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

Alexopoulus, C. J. and C. W. Mims, 1979. Introductory Mycology John Wiley & Sons Co., New York, 632 pp.
Anders, R. J. J. G. Cervency, and A. L. Milkowski, (Dec. 19, 1989). U.S.
Patent 4,888,191, ( to Oscar Mayer Foods Corp., U.S.A.).
Anders, R. J. J. G. Cervency, and A. L. Milkowski, (May. 21, 1991). U.S.
Patent 5,017,391, ( to Oscar Mayer Foods Corp., U.S.A.).
Bai, D., M. Jia, X. Zhao, R. Ban, F. Shen, X. Li and S. Xu, 2003. L(+)-lactic acid production by pellet-form Rhizopus oryzae R1021 in a stirred tank fermentor. Chem. eng. sci. 58: 785-791.
Benthin, S. and J. Villadsen, 1995. Production of optically pure D− lactate
by Lactobacillus bulgaricus and purification by crystallization and liquid/liquid extraction. Appl. microbiol. biotechnol. 42: 826−829.
Bottone, E. J., I. Weitzman and B. A. Hanna, 1979. Rhizopus rhizopodiformis: Emerging etiological agent of Mucormycosis. J. Clin. Microbiol. 9: 530-537.
Datta, R., S. P. Tsai, P. Bonsignore, S. H. Moon, and J. R. Frank, 1995. Technological and economic potential of poly (lactic acid) and lactic acid derivatives. FEMS microbial. rev. 16: 221-231.
Dominguez, J. M., and Vazquez M., 1999. Effect of the operational conditions on the L-lactic acid production by Rhizopus oryzae. Cienc. Tecnol. Aliment. 2(3):113−118.
Ellis, J. J., L. J. Rhodes and C. W. Hesseltin, 1976. The genus Amylomyces. Mycologia 68: 131-143.
Espinel-ingroff, A., L. A. Oakley, and T. M. Kerkering, 1987.Opportunistic Zygomycotic infections. Mycopathologia .97:33−41.
Frye, C. B. and D. J. Reinhardt, 1993. Characterization of groups of the Zygomycete genus Rhizopus. Mycopathologia.124:139−147.
Harris, J. E. and C. Dennis, 1980. Distribution of Mucor piriformis, Rhizopus sexualis and Rhizopus stolonifer in relation to their spoilage of strawberries. Trans. Brit. Mycol. Soc. 75: 445-450.
Heights, A., 1989. Lactic Acid and Lactates. Product Bulletin, Purac. Inc., IL.
Hesseltine, C.W. and J. J. Ellis, 1961. Notes on mucorales, especially Absidia. Mycologia 53: 406-426.
Holten, C.H., A. Muller, D. Rehbinder. 1971. Lactic acid, Verlag Chemie, Internation Research Association, Copenhagen, Denmark.
Kandler, O., 1982. Garungsmechansimen bei Milchsaurebakterien. Forum mikrobiol. 5: 16.
Kascak, J. S., J. Kominek, and M. Roshr, 1996. Lactic acid. In: Biotechnology, Vol.6, Rehm, H. J., G. Reed, A. Puhler, and P. Stadler, Eds, pp. 293-306. Weinheim: VCH Press.
Keller, A. K. and Gerhardt, P., 1975. Continuous lactic acid fermentation of whey to produce a ruminant feed supplement high in crude protein. Biotechnol. Bioeng., 17: 997-1018.
Keshavarz, T., R. Eglin, E. Walker, C. Bucke, G. Holt, A.T. Bull, and
Liou G. Y., Chann C. C. and W. H. Hsu, 1990. Atlas of the genus Rhizopus and its allies.
M.D. Lilly, 1990. The large−scale immobilization of Penicillium chrysogenum: Batch and continuous operation in an air−lift reactor. Biotechnol. bioeng. 36: 763 −700.
Kosakai, Y., YS. Park, and M. Okabe, 1997. Enhancement of L (+)−lactic acid production using mycelial flocs of Rhizopus oryzae. Biotechnol. bioeng. 55: 461-470.
Lipinsky, E. S. and R. G. Sinclair, 1986. Environmentally benign lactic acid polymers can be produced in large quantities at low process for packaging and consumer goods. Chem. eng. prog. 82(8): 26−32.
Lockwood, L. B., G. B. Ward, and O. E. May, 1936. The physiology of Rhizopus oryzae. J. agric. res. 53: 849−857.
