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研究生:連育賢
研究生(外文):Yu-Hsien Lien
論文名稱:不同前處理在水生植物纖維素酵素水解之探討-以大萍纖維素為例
論文名稱(外文):Different Pretreatment of Aquatic Plant in the Hydrolysis of Cellulose Enzyme – An Example of Water Lettuce Cellulose
指導教授:許哲奇
指導教授(外文):Che-Chi Shu
口試委員:李瑞元、林仁斌
口試日期:2016-07-29
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
畢業學年度:104
中文關鍵詞:水芙蓉、纖維素、酵素水解、乳酸球菌、綠色能源
外文關鍵詞:Pistia stratiotes L.CelluloseEnzymatic hydrolysisL. lactisRenewable energy
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地球資源即將耗竭,在人類長期下來造成的環境汙染,已造成極端氣候頻傳、臭氧層出現破洞等不良後果,因此近年來有許多綠色能源的出現。本文以綠色能源為出發點,主要實驗以常見的水生植物 (大萍) 作為纖維素的來源,預先處理先改變生物質的尺寸與蒸汽爆裂、鹼處理等前處理,再以酵素水解的方式來獲得醣類,作為提供細菌生長時所須的碳源。實驗中以水生植物 (大萍) 為基底製成的培養液,製作成能讓細菌培養的環境,同時培養經基因改造具有抑制糞腸球菌 (E. faecalis) 的乳酸球菌 (L. lactis NZ9000-pBk1:Bac),檢測乳酸球菌是否與商業版培養液同樣具有抑制糞腸球菌之能力。
由大萍為原料製作成培養基與其他不同培養液比較也能提供較佳的培養環境供細菌生長。以檢測乳酸球菌 (L. lactis NZ9000-pBk1:Bac) 抑制糞腸球菌之能力,可能來確認由大萍製作的培養基液體培養之細菌並沒有使得細菌失去原有的功能與功用,其中對於糞腸球菌 OG1RF (E. faecalis OG1RF) 有較佳的抑制能力。
The resources of earth are going to be depleted. Because of environmental pollution which the human caused, has resulted in frequent extreme weather, ozone holes and other adverse consequences. In recent years there are many green energy have appeared to against those conditions. In this paper, we take the green energy as a starting point. We use common aquatic plants (Water Lettuce) as a source of cellulose, pre-treat with sodium hydroxide and high temperature and pressure to change the size of the biomass and then we hydrolyze cellulose by a hydrolyzing enzymes to get carbohydrates as the carbon source to grow the bacteria. Experiments used medium based on aquatic plants (Water Lettuce) to cultivate bacteria, while cultivated genetically modified Lactococcus lactis that can inhibit the growth of Enterococcus faecalis. We detected L. lactis (L. lactis NZ9000-pBk1: Bac) to find whether the medium has the ability to inhibit the growth of E. faecalis like the medium of commercial version. Medium based Water Lettuce can provide a better culture environment for bacterial growth in comparison with other medium. By detecting Lactococcus lactis (L. lactis NZ9000-pBk1: Bac) inhibiting ability can identify that medium produced from Water Lettuce would not lose its original function, wherein L. lactis have better ability to inhibit the growth of Enterococcus faecalis (E. faecalis OG1RF).
