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研究生:鄧欣宜
研究生(外文):TENG, HSIN-YI
論文名稱:Actinobacillus succinogens突變菌株醱酵木質纖維素水解液之研究
論文名稱(外文):Study on the Fermentation of Actinobacillus Succinogens Mutant Using the Lignocellulose Hydrolysate as the Substrate
指導教授:李文乾
指導教授(外文):LEE,WEN-CHIEN
口試委員:黃光策段國仁
口試委員(外文):Huang,Kuang-tseDuan, Kow Jen
口試日期:2017-07-14
學位類別:碩士
校院名稱:國立中正大學
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
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:101
中文關鍵詞:木質纖維素象草丁二酸Actinobacillus succinogenes醱酵
外文關鍵詞:lignocelluloseNapier grasssuccinic acidActinobacillus succinogenesfermentation
相關次數:
  • 被引用被引用:1
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各國為了降低溫室氣體排放和降低全球溫度利用生物質來生產生質化學品和生質燃料的使用便成大多數國家的趨勢。從木質纖維素製造生質化學品與第二代生物燃料的技術日漸重要。丁二酸是TCA循環的產物之一,在食品、化學、醫藥工業等有的廣泛的應用,近幾年來丁二酸隨著新的應用領域不斷的開拓,讓國際市場對於丁二酸的需求量有大量的提升。
本研究將象草透過鹼處理後利用纖維素酵素水解獲得含有豐富葡萄糖與木醣的水解液,把水解液當作碳源來生產丁二酸,本研究利用Actinobacillus succinogenes 130Z BCRC 80310,經過NTG處理,挑選出產能較高,且無酒精生成之突變菌株當作出發菌株來醱酵丁二酸。經過定序,確認突變菌株的基因ADH(Asuc_0403)有突變點。
但象草水解液做醱酵時,丁二酸產量為0.59±0.03 g/g,與甘油醱酵相比,減少了44.4%的產量,且醋酸也有所增加。所以添加甘油,希望可以透過其還原力,來增加丁二酸產量,減少醋酸生成,發現丁二酸產量有因甘油添加量逐漸增加,而且其生產力也較純甘油佳,但醋酸生成並沒有因此有所減少。尋找代謝途徑後發現,當碳源為木醣時,所消耗之ATP比葡萄糖與甘油來得多,且產醋酸時會增加ATP。因此我們最後將木醣當作碳源做醱酵,其醱酵結果,丁二酸產量為0.59±0.04 g/g,而醋酸產量也有所增加,證實了木醣的存在為丁二酸產率下降與醋酸產生的原因。
象草水解液添加甘油雖然無法減少醋酸之生成,可提高丁二酸產率與文獻中不同水解液的醱酵相比,其丁二酸產率較佳,且生產力也較高,有一定的競爭力。

In order to reduce the emissions of the greenhouse gases and the global temperature, the use of biomass as the raw material to produce chemicals and biofuels become a trend in most countries. The production techniques for generating chemicals and second generation biofuels from lignocellulosic feedstock are crucial. Succinic acid is one of the metabolites in the TCA cycle and has been widely used in food, chemical and medical industry. In the recent years, as the expansion of its applications, succinic acid becomes highly demanded in the global market.
This study used Napier grass as the source of biomass. After alkali pretreatment, Napier grass was subjected to hydrolysis with cellulosic enzymes to gain the hydrolysate, which was enrich of glucose and xylose. The hydrolysate was then used as the carbon source for the production of succinic acid by a mutant of Actinobacillus succinogenes 130Z (BCRC 81310) which was previously established by using NTG method. The mutant can produce succinic acid with a higher yield and without producing ethanol. Results from gene sequencing showed that there is a mutant point in ADH gene (Asuc_0403).
When the hydrolyzate of pretreated Napier grass was used as the carbon source, the fermentation by the A. succinogenes mutant resulted in producing 0.59±0.03 g succinic acid per unit gram of reducing sugar. In comparison with the fermentation on glycerol, the succinic acid yield was reduced by 44.4% using the hydrolysate as the carbon source. Also, more amounts of acetic acid were produced when fermented on the Napier grass hydrolysate. In view of this finding, we tried to add some glycerol to enhance the reducing power of the carbon source during the fermentation on the hydrolysate. We discovered that the titer of succinic acid increased with glycerol amount and the productivity of succinic acid also increased but the formation of acetic acid remained at the same level. However, after taking a look at the metabolic pathways, we found that cells need to consume more ATP when xylose is used, in comparison with other carbon source like glucose or glycerol. And the production of acetic acid can generate ATP in the cells. Therefore, we tested the use of xylose as carbon source for succinic acid fermentation. The production yield of succinic acid was 0.59±0.04 g/g, almost the same as the Napier grass hydrolysate as the carbon source, confirming that the presence of xylose was the reason of low succinic acid yield.
Although adding glycerol into the hydrolysate of Napier grass cannot reduce the formation of acetic acid, but the succinic acid yield can be promoted. Compared with fermentation on different kinds of hydrolysate in the literatures, the yield and productivity of succinic acid obtained in the present work are very competitive.

