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研究生:黃新翔
研究生(外文):Huang, Shins shiang
論文名稱:利用固定化菌株顆粒以提升酒精產量之研究
論文名稱(外文):Study on Enhancement of Ethanol Production by Immobilized Bacterial Beads
指導教授:吳建一顏裕鴻顏裕鴻引用關係
指導教授(外文):Wu, Jane yiiYen, Yue horng
口試委員:吳建一顏裕鴻蔡明勳陳晉照
口試日期:2012-07-10
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:273
中文關鍵詞:酸水解纖維素還原糖固定化填充床酒精生產
外文關鍵詞:Acid hydrolysisCelluloseReducing sugarImmobilizedFixed-Bed ReactorsEthanol production
相關次數:
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在過去十年之間,生物工藝主要致力以木質纖維素做為生產酒精燃料之運用,除具有經濟效益也能和能源危機問題競爭發展。由於酵素水解和酒精醱酵的環境相似,所以可選擇將兩個步驟同時在單一容器裡一起反應,因此,這種應用糖化和醱酵(Simultaneous sccharification and fermentation,SSF)或者利用分開水解與醱酵 (Separate Hydrolysis and Fermentation,SHF)來將木質纖維素轉化為酒精的共醱酵程序,更具節省成本效益。本研究中,從麝香貓(果子狸)的糞便中,篩選出一株具有高酒精生產能力的酵母菌,根據16S rDNA的基因序鑑定後,命名為 Saccharomyces cerevisiae Wu-Y2。為了能提昇酒精的生產能力,實驗中將利用包埋法把Wu-Y2菌株固定化於PVA膠體顆粒。此外,在利用固定化細胞來進行反應器之擴大酒精生產時,我們設計了不同的醱酵參數來作為因子探討,包括顆粒填充比、水力滯留時間(HRT)及入流的glucose濃度。由實驗結果得知,當使用反應器在3.34%的顆粒填充比,以HRT為8 hr做為系統的生產條件時,可達有的最大酒精生產量則為19.8 g/L (47%)。除此之外,實驗中也研究利用細菌纖維膜 (BCM)來當作一種新型固定化技術的載體材料,由研究成果得知,利用BCM來固定化酵母菌細胞,相較於懸浮的醱酵系統,則是有較佳的生產操控性和酒精產力表現(9.2 g/L)。另一方面,本研究也特別設計了一組屬於懸吊式類型的固定化反應器,透過這反應器的運用而可順利將羧甲基纖維素(Carboxymethyl cellulose,CMC)轉化成能被醱酵利用的glucose。實驗中所使用的PVA固定化顆粒,其顆粒結構的顯微觀察和機械強度表現,分別利用電子顯微鏡(Scanning Electron Microscopy,SEM) 和強度測定儀(Rheometer)來完成檢測。
The application of simultaneous saccharification and fermentation (SSF) or separate hydrolysis and fermentation for the conversion of lignocellulosics to alcohol would result in a more cost-effective process. In this study, the ability to production ethanol of strains from civet faeces were screened. One strain with high ethanol production capability was identified as Saccharomyces cerevisiae Wu-Y2 (Wu-Y2), according to its 16S rDNA gene sequences. To enhance this isolated srain fermentative ability, the cells of Saccharomyces cerevisiae Wu-Y2, were entrapped in PVA gel bead. Additionally, to study the effects of the fermentation parameters (such as beads loading volume, hydraulic retention time (HRT) and glucose concentration) on the ethanol production using immobilized-cell beads in fixed bed reactor. The maxium ethanol production was 19.8 g/L (47%) at HRT of 8h and beads loading of 3.34%. Otherwise, bacterial cellulose membrane (BCM) was used as a novel carrier for immobilization. Initial experiment shows this immobilized yeast cell is allowed better operational control and metabolic activities than those of the free yeast (9.2 g/L). On the other hand, we have designed a hanging type immobilized reactor for the conversion of Carboxymethyl cellulose (CMC) to fermentable glucose. Obserbation of structure and determine of mechanical strength of PVA-immobilized beads by Scanning Electron Microscopy (SEM) and Rheometer, respectively.
