臺灣博碩士論文加值系統

生物產氫研究近年來蓬勃發展，暗醱酵厭氧產氫為眾熱門研究之一，研究方向以不同菌種、碳源、調控pH值、溫度設置、反應槽設計、光照強度等嘗試提升氫氣產值。近期有研究顯示當添加不同奈米金屬有助於氫氣之生產，但因為奈米金屬本身的特性，對於微生物產氫表現之作用尚有負面影響；另一方面也有研究透過固定化技術將產氫微生物包埋於PVA-硼酸凝膠顆粒之中，其目的於保護生物於不佳的環境條件可以更穩定的生長。故本研究提議將兩方法合併使用，將不同的奈米金屬和暗醱酵細菌共同固定在PVA-硼酸凝膠顆粒中進行批次實驗，選用純菌Clostridium pasteurianum (CH5)作為暗醱酵產氫微生物，以探討奈米金屬和暗醱酵細菌共同固定產氫的效果。

本研究將分別添加四種不同奈米金屬NP-Fe、NP-Ni、NP-Ag及NP-Zn與C. pasteurianum進行共固定化厭氧醱酵產氫，每批次添加0、200、600、800、1000 mg/L金屬劑量以找到最利產氫濃度，實驗過程針對每一系統之Hydrogen Production Yield (HPY)、Hydrogen Production Rate (HPR)、Volatile Fatty Acids(VFAs)、氣體組成、殘餘糖量進行分析，以評估於此環境C. pasteurianum的生長狀態以及數據之合理性。再藉由分生技術觀察不同培養條件下之酵素活性。最後透過顆粒穩定性測試以探討固定化技術是否從頭到尾確實將C. pasteurianum包埋於PVA-硼酸凝膠顆粒之中，以及透過強酸破壞PVA-硼酸凝膠顆粒將奈米金屬回收之試驗。

從實驗結果得知，添加NP-Fe、NP-Ni、NP-Ag 於800 mg/L時，皆可得到最佳的HPY表現，分別為1.49、1.71及1.54 mol H2/mol glucose，相當於控制組增加27.4%、21.3%及11.6%，說明添加此三種金屬能使產氫表現更趨於理論值；添加NP-Zn時唯有 200 mg/L時有產氣表現，其HPY為1.17 mol H2/mol glucose，低於控制組3.3%。整體而言，產氫表現由優至劣分別為添加NP-Fe、NP-Ni、NP-Ag及NP-Zn，除NP-Zn以外，共同固定奈米金屬與C. pasteurianum應用於暗醱酵厭氧產氫是可行的。而HPR的部分各批次幾乎皆小於控制組，說明添加奈米金屬可能會導致產氫速率下降，但由於添加奈米鐵、鎳及銀仍然有助於產氣，加上先前實驗已確定奈米金屬是有助於電子的傳遞，故推測添加奈米金屬有助於生物產氫，可能是作用在增加電子傳遞的數量，而不是增加電子傳遞的速率。至於代謝途徑的部分，結果顯示添加NP-Fe、NP-Ni及NP-Ag可以增加HAc/HBu比值，使代謝副產物較趨於乙酸，亦說明了能產生較多的理論產氫量，而其中NP-Fe與NP-Ag之HAc/HBu甚大於1；添加NP-Zn的部分並不能改善其HAc/HBu比值，此結果與其產氫表現不佳一致。

共固定化與懸浮態培養CH5添加高濃度奈米鐵以探討兩者DNA/RNA和產氣之關係實驗部分，懸浮培養兩批次實驗之產氣表現RNA、DNA濃度數值具有相關性，添加NP-Fe 200 mg/L之RNA濃度與添加NP-Fe 800 mg/L皆在同一個級數，說明了添加更高濃度之NP-Fe也無法改變酵素活性的表現，與過去有無添加奈米鐵之研究結果一致，因為NP-Fe只是促進電子傳遞的媒介。而固定化培養兩批次實驗之DNA、總RNA濃度與其累積產氣量較無相關性，故推測此試驗結果因由實驗採樣方法不完善所致。