摘要 I
Abstract III
目錄 V
圖目錄 X
表目錄 XII
第一章:緒論 1
1.1前言 1
1.2研究動機與目的 4
第二章 文獻回顧 5
2.1丁二酸 5
2.1.1丁二酸介紹 5
2.1.2 丁二酸應用 6
2.1.3 丁二酸製備方式 7
2.1.3 丁二酸生產菌種選擇 8
2.1.4 Actinobacillus succinogenes的代謝途徑 9
2.1.5 Actinobacillus succinogenes醱酵原料 13
2.2.木質纖維素 15
2.2.1木質纖維素之介紹 15
2.2.2象草 16
2.2.3前處理 17
2.2.4纖維素分解酵素 25
第三章 實驗藥品與設備 27
3.1實驗藥品 27
3.2實驗設備(*含型號及廠商) 29
第四章 實驗方法 31
4.1 實驗使用之培養基與引子 31
4.1.1 實驗培養基與緩衝液 31
4.1.2實驗用引子 33
4.2 Actinobacillus succinogenes菌種培養與凍存 33
4.3 Actinobacillus succinogenes ADH與pta gene定序 34
4.3.1 抽取Actinobacillus succinogenes染色體 34
4.3.2 利用聚合酶連鎖反應放大ADH與pta 片段 35
4.3.3利用DNA電泳確定片段大小 38
4.4象草水解液備製 40
4.4.1鹼處理 40
4.4.2酵素水解 41
4.5利用突變菌Mu-B7 醱酵各種碳源 42
第五章 結果與討論 43
5-1 Actinobacillus succinogenes Mu-B7突變菌定序 43
5-2象草水解液 44
5-3不同碳源批次醱酵 45
5-3-1利用甘油批次醱酵 45
5-3-2利用水解液批次醱酵 46
5-3-3利用不同比例水解液與甘油批次醱酵 47
5-3-4不同比例水解液與甘油批次醱酵比較 50
5-4木醣批次醱酵 51
5-5不同碳源批次醱酵之比較 52
第六章 結論 55
第七章 建議與後續實驗 57
附錄A 58
附錄B 62
附錄C 64
附錄D 68
附綠E-pta的基因剃除實驗 71
E-1實驗用引子 71
E-2實驗方法 72
E-2-1. 質體抽取 72
E-2-2. PCR產物純化 73
E-2-3. Double digestion 74
E-2-4. 接合作用(Ligation) 75
E-2-5熱休克轉形法 (heat-shock transformation) 76
E-2-6. 利用OE-PCR製備Frt-PGK-gb2-cm-Frt 77
E-2-7 Frt-PGK-gb2-cm-Frt casstte 製備 80
E-3結果與後續實驗建議 81
E-3-1製備Frt-PGK-gb2-cm-Frt與Frt-PGK-gb2-cm-Frt casstte 81
參考文獻 85

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