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研究生:林怡君
研究生(外文):I-Chun Lin
論文名稱:以米根黴生產L型乳酸
論文名稱(外文):Production of L (+)�{lactic acid by 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:生物工程學系(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:2004
畢業學年度:93
語文別:英文
論文頁數:121
中文關鍵詞:生物反應器L型乳酸米根黴
外文關鍵詞:bioreactorL-lactic acidRhizopus oryzae
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L型乳酸,分子式C3H6O3,為含有氫氧基的有機酸,是生物的代謝中間產物。L型乳酸可以在動物和人類細胞中新陳代謝因為這些細胞含有L型乳酸去氫酵素。L型乳酸還可用於生産L型乳酸聚合物。L型乳酸聚合物屬於無毒的高分子化合物,具有生物相容性,可用於製造生物可分解的塑膠、纖維以及生醫材料等。本研究以黴菌Rhizopus oryzae CCRC 9363為L型乳酸生產菌,針對生物反應器的種類與最適操作條件來進行探討。最後由實驗得知以改良型的生物反應器為最適生產L型乳酸的反應器。
L-lactic acid, C3H6O3, an organic acid with hydroxy group is a metabolic intermediate in living organisms. Only the L-form of lactic acid is metabolized in animal and human cells. This is due to the fact that only L-lactate dehydrogenase is synthesised by these cells. L-lactic acid can be polymerized to form Poly�{lactic acid (PLA). L-form PLA is a non�{virulent macromolecular compound with biocompatibility, which used in the manufacture of new biodegradable plastics, fiber, and biomedical material. This experiment used fungus, Rhizopus oryzae CCRC 9363 to produce L-lactic acid and discussed the types of bioreactor and best conditions. Final, it is discovered that the modified bioreactor is the best bioreactor to produce L-lactic acid.
Content

ABSTRACT I

中文摘要 II

List of Figure VI

List of Table XI

List of Picture XII

CHAPTER 1. INTRODUCTION 1

CHAPTER 2. MATERIALS AND METHODS 24
2.1. Materials 24
2.1.1. Instruments 24
2.1.2. Fungal Strain 26
2.1.3. Chemicals 26
2.1.4. Culture medium 27
2.2. Production of L�{lactic acid in by fermentation in a stirred tank bioreactor 29
2.2.1. Experiment (1): 5�{L fermenter 29
2.2.2. Experiment (2): Batch fermentation in a 5�{L bioteactor equipped with the septum made of stainless steel net 34
2.2.3. Experiment (3): A modified type bioreactor with central downward-dring propelles 39
2.3. Assay methods 44
2.3.1. Analysis of glucose, L�{lactic acid and ethanol by HPLC 44
2.4. Control of fermentation 46

CHAPTER 3. RESULTS AND DISCUSSIONS 49
3.1. Shake culture of Rhizopus oryzae 49
3.2. Large scale production of L�{lactic aicd by Rhizopus oryzae 54
3.2.1. Production of L�{lactic acid by Rhizopus oryzae using a 5�{L jar fermenter. 55
3.2.2. Production of L�{lactic acid by Rhizopus oryzae using a 5�{L bioreactor equipped with the septum made of stainless steel net 65
3.2.3. Production of L�{lactic acid by Rhizopus oryzae using an air�{lift bioreactor with central downward�{driving propells 74

