臺灣博碩士論文加值系統

利用微生物將廢棄有機物或生質體，經由生物轉化成乾淨能源氫氣，是一項創新且可提供永續能源的生物技術。本研究主要目的在開發適合連續式反應器操作之高產氫速率固定化細胞技術，希望藉由固定化產氫菌群，能夠穩定生物產氫反應及避免菌體洗出而造成反應器操作失敗的現象，並進一步提升醱酵產氫速率。
在固定化細胞技術的開發過程中，首先必須考量固定化細胞擔體的機械強度與產氫穩定性。因此經過多方嘗試後，選用矽膠合成高分子為主要固定化細胞擔體材料，再以矽膠(Silicone gel, SC)固定化細胞作為生物觸媒進行批次暗醱酵產氫實驗，探討碳源(葡萄糖、果糖、蔗糖及木糖)、基質濃度(5-40 g COD/l)及溫度效應(30-50 ℃)對產氫之影響。結果顯示，碳源以葡萄糖在基質濃度20 g COD/l下，有最佳的產氫速率(vH2)與氫氣產率(YH2)分別為130.7 ± 7.1 ml H2/h/g VSS和0.76 ± 0.04 mol H2/mol glucose，同時最佳的產氫操作溫度為40 ℃。SC固定化細胞經過重複批次馴養後能夠提升約1.1-2.3倍的產氫速率(vH2)。產氫生物動力學可用Andrews修正模式描述產氫速率與限量碳源濃度的關係。液相代謝物的組成分析結果顯示，矽膠固定化細胞能夠有效的進行生物產氫。
為了要提升固定化細胞產氫效率，因此將矽膠固定化細胞置入於上升管式流化床反應器內(Draft tube fluidized bed reactor, DTFBR)進行連續式醱酵產氫程序，並改變水力滯留時間(Hydraulic retention time, HRT) 8.9-2.2 h與蔗糖進料濃度(Cs)為5-40 g COD/l，探討對生物產氫之影響。結果顯示，隨著蔗糖濃度的增加或是HRT 的調降，產氫速率(vH2)也隨之增加，而氫氣產率(YH2)則是逐漸的下降。系統在Cs 40 g COD/l、HRT 2.2 h時有最佳vH2 為2.27 ± 0.13 l/h/l，另外，系統在Cs 40 g COD/l及HRT 8.9 h時有最佳YH2 為 4.98 ± 0.18 mol H2/mol sucrose)。另外， vH2 與有機進料速率(Organic loading rate, OLR)之關係式可用Monod-type 修正模式描述。產氫過程中氫氣濃度可保持在40%以上，主要之液態代謝物以乙酸(Acetic acid, HAc)與丁酸(Butyric acid, HBu)為主，分別佔總可溶性代謝物(Soluble microbial product, SMP)的16-22%、62-73%。利用DTFBR進行產氫長期操作時擁有穩定的產氫狀態與產氫再現性，當產氫不穩定時可利用熱處理方法(70-80 ℃、1 h)快速恢復產氫效能。
為了要實際應用生物氫氣於燃料電池上，因此設計一個連續攪拌式厭氧生物反應器(Continuously stirred anaerobic bioreactor, CSABR)，並填入以矽膠固定化細胞為生物觸媒進行醱酵產氫。系統進料以蔗糖(Sucrose)為單一碳源，HRT與基質濃度分別控制在6 h與30 g COD/l。結果顯示，系統之最佳產氫速率(vH2)與氫氣產率(YH2)分別為1.15 ± 0.08 l/h/l和3.71 ± 0.18 mol H2/mol sucrose，另外系統也可操作達300天以上。CSABR所產生之生物氣體經由CO2吸收劑與矽膠除水裝置後，可得純度達> 99%之氫氣，氫氣(1.72 l/h)再經由燃料電池系統(Proton exchange membrane fuel cell, PEMFC)可產生3.30 ± 0.04 V的穩定電壓，如果接上一個小型的發光二極體(Light emitting diode, LED)面板，就可產生0.87W (25 ℃)的能量，其中電壓與電流分別為2.28 V與0.38 A。

