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LACTIC ACID PRODUCTION by IMMOBILIZED RHIZOPUS ORYZAE in a ROTATING FIBROUS BED BIOREACTOR DISSERTATION Presented in Partial

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LACTIC ACID PRODUCTION BY IMMOBILIZED RHIZOPUS ORYZAE IN A ROTATING FIBROUS BED BIOREACTOR DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nuttha Thongchul, M.S. ***** The Ohio State University 2005 Dissertation Committee: Approved by Professor Shang-Tian Yang, Advisor Professor Jeffrey J. Chalmers __________________________________ Professor James F. Rathman Advisor Graduate Program in Chemical Engineering ABSTRACT Lactic acid has long been widely used in food and pharmaceutical industries. Due to the evolution of new applications in solvent replacement, biodegradable polymer, and oxygenated chemicals, the estimated worldwide market of lactic acid, especially the L(+)-isomer, tends to increase rapidly. Lactic acid bacteria have been extensively used in industrial production of lactic acid because of their high growth rate and product yield. However, the major limitations including costly substrates and complicated product recovery make bacterial fermentation economically unattractive. In contrast, filamentous fungi, Rhizopus oryzae, can produce the optically pure L(+)-lactic acid from complex carbohydrates present in agricultural residues and plant biomass without prior treatment; therefore, can overcome the problems in bacterial fermentation. However, cultivation of filamentous fungi in a stirred tank bioreactor is usually troublesome because of the diversity and change in morphology during fermentation which in turn affects lactic acid production. Therefore, in this research, fungal morphology was controlled by immobilization. Contrary to free cell culture in the stirred tank bioreactor, the fermentation carried out in a Rotating Fibrous Bed bioreactor (RFBB) resulted in good control of morphology, and improved oxygen transfer and lactic acid production from glucose. The improved oxygen transfer obtained in the RFBB not ii only increased lactic acid production rate, but also limited undesirable ethanol production and allowed the bioreactor to be operated for long-term production. To minimize the production cost, the feasibility of using low-value substrates derived from agricultural residues and plant biomass was studied. It was found that R. oryzae was capable of utilizing both starchy materials present in agricultural residues and pentose sugars which were abundant in plant biomass. Lactic acid yields obtained from these substrates were comparable to the yield from glucose. However, the production rate obtained from fermentation of pentose and insoluble starch was lower than that obtained from fermentations of glucose and soluble starch because of the complicated pentose metabolism and poor oxygen transfer in the cultivation with insoluble starch. Process engineering techniques were also used to improve lactic acid production in the RFBB. Previous study reported the critical demand of oxygen for lactic acid production. In this research, it was found that increasing oxygen transfer rate led to the increase in lactic acid productivity. Although high oxygen transfer rate was maintained, ethanol production and the estimation of the critical biofilm thickness indicated an anoxic condition in the overgrown immobilized fungal cells on the rotating fibrous matrix. In this study, growth of immobilized cells was controlled by shaving-off the mycelia with rotational shear rate and by limiting the concentration of the nitrogen source in the medium. iii In order to achieve controlled growth and immobilization of productive cells for stable long-term operation, spore germination and cell immobilization at the initial phase was studied. The effect of rotational speed on spore immobilization on different fibrous matrices was investigated. The mechanisms of spore immobilization on different fibrous matrices were elucidated. The knowledge gained in these process engineering techniques is important to the development of the RFBB and its scale-up for lactic acid production from sugars. iv Dedicated to my mother v ACKNOWLEDGMENTS My great appreciation goes to my advisor, Dr. Shang-Tian Yang, for intellectual support, encouragement, and enthusiasm throughout my study, and for his patience in correcting both my stylistic and scientific errors. I have gained a lot from his insights and I have enjoyed my staying at Ohio State as his student. I would like to acknowledge with sincere gratitude to the members of my dissertation committee, Dr. Jeffrey J. Chalmers and Dr. James F. Rathman. I am grateful for their helpful advices on various topics. I wish to thank Dr. Nalin Nilubol, Dr. Suraphong Nawankasattusat, and Dr. Amorn Petchsom for providing me help and opportunity to pursue my