CHAPTER 4. CONCLUSION 95

REFERENCE 97

APPENDIXES 105
Appendixes 1: Referential Table of JIS, A.S.T.M and TYLER 105













List of Figure
Figure1.1. Hypothesis of formation of the cotton-like flocs among the mycelia, supports, and PEO. 23
Figure 2.1. The 5�{L fermentor. The schematic diagram of the system for batch fermenter. 31
Figure 2.2. Batch fermentation in a 5�{L fermenter. Scheme the experimental design of fermentation by Rhizopus oryzae for the production of L�{lactic acid. 33
Figure 2.3. The 5�{L bioreactor equipped with the septum made of stainless steel net and an air�{supply ring at the bottom. The air bubbles were released next to glass wall of the bioreactor. 36
Figure 2.4. Batch fermentation in a 5�{L bioreactor equipped with the septum made of stainless steel net. Scheme the experimental design of fermentation by R. oryzae for the production of L�{lactic acid. 38
Figure 2.5. A modified type bioreactor equipped with central downward�{driving propells and air�{supply ring at the bottom. The air bubbles were released next to glass wall of the bioreactor. 41
Figure 2.6. A modified type bioreactor with central downward�{driving propells. Scheme the experimental design of fermentation by Rhizopus oryzae for the production of L�{lactic acid. 43
Figure 2.7. Scheme of the procedures for analysis of glucose, L�{lactic acid and ethanol by HPLC 45
Figure 2.8. A display of the original ADVENTECH GENIE strategy of fermentation (mainboard) 47
Figure 2.9. A display of original ADVENTENCH GENIE strategy for fermentation (connecting system) 48
Figure 3.1. Production of L�{lactic acid in the shake culture. (glucose, 50 g/L, yeast extract, 10 g/L) 50
Figure 3.2. Production of L�{lactic acid in the shake culture. (glucose, 50 g/L, yeast extract 10 g/L , 5% w/v CaCO3) 51
Figure 3.3. Production of L�{lactic acid in the shake culture. (glucose, 100 g/L, yeast extract 10 g/L) 52
Figure 3.4. Production of L�{lactic acid in the shake culture. (glucose, 100 g/L, yeast extract 10 g/L , 5% w/v CaCO3) 53
Figure 3.5. Production of L�{lactic acid using a two-stage fermentation in a 5�{L jar fermenter (glucose 100 g/L, yeast extract 10 g/L, aeration rate 10→2 L/min; temp. 30°C) 57
Figure 3.6. Production of L�{lactic acid in a 5�{L jar fermenter with aeration rate unchanged (1st�{run). (glucose 100 g/L, yeast extract 10 g/L, aeration rate 10 L/min, temp. 30°C) 59
Figure 3.7. Production of L�{lactic acid in a 5�{L jar fermenter with aeration unchanged (2nd�{run). (glucose 100 g/L, yeast extract 10 g/L, aeration rate 10 L/min, temp. 30°C) 60
Figure 3.8. Production of L�{lactic acid in a 5�{L jar fermenter with aeration rate unchanged (3rd�{run) (glucose 100 g/L, yeast extract 10 g/L, aeration rate 10 L/min, temp. 30°C) 61
Figure 3.9. Production of L�{lactic acid in a 5�{L jar fermenter (the fermenter was equipped with five pieces of propellers)(glucose 100 g/L, yeast extract 10 g/L, aeration rate 10 L/min, temp. 30°C) 63
Figure 3.10. Production of L�{lactic acid in a 5�{L air�{lift bioreactor with the septum made of stainless steel net (glucose 100 g/L, yeast extract 10 g/L, aeration rate 10 L/min, temp. 30°C, 35 mesh) 66
Figure 3.11. Production of L�{lactic acid in a 5�{L air�{lift bioreactor with septum made of stainless steel net (air supplied by a ring structure). (glucose 100 g/L, yeast extract 10 g/L, aeration rate 10 L/min, temp. 30°C, 35 mesh) 68
Figure 3.12. Production of L�{lactic acid in a 5�{L air�{lift bioreactor with septum made of stainless steel net (air supplied by a ring structure). (glucose 100 g/L, yeast extract 10 g/L, aeration rate 10 L/min, temp. 30°C, 16 mesh) 70
Figure 3.13. Production of L�{lactic acid in the 5�{L air-lift bioreactor with septum made of stainless steel net (air supplied by a ring structure). (glucose 100 g/L, yeast extract 10 g/L, aeration rate 10 L/min, temp. 30°C, 5 mesh) 72
Figure 3.14. Production of L�{lactic acid in the bioreactor with central downward-driving propells. (glucose 100 g/L, yeast extract 10 g/L, aeration rate 10 L/min, stir rate 200rpm, temp. 30°C) 75
Figure 3.15. Production of L�{lactic acid in the bioreactor with central downward�{driving propells. (glucose 200 g/L, yeast extract 10 g/L, aeration rate 10 L/min, stir rate 200rpm, temp. 30°C) 77
Figure 3.16. Production of L�{lactic acid in the bioreactor with central downward�{driving propells. (glucose 150 g/L, yeast extract 10 g/L, aeration rate 10 L/min, stir rate 200rpm, temp. 30°C) 79
Figure 3.17 Production of L�{lactic acid in the bioreactor with central downward�{driving propells. (glucose 100 g/L, yeast extract 5 g/L, aeration rate 10 L/min, stir rate 200rpm, temp. 30°C) 81
Figure 3.18. Production of L�{lactic acid in the bioreactor with central downward�{driving propells. (glucose 100 g/L, corn steep liquor 5 g/L, aeration rate 10 L/min, stir rate 200rpm, temp. 30°C) 83
Figure 3.19. Production of L�{lactic acid in the bioreactor with central downward�{driving propells. (glucose 100 g/L, corn steep liquor 2 g/L, aeration rate 10 L/min, stir rate 200rpm, temp. 30°C) 85
Figure 3.20. Production of L�{lactic acid in the bioreactor with central downward�{driving propells. (glucose 100 g/L, corn steep liquor 1 g/L, aeration rate 10 L/min, stir rate 200rpm, temp. 30°C) 87
Figure 3.21. Production of L�{lactic acid in the bioreactor with central downward�{driving propells. (glucose 100 g/L, corn steep liquor 2 g/L, aeration rate 10 L/min, stir rate 200rpm, temp. 35°C) 89
Figure 3.22. Production of L�{lactic acid in the bioreactor with central downward�{driving propells. (support and PVA added, glucose 100 g/L, corn steep liquor 2 g/L, aeration rate 10 L/min, stir rate 200rpm, temp. 35°C) 91
Figure 3.23. Production of L�{lactic acid in the bioreactor with central downward�{driving propells. (support and PVA added, glucose 100 g/L, NH4NO3 2 g/L, aeration rate 10 L/min, stir rate 200rpm, temp. 35°C) 93
List of Table

Table 1.1 Lactic acid: potential products, volumes, and value 12

Table 1.2 Lactide copolymer vs. vinyl polymers 14

Table 1.3 Lactide copolymer vs.polystyrene 15

Table 3.1 Production of L –lactic acid 94












List of Picture

Picture 2.1: 5-L jar fermenter 32

Picture 2.2: 5-L bioreactor equipped with the septum made of stainless steel net 37

Picture 2.3: A bioreactor with central downward-driving propells 42
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