目錄

封面內頁
簽名頁
中文摘要 iii
英文摘要 v
誌謝 vi
目錄 viii
圖目錄 xv
表目錄 xxii

1. 緒論 1
1.1前言 1
1.2 研究動機與目的 9
2. 文獻回顧 12
2.1 纖維素的應用 12
2.1.1 生質酒精於全世界能源的市佔情形 12
2.1.2 歐洲聯盟之發展體系 15
2.2 生質能源和酒精燃料的應用 16
2.3 纖維素對於環境的衝擊影響 18
2.4 酒精生產原料 20
2.4.1 木質纖維素 (Lignocelluloses) 20
2.4.1.1 纖維素 (Cellulose) 24
2.4.1.2 半纖維素 (Hemicellulose) 26
2.4.1.3 木質素 (Lignin) 27
2.4.2 海洋微藻 (Microalgae) 29
2.5 纖維素的前處理 30
2.5.1 酵素的水解處理 (Enzymatic hydrolysis) 36
2.5.2 酸溶液水解處理 (Acid hydrolysis) 37
2.6 天然分解纖維之菌株 42
2.7 天然生產酒精之微生物 46
2.8 固定化技術於生質酒精的生產應用情形 51
2.8.1 固定化技術應用於生質酒精的生產 51
2.8.1.1 利用無機擔體做為細胞固定化材料來 生產酒精 52
2.8.1.2 利用有機擔體做為細胞固定化材料來 生產酒精 53
2.8.2 利用薄膜做為細胞固定化材料生產酒精 54
2.8.3 利用天然的載體做為細胞固定化材料生產酒精 55
2.9 固定化微生物反應器應用 59
2.9.1固定化細胞反應器的優點 61
2.9.2固定化細胞反應器的類型 63
3. 材料與方法 69
3.1 實驗使用材料 69
3.1.1 實驗藥品 69
3.1.2 儀器設備 71
3.2 菌株來源與鑑定 72
3.2.1 纖維分解菌株篩選 72
3.2.2 酒精生產菌株篩選 75
3.2.3純化菌體之DNA鑑定 76
3.3 實驗方法 81
3.3.1 纖維水解試驗 81
3.3.1.1 物理水解方法 81
3.3.1.2 化學水解方法 81
3.3.1.3 微生物降解方法 83
3.3.1.4 葉綠素與胡蘿蔔素的測定 83
3.3.1.5 微藻細胞破碎的效率測定 84
3.3.2 培養基組成對懸浮及固定化之菌株生產酒精及 纖維分解能力探討 85
3.3.2.1 碳源濃度之影響 85
3.3.2.2 氮源種類之影響 86
3.3.2.3 氮源濃度之影響 86
3.3.3 環境因子對懸浮及固定化之菌株生產酒精及 纖維分解探討 87
3.3.3.1 pH值變化之影響 87
3.3.3.2 溫度變化之影響 87
3.3.3.3 攪拌速率之影響 87
3.3.4 微生物醱酵液之分析方法 87
3.3.4.1 還原糖定性與定量之分析 87
3.3.4.2 氨氮濃度之分析 89
3.3.4.3 酒精和丙酮酸之高效能液相層析儀 (High Performance Liquid Chromatography, HPLC) 之分析 90
3.3.5固定化菌株之製備 91
3.3.5.1 菌體收集 91
3.3.5.2 菌體量之量測 91
3.3.5.3 固定化菌株之製備 92
3.3.5.4 PVA顆粒製備方法和物性測定 93
3.3.5.5 以Bacterial cellulose membrane (BEM) 作為固定化擔體 95
3.3.6 固定化生化反應器設計與操作 97
3.3.7 物質結構之分析 99
3.3.7.1 傅立葉轉換紅外線光譜 (Fourier Transform Infrared Spectroscopy, FT-IR) 99
3.3.7.2 掃描式電子顯微鏡 (Scanning Electron Microscopy, SEM) 100
4. 結果與討論 102
4.1 微藻纖維分解處理 102
4.1.1 物理方式處理之結果 102
4.1.2 化學方式水解微藻纖維 117
4.1.3 不同光源培養生長之微藻纖維進行單醣轉化 123
4.2 纖維分解部份 127
4.2.1 纖維分解菌株之篩選 127
4.2.1.1 剛果紅試驗結果 127
4.2.1.2 液態培養試驗之比較 132
4.2.2 利用懸浮和固定化系統進行纖維分解菌株Acinetobacter sp. Wu-4-4最適生長條件 134
4.2.2.1 pH值變化對菌株分解纖維之影響 134
4.2.2.2 培養溫度對菌株分解纖維之影響 136
4.2.2.3 攪拌速率對菌株分解纖維之影響 139
4.2.2.4 CMC濃度對菌株分解纖維之影響 142
4.2.2.5 氮源種類及濃度對菌株分解纖維之影響 145
4.2.2.6 比較固定化和懸浮細胞系統對於分解 纖維之影響 150
4.2.2.7 纖維菌株分解纖維活性測試 153
4.3 酒精醱酵部份 156
4.3.1 生產菌株之篩選 156
4.3.2 利用懸浮和固定化系統進行酵母菌株 Saccharomyces cerevisiae Wu-Y2之最適生產 酒精條件 158
4.3.2.1 pH值變化對菌株之生產酒精能力影響 158
4.3.2.2 培養溫度對菌株之生產酒精能力影響 163
4.3.2.3 攪拌速率對對菌株之生產酒精能力影響 168
4.3.2.4氮源種類及濃度對菌株之生產酒精能力 影響 172
4.3.2.5 Glucose濃度對菌株之生產酒精能力影響 181
4.4 篩選菌種之16S rDNA 鑑定結果 184
4.5 利用PVA材料作為菌株固定化使用之擔體 189
4.6新型固定化技術之開發-酵母菌菌株固定於生物纖維 薄膜上之酒精生產能力測試 197
4.6.1生物纖維膜(簡稱BCM)之固定化 197
4.6.2 Saccharomyces cerevisiae Wu-Y2固定於BCM上 之生長觀察 198
4.6.3 利用固定於BCM之Saccharomyces cerevisiae Wu-Y2菌株進行酒精生產測試 206
4.6.4利用固定於生物纖維膜之Saccharomyces cerevisiae Wu-Y2菌株進行酒精重覆批次試驗 211
4.7 以PVA作為固定化擔體在單一菌株下進行填充床 反應器之連續生產測試 213
4.7.1填充顆粒比例對於固定化酒精菌株之生產 能力影響 213
4.7.2 水力滯留時間對於固定化酒精菌株之生產 能力影響 216
4.7.3 初始glucose濃度對於固定化酒精菌株之生產 能力影響 218
4.7.4不同CMC濃度對於固定化纖維菌株分解纖維 能力之穩定性測試 220
4.8 同時結合纖維分解和酒精生產菌株在SHF生產模式下 進行酒精生產測試 223
4.8.1 篩選菌株在懸浮培養系統下利用搖瓶進行之 酒精批次生產試驗 223
4.8.2以PVA固定化之篩選菌株在填充床反應器下 進行酒精連續生產試驗 227
4.9 同時結合纖維分解和酒精生產菌株在SSF生產模式下 進行酒精生產測試 229
4.10 利用減壓濃縮系統進行酒精產物之回收應用 232
5. 結論 234
參考文獻 236


圖目錄

Figure 1-1 Schematic of this study procedure 8
Figure 2-1 Annual ethanol production by the major producers 12
Figure 2-2(a) Diagrammatic representation of Lignocelluloses tissue 22
Figure 2-2(b) Composition of lignocellulosic materials and their potential hydrolysis products 22
Figure 2-3 Chemical structure of cellulose 25
Figure 2-4 The structure of Micelle 25
Figure 2-5 The building units of lignin 28
