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... 40 Photographs of (a) MA-SCA and (b) MA-TEY microbioreactors after 14 days of fermentation 128 Figure 41 Photographs of (a) GG-SCA and (b) GG-TEY microbioreactors after 14 days of fermentation. .. Bio- ethanol B1 Application of bio- ethanol in transportation B2 Production of bio- ethanol Saccharomyces cerevisiae D Bioreactors D1 E F Advantages of using bioreactors in fermentation processes Microencapsulation... microencapsulation Part Two: Fermentation efficiency of free yeast and 102 micro- bioreactors A Assay of ethanol A1 Optimisation of gas chromatography-mass spectrometry 102 102 conditions for assay of ethanol
DEVELOPMENT OF MICRO-BIOREACTORS FOR A MORE EFFICIENT FERMENTATION PROCESS TO PRODUCE BIO-ETHANOL TAN SOOK MUN NATIONAL UNIVERSITY OF SINGAPORE 2010 DEVELOPMENT OF MICRO-BIOREACTORS FOR A MORE EFFICIENT FERMENTATION PROCESS TO PRODUCE BIO-ETHANOL TAN SOOK MUN (B Sc Microbiology (Hons.), UPM) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements ACKNOWLEDGEMENTS I wish to express my heartfelt gratitude to my supervisors, Associate Professor Chan Lai Wah and Associate Professor Paul Heng Wan Sia for their guidance, encouragement, patience and tireless effort throughout the course of this study I am especially grateful for the priceless experience and opportunities they have given to me to learn and improve myself It has been a wonderful experience to work closely and sharing constructive and innovative research ideas with them Thank you once again for making me a part of the GEA-NUS research team and I am proud to have been a part of the GEA-NUS family I wish to acknowledge the National University of Singapore for providing the research scholarship and facilities to carry out the research work My appreciation also extends to the Laboratory Technologists, Mdm Wong Mei Yin, Mdm Teresa Ang Swee Har, Mr Peter Leong, Mdm Ng Sek Eng, Mr Tang and Ms Yong Sock Leng for their assistance and support in my research study My sincere appreciation goes to my colleagues and friends in GEA-NUS and the Department of Pharmacy for their comfort, encouragement, motivation and humor Special thanks to my beloved family for their love, confidence and unfailing support Thank you all Sook Mun January 2010 ii Contents CONTENTS ACKNOWLEDGEMENTS ii CONTENTS iii SUMMARY xii LIST OF TABLES xiv LIST OF FIGURES xv LIST OF ABBREVIATIONS xx I INTRODUCTION A Bio-fuels A1 Bio-fuels as alternative renewable and sustainable energy A2 Advantages of bio-fuels B C Bio-ethanol B1 Application of bio-ethanol in transportation B2 Production of bio-ethanol Saccharomyces cerevisiae D Bioreactors D1 E F Advantages of using bioreactors in fermentation processes Microencapsulation E1 Microencapsulation of microbial cells E2 Production of micro-bioreactors by emulsification method 11 Biopolymers for encapsulation of cells 12 F1 Alginates 13 F1.1 