Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3766
標題: 類芽孢桿菌液態發酵培養生產幾丁質酵素之研究
Studies on Chitinase Production in Submerged Culture of Paenibacillus taichungensis
作者: 高博敏
Kao, Po-Min
關鍵字: Chitinase;幾丁質酵素;Paenibacillus taichungensis;Shear stress;Shear rate;Stirred-tank bioreactor;Membrane bioreactor;Net-draft tube air-lift bioreactor;Oxygen mass transfer;Microfiltration;Ceramic membrane;類芽孢桿菌;剪切應力;剪切率;攪拌式發酵槽;膜式發酵槽;網狀內管氣舉式發酵槽;氧氣質傳;微過濾;陶瓷過濾膜
出版社: 化學工程學系所
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摘要: 
類芽孢桿菌台中種(Paenibacillus taichungensis)為一種新品種細菌,此菌也是一株具有高幾丁質酵素活性之生產菌。本研究的主要目的在探討液態發酵培養類芽孢桿菌生產幾丁質酵素並提高其酵素生產力。其中主要包含下列三部份為
第一部分,應用5 L攪拌式發酵槽培養P. taichungensis生產幾丁質酵素之研究。其操作條件的影響,包括不同的攪拌轉速及通氣量,並觀察菌體生長和幾丁質酵素活性的變化。當發酵槽之攪拌轉速低於100 rpm時,氧氣質傳限制是顯著的。而攪拌轉速在300 rpm時,具有過高的剪切應力,其高剪切應力會造成菌體分解死亡,並且會產生大量的孢子繁殖,因而影響到幾丁質酵素的生產。而5 L攪拌式發酵槽得最佳操作條件是在通氣量3 vvm,攪拌轉速200 rpm和不調控pH值,其最佳的幾丁質酵素活性可達11.8 U/mL。
第二部分,應用陶瓷過濾膜貫流式發酵槽進行連續式培養P. taichungensis生產幾丁質酵素之研究。在發酵槽外部環路上,配合一陶瓷過濾膜,進行細胞回流操作,藉此將菌體留在發酵槽內,而排除發酵液中部分之幾丁寡糖及其他抑制菌體生產幾丁酵素之物質,以期能提高菌體之幾丁質酵素產量。並且探討在不同陶瓷薄膜過濾膜孔隙大小對於菌體保留效率、過濾流速、膜阻塞和幾丁質酵素濾液回收等方面的影響。實驗結果顯示,應用陶瓷過濾膜貫流式操作,在穿膜壓力 0.9 kg/cm2,以分子量300 kDa之孔隙大小的M 9之微過濾膜,具有最好的微過濾特性。並藉由置換添加去離子水,進行陶瓷過濾膜貫流式操作與批式操作之結果比較,在132 h其幾丁質酵素總活性可達到42.8 U,比起批式操作可提高78 %的幾丁酵素產量。更近一步探討,每3-4天以500 mL/day之流速添加幾丁質補充營養源進行連續式培養。實驗結果顯示,能穩定連續生產幾丁質酵素,且幾丁質酵素活性可維持在13-15 mU/mL的範圍。由此可見,應用陶瓷過濾膜貫流式微過濾操作,在發酵過程中是可提升幾丁質酵素活性及產量。
第三部分,應用不同型式之管柱式發酵槽培養P. taichungensis生產幾丁質酵素。在3種不同管柱式發酵槽之實驗結果發現,以網狀內管氣舉式發酵槽效果最好。並探討此發酵槽在不同通氣量下,細胞生長及幾丁質酵素生產之影響。實驗結果發現,在2 vvm下,其幾丁質酵素活性最佳可達21.1 mU/mL。為了暸解其原因,發酵槽裡的剪應速率(Shear rate)和氧氣質傳被探討。同時,菌體形態與幾丁質酵素生產之關聯性也一併被研究。

Paenibacillus taichungensis is a new strain bacterium, which is also a major chitinase production microorganism. The main purpose of this research was to improve the productivity of chitinase from Paenibacillus taichungensis by submerged fermentation. The study contains three pasts.
In the first part, the chitinase fermentations with strain Paenibacillus taichungensis were carried out in a 5 L stirred-tank bioreactor. The effects of operation variables including agitation rates and aeration rates, on cell growth and chitinase activity yield were evaluated. Oxygen transfer limitation was predominant when the fermentation was carried out at low agitation rate 100 rpm. At agitation rate of 300 rpm, the high shear stress was dominant. Decrease chitinase production and extensive sporulation along with cell degradation were observed. The operation conditions at an aeration rate of 3 vvm, an agitation rate of 200 rpm, and without pH value control could yield an optimal chitinase activity level of 11.8 U/mL.
In the second part, the feasibility of using membrane mode fermentation operations for the continuous chitinase production by Paenibacillus taichungensis was investigated. The bioreactor with a membrane outer recycling loop was used to evaluate the effect of membrane pore size on cell retention efficiency, permeate flow rate, fouling, and chitinase recovery in permeate. The results showed that at a transmembrane pressure of 0.9 kg/cm2, M 9 microfiltration column with a nominal pore size of 300 kDa exhibited the best microfiltration characteristics and was used for the membrane mode operation. As comparing the chitinase production in the membrane mode operation by feeding deionized water with that in batch mode, the total chitinase activity obtained in membrane mode operation could reach 42.8 U for 132 h, about 78 % higher than that obtained in batch mode operation. Further improvement by feeding chitin every 3-4 days showed a steadily continuous chitinase production with the activity ranging from 13-15 mU/mL at a flow rate of 500 mL/day. The membrane-based microfiltration operation appears to be useful for enhancing the chitinase activity production in fermentation.
Finally, various bioreactors were employed to investigate the chitinase production with Paenibacillus taichungensis. It was found that among the three column bioreactors, the net-draft tube air-lift bioreactor gave the best performance. The aeration condition for chitinase production of this bioreactor was carried out to evaluate the cell growth and chitinase production. It was found that at 2 vvm an optimal chitinase activity level of 21.1 mU/mL was produced. To exploit the result, the shear rates and oxygen transfer rates in these bioreactors were evaluated. Meanwhile, the morphologies of the strain associated to the chitinase production were also investigated.
URI: http://hdl.handle.net/11455/3766
其他識別: U0005-2107200915394200
Appears in Collections:化學工程學系所

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