Figure 2-6 Effect of immobilized bioreactor and chemostat on operation stability 63
Figure 2-8 Schematic diagram of immobilized cell reactor for fixed bed reactors 65
Figure 2-9 Schematic diagram of immobilized cell reactor for fluidized bed reactors 67
Figure 2-10 Schematic diagram of immobilized cell reactor for flocculated cell reactors 68
Figure 3-1 Scheme of screening strategies of cellulose-degrading microorganisms 75
Figure 3-2 Scheme of hydrolysis of cellulose by physical/chemical methods 83
Figure 3-3 The standard calibration curve of glucose 89
Figure 3-4 Schedule of PVA immobilized process 92
Figure 3-5 Experimental apparatus for the measurement of immobilize bead strength 94
Figure 3-6 Schematic diagram of immobilization of cell using BCM support by injection method 96
Figure 3-7 Schematic process of reactor operation (Fix Bed Reactor) 98
Figure 3-8 Schematic process of reactor operation (Hanging Type Reactor) 99
Figure 4-1 Effect of beading during bead beating on C/C0 values 111
Figure 4-2 Effect of beading during bead beating on D/D0 values 111
Figure 4-3 Effect of acoustic power of ultrasonication on C/C0 values 112
Figure 4-4 Effect of acoustic power of ultrasonication on D/D0 values 112
Figure 4-5 Scanning electron micrographs of Chlorella sp. Y8-1 113
Figure 4-6 Effects of boiling waterbath and autoclaveing on Hydrolysis of Chlorella sp. untisruption and tisruption algae powder to glucose 120
Figure 4-7 FT-IR spectra of Chlorella sp. Y8-1 algae cell disruption powder 121
Figure 4-8 FT-IR spectra of Chlorella sp. Y8-1 algae undisruption powder 122
Figure 4-9 Effects of treat methods by adding acid and alkaline solution on hydrolysis of Chlorella sp. Y8-1. 126
Figure 4-10 Production active of reducing sugar by different strain isolation from marine and terrestrial region 133
Figure 4-11 The time course of reducing sugar product by Acinetobacter sp. Wu-4-4 at different pH in batch cultures. 135
Figure 4-12 The time course of reducing sugar product by Acinetobacter sp. Wu-4-4 at different temperature in batch cultures 138
Figure 4-13 The time course of reducing sugar product by Acinetobacter sp. Wu-4-4 at different stirrer speed in batch cultures 141
Figure 4-14 The time course of reducing sugar product by Acinetobacter sp. Wu-4-4 at different CMC concentration in batch cultures 144
Figure 4-15 The time course of reducing sugar product by Acinetobacter sp. Wu-4-4 at different nitrogen source in batch cultures 148
Figure 4-16 The time course of reducing sugar product by Acinetobacter sp. Wu-4-4 at different concentration of yeast extract in batch cultures 149
Figure 4-17 The time course of reducing sugar product by Acinetobacter sp. Wu-4-4 at different cultures system 152
Figure 4-18 Time course of cellulolytic enzyme production by Acinetobacter sp. Wu-4-4 155
Figure 4-19 The time course ethanol product and glucose consuming concentration for free yeast at batch cultures using glucose carbon source 157
Figure 4-20 The time course of ethanol product by suspended Saccharomyces cerevisiae Wu-Y2 at different pH in batch cultures 161
Figure 4-21 The time course of ethanol product by immobilized Saccharomyces cerevisiae Wu-Y2 cell beads at different pH in batch cultures 162
Figure 4-22 The time course of ethanol product by suspended Saccharomyces cerevisiae Wu-Y2 at different temperature in batch cultures 166
Figure 4-23 The time course of ethanol product by immobilize Saccharomyces cerevisiae Wu-Y2 cell beads at different temperature in batch cultures 167
