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本研究之目的為利用含腈水合酶(nitrile hydratase, NHase)與醯胺酶(amidase)兩酵素系統的丙烯醯胺生成菌，以生物處理法將丙烯腈轉換至高價值的商業產品丙烯醯胺，並控制不同的生長環境因子，使菌株具有較佳轉換丙烯腈速率，且藉由添加化學物質以抑制醯胺酶的活性，使所獲得的丙烯醯胺能持續累積不易轉換至丙烯酸與氨。實驗結果顯示由PAN人造纖維製造廠廢水處理廠活性污泥中所分離篩選出菌株Mesorhizobium sp.具有轉換丙烯腈累積丙烯醯胺的能力，且該菌不具有質體；其最佳生長溫度在以R2A作為培養基時是37oC。
由菌株Mesorhizobium sp.轉換丙烯腈累積丙烯醯胺的特性可知，於丙烯腈濃度小於976.2 mg/l與培養環境的pH值為7.5~8.2時，菌株具有較佳轉換丙烯腈累積丙烯醯胺的能力。由額外添加基質之實驗結果顯示，添加濃度494.0 mg/l酵母抽出物對菌株生長有所助益，可增加菌株轉換丙烯腈累積丙烯醯胺效率，於26.7小時濃度964.5 mg/l丙烯腈的轉換率約100%；但添加葡萄糖與丙酮酸鈉卻會降低菌株Mesorhizobium sp.轉換丙烯腈的效率，於反應時間45.4小時濃度964.5 mg/l丙烯腈之轉換率約52.6%。
促進菌株腈水合酶與抑制其醯胺酶的活性之不同影響因子研究結果發現，所添加化學物質中，尿素、丙醛與丁醛皆能抑制醯胺酶活性，亦能誘導促進腈水合酶的活性，使丙烯腈轉換丙烯醯胺百分率增加，其中以丙醛效果最好，添加濃度207.4 mg/l丙醛時，於3.8小時丙烯腈轉換丙烯醯胺百分率約近似100%；而添加苯基醛則會輕微抑制菌株腈水合酶活性，對抑制其醯胺酶活性則無助益。對菌株腈水合酶而言，其以鈷離子作為輔助因子時具有較佳活性；而添加0.1 mg/l Ag2SO4時會輕微抑制腈水合酶的活性影響其轉換丙烯腈能力，濃度492.9 mg/l丙烯腈於29.1小時其轉換率為54.9%，當Ag2SO4濃度增加至0.5 mg/l時菌株轉換丙烯腈能力完全喪失；此外，添加EDTA、己內醯胺與丙烯醯胺對菌株腈水合酶沒有影響。
綜合上述研究結果，若要將菌株Mesorhizobium sp.運用於處理含丙烯腈(濃度約495.0 mg/l)的廢水以產生丙烯醯胺，可將反應條件設定為：添加丙醛於含鈷離子之磷酸鹽緩衝溶液中，並控制培養環境的pH值在7.5~8.2間，則菌株Mesorhizobium sp.應能於短時間內將丙烯腈轉換成丙烯醯胺，且丙烯醯胺不易轉換至丙烯酸與氨。
Acrylamide, a commodity chemical widely used to produce polymers for application in sewage treatment, petroleum recovery papermaking and textile sizing industrial processes, is manufactured from acrylonitrile mainly by chemical processes. Nevertheless, acrylonitrile biotransformation to acrylamide has been also developed up to the industrial scale.
Based on the high-value of acrylamide, the purpose of this study was to use the microorganisms that have both enzymes of nitrile hydratase (NHase) and amidase to produce acrylamide from acrylonitrile. In order to obtain acrylamide, it was necessary to realize the physiological conditions of the bacteria and adjust the suitable environmental factors for the bacteria to convert acrylonitrile into acrylamide effectively. Furthermore, the percentage of acrylonitrile converted to acrylamide could be raised by adding chemicals to enhance the activity of NHase and inactivate amidase.
Mesorhizobium sp., which was isolated from the activated sludge of the polyacrylonitrile (PAN) fiber manufacture wastewater treatment system, contained both enzymes NHase and amidase, and could convert acrylonitrile to acrylamide. The optimum growth temperature of Mesorhizobium sp. was 37oC by utilizing R2A as growth medium. When the pH values were between 7.5 and 8.2, Mesorhizobium sp. had the better ability to convert acrylonitrile to acrylamide.
The growth of Mesorhizobium sp. and the percentage of acrylonitrile converted to acrylamide could be increased by adding yeast extract. In the presence of yeast extract concentration was added about 494.0 mg/l, 964.5 mg/l acrylonitrile could be completely converted within 26.7 hours. On the other hand, the conversion rate of acrylonitrile would decrease as adding glucose and sodium pyruvate; 964.5 mg/l acrylonitrile was converted about 52.6% within 45.4 hours.
Addition of urea, propionaldehyde, or butyraldehyde could not only enhance the activity of NHase but inhibit the activity of amidase, therefore, acrylamide could be accumulated effectively and the percentage of acrylonitrile converted to acrylamide increased. Propionaldehyde was the most effective one among them. The percentage of acrylonitrile converted to acrylamide was almost 100% at 3.8 hours when propionaldehyde was added about 207.4 mg/l. Addition of benzaldehyde was unable to increase the percentage of acrylonitrile converted to acrylamide.
For the NHase of Mesorhizobium sp., cobalt ion was its cofactor and could increase the activity of NHase. EDTA, ε- caprolactam, and acrylamide were of no effect on the activity of NHase. However, 0.1 mg/l silver thiosulphate would slightly inhibit the activity of NHase and the conversion rate of 492.9 mg/l acrylonitrile was 54.9% at 29.1 hours. Moreover, the ability of the acrylonitrile biotransformation was completely inhibited if the concentration of the silver thiosulphate was above 0.5 mg/l.
If Mesorhizobium sp. was used in real wastewater treatment and acrylonitrile concentration of the wastewater was assumed as 495.0 mg/l, the optimal conditions could be set as follows:
First, remain the pH of the wastewater between 7.5 and 8.2, then add cobalt chemicals to activate NHase, and finally add propionaldehyde to inactivate amidase. Therefore, 605.6 mg/l acrylamide could be obtained within 3.8 hrs. By this means, Mesorhizobium sp. could convert acrylonitrile effectively and high- value acrylamide could be acquired.
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