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研究生(外文):Shih-Nung Chou
論文名稱(外文):Effects of Nitrogen Source on Proliferation of Transformed Hairy Root and Secondary Metabolite Production for Promoting the Performance of Mist Trickling Bioreactor
指導教授(外文):Shih-Yow Huang
外文關鍵詞:L-DOPANitrogen sourceMist trickling bioreactorTransformed hairy root
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藉由農桿菌(Agrobacterium rhizogenes)所誘導的轉殖毛狀根,除了具有高基因生化穩定性、高生長速率以及不須添加荷爾蒙即可培養等優點外,也能夠生產高經濟價值的二次代謝產物。本研究利用轉殖藜豆毛狀根生產二次代謝產物L-DOPA,並以250mL錐形瓶及9L噴霧滴流式生物反應器(mist trickling bioreactor, MTB)進行培養。基於培養末期氮源不足的問題,本研究將進一步地探討無機氮源(硫酸銨,硝酸鉀)中期添加的最適濃度以及如何藉由無機氮源與其它養分(蔗糖,ascorbic acid)的添加來達到提高生質量以及L-DOPA產率的目的。此外,對於有機氮源(Tyrosine,Glycine)的添加也將一併探討。錐形瓶培養結果顯示,無機氮源中期最適添加濃度方面,以添加2×B5濃度之硫酸銨以及硝酸鉀所得到的L-DOPA產量最多,並發現此硫酸銨濃度的添加會造成蔗糖不足的現象。若同時補充2×B5濃度之硫酸銨以及蔗糖,則會更提高L-DOPA的總產量(提高9.54%)。有機氮源中期添加0.33 M Glycine對於L-DOPA總產量的提高有利,而1.38mM Tyrosine則否。反應器培養結果顯示,多量接種會使根與根纏結叢密,阻抑根之生長,造成根的生質量減少。氮源添加方面,2倍(NH4)2SO4的添加對於培養液之pH、導電度、ORP以及蔗糖利用率的影響很大,而從rH的變化可知NH4+的加入會再一次地抑制NO3-代謝,並且也伴隨其它的氧化反應(如碳源代謝等),造成培養液之氧化反應大於還原反應再次使得rH上升;而當NH4+濃度下降至一定時,NO3-代謝不再受到抑制,並且此時其它如碳源代謝等氧化反應減少,以致於培養液的還原反應大於氧化反應,再次使得rH下降。
The transformed hairy roots induced by Agrobacterium rhizogenes can have several advantages, including highly genetic stability, high growth rate, cultivation without hormones and production of high value of secondary metabolites. In this work the transformed Stizolobium hassioo hairy roots was cultivated in 250 ml of shake flasks as well as in a 9 L of mist trickling bioreactor (MTB) for producing L-DOPA. To overcome the shortage of nitrogen source in the later stage of culture, the optimal dosage and feeding time of inorganic nitrogen source as well as other nutrients were investigated. This is aimed at the enhancement of hairy root proliferation and L-DOPA production. In addition, the effects of addition of organic nitrogen source have also been studied.

In the shake flask study, the optimum concentration of the inorganic source fed at a mid-term stage was a 2-fold dosage of (NH4)2SO4 and KNO3 to that of B5 basal medium. The highest L-DOPA concentration in the culture medium was obtained in this dosage. It was found that the sucrose during the culture medium was exhausted. The overall productivity of L-DOPA can be increased further if the above-mentioned concentration of ammonium sulfate together with 0.75% sucrose was dosed at the mid-term stage of culture. The dosage of 0.33M glycine in a mid-term stage of culture enhanced the total L-DOPA productivity, nevertheless, the dosage of 1.38mM of tyrosine did not.

In the MTB study, less inoculum density promoted the hairy root proliferation owing to the larger growing space. For nitrogen source supplementation by feeding a 2-fold B5 ammonium sulfate greatly influenced the pH, conductivity, redox potential and sucrose consumption rate of the medium. Throughout an observation by rH, it was elucidated that ammonium sulfate feeding would inhibit the metabolism of nitrate as well as the other oxidation reaction (e.g. carbon source metabolisms) which resulted in the predominance of oxidation reaction over reduction reaction and increased the rH. As concentration of ammonium ions decreased to a critical value, the metabolism of nitrate triggered. Consequently, other metabolisms of carbon source were retarded resulted in the decrease of rH.
誌謝 I
Abstract II
英文摘要 III
目錄 V
圖目錄 VII
表目錄 X

第一章 緒論 1

第二章 文獻回顧 2
2-1轉殖毛狀根 2
2-2二次代謝產物 6
2-3生物反應器 9
2-4植物細胞之氮源代謝 15
2-5氧化還原電位 22

第三章 實驗 25
3-1 實驗流程 25
3-2 實驗藥品 26
3-3 實驗儀器 28
3-4 實驗步驟 29

第四章 結果與討論 39
4-1 250 mL錐形瓶之養分中間饋料實驗 39
4-1-1 氮源之添加 39
4-1-1-1 氮源初期饋料與中期饋料之影響實驗 40
4-1-1-1-1 KNO3在初期饋料與中期饋料之影響實驗 41
4-1-1-1-2 (NH4)2SO4在初期饋料與中期饋料之影響實驗 43
4-1-1-2 氮源饋料對根之生長及培養液之pH的影響 44
4-1-1-3 中期饋料之氮源最適化實驗 47
4-1-1-3-1 中期饋料之KNO3濃度最適化實驗 48
4-1-1-3-2 中期饋料之(NH4)2SO4濃度最適化實驗 49
4-1-1-3-3 中期饋料之KNO3及(NH4)2SO4最適氮源實驗 52
4-1-2 胺基酸之添加 59
4-2 噴霧滴流式生物反應器(MTB)之放大研究 64
4-2-1 9 L MTB反應器之最適化研究 64
4-2-2 導電度、pH、DO、ORP及蔗糖之變化 69
4-2-3 培養中期(10天)之氮源添加研究 74

第五章 結論 77

第六章 參考文獻 79
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