Miura, S., T. Arimura, M. Hoshino, M. Kojima, L. Dwiarti, and M. Okabe, 2003. Optimization and scale-up of L−lactic acid fermentation by mutant strain Rhizopus sp. MK-96-1196 in airlift bioreactors. J. biosci. bioeng. 96(1): 65−69.
Norton, S.; Lacroix, C. & Vuillemard, J. C., 1994. Kinetic study of continuous whey permeate fermentation by immobilized Lactobacillus helveticus for lactic acid production. Enzyme Microb. Technol., 16: 457-466.
Ohara, H., 1994. Poly−L−lactic acid as biodegradable plastic. Biosci. ind. 52: 642-644.
Parajó, J. C., Alonso, J. L. & Santos, V., 1996. Lactic acid from wood. Process Biochem. 31: 271-279.
Park, E. Y., Y. Kosakai, and M. Okabe, 1998. Efficient Production of L −(+)−lactic acid using mycelial cotton-like flocs of Rhizopus oryzae in an air−lift bioreactor. Biotechnol. prog. 14: 699−704.
Peter, C. F., and P. A. Sullivan, 1998. The Rhizopus oryzae secreted aspartic proteinase gene family: an analysis of gene expression. Microbiol. 144: 2355−2366.
Roble, N. D., J. C. Ogbonna and H. Tanaka, 2003. L−lactic acid production from raw cassava starch in a circulating loop bioreactor with cells immobilized in loofa (Luffa cylindrical). Biotechnol. Lett. 25: 1093-1098
Skory, C. D., S. N. Freer, and R. J. Bothast, 1998. Production of L−lactic acid by Rhizopus oryzae under oxygen limiting conditions. Biotechnol. lett..20(2): 191−194.
Soccol, C. R., B. Marin, M. Raimbault, and JM. Lebault, 1994a. Potential of solid state fermentation for production of L (+)−lactic acid by Rhizopus oryzae . Appl. micribio. biotechnol. 41: 286−290.
Soccol, C. R., VI. Stonoga, and M. Raimbault, 1994b. Production of L (+)− lactic acid by Rhizopus species. World. j. microbio. biotechnol.10: 433−435.
Tay, A. and S. P. Yang, 2002. Production of L (+)−lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor. Biotechnol. bioeng. 80: 1−12.
Tsai, S.P. and S.H. Moon, 1998. An Integrated Bioconversion Process for Production of L− lactic acid from Starchy Potato Feedstocks. Appl. biochem. biotechnol. 70−72: 417−428.
Van Ness, J.H. 1981. Hydroxy Carboxylic Acids. In Kirk Othmer Encyclopedia of Chemical Technology, 3rd Ed., Vol. 13, 80−103. J. Wiley and Sons, New York.
Vickory, TB. 1985. Lactic acid. In: Blanch HW, Drew S, Wang DIC, eds. The Practice of Biotechnology: Commodity Products, Vol.3. Elmsford, pp. 761−776.
Wang, C. W., Z. Lu, and G. T. Tsao, 1995. Lactic acid production by pellet-form Rhizopus oryzae in a submerged system. Appl. biochem. biotechnol. 51/52: 57−71.
Webster, J. 1980. Introduction to Fungi. 2nd edn, p.213. Cambridge: Cambridge University Press.
Wright, B. E., A. Longacre, and J. Reimers, 1996. Models of metabolism in Rhizopus oryzae. J. theor. biol.182: 453−457.
Yang, C. W., Lu, Z. J., & Tsao, G. T., 1995. Lactic acid production by pellet-form Rhizopus oryzae in a submerged system. Biotechnol. bioeng. 51/52: 57−71.
Yin, P., K. Yahiro, T. Ishigaki, Y. Park, and M. Okabe, 1998. L (+)− lactic acid production by repeated batch culture of Rhizopus oryzae in an air−lift bioreactor. J. ferment. bioeng .85: 96-100.
Yoneya, T. and Y. Sato, 1980a. Effect of growth condition on the formation of Rhizopus oryzae javanicus, Funfi alcohol dehydrgenase. Agric. biol. chem.44: 1949-1959.
Yoneya, T. and Y. Sato, 1980b. Comparison of two alcohol dehydrogenases in the fungus Rhizopus javanicus. Appl. environ. microbiol.40: 967-969.