Ph.D. study. The scholarship from Chulalongkorn University is gratefully acknowledged. Financial supports from the U.S. department of agriculture (SBIR), Consortium for Plant Biotechnology Research, Inc., and Environmental Energy, Inc. to various phases of this work are also acknowledged. I also appreciate Mr. Carl Scott, Mr. Paul Green, and Mr. Leigh Evrard for their technical assistances. My colleagues in my research group, especially Ms. Supaporn Suwannakham, Ms. Suwattana Pruksasri, Dr. Liping Wang, and Mr. Anli Ouyang, offered many kinds of support and help over the last 4 years. I benefited a lot from discussions with them. vi I feel indebted to my best friend, Mr. Kovit Visessompak, for his helps in many ways during the last 4 years. I also wish to thank Mr. Taradon Luangtongkum, who is always by my side, for his warm support and understanding. Special thank goes to my family for their love and support. Without their love, I would not be able to deal with ups and downs during my study. Finally, my heartfelt gratitude goes to my mother, Ms. Sudathip Thongchul, for her love and inspiration. My graduation could have not been possible without her warm support and understanding. vii VITA October 5, 1974…………………….. …………..Born – Bangkok, Thailand July, 1995…………………………………….....B.S. Chemical Engineering Chulalongkorn University Bangkok, Thailand August, 1998……………………………………M.S. Bioprocess Technology Asian Institute of Technology Pathumthani, Thailand September, 1998 – present……………………...Researcher Chulalongkorn University Bangkok, Thailand PUBLICATION Thongchul, N. and Yang, S.-T., Controlling filamentous fungal morphology by immobilization on a rotating fibrous matrix to enhance oxygen transfer and L(+)-lactic acid production by Rhizopus oryzae. In Fermentation Biotechnology, ed. B.C. Saha. Oxford University Press, MA, 2003, pp. 36-51. FIELD OF STUDY Major Field: Chemical Engineering Specialty: Biochemical Engineering viii TABLE OF CONTENTS Page Abstract…………………………………………………………………….................……ii Dedication……………………………………………………………………….........……v Acknowledgments……………………………………………………………………..….vi Vita…………………………………………………………………………………....…viii List of Tables……………………………………………………………....................….xiii List of Figures………………………………………………………………………...…..xv Chapters: 1. Introduction………………………………………………………………………..…. …..1 2. Literature Review…………………………………………………………………..….13 2.1 Significance of Filamentous Fungal Fermentation…………………………...…..13 2.2 Fungal Morphology in Submerged Fermentation…………………………….…..15 2.2.1 Substrate………………………………………………………………...…..20 2.2.2 pH……………………………………………………………………….…..24 2.2.3 Shear rate……………………………………………………………….…..26 2.2.4 Dissolved oxygen……………………………………………………….….31 2.3 Reaction Systems Used in Submerged Filamentous Fungal Fermentation…..…..37 2.4 L(+)-Lactic Acid Production……………………………………………………..41 2.4.1 Metabolic pathway of R. oryzae………………………………………..…..42 2.4.2 L(+)-lactic acid production by R. oryzae……………………………….…..43 2.5 Conclusion………………………………………………………………………..44 2.6 References…………………………………………………………………….…..46 3. Controlling Filamentous Fungal Morphology by Immobilization on a Rotating Fibrous Matrix to Enhance Oxygen Transfer and L(+)-Lactic Acid Production by Rhizopus oryzae……………………………………………………………….…..69 Summary………………………………………………………………………….…..69 3.1 Introduction…………………………………………………………………...…..71 3.2 Materials and Methods………………………………………………………..…..74 3.2.1 Culture, inoculum preparation, and medium compositions…………….…..74 3.2.2 Rotating Fibrous Bed Bioreactor……………………………………….…..74 3.2.3 Determination of volumetric oxygen transfer coefficient…………………..75 ix 3.2.4 Analytical methods……………………………………………………..…..76 3.3 Results and Discussion……………………………………………………….…..77 3.3.1 Effect of immobilization on fungal morphology……………………….…..77 3.3.2 Fermentation kinetics…………………………………………………...…..77 3.3.3 Long-term lactic acid production in the RFBB……………………………..79 3.3.4 Effects of aeration on fermentation…………………………………….…..82 3.4 Conclusion………………………………………………………………………..84 3.5 References…………………………………………………………………….…..85 4. Preference of Carbon Assimilation in Lactic Acid Fermentation by Rhizopus oryzae in a Rotating Fibrous Bed Bioreactor…………………………………….…..94 Summary………………………………………………………………………….…..94 4.1 Introduction…………………………………………………………………...…..96 4.2 Materials and Methods………………………………………………………..…..98 4.2.1 Culture, inoculum preparation, and medium compositions…………….…..98 4.2.2 Bioreactor setup………………………………………………………... .…99 4.2.3 Lactic acid fermentation……………………………………………….. ….99 4.2.4 Acid hydrolysis of corn fiber…………………………………………...…100 4.2.5 Oxygen transfer determination……………………………………………100 4.2.6 Analytical methods……………………………………………………..…101 4.3 Results and Discussion……………………………………………………….…104 4.3.1 Fermentation of glucose and corn starch……………………………….…104 4.3.2 Fermentation
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