李篤中臺灣大學：化學工程學研究所丁健弘Ting, Chien-HungChien-HungTing2007-11-262018-06-282007-11-262018-06-282004http://ntur.lib.ntu.edu.tw//handle/246246/52162中文摘要 本論文討論前處理及加入不同微生物群對污泥厭氧消化程序之影響。前處理包含增溫、凍融及調整酸鹼值等；加入微生物菌群則包含梭狀芽孢桿菌群(Clostridium)及自基隆河底泥採集之甲烷生成優勢菌種(K8)。由代謝物分析結果可了解厭氧消化的代謝現象，而分子生物技術(PCR & DGGE)則用來追蹤消化過程中菌群之變化。 研究結果發現，經過前處理後污泥的確有加快代謝的效果。樣品在厭氧消化初期進行水解和酸化時會累積少陴B氣(0.6 g H2/kg Dry Solid)，但稍後即快速消失。待累積一段時間的揮發酸後，甲烷化才會開始進行，且都以代謝乙酸開始，接著再進行丙酸或丁酸代謝，代謝發生同時都可以發現甲烷累積量明顯的增加。 原始污泥加入clostridium培養液後，即可加速污泥厭氧消化，其產氫和甲烷化速度與鹼化後污泥差不多，此兩種樣品在168小時後就會快速的累積甲烷。而凍融後污泥則在約240小時時才會開始累積甲烷，前處理加速代謝的效果在加入梭狀芽孢桿菌培養液的樣品中並不明顯。 但前處理對加入K8後知污泥則有顯著的效果。因為前處理幫助水解，使污泥甲烷化速度加快，而污泥(加K8)又比原始污泥厭氧代謝速度快，證明前處理及加入厭氧菌群都可以加速甲烷化。 微生物菌群分析發現，厭氧消化初期污泥內以消化水解產物生成酸類之微生物菌群為主，如:Clostridium sp.、Lactobacillus sp.等。Abstract This thesis discusses the influence of pretreatment and microbial inoculums on sludge. The pretreatments include sterilization, frozen/thawed, acidification, and alkalization. The microbial inoculums include clostridium and K8 inoculums. Based on the HPLC results we analyzed, the digestion pathway of pathway and biotechnology experiments (PCR&DGGE) assisted to show the microbial society change. The results show that the pretreated sludge increases digestion rate. The sample accumulates hydrogen initially, but was consumed fast. The volatile acid accumulates for a period of time, then the acetic acid was consumed ollowed by propionic acid. The methane was also produced at the same time. The original sludge, which was added clostridium inoculums, increases anaerobic digestion rate. The rate is almost the same with the alkalized sludge, and both of the samples accumulates methane faster after 168 hours of digestion. The frozen/thawed sludge accumulates methane after 240 hrs. Therefore the avail is not very clear by pretreatment in this case. The avail is very clear when added the K8 into the sludge. The pretreatments help hydrolysis, and the sludge added K8 produces methane faster than the original sludge. This result proves that the pretreatments and sludge added K8 can make the anaerobic digest faster. The DGGE results show that the pretreatment will influence the growth of bacteria. In the hydrolysis pretreatments (sterilization, alkalization), the bacterium grew very well; and the destructive pretreatments (like acidification, sterilization) kills the bacterium. When the initial sludge digestion, the bacteria are mainly acidgenic bacterium, like clostridium sp., and lactobacillus sp.目錄 頁次 中文摘要……………………………………………………………………….. Ⅰ 英文摘要……………………………………………………………………….. Ⅱ 目錄 …………………………………………………………………………… Ⅲ 圖目錄 ………………………………………………………………………… Ⅴ 表目錄 ………………………………………………………………………… Ⅸ 第一章 前言…………………………………………………………………… 01 第二章 文獻回顧……………………………………………………………… 02 2-1污泥問題與再利用 ………………………………………………….... 02 2-2厭氧消化 …………………………………………………………….... 03 2-2-1 厭氧消化產甲烷代謝路徑……………………………………... 04 2-2-2 產甲烷菌群……………………………………………………… 10 2-3利用分子生物技術追蹤污泥中微生物菌群…………………………. 11 2-3-1 聚合酵素鏈鎖反應(PCR)的原理與應用………………………. 11 2-3-2變性梯度凝膠電泳(DGGE) ……………………………………. 13 第三章 實驗方法 …………………………………………………………….. 15 3-1 污泥與菌種 ………………………………………………………….. 15 3-1-1污泥來源與性質 ………………………………………………. 15 3-1-2菌種來源………………………………………………………... 15 3-2 前處理方法…………………………………………………………… 17 3-3 厭氧消化實驗 ……………………………………………………….. 18 3-4分析方法………………………………………………………………. 19 3-5 分子生物技術（PCR&DGGE）……………………………………… 23 3-5-1污泥中微生物DNA萃取……………………………………….. 24 3-5-2 PCR之操作條件………………………………………………… 24 3-5-3 變性梯度凝膠電泳DGGE……………………………………… 26 第四章 結果與討論…………………………………………………………… 30 4-1 前處理對樣品的影響……………………………………..…………... 30 4-2 加入不同菌群對原始污泥之影響………………………..…………... 34 4-3 污泥厭氧消化實驗Ⅰ……………………………………..…………... 39 4-3-1 厭氧消化產氣…………………………………………………… 39 4-3-2 代謝物分析……………………………………………………… 42 4-3-3 鹼化麵條實驗…………………………………………………… 52 4-4 污泥厭氧消化實驗Ⅱ………………………………………..………... 55 4-4-1 厭氧消化產氣…………………………………………………… 55 4-4-2 厭氧消化代謝物分析…………………………………………… 59 4-5樣品微生物菌群監測………………………………………..………… 72 4-5-1厭氧微生物之培養液菌群分析…………………………………. 72 4-5-2 厭氧消化實驗Ⅰ之微生物菌群………………………………… 75 4-5-3厭氧消化實驗Ⅰ之微生物菌群………………………………… 80 第五章 結論…………………………………………………………………… 89 第六章 參考文獻……………………………………………………………… 91 附錄一 氣體產量計算公式……………………………………………………. 9511049772 bytesapplication/pdfen-US甲烷厭氧污泥methanePCRsludgeDGGEanaerobicthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/52162/1/ntu-93-R91524040-1.pdf