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dc.description.abstract近年來對於暗醱酵生物產氫之研究理論基礎已趨於完備,為了加強其實際應用,開發模場規模之反應槽操作技術,並且增加甲烷回收再利用便成為學術界積極研究之目標。反應槽中微生物族群對系統產氫與產甲烷效能有著密切的關係,其中Clostridium sp.被認定為暗醱酵產氫系統中最具有產氫潛力之微生物;而在產甲烷系統中主要為甲烷菌,但會因操作條件使菌群種類改變而影響效能。本研究乃利用PCR、DGGE、cloning、Real-time PCR等分子生物技術,針對「逢甲大學產氫團隊」所架設之具商業規模串聯CSTR 0.4 m3產氫槽與2.4 m3甲烷槽進行菌群結構以及目標菌群數量分析,以探討對其產氣效能之影響關係。此外,利用E/PMA-PCR方法探討本研究中微生物之活性進行可行性試驗。 菌群結果顯示,產氫槽所存在的微生物,Clostridium族群 以C. butyricum與C. tyrobutyricum為主,其他微生物包含有Klebsiella pneumoniae、Clostridiaceae bacterium、Desulfovibrio sp.、Dialister succinatiphilus、Bifidobacterium minimum、Ruminococcus sp.。在微生物數量結果顯示,前期Clostridium sp.數量約為105至106 gene copies/mL,其餘微生物族群數量約104至106 gene copies/mL,且產氫率隨著Clostridium sp.數量而受影響;後期Clostridium sp.數量降至105 gene copies/mL以下,其中Klebsiella族群數量甚至高於Clostridium族群,導致產氫速率下降,由此可知,產氫槽中微生物族群會相互競爭,但數量高於主要產氫族群Clostridium將會影響產氫效率。 另外在甲烷槽部分,隨著HRT降低產甲烷速率隨之上升,甲烷菌種類亦隨之變動,於HRT 67hr,主要以Methanosarcina mazei與Methanosaeta harundinacea為主,操作至76天前整體甲烷菌數量無明顯變化,約為104至105 gene copies/mL,當HRT降至24hr時,數量減少至104 gene copies/mL以下,顯示有部分甲烷菌被洗出(wash out)導致產甲烷能下降,但隨後又立即恢復甲烷菌數量以及產氣效能,表示本系統內甲烷菌之甲烷化恢復能力佳。 在E/PMA-PCR活性試驗中結果顯示,EMA與PMA皆可用於本研究活性偵測,且應用於產氫系統中可得知微生物皆有活性;此外,微生物會因取樣後隨時間增加而失去活性,並會使定性結果造成誤判,若以E/PMA進行活性判斷時,務必取樣後立即分析。zh_TW
dc.description.abstractIn recent years, theory of dark fermentation biohydrogen production has been well established. Focuses have been shifted to pilot-scale reactor operation as well as to increase methane production using effluent of biohydrogen reactor. System performance strongly correlates to the interaction of microbial community within the reactor. In dark fermentation system, Clostridium sp. is the predominant microorganism responsible for hydrogen production and methanogens is responsible for methane production. However, the microorganism community changes constantly as the operation condition differs. In order to understand the relationship between system performance and microorganism community, molecular biological techniques, including PCR-DGGE, cloning and real-time PCR were applied on sludge samples collected from a pilot-scale sequencing hydrogen/methane producing system operated by Fen Chia University. According to the results from PCR-DGGE, C. butyricum and C. tyrobutyricum were the major clostridia, which is responsible for the biohydrogen production. Others existed microorganisms included Klebsiella pneumonia, Clostridiaceae bacterium, Desulfovibrio sp., Dialister succinatiphilus, Bifidobacterium minimum, and Ruminococcus sp. Amount of Clostridium sp. cell count was around 105-106 gene copies/mL and the rest is about 104-106 gene copies/mL. Hydrogen production rate correlated with the number of Clostridium sp. On the late operational stage, the number of Clostridium sp. decrease to less than 105 gene copies/mL and the number of Klebsiella sp. cell count was higher than Clostridium sp. in agreement with a decreasing of hydrogen production. Clearly, the interaction of different microorganisms strongly affect the performance of biohydrogen production system. As for the methane tank, production rate increased as the HRT decreased. Composition of methanogens changed as well. The results showed that the Methanosarcina mazei and Methanosaeta harundinacea were the predominant methanogens when operated at HRT 67hr. No significant change on the methanogens number was observed for the first 79 days of operation. The amount of methanogens cell count was around 104-105 gene copies/mL. When changed to HRT 24hr, the number of methanogens decrease to less than 104 gene copies/mL but rebounded to the previous cell counts immediately. This is a demonstration that even though possible washed out happened, the system could be re-established in no time. In the E / PMA-PCR experiments on bacteria activity, the results showed that EMA and PMA can be used in hydrogen production system to reveal microbial activity. Furthermore, storage could be a strong factor on misleading the bacterial activity results.en_US
dc.description.tableofcontents摘要 i Abstract ii 目錄 iv 圖目錄 vii 表目錄 viii 第一章 前言 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 能源發展 3 一、 能源分類 4 二、 生質能 5 第二節 氫氣及甲烷 8 一、 氫氣介紹 8 二、 氫氣製備方法 12 三、 甲烷介紹 13 四、 甲烷製備方法 15 第三節 微生物產氣 16 一、 生物產氫 16 二、 產氫微生物 17 三、 生物產氫原理及機制 19 四、 暗醱酵產氫微生物 27 五、 厭氧生物產氫機制 29 六、 產甲烷微生物 34 七、 生物產甲烷原理及機制 34 第四節 影響厭氧微生物之因素 37 一、 pH值 37 二、 溫度 39 三、 有機酸 40 四、 氧氣 40 五、 氫氣 40 六、 醇類 41 七、 有機負荷 41 八、 水力停留時間 42 第五節 厭氧醱酵產氫系統 43 一、 CSABR反應器 43 二、 UASB反應器 43 三、 CIGSB反應器 44 第六節 分子生物技術對微生物活性之應用 46 第七節 文獻閱讀心得及研究方向擬定 47 第三章 材料與方法 48 第一節 實驗架構 48 第二節 實驗設備 49 第三節 實驗方法 50 一、 0.4 m3暗醱酵生物產氫反應槽 50 二、 2.4 m3暗醱酵生物產甲烷反應槽 52 第四節 分析方法 53 一、 樣本污泥前處理 53 二、 DNA萃取 53 三、 微生物菌群結構分析 54 四、 16S rDNA基因選殖 58 五、 親緣分析 60 六、 目標標菌群數量分析 60 第四章 結果與討論 63 第一節 0.4 m3暗醱酵產氫槽菌群結構分析 63 一、 總菌群結構分析 63 二、 Clostridium sp.菌群結構分析 69 三、 Real-time PCR檢測產氫槽目標菌群數量分析 72 四、 菌群結構變化與代謝副產物之探討 77 第二節 2.4 m3暗醱酵甲烷槽菌群結構分析 81 一、 Methanogens菌群結構分析 81 二、 Methanogens菌群數量分析 86 第三節 E/PMA對產氫槽微生物活性之可行性分析 89 第四節 綜合討論 91 一、 產氫系統 91 二、 產甲烷系統 92 三、 E/PMA活性試驗 92 第五章 結論與建議 93 第一節 結論 93 第二節 建議 94 參考文獻 95zh_TW
dc.subjectDark fermentationen_US
dc.titleThe Effect of Microbial Community Composition on a Pilot-Scale Sequencing Hydrogen/Methane Producing Systemen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
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