, HPR)是當中最大值0.92 H2 L/L/day。此外，添加MWCNT-COOH能促使CH5之代謝途徑趨向乙酸發展，且其對CH5的作用為可以提升產氫代謝的電子傳遞速率。故添加MWCNT-COOH於CH5中能提升其產氫表現。

Biohydrogen could be produced by microbial decomposition of organic matter. These metabolic processes include biophotolysis, photofermentation and dark fermentation. Under anerobic conditions, slected bacteria can convert glycolysis to hydrogen and carbon dioxide through dark fermentation, and even produce valuable alcohols and volatile organic acids, which have development potential. However, the efficiency of biohydrogen production is limited due to limited operation condition. Some researchers have successfully improved hydrogen production efficiency by adding nanoparticles to grow/stimulate with microorganism. Carbon nanotube (CNT) as unique form of carbon material has potential for biocatalysts, such as good electrical conductivity, high surface area, and high resistance of mechanical, thermal, and chemical.

In this study, it is desirable to investigate the hydrogen production by co-culture of Clostridium pasteurianum (CH5) and multi-walled carbon nanotubes with carboxylic acid groups (MWCNT-COOH). The effect of different MWCNT-COOH concentrations (0, 200, 600, 800 and 1000 mg/L) on hydrogen production is investigated. Analyzing accumulative biogas production, biogas composition, Volatile Fatty Acids (VFAs), and glucose utilization were conducted to find out the best operation concentration. Also, endospore stain as well as activity of enzyme performance were tested to reveal the effect of nanotube on the growth of CH5.

The results showed that maximum total biogas production of 175.4 mL can be obtained when adding 1000 mg/L of MWCNT-COOH. Adding the MWCNT-COOH of 200 and 1000 mg/L would obtain maximum hydrogen gas production of about 66 mL, and also have the better hydrogen production yield (HPY),1.06 and1.07 mol H2/mol glucose, respectively. Then, when adding the MWCNT-COOH of 200 mg/L have the maximum hydrogen production rate (HPR) 0.92 H2 L/L/day.

In addition, addition of MWCNT-COOH can promote the metabolic pathway to produce acetic acid, and it even can increase the electron transfer rate for hydrogen production. Therefore, the addition of MWCNT-COOH to CH5 can improve its hydrogen production performance.

摘要 i
Abstract ii
目錄 iii
表目錄 v
圖目錄 vi
第一章 緒論 1
第一節 研究緣起 1
第二節 研究目的 2
第二章 文獻回顧 3
第一節 能源現況與發展 3
一、 非再生能源(Non- Renewable Energy Source, NRES) 4
二、 再生能源(Renewable Energy Source, RES) 5
第二節 生物產氫 7
一、 生物光解(Biophotolysis) 7
二、 光醱酵(Photofermentation) 8
三、 暗醱酵(Dark fermentation) 9
第三節 Clostridium sp.產氫機制 10
第四節 暗醱酵產氫影響因子 12
一、 基質 12
二、 pH值 12
三、 副產物 12
四、 溫度 13
五、 營養源(氮、磷、硫) 13
六、 金屬離子 14
第五節 奈米材料對生物產氫的影響 15
一、 無機奈米材料對暗醱酵產氫影響 15
二、 有機奈米材料對暗醱酵產氫影響 16
第六節 文獻閱讀心得 18
第三章 材料與方法 19
第一節 實驗架構 19
第二節 實驗設備 20
第三節 實驗方法 21
一、 菌種來源與培養條件 21
二、 奈米碳管添加 22
三、 固定化顆粒製備 23
第四節 分析方法 24
一、 累積生物產氣量檢測 24
二、 生物氣體組成比分析 24
三、 酸鹼值(pH)檢測 25
四、 揮發性有機酸分析 25
五、 葡萄糖利用率分析 27
六、 總有機碳分析 28
第五節 分子生物技術 29
一、 DNA萃取 29
二、 RNA萃取 30
三、 產氫酵素基因表現 31
第六節 顯微鏡觀察 32
一、 內胞子染色法(endospore stain) 32
第四章 結果與討論 33
第一節 添加不同濃度之奈米碳管對CH5產氫影響 33
第二節 添加不同濃度奈米碳管之內胞子染色觀察 40
一、 控制組 40
二、 添加200 mg/L之奈米碳管 41
三、 添加600 mg/L之奈米碳管 42
四、 添加800 mg/L之奈米碳管 43
五、 添加1000 mg/L之奈米碳管 44
六、 綜合討論 45
第三節 奈米碳管添加對CH5之基因與活性酵素表現 46
一、 DNA與RNA濃度比 46
二、 qPCR檢測 48
三、 綜合討論 49
第四節 共固定奈米碳管與CH5之可行性 50
一、 共固定化培養CH5與MWCNT-COOH 50
二、 懸浮與固定化產氫表現比較 53
第五章 結論與建議 55
第一節 結論 55
第二節 建議 57
第六章 參考文獻 58

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