Figure 4-24 The time course of ethanol product by Saccharomyces cerevisiae Wu-Y2 at different stirred speed in batch cultures 170
Figure 4-25 The time course of ethanol product by immobilize Saccharomyces cerevisiae Wu-Y2 cell beads at different stirrer speed in batch cultures 171
Figure 4-26 The time course of ethanol product by Saccharomyces cerevisiae Wu-Y2 at different nitrogen source in batch cultures. 174
Figure 4-27 The time course of ethanol product by Saccharomyces cerevisiae Wu-Y2 at different yeast extract concentration in batch cultures 175
Figure 4-28 The time course of ethanol product by immobilized Saccharomyces cerevisiae Wu-Y2 cell beads at different nitrogen source in batch cultures. 178
Figure 4-29 The time course of ethanol product by immobilized Saccharomyces cerevisiae Wu-Y2 cell beads a different Yeast extract concentration in batch cultures 180
Figure 4-30 The time course of biomass and ethanol product by suspended and immobilized Saccharomyces cerevisiae Wu-Y2 cell beads at different Glucose concentration in batch cultures 183
Figure 4-31 Phylogenetic dendrogram showing a comparison of the aligned 16S rDNA sequences of known Saccharomyces species with that of strain Saccharomyces cerevisiae Wu-Y2 187
Figure 4-32 Phylogenetic dendrogram showing a comparison of the aligned 16SrDNA sequences of known Acinetobacter species with that of strain Acinetobacter sp. Wu-4-4 187
Figure 4-33 Microscopic observation of immobilized growing cells for Saccharomyces cerevisiae Wu-Y2 193
Figure 4-34 Microscopic observation of immobilized growing cells for Acinetobacter sp. Wu-4-4 193
Figure 4-35 The physical characteristics of Saccharomyces cerevisiae Wu-Y2 immobilized-cell beads on particle average compressive strength by strength recognizer. 194
Figure 4-36 The physical characteristics of Acinetobacter sp. Wu-4-4 immobilized-cell beads on particle average compressive strength by strength recognizer 194
Figure 4-37 The time course of biomass and ethanol product by immobilized Saccharomyces cerevisiae Wu-Y2 cell in various thicknes of BCM in batch cultures 208
Figure 4-38 Kinetic properties of the immobilized and free cells. 210
Figure 4-39 The production of ethanol by immobilized Saccharomyces cerevisiae Wu-Y2 in bacterial cellulose support during I-VI cycles of the repeated batch fermentation 212
Figure 4-40 Time course of ethanol production and glucose consumption by immobilized Saccharomyces cerevisiae Wu-Y2 cell beads at different beads volume ratio in fixed bed reactor 215
Figure 4-41 Time course of ethanol production and glucose consumption by immobilized Saccharomyces cerevisiae Wu-Y2 cell beads at different hydraulic retention time in fixed bed reactor 217
Figure 4-42 Time course of ethanol production and glucose consumption by immobilized Saccharomyces cerevisiae Wu-Y2 cell beads at different glucose concentration in fixed bed reactor 219
Figure 4-43 Performance of production stability during scale-up operations of reducing sugar product with CMC loading by immobilized Acinetobacter sp. Wu-4-4 cell beads in fixed bed reactor 222
Figure 4-44 Effect of different cellulose substrate on reducing sugar and ethanol product by suspended Saccharomyces cerevisiae Wu-Y2 and Acinetobacter sp. Wu-4-4 225
Figure 4-45 Separate hydrolysis and fermentation of glucose for the production of ethanol by immobilize Saccharomyces cerevisiae Wu-Y2 and Acinetobacter sp. Wu-4-4 cell beads in fixed reactor 228
