臺灣博碩士論文加值系統

衣康酸(itaconic acid, IA)為一種不飽和雙碳有機酸，因衣康酸具有可將單體聚合成聚合體的特性，故可應用於合成樹脂、纖維、塑料及橡膠的原料，廣泛應用於化學、高分子工業上。衣康酸通常可藉由乾餾法或微生物合成，此篇研究將著重利用微生物法合成衣康酸。本研究主要目的，係探討Aspergillus terreus合成衣康酸之相關生產製程，以增加其日後之產業應用。
由實驗結果顯示：A.terreus合成衣康酸最佳醱酵條件為轉速150 rpm、溫度30℃、pH值控制在3.4，在上述條件下，衣康酸產量可達2106 ppm。其次，前培養方面以適合真菌生長的培養基M1(Malt extract broth)為佳，主培養基最佳碳源為葡萄糖、無機氮源為硝酸銨、有機氮源為Corn steep liquor時產量為佳，且氮源對於衣康酸的代謝是非常重要。研究進一步顯示：若將氮源刪除，無論是有機氮源或無機氮源，則衣康酸產量可從2106 ppm降至14 ppm。另外，發現額外添加鈣離子對促進衣康酸之合成非常重要，當培養基存在5 mM之CaSO4．2H2O，可將衣康酸產量從2106 ppm提升至3641 ppm，藉由回應曲面實驗設計法，進行培養基最適化方面，研究結果顯示：硝酸銨、硫化鈣為影響衣康酸合成之兩個相關參數，當使用硝酸銨15.41g/L及硫酸鈣 7.39 mM，主培養基時，可獲得衣康酸理論產量達3163.63ppm。

Itaconic acid is an unsaturated dicarbonic acid, because it can easily be incorporated into polymers. For this reason, it can be applied to manufacturing of polyester resins or other polymeric materials and has been widely utilized in chemical industries. Itaconic acid can be produced by chemical method or bio-synthesis method. This study will be focused on the production of itaconic acid by bio-synthesis method. The major synthetic pathway of itaconic acid (IA) is the conversion of carbohydrate via glycolysis and TCA cycle, the transference of citrate to cis-aconitate by aconitase, followed by the decarboxylation of cis-aconitate by cis-aconitate decarboxylase (CAD). The purpose of this thesis is studying the process for the production of itaconic acid by Aspergillus terreus.
Our results shows that the optimal culture conditions for itaconic acid production were 30 oC, pH 3.4 and agitation speed of 150 rpm. It is clear that the best source of carbohydrate, nitrogen and ion were glucose, ammonium nitrate, corn steep liquor and calcium sulfate dihydrate. This study also found ammonium nitrate plays an important role in itaconic acid production. When the media lack ammonium nitrate, the production of itaconic acid decreased from 2106 ppm to 14 ppm. In this study, we found that the addition of calcium is very helpful for itaconic acid production. It could raise the itaconic acid concentration from 2106 ppm to 3641 ppm.
Furthermore, statistical experimental design methodology was used to optimize the culture media composition. Two key parameters (NH4NO3, CaSO4．2H2O ) were selected by two-level factorial design. Response surface methodology was then applied to identify the optimum composition of the two key parameters. As the results, an optimal concentration of NH4NO3 and CaSO4．2H2O were 15.41g/L, and 7.39 mM, respectively. With this optimal medium, the itaconic acid production could be markedly elevated to a yield of 3163.63 ppm

中文摘要 IV
英文摘要 V
致謝 VII
目錄 VIII
表目錄 XI
圖目錄 XII
第一章 前言 1
1-1菌株Aspergillus terreus 1
1-2 衣康酸物理及化學性質 1
1-3 衣康酸之用途及市場需求 3
1-4 衣康酸生產方式及生合成途徑 4
1-5 影響衣康酸產率因素 7
1-5-1 pH值及菌體型態 7
1-5-2 培養液溶氧量 7
1-6 回應曲面法 (Response surface methodology, RSM) 8
1-6-1 二水準因子設計 (Two-level factorial design) 9
1-6-2 陡升路徑法 (Method of path steepest ascent) 9
1-6-3 中心混合程式設計 (Central composite design, CCD) 9
1-7 論文研究動機 10
第二章 材料與方法 11
2-1 實驗藥品及分析儀器 11
2-1-1 實驗藥品 11
2-1-2 分析儀器 12
2-2 菌株及培養基 13
2-2-1 菌株 13
2-2-2 培養基 13
2-3 實驗與方法 15
2-3-1 前培養條件測試之實驗流程 15
2-3-2 培養條件測試 16
2-3-3衣康酸定性分析 17
2-3-4衣康酸定量分析 17
2-3-5微生物生長分析 18
2-3-6培養基葡萄糖殘留分析 (Trinder, 1969) 19
第三章 結果與討論 20
3-1論文架構 20
3-2培養條件探討 21
3-2-1其他文獻前培養配方測試 21
3-2-2 培養轉速測試 23
3-2-3 pH值測試 25
3-2-4 溫度測試 27
3-2-5 接菌量測試 29
3-2-6 不同主培養基之測試 30
3-3 開發最適化培養基前置步驟 – 篩選因子 31
3-3-1 碳源測試 31
3-3-2 氮源測試 32
3-3-3 離子測試 34
3-4 依實驗設計法探討最適化培養基之組成 36
3-4-1 二水準因子設計(Two-level factorial design) 36
3-4-2 陡升路徑法 (Method of path of steepest) 38
3-4-3 回應曲面法 (Response surface methodology, RSM) 39
3-5 衣康酸初步分離之分子量確認 42
第四章 結論 44
4-1結論 44
參考文獻 45

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