為探討固定化C. pasteurianum是否會隨實驗進行而流出，將培養液萃取DNA並進行PCR水平電泳之後，顯示在培養第16hr時有出現訊號，即代表菌液已從PVA硼酸凝膠顆粒中流出，而第16hr是開始產氣之時間點，故推測可能是產氣或是揮發性有機酸的生成以導致PVA-硼酸凝膠顆粒破裂，未來會再對於固定化技術進行改良研究。由奈米金屬回收實驗可得知，添加濃硫酸可以將PVA-硼酸凝膠顆粒溶解去除，但推測由於高濃度之硫酸亦會與NP-Fe起化學反應形成硫酸鐵及硫酸亞鐵，為避免奈米金屬溢出至環境，可以考慮利用添加適當濃度的硫酸以利於奈米金屬回收。

Anaerobic hydrogen production by dark fermentation has been commonly used for biogas production system recently. Most researchers discussed different types of bacteria, carbon sources, pH, temperature, reaction tank, light intension, etc. to increase the hydrogen production. Moreover, some scholars added nano-metals that proved to be helpful for hydrogen production. On the other hand, some scholars immobilized bacteria with PVA-boric gel granule, which can protect bacteria from environment. Therefore, in this study, different nano-metals and one selected dark-fermentation bacteria were co-immobilized in PVA-boric acid gel granules for possible hydrogen production in bactch experiments.

Batch experiments were carried out in the concentration of 0-1000 mg/L nano-metals including NP-Fe, NP-Ni, NP-Ag, and NP-Zn, to analyse Hydrogen Production Yield(HPY), Hydrogen Production Rate(HPR), Volatile Fatty Acids(VFAs), biogas composition, and residual sugar from glucose by co-immobilized nano-metals and C. pasteurianum in obligated anaerobic environments. Furthermore, the degree of enzyme performence can be analysed by biotechnologies. Finally, the integraty of granules would indicate whether CH5 and NP-metals be keeped in PVA-boric gel granules to improve hydrogen production.

In this study, HPY and HPR were measured to indicate that whether the amount or the speed of electron transfer could improve hydrogen production. The results showed that, in the concentration of 800 mg/L, the HPY were enhaced to be 1.49, 1.71, and 1.54 mol H2/mol glucose and 27.4%, 21.3%, and 11.6% higher than the control groups by NP-Fe, NP-Ni, and NP-Ag respectively, which means these NP-metals could make the hydrogen production performance approach to the theorical value. However, HPY was inhibited when nano-metals used was upto the 1000 mg/L. In addition, HPY decreased to 1.17 mol H2/mol glucose, which was 3.3% lower than the control when 200 mg/L of NP-Zn was added. Overall, in HPY analysis, NP-Fe was the best choice of nano-metal addition, NP-Ni was better than NP-Ag, and NP-Zn was the worst. Apart from NP-Zn, co-immobilizing specific nano-metal and anaerobic CH5 would be a feasibile study in hydrogen production. Moreover, in HPR analysis, the results were lower than the control groups in every batch experiments. It suggested that the amount of electron transfer, instead of the speed was the reason for stimulated biohydrogen production. On the other hand, the results showed the ratio of HAc/HBu with nano-metal addition was higher than the control groups in NP-Fe, NP-Ni, and NP-Ag additions and VFAs was mainly composed of acetic acid, rather than butyric acid. The ratio of HAc/HBu in NP-Fe and NP-Ag culture were both higher than one, which means the hydrogen production performance approached to the theorical value. Beside, the results showed the ratio of HAc/HBu was lower than the control group with NP-Zn addition.

The results showed the concentration of DNA and RNA were not only a positive correlation with bigas production but also in the same order with 200 mg/L and 800 mg/L of NP-Fe addition, which means nano-metal addition did not directly improve enzyme performance but did promote the transfer of electrons. The result was same as previous study. In addition, the results showed the concentration of DNA and RNA were non-correlation with bogas production with 200 mg/L and 800 mg/L of NP-Fe added in immobilization culture. The reason might due to improper sampling.