Using anaerobic microorganisms for hydrogen production from waste organics or biomass have become an innovation and promising biotechnology. Moreover, biohydrogen production combined with fuel cells could commit the increased demand of renewable hydrogen for the transition to the Hydrogen Economy. The object of this study was to find a new material to immobilized H2-producing bacteria, which also can be applied in continuously bioreactors. The immobilized cells can not only maintain or increase the hydrogen production rate, but also avoid the biomass washout at low hydraulic retention time (HRT), which results in the failed operation during the long-term fermentative hydrogen production.
Under the development of cell immobilization researches, the mechanism and the stability of immobilized cells for hydrogen production have to be considered. In this study, a novel synthetic polymer (Silicone gel, SC) was used to immobilize acclimated sewage sludge for fermentative hydrogen production. Using glucose, fructose, sucrose and xylose as the sole carbon substrate, respectively, our laboratory developed SC immobilized cells achieved a higher hydrogen production rate (vH2) of 130.7 ± 7.1 ml H2/g VSS and the best substrate-based yield (YH2/glucose) of 0.76 ± 0.04 mol H2/mol glucose at substrate concentration of 20 g COD/l. Operation at temperature 40 oC resulted in the highest hydrogen production rate. Acclimation of the sewage sludge allowed up to 1.1-2.3 folds enhancement on the performance of hydrogen production. Kinetic studies showed that modified Andrews model was able to describe the dependence of specific hydrogen production rate on substrates concentration. The composition of soluble metabolites was found to be a reliable indicator for the efficiency of biohydrogen production.
For practical application of SC immobilized cells in a continuously bioreactor, a draft tube fluidized bed reactor (DTFBR) was designed to produce H2. The SC immobilized cells were used as the biocatalyst for H2 production in DTFBR with a working volume of 8 l. The DTFBR system was operated at HRT of 2.2-8.9 h and an influent sucrose concentration (Cs) of 5-40 g COD/l. The results showed that in general decreasing HRT or increasing sucrose concentration led to a marked increase in the volumetric H2 production rate (vH2), but a gradual decrease in the H2 yield (YH2). The best vH2 (2.27 ± 0.13 l/h/l) occurred at Cs 40 g COD/l and HRT 2.2 h, whereas the highest YH2 (4.98 ± 0.18 mol H2/mol sucrose) was obtained at Cs 40 g COD/l and HRT 8.9 h. The correlation between the production rate and the organic loading rate (OLR) could be successfully described by modified Monod-type models. The H2 content in the biogas was stably maintained at over 40%. The major soluble products were butyric acid and acetic acid, as they accounted for 62-73% and 16-22% of total soluble microbial products (SMP), respectively. The H2-producing performance in the DTFBR system could be stably maintained and reproducible in long-term operations, while unstable operations could be quickly recovered via proper thermal treatment at 70-80 oC for 1 h.
For further practical application of biohydrogen in proton-exchange-membrane fuel cell (PEMFC) system, a dark H2 fermentation process was designed and integrated with PEMFC for on-line electricity generation. The H2 producing system was a continuously stirred anaerobic bioreactor (CSABR) seeded with SC immobilized sludge. The CSABR system, using sucrose as the sole carbon substrate, was able to continuously and stably produce H2 for over 300 days at HRT of 6 h and an influent sucrose concentration of 30 g COD/l. The maximum H2 production rate and the best H2 yield were 1.15 ± 0.08 l/h/l and 3.71 ± 0.18 mol H2/mol sucrose, respectively. The H2 produced from the CSABR system was purified via a CO2 absorber and a silica-gel desiccator, and then the > 99 % pure H2 was fed into a PEMFC system at a rate of 1.72 l/h, generating electricity with a stable electromotive force of 3.30 ± 0.04 V. While connecting to a small light emitting diode (LED) panel, the output power was ca. 0.87 W (at 25 oC), and the output voltage and current were stably maintained at 2.28 V and 0.38 A, respectively.

中文摘要 ………………………………………………………………I
英文摘要 ………………………………………………………………III
目錄 …………………………………………………………………VI
圖目錄 …………………………………………………………………X
表目錄 ………………………………………………………………XII
符號說明 ……………………………………………………………XIII

第一章 序論 …………………………………………………………1
1-1 前言 ………………………………………………………………1
1-2 研究目的與反應器演化流程 ……………………………………3