Figure 4-46 Simultaneous saccharification and fermentation of glucose for the production of ethanol by immobilize Saccharomyces cerevisiae Wu-Y2 and Acinetobacter sp. Wu-4-4 cell beads with beads weight proportions in hanging type reactor 231
Figure 4-47 Ethanol collection content by suspended Saccharomyces cerevisiae Wu-Y2 at different glucose concentration in batch cultures 233

表目錄

Table 2-1 Cellulose, hemicelluloses and lignin content in common agricultural residues and wastes 23
Table 2-2 Common pretreatment methods for lignocellulosic materials 35
Table 2-3 Ethanol yield from different biomass sources 42
Table 2-4 Microorganism species which produce reducing sugar as the main fementation product. 44
Table 2-5 Microorganism species which produce reducing sugar as the main fementation product 45
Table 2-6 Yeast species which produce ethanol as the main fementation product 48
Table 2-7 Yeast species which produce ethanol as the main fementation product 49
Table 2-8 Yeast species which produce ethanol as the main fementation product 50
Table 3-2 The Composition of SL7 74
Table 3-3 Yeast Proteome Database (YPD) medium 76
Table 3-4 The Composition of PCR formula 78
Table 3-5 The operating conditions of PCR 79
Table 3-6 The Composition of SDS-PAGE 80
Table 3-7 5X TBE (Tris-Borate-EDTA) Buffer. 80
Table 4-1 Optical microscope observation of Chlorella sp. Y8-1 algae cell treated by homogenizers disrupt 107
Table 4-2 Photographs of chlorophyll extraction solutions. ( homogenizers disrupt method) 108
Table 4-3 Optical microscope observation of Chlorella sp. Y8-1 algae cell treated by suspersonic disrupt 109
Table 4-4 Photographs of chlorophyll extraction solutions. ( suspersonic disrupt method) 110
Table 4-5 Characterization of cell mass compositiom for Chlorella fusca 125
Table 4-6 Compare with celluloose degrading ability of different cellulose degrading bacteria 129
Table 4-7 The congo red test:cellulose degradation activities of experimental cellulolytic microbes isolates on SEA-CMC agar plate. 130
Table 4-8 DNA sequence alignment of the isolated ethanol product bacteria Saccharomyces cerevisiae Wu-Y2 in database of NCBI blast. 186
Table 4-9 DNA sequence alignment of the isolated cellulose-degrading bacteria Acinetobacter sp. Wu-4-4 in database of NCBI blast. 186
Table 4-10 Different physiological of microbial at Saccharomyces cerevisiae Wu-Y2 and Acinetobacter sp. Wu-4-4 188
Table 4-11 The colony morphology of isolated bacterial strain 189
Table 4-12 SEM observation on immobilized beads of cell growth by Saccharomyces cerevisiae Wu-Y2 195
Table 4-13 SEM observation on immobilized beads of cell growth by Acinetobacter sp. Wu-4-4 196
Table 4-14 Electron micrographs of the immobilized yeast in bacterial cellulose support.(immersion method) 201
Table 4-15 Electron micrographs of the immobilized yeast in bacterial cellulose support.(injection method) 205
Table 4-16 The appearance of different thickness bacterial cellulose membrane 209
Table 4-17 Comparisons of fermentation performances of immobilized and free yeast cells in the batch culture 210
Table 4-18 Stability of the immobilized yeast in BC membrane for
alcohol fermentation. 212
Table 4-19 Comparison of content at the reducing sugar and ethanol product at different cellulose substrate 226

參考文獻

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