The results from structural stability test showed that after 16 hr operation there is DNA signal detected by PCR-electrophoresis. It represented that CH5 would leak out from the PVA-boric gel granule after 16 hr operation. Moreover, since 16 hr was also the time for significant gas production. It is possible that gas production loosen up the structure of granular. Recyling NP-metals test results showed that sulfic acid would dissolve PVA-boric gel granule to recover nano-metal. However, higher concentration of sulfic acid could promote chemical reaction to form ferric sulfate or ferrous sulfate. Therefore, an appropriate concentration of sulfuric acid to facilitate the recovery of metals is still not clear at this stage.

摘要 i
Abstract iii
目錄 v
表目錄 viii
圖目錄 ix
第一章 緒論 1
第一節 研究緣起 1
第二節 研究目的 3
第二章 文獻回顧 4
第一節 能源介紹 4
ㄧ、能源發展 4
二、環境衝擊 5
三、能源種類 6
四、永續發展 9
第二節 氫能介紹 11
一、氫能 11
二、生物產氫(Biohydrogen) 15
三、固氮酶(nitrogenase)與氫化酶(hyrogenase) 26
四、產氫微生物的種類 29
五、光暗醱酵菌共同培養產氫 30
第三節 暗醱酵產氫 31
一、暗醱酵產氫菌 31
二、梭菌屬(Clostridium sp.) 31
三、Clostridium sp.產氫機制 31
第四節 影響暗醱酵產氫之因素 33
一、基質(Substrate) 33
二、酸鹼值(pH) 34
三、揮發性有機酸(Volatile Fatty Acids ,VAFs) 34
四、溫度(Temperature) 34
五、氮源(Nitrogen source) 35
六、光照強度(Lihgt Intensity) 35
七、奈米金屬(Nano-partcles-metal, NPs-metal) 35
第五節 微生物固定化 36
一、微生物固定化 36
二、生物固定化方法 36
三、包埋法之凝膠機制 39
四、固定化技術的產氫應用 40
第六節 奈米材料對暗醱酵產氫影響 42
一、不同奈米材料顆粒於產氫之影響 42
二、奈米材料顆粒對於暗醱酵產氫影響之機制 46
第七節 閱讀文獻心得 47
第三章 材料與方法 48
第一節 實驗架構圖 48
第二節 實驗設備 49
第三節 實驗方法 50
一、菌種來源及培養條件 50
二、固定化方法 51
三、添加奈米金屬 52
第四節 分子生物技術 53
一、樣本前處理 53
二、去氧核醣核酸萃取(DNA Extraction) 53
三、核醣核酸萃取(RNA Extraction) 53
三、DNA(Deoxyribonucleic Acid)濃度測定 54
四、RNA(Ribonucleic Acid)濃度測定 54
五、聚合酶鏈鎖反應(Polymerase Chain Reaction, PCR) 54
六、瓊脂凝膠電泳(Agar Gel Electrophoresis) 55
七、變性梯度凝膠電泳(DGGE) 56
八、DNA(Deoxyribonucleic Acid)序列分析 57
第五節 分析方法 58
一、樣本採集及保存 58
二、O.D.(Optical Density)值與細胞乾重 58
三、累積氣體產量測定 59
四、氣體組成成分析 59
五、酸鹼值測定 61
六、揮發性脂肪酸測定 61
七、3,5-二硝基水楊酸(DNS)檢測還原糖 64
第四章 結果與討論 66
第一節 菌株菌種鑑定 66
第二節 共固定奈米金屬與CH5以探討奈米金屬濃度之影響 68
一、添加不同濃度奈米金屬-鐵(NP-Fe) 68
二、添加不同濃度奈米金屬-鎳(NP-Ni) 72
三、添加不同濃度奈米金屬-銀(NP-Ag) 76
四、添加不同濃度奈米金屬-鋅(NP-Zn) 80
五、綜合討論 84
第三節 不同培養條件下添加NP-Fe之產氫基因表現 86
一、懸浮態培養CH5與NP-Fe 86
二、共固定化培養CH5與NP-Fe 88
第四節 PVA-硼酸凝膠顆粒結構穩定性測試 90
第五節 奈米金屬回收測試 92
第五章 結論與建議 94
一、結論 94
二、建議 96
第六章 參考文獻 97

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