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研究生:吳靜怡
研究生(外文):Ching-Yi Wu
論文名稱:用米根黴的菌絲團生產L型乳酸
論文名稱(外文):Production of L-Lactic Acid Using Mycelia Flocs of Rhizopus oryzae.
指導教授:許垤棊許垤棊引用關係
指導教授(外文):Dey-Chyi Sheu
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:英文
論文頁數:100
中文關鍵詞:絮狀菌絲補充氮源L型乳酸米根黴
外文關鍵詞:L(+)-lactic acidRhizopus oryzaemycelial flocsreplenish of nitrogen source
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在米根黴( Rhizopus oryzae BCRC 9363 )的傳統培養液中,菌絲會凝聚成菌塊,我們發現是因為培養液中的氮源耗盡的因素。因此在液態培養的過程定時補充氮源,可防止菌塊的產生,而形成棉絮狀的菌絲。因此,L型乳酸產量大大地提高。當在5公升攪拌式生物反應器中進行醱酵,用碳酸鈣乳液控制pH值在 4.3-4.5 之間,且每隔8小時補充少量的硫銨,以維持氨氮的濃度在每公升0.3-0.5公克之間。在此條件下,以每公升120克的葡萄糖為受質,L型乳酸產量為105公克,產率為0.88,生產速率為每小時每公升2.63公克。若沒有補充氮源,菌塊會纏繞在生物反應器內的裝置上面,而且L型乳酸產量顯著的下降。本研究展示一個簡單的方法使米根黴產生棉絮狀菌絲,而且L型乳酸的生產量達到最適化。
The formation of mycelial clumps in conventional cultures of Rhizopus oryzae resulted mainly from the depletion of nitrogen source in the cultural medium. Based on this behavior, L(+)-lactic acid production could be enhanced in the submerged culture using mycelial flocs induced by replenishing ammonium-nitrogen in the cultural medium, thereby the formation of mycelial clumps or pellets which often occurred in conventional cultures of R. oryzae was prevented. When the fermentation was performed in a 5-L stirred tank bioreactor with the pH being controlled in the range of 4.3 – 4.5 by adding calcium carbonate slurry, and small amounts of ammonium sulfate was added at 8-h intervals to sustain ammonium level of the culture in the range of 0.3 – 0.5 g/L, lactic acid production was optimized. The lactic acid concentration produced was 105 g/L, with the yield of 0.88 and the productivity of 2.63 g/L-h, using 120 g/L of glucose as substrate. Without replenish of ammonium-nitrogen, mycelial clumps attached everywhere in the bioreactor and lactic acid production dramatically declined. This article demonstrates a simple way to obtain mycelial flocs of R. oryzae thereby an optimized lactic acid production can be achieved.
ABSTRACT................................................................... I
中文摘要.......................................................................III
List of Figure .............................................................. IX
List of Table ..............................................................XIII
List of Picture ...........................................................XIV
CHAPTER 1. INTRODUCTION.................................1
1.1. Lactic acid…………………………………………………1
1.1.1. Properties of lactic acid…………………………………………1
1.1.2. Manufacturing technologies…………………………………….2
1.1.2.1. Chemical synthesis……………………………………..3
1.1.2.2. Carbohydrate fermentation……………………………..4
1.1.3. Current uses……………………………………………………..7
1.1.3.1. Food processing applications…………….…………..…8
1.1.3.2. The industrial applications…………………………….10
1.1.4. Potential productions and markets……………………………..10
1.2. L-lactic acid………………………………….…………...13
1.3. Poly-L-lactic acid ( PLLA )……………………………..13
1.4. Rhizopus oryzae………………………………………….18
1.4.1. Rhizopus species……………………………………………….18
1.4.2. Rhizopus oryzae………………………………………………..19
1.4.3 The mycelial morphology………………………………………23
CHAPTER 2. MATERIALS AND METHODS........29
2.1. Materials…………………………………………………29
2.1.1. Microorganisms………………………………………………..29
2.1.2. Instruments…………………………………………………….29
2.1.3. Chemicals……………………………………………………...31