第二章 文獻回顧 ………………………………………………………6
2-1 微生物產氫研究 ……………………………………………6
2-2 產氫微生物 ……………………………………………………7
2-3 暗醱酵產氫 …………………………………………………11
2-3-1 暗醱酵生物產氫代謝途徑 ………………………………11
2-3-2 暗醱酵生物產氫反應機制 ………………………………13
2-4 Clostridia ……………………………………………………15
2-4-1 Clostridia的特徵 …………………………………………15
2-4-2 Clostridium的產氫能力 …………………………………17
2-5 Klebsiella ……………………………………………………18
2-5-1 兼性厭氧菌Klebsiella的特徵 …………………………18
2-5-2 兼性厭氧菌Klebsiella的產氫能力 ……………………19
2-6 固定化細胞技術 ……………………………………………22
2-6-1 固定化細胞的定義 ………………………………………22
2-6-2 固定化細胞的方法與分類 ………………………………22
2-6-3 固定化細胞擔體比較 ……………………………………24
2-6-3-1 天然固定化擔體 ……………………………………24
2-6-3-2 合成擔體 ……………………………………………26
2-6-4 固定化細胞應用於生物反應器特性 ……………………29
2-7 生物產氫動力學 ……………………………………………31
2-8 生物產氫反應器 ……………………………………………33
2-8-1 光生物反應器 ……………………………………………33
2-8-2 暗醱酵生物反應器 ………………………………………36

第三章 批次醱酵產氫 ………………………………………………61
摘要 …………………………………………………………………61
3-1 研究前言 ……………………………………………………62
3-2 研究方法與步驟 ……………………………………………63
3-2-1 產氫菌群 ………………………………………………63
3-2-2 產氫菌群固定化細胞製備 ……………………………64
3-2-3 基質配方 ………………………………………………64
3-2-4 產氫菌群固定化細胞批次產氫操作步驟 ……………67
3-2-5 分析方法 ………………………………………………68
3-3 結果與討論 …………………………………………………68
3-3-1 未經馴養之矽膠固定化細胞產氫表現 ………………68
3-3-2 馴養後之矽膠固定化細胞產氫表現 …………………69
3-3-3 碳源與基質濃度對矽膠固定化細胞產氫之影響 ……71
3-3-4 固定化細胞產氫動力學 ………………………………73
3-3-5 溫度對產氫之影響 ……………………………………75
3-3-6 不同碳源與濃度下之液相
可溶性代謝物與糖利用率變化情形 …………………75
3-3-7 六碳糖/五碳糖之菌相分析 ……………………………78
3-4 結論 …………………………………………………………79

第四章 上升管式流體化床醱酵產氫 ………………………………83
摘要 …………………………………………………………………83
4-1 研究前言 ……………………………………………………84
4-2 研究方法與步驟 ……………………………………………85
4-2-1 產氫活性污泥 …………………………………………85
4-2-2 固定化細胞製備 ………………………………………85
4-2-3 基質配方 ………………………………………………85
4-2-4 分析方法 ………………………………………………85
4-2-5 DTFBR啟動及生物產氫操作 …………………………86
4-3 結果與討論 …………………………………………………88
4-3-1 DTFBR反應器特性 ……………………………………88
4-3-2 熱處理對產氫之影響 …………………………………..90
4-3-3 生物產氫再現性 ………………………………………91
4-3-4 蔗糖濃度與HRT對產氫之影響 ……………………….94
4-3-5 DTFBR中可溶性代謝物與糖利用率變化情形 ……….98
4-4 結論 …………………………………………………………100

第五章 暗醱酵產氫與燃料電池之即時發電系統 …………………101
摘要 …………………………………………………………………101
5-1 研究前言 ……………………………………………………102
5-2 研究方法與步驟 …………………………………………103
5-2-1 產氫活性污泥 …………………………………………103
5-2-2 固定化細胞製備 ………………………………………103
5-2-3 基質配方 ………………………………………………103
5-2-4 分析方法 ………………………………………………103
5-2-5 CSABR啟動及生物產氫操作 ……………………104
5-2-6 質子交換膜燃料電池(PEMFC) ………………………104
5-3 結果與討論 …………………………………………………106
5-3-1 CSABR生物產氫系統 ………………………………106
5-3-2 PEMFC發電 …………………………………………109
5-3-3 生物產氫發電可行性 …………………………………110
5-4 結論 ………………………………………………………115
第六章 總結與未來展望 ……………………………………………117
6-1 總結 ………………………………………………………117
6-2 未來展望 ……………………………………………………120

參考文獻 ……………………………………………………………123
附錄一 個人簡歷(Curriculum Vitae) ………………..…………………i

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