摘要 i
ABSTRACT ii
誌謝 iv
目錄 v
表目錄 ix
圖目錄 xi
第一章 緒論 1
1.1 前言 1
1.2 研究背景及動機 3
1.3 研究目的 4
第二章 文獻回顧 5
2.1 植物細胞壁成分 5
2.2 木質纖維素 (Lignocellulose) 6
2.2.1 纖維素 (Cellulose) 8
2.2.2 半纖維素 (Hemicellulose) 10
2.2.3 木質素 (Lignin) 12
2.3 前處理 (Pretreatment) 13
2.3.1 酸處理 (Acidic Pretreatment) 15
2.3.2 鹼處理 (Alkaline Pretreatment) 15
2.3.3 離子液體 (Ionic Liquids) 16
2.3.4 氧化脫去木質素 (Oxidative Delignification) 16
2.3.5 有機溶劑法 (Organosolv) 17
2.3.6 物理方式前處理 (Physical Pretreatment) 17
2.3.7 蒸汽爆裂前處理 (Stream Explosion Pretreatment) 17
2.3.8 高壓熱水前處理 (Liquid Hot Water Pretreatment) 18
2.4 纖維素酵素水解 (Cellulose Enzymatic Hydrolysis) 19
2.4.1 內切型纖維素纖維分解酶 (Endo-β-1,4-glucanase) 19
2.4.2 外切型纖維素纖維分解酶 (Exo-β-1,4-glucanase) 19
2.4.3 葡萄糖苷酶 (β-1,4-glucosidase) 19
2.4.4 纖維酵素活性 (Cellulase Activity) 20
2.5 酵素水解過程 (Enzyme Hydrolysis Process) 22
2.6 大萍 (Water Lettuce) 24
2.7 乳酸菌 (Lactic acid bacteria) 25
2.7.1 乳酸球菌屬 (Lactococcus lactis) 26
2.7.2 乳酸球菌NZ9000-pBK1:Bac (L. lactis NZ9000-pBK1:Bac) 26
2.8 糞腸球菌 (Enterococcus faecalis) 27
第三章 材料與方法 28
3.1 實驗流程 28
3.1.1 木質纖維素前處理流程 28
3.2 實驗材料與藥品 29
3.2.1 實驗材料 29
3.2.2 實驗藥品 32
3.2.3 實驗儀器 33
3.2.4 使用菌株 34
3.2.5培養基配方 34
3.3 實驗方法 35
3.3.1 原料之基本成分檢測分析 35
3.3.2 原料之基本成分檢測分析方法 35
3.3.2.1 含水率測定 35
3.3.2.2 灰分含量之測定 35
3.3.2.3 醇苯萃取物之測定 36
3.3.2.4 木質素之含量測定 36
3.3.2.5 纖維素、半纖維素之含量測定 37
3.3.3前處理 38
3.3.3.1 NaOH前處理 38
3.3.3.2 蒸汽爆裂前處理 38
3.3.4 酵素水解 39
3.3.4.1 使用Cellulase、β-glucosidase酵素進行水解 39
3.3.4.2 使用CTec2酵素進行水解 39
3.3.5 葡萄糖、木糖、其他成分之濃度分析 40
3.3.5.1 檢量線之建立 40
3.3.5.2 葡萄糖檢量線之建立 41
3.3.6 培養液之製作 42
3.3.6.1製作大萍之培養液 42
3.3.6.2 製作大萍前處理之培養液 42
3.3.6.3 製作大萍酵素水解之培養液 43
3.3.7 培養液比較 43
3.3.7.1 培養菌株 44
3.3.7.2 作為輔助培養液 44
3.3.8 抑菌測試 45
3.3.8.1 糞腸球菌之培養 46
第四章 結果與討論 47
4.1 大萍之組成分析 47
4.1.1 含水率之測定 47
4.1.2 灰分含量之測定 47
4.1.3 醇苯萃取物含量之測定 49
4.1.4 木質素含量之測定 49
4.1.5 纖維素含量之測定 50
4.1.6 大萍乾重總組成比例 51
4.2 纖維素水解分析 52
4.2.1 不同前處理比較 52
4.2.2 不同酵素量比較 54
4.2.3 使用不同種酵素比較 55
4.3 養菌測試 56
4.3.1 培養液測試 57
4.3.2 作為輔助培養液測試 58
4.3.3 培養菌株 62
4.4 抑菌測試 65
4.4.1 抑制E. faecalis V583測試 66
4.4.2 抑制E. faecalis OG1RF測試 67
第五章 結論 68
參考文獻 69
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