2.2. Cultivations………………………….…………………...31
2.2.1. Culture medium………………………………………………..32
2.2.2. Culture method………………………………………………...32
2.3. Assay methods…………………………………………...38
2.4. Control of fermentation…………….…………………...40
CHAPTER 3. RESULTS AND DISCUSSIONS........43
3.1. Effect of Two Nitrogen Sources on Mycelial Morphology of the Seed Culture……………………………………..43
3.2. Effect of Replenish of Ammonium Sulfate on Floc Formation in a Bioreactor…………………………………………………..……..47
3.3. Effect of Phosphate Concentration on Lactic acid Production using mycelial flocs in a Bioreactor……………………………...49
3.3.1. Production of L-lactic acid by Rhizopus oryzae with 0.25 g/L of K2HPO4 at pH 4.3 – 4.5 in the 5-L stirred tank bioreactor………………………………………………….….50
3.3.2. Production of L-lactic acid by Rhizopus oryzae with 0.5 g/L of K2HPO4 at pH 4.3 – 4.5 in the 5-L stirred tank bioreactor……………………………………………………..52
3.3.3. Production of L-lactic acid by Rhizopus oryzae with 0.75 g/L of K2HPO4 at pH 4.3 – 4.5 in the 5-L stirred tank bioreactor…………………………………………………….54
3.4. Effect of Controlled pH on Mycelial Morphology and Lactic acid Production in a Bioreactor…………………………………...…..56
3.4.1. Production of L-lactic acid by Rhizopus oryzae with 0.5 g/L of K2HPO4 at pH 4.0 – 4.2 in the 5-L stirred tank bioreactor………………………………………………….….57
3.4.2. Production of L-lactic acid by Rhizopus oryzae with 0.5 g/L of K2HPO4 at pH 4.3-4.5 in the 5-L stirred tank bioreactor……………………………………………………..59
3.4.3. Production of L-lactic acid by Rhizopus oryzae with 0.5 g/L of K2HPO4 at pH 4.6-4.8 in the 5-L stirred tank bioreactor……………………………………………………..61
3.4.4. Production of L-lactic acid by Rhizopus oryzae with 0.5 g/L of K2HPO4 at pH 4.9-5.1 in the 5-L stirred tank bioreactor………………………………………………….….63
3.4.5. Production of L-lactic acid by Rhizopus oryzae with 0.5 g/L of K2HPO4 at pH 5.2-5.4 in the 5-L stirred tank bioreactor………………………………………………..……65
3.4.6. Effect of controlled pH on mycelial morphology and lactic acid production by submerged cultures of R. oryzae in the 5-L stirred tank bioreactor………………………………………...67
3.5. Repeated Batch Fermentation for Lactic acid Production in a Bioreactor…………………………...…………………………….71
3.5.1. Production of L-lactic acid by R. oryzae with 0.5 g/L of K2HPO4 at pH 4.3-4.5 in the 5-L stirred tank bioreactor. ( 1st-run)...72
3.5.2. Production of L-lactic acid by R. oryzae with 0.5 g/L of K2HPO4 at pH 4.3-4.5 in the 5-L stirred tank bioreactor. ( 2nd-run)..74
3.5.3. Production of L-lactic acid by R. oryzae with 0.5 g/L of K2HPO4 at pH 4.3-4.5 in the 5-L stirred tank bioreactor. ( 3rd-run)..76
3.5.4. Production of L-lactic acid by R. oryzae with 0.5 g/L of K2HPO4 at pH 4.3-4.5 in the 5-L stirred tank bioreactor. ( 4th-run)..78
3.5.5. Production of L-lactic acid by R. oryzae with 0.5 g/L of K2HPO4 at pH 4.3-4.5 in the 5-L stirred tank bioreactor. ( 5th-run)..80
3.5.6. Production of L-lactic acid by R. oryzae with 0.5 g/L of K2HPO4 at pH 4.3-4.5 in the 5-L stirred tank bioreactor. ( 6th-run)..82
3.5.7 Results of six cycle of repeated batch fermentation in a stirred tank fermentation………………………………………………84
3.7 Summary of lactic acid production by R. oryzae…………….......87

CHAPTER 4. CONCLUSION....................................90
REFERENCE.........................................................94
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