臺灣博碩士論文加值系統

隨著人口的增長，環境及能源短缺問題逐漸引起人們關注，而以木質纖維素為原料產生之生質能源取代化石燃料的研究被提起並被深入討論。然而，因木質纖維素結構複雜而使用的預處理，產生了會對微生物發酵生產造成抑制的副產物。在這些副產物之中，酚類化合物比另外兩種的副產物抑制效果強、種類也最繁雜。酚類副產物的影響已經在生質乙醇中已被廣泛討論，但對Clostridia 的 acetone-butanol-ethanol (ABE)發酵影響一直沒有詳細的研究。

因此，本研究目的為探討酚類化合物對饋料批次發酵(fed-batch fermentation)中的Clostridium saccharoperbutylacetonicum N1- 4 (ATCC 27021)抑制效果。本篇使用Vanillin(香蘭素), 4-hydroxybenzoic acid (4-HBA), and syringaldehyde(丁香醛)等三個，作為酚類副產物中三種以官能基區分 guaiacyl, hydroxyl, syringyl的其中一種。 結果發現，與控制組相比，丁醇生產分別在0.5 g/L的香蘭素，4-HBA和丁香醛下分別抑制了60％，37％和52％；當濃度至1.0 g/L時三種分別抑制了90％，80％和93％；而在5.0 g/L對丁醇生產抑制達 99％以上。三種抑制性副產物對ABE(丙酮、丁醇、乙醇濃度總和)生產抑制趨勢與丁醇生產相似，尤其在5.0 g/L丁香醛中未觀察到ABE產生。由此明顯發現，在ABE發酵中，丁香醛相較於香蘭素、4-HBA的毒性更強。
此外，從結果也觀察出酚類化合物對C. saccharoperbutylacetonicum N1- 4 (ATCC 27021)的生長產生抑制: 與控制組相比在0.5 g/L 香蘭素、4-HBA、丁香醛存在下，細胞濃度分別減少了 42％、31％、59％；濃度 1.0 g/L時降低了 78％、65％、80％；而在濃度達 5.0 g/L時對細胞生長抑制均達 99％
以上，近乎完全抑制，此一結果也與ABE生產雷同。而對於葡萄糖的消耗，當三種酚類化合物濃度增加到5.0 g/L時，消耗量均低於 10％(控制組為 77％)，其趨勢與丁醇生產、細胞生長相同。
總而言之，酚類衍生物對ABE發酵的抑制效果隨其種類變化而不同。當濃度超過5.0 g/L酚類衍生物便對丁醇生產造成完全抑制。酚類衍生物的存在對
C. saccharoperbutylacetonicum N1- 4 (ATCC 27021)的ABE發酵性能造成顯著的降低。

With the growth of population, the problem of environment and energy shortage has attracted people's attention, and bioenergy is one of the most effective way to solve the problem. Bioethanol is the first to be studied and discussed in bioenergy, but biobutanol has more advantages than bioethanol such as more rich energy , less volatile , less corrosive and easier to purify than bioethanol。It can solve the environmental problems and re-use it for energy production by using waste containing lignocellulose as a substrate. Lignocellulose has a structure that is difficult to be decomposed by microorganisms , so before use must be subjected to hydrolysis pretreatment. In addition to saccharides for microorganisms by hydrolysis , other inhibitory byproducts are also produced. These inhibitory by-products are classified as furan derivatives, carboxylic acid compounds and phenolic compounds. Among them, the phenolic compounds than the other two byproducts to suppress the effect of strong, the most complex types. The effects of phenolic byproducts have been extensively discussed in biomass ethanol, but not in biomass butanol.
In this study, common lignocellulose hydrolyzed phenolic compounds vanillin, 4-hydroxyhydroxyoic acid (4-HBA) and syringaldehyde were added to different concentrations of C.saccharoperbutylacetonicum N1- 4 (ATCC 27021) fed-batch culture. The results of the study found that: The inhibitory effect of vanillin on the product increased with increasing concentration , at 0.5 g / L, the cell growth was increased by 9.5% compared with the control group , the 1.5 g / L cell growth ratio higher than 8% at 1.0 g/L; Until the complete inhibition of the product and cells was achieved at 8.0 g / L. The inhibition of cell growth was increased with the increase of 4-HBA concentration. The inhibitory effect of product and cell growth did not increase with the concentration of clove aldehyde , such as syringaldehyde concentration 1.0 g/L and 1.5 g/L were 12.1% , 42.2% and 33.7% , 50.1%；at 0.5 g/L glucose utilization increased 13% than the control group.
According to the results, the vanillin inhibition mechanism was judged to inhibit the production of coenzyme by inhibiting the production of the product; Syringaldehyde inhibit the production of cells by inhibiting cells. Three compounds in the 1.0 g / L production inhibition of more than 80%, the cell growth inhibition of more than 60%, but less than or greater than 1.0 g / L concentration will be different according to different compounds have different inhibitory effect.

摘要............................I
Abstract............................II
目錄............................IV
表目錄............................VII
圖目錄............................VIII
第一章 緒論............................1
1-1 研究背景............................1
1-2 研究目的............................2
1-3 研究架構............................3
第二章 文獻回顧..........................4
2-1 生質能...............................4
2-1.1 生質能發展............................4
2-1.2 生質丁醇............................4
2-2 ABE (Acetone–butanol–ethanol) 發酵....5
2-2.1 ABE發酵史............................5
2-2.2 發酵菌株............................6
2-2.3 代謝途徑............................6
2-3 木質纖維素............................7
2-3.1 結構............................7
2-3.2 纖維素............................8
2-3.3 半纖維素............................9
2-3.4 木質素............................9
2-3.5 預處理種類............................10
2-4 抑制性副產物............................11
2-4.1 抑制性副產物形成............................11
2-4.2 抑制原理............................19
第三章 材料與方法............................21
3-1 實驗流程............................21
3-2 實驗器材............................22
3-2.1 儀器設備............................22
3-2.2 實驗藥品............................23
3-3 實驗步驟..................................25
3-3.1 菌種來源...............................25
3-3.2 Peptone-Yeast-Glucose (PYG)培養液配製..25
3-3.3 種源培養............................26
3-3.4 植種培養............................27
3-3.5 批次培養液配製.........................27
3-3.6 饋料批次發酵............................28
3-4 分析方法............................29
3-4.1 細胞濃度分析............................29
3-4.2 產物分析............................29
3-4.3 抑制物分析............................30
3-4.4 碳水化合物分析............................30
3-4.5 數據分析............................31
3-5 實驗參數............................31
第四章 結果與討論............................32
4-1 酚類化合物之影響............................32
4-1.1 丁醇生產............................32
4-1.2 丙酮生產............................35
4-1.3 ABE生產............................37
4-1.4 細胞生長............................40
4-1.5 葡萄糖消耗............................43
4-2 動力學分析............................45
4-2.1 丁醇分析............................45
第五章 結論與建議............................47
5-1 結論............................47
5-2 建議............................47
參考文獻 ............................51
附錄............................59

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