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研究生:呂媚君
研究生(外文):Lu Mei-Chun
論文名稱:利用篩選之菌株BacillussubtilisDYU1生產聚麩胺酸之研究
論文名稱(外文):Production of Poly(glutamic acid) Using the Isolated Strain Bacillus subtilis DYU1
指導教授:吳建一
指導教授(外文):Wu Jane-Yii
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:2007
畢業學年度:95
語文別:中文
論文頁數:205
中文關鍵詞:Bacillus subtilis聚麩胺酸動力學模式流變學特性糖蜜廢液
外文關鍵詞:Bacillus subtilispoly(glutamic acid)kinetic modelrheological propertymolasses wastewater
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  • 被引用被引用:1
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Bacillus subtilis DYU1於含有70 g/L L-glutamic acid、30 g/L maltose和5 g/L peptone的培養基中有最大poly-glutamic acid (PGA)產量61 g/L。C/N比會顯著地影響PGA產量及產率。當固定maltose濃度(30 g/L)時,最適C/N比為0.6-7.5;固定peptone濃度(5 g/L)時,最適C/N比為6-10。另外,本研究亦探討其他影響因子,如pH、振盪速度、金屬離子、NaCl和Biotin對PGA生產之影響。另外,nuclear magnetic resonance (NMR) spectrometry的結果證實所純化之產物為PGA。而胺基酸分析結果顯示PGA之純度高達99%。以gel permeation chromatography (GPC)的結果顯示,PGA之分子量超過1,000 kDa;光學異構物分析之結果顯示PGA是以98% D-glutamic acid所組成,證明在B. subtilis DYU1代謝路徑中,可將L-glutamic acid轉化為D-glutamic acid。另外,本研究亦以動力學模式來解析B. subtilis DYU1生長、PGA生長和maltose消耗。Monod和Michaelis-Meten models的結果顯示,當glutamic acid和maltose濃度分別高於70和50 g/L時,有基質抑制PG生產之情形產生。此外,logistic model除了可合理且精確地模擬B. subtilis DYU1生長、PGA生產及maltose消耗之情形,並證實PGA生產行為屬於混合相關模式。另外,本研究探討不同pH值和溫度之PGA溶液流變學行為,亦發展一套與溫度和PGA濃度相關,且對PGA溶液外觀黏度影響之數學模式。本研究亦以活性碳來去除糖蜜廢液之色度,並探討稀釋倍數、吸附劑濃度、pH值和溫度對色度去除之影響。最後,比較處理前後之廢液作為基質來生產PGA之可行性,以達到減廢之效果。
A newly isolated strain from soil which was identified taxonomically as Bacillus subtilis (called B. subtilis DYU1) with high extracellular poly-glutamic acid (PGA) productivity. PGA production by B. subtilis DYU1 was strongly dependent on the concentration of additional exogenous L-glutamic acid and carbon source in the fermentation medium. Most favorable carbon and nitrogen sources for PGA production were maltose and peptone, respectively. The maximum PGA production (61 g/L) was obtained when it was grown in a medium containing 70 g/L L-glutamic acid, 30 g/L maltose and 5 g/L peptone at 37℃ for 96 h with shaking (150 rpm). The maximum yield and productivity rate of PGA were both markedly depended on the ratio of carbon to nitrogen (C/N). An optimal C/N ratio range from 0.6 to 7.5 and 6 to 10 were obtained for maltose-based (30 g/L) and peptone-based (5 g/L) culture, respectively. In addition, other effect factor such as pH, agitation speed, metal ions, NaCl and biotin was also discussed to found the optimization condition for PGA production. The analysis with nuclear magnetic resonance (NMR) spectrometry and amino acid identification shows that the purified biopolymer possesses the structure of a PGA. However, the product produced by B. subtilis DYU1 was characterized by amino acid analysis to be composed of solely glutamic acid (99%). The average molecular weight of purified PGA was over 1,000 kDa determined by gel permeation chromatography, and 98% of the purified PGA was D-glutamate indicating that the B. subtilis DYU1 possesses a metabolic pathway to produce D-glutamate from L-glutamate. Additionally, the fermentation kinetic of PGA by B. subtilis DYU1 were also investigated in this study. By using Monod and Michaelis-Meten models, it was found that substrate inhibition for PGA production when glutamic acid and maltose was greater than 70 and 50 g/L, respectively. Moreover, a model based on the logistic and Luedeking-Piret equation of B. subtilis DYU1 growth, PGA accumulation combined non-growth-assocaited and growth-assocaited contributions, and consumprion of maltose were developed. The results predicted by the model were good agreement with the experimental observations. The rheological properties of PGA broth were studied using a rotational viscometer at several pHs (1.0-13.0) and temperatures (5-75℃). The mathematical models were developed for determining the apparent viscosity of PGA broth as affected by temperature and PGA concentration. Additionally, carbon activated was used as an adsorbent for the removal of molasses wastewater. Batch adsorption studies were conducted to explore the effect of dulition times, adsorbate concentration, pH and temperature. Finally, using molasses wastewater without or with pretreatment as a substrate for the production of PGA by B. subtilis DYU1 and lessening the pollution of molasses wastewater.
封面內頁
簽名頁
授權書.........................................................iii
中文摘要.......................................................iv
英文摘要.......................................................vi
誌謝...........................................................viii
目錄...........................................................ix
圖目錄.........................................................xiv
表目錄.........................................................xx
符號說明.......................................................xxii
1.前言.........................................................1
2.文獻回顧......................................................4
2.1麩胺酸鈉鹽製程概述............................................4
2.1.1發酵廢液分析...............................................4
2.1.2發酵廢液處理概述............................................5
2.2聚醯胺(polyamide)之簡介......................................12
2.3聚麩胺酸(PGA)之特性、生合成與降解..............................15
2.3.1聚麩胺酸之特性.............................................15
2.3.2聚麩胺酸之生合成...........................................16
2.3.3合成聚麩胺酸有關之基因......................................19
2.3.4生化代謝上與合成聚麩胺酸有關之酵素............................21
2.3.5聚麩胺酸之降解作用..........................................24
2.4聚麩胺酸之生產菌株及其特性.....................................26
2.4.1需額外補充L-glutamic acid之菌株.............................31
2.4.2不需額外補充L-glutamic acid之菌株...........................40
2.5聚麩胺酸生產之環境影響因子.....................................44
2.5.1碳源......................................................44
2.5.2氮源......................................................48
2.5.3氧氣......................................................49
2.5.4金屬離子...................................................52
2.6聚麩胺酸之應用...............................................53
2.6.1食品工業上之應用............................................55
2.6.2生物醫藥/醫學材料上之應用....................................56
2.6.3環境上之應用...............................................60
2.6.4其他之應用.................................................62
3.材料與方法....................................................64
3.1實驗材料....................................................64
3.1.1藥品......................................................64
3.1.2儀器設備..................................................66
3.2菌株培養....................................................67
3.2.1菌株來源..................................................67
3.2.2菌株活化..................................................68
3.2.3聚麩胺酸生產培養...........................................68
3.3分析方法....................................................68
3.3.1Maltose分析..............................................68
3.3.2多醣分析-酚-硫酸法........................................69
3.3.3NH4+-N濃度之測定..........................................70
3.3.4黏度.....................................................71
3.4聚麩胺酸之分析..............................................71
3.4.1膠體滲透層析..............................................71
3.4.2高效能液相層析儀(HPLC)分析.................................72
3.4.3胺基酸分析................................................73
3.4.4核磁共振(NMR)分析.........................................74
3.4.5PGA中D/L-光學異構物之比率分析..............................74
3.5聚麩胺酸之回收與純化.........................................74
3.6糖蜜廢液之批次吸附試驗.......................................77
3.7色度分析...................................................78
3.8COD分析....................................................78
3.9BET (Brunauer-Emmett-Teller)表面積測定......................79
4.動力學模式解析...............................................80
4.1 Monod and Michaelis-Menten models........................80
4.2 Logistic and Luedeking-Piret model.......................82
5.結果與討論..................................................88
5.1探討B. subtilis DYU1生產PGA之最適培養基及環境因子.............88
5.1.1不同L-glutamic acid濃度對B. subtilis DYU1生產PGA之影響.....88
5.1.2不同碳源及濃度對B. subtilis DYU1生產PGA之影響...............92
5.1.3不同氮源及濃度對B. subtilis DYU1生產PGA之影響...............97
5.1.4 C/N比對B. subtilis DYU1生產PGA之影響.....................101
5.1.5 pH對B. subtilis DYU1生產PGA之影響........................103
5.1.6振盪速度對B. subtilis DYU1生產PGA之影響....................105
5.1.7金屬離子對B. subtilis DYU1生產PGA之產量及D/L glutamic acid組成之影響............................................................107
5.1.8 NaCl和biotin對B. subtilis DYU1生產PGA之影響...............111
5.2PGA之基本特性...............................................115
5.3動力學模式..................................................120
5.3.1 Monod and Michaelis-Menten models.......................120
5.3.2 Logistic model..........................................133
5.4 PGA溶液之流變學特性.........................................146
5.4.1 pH對黏度的影響............................................148
5.4.2溫度和PGA濃度對PGA溶液黏度的影響.............................149
5.4.3溫度和PGA濃度與PGA溶液外觀黏度之關係.........................150
5.5以活性碳去除糖蜜廢液色度之研究.................................166
5.5.1不同稀釋度對糖蜜廢液色度去除之影響............................166
5.5.2不同活性碳吸附劑量對糖蜜廢液色度去除之影響.....................166
5.5.3不同pH對糖蜜廢液色度去除之影響...............................167
5.5.4不同溫度對糖蜜廢液色度去除之影響..............................168
5.5.5活性碳之BET分析.............................................169
5.6利用糖蜜廢液生產PGA之可行性.....................................177
6.結論..........................................................180
參考文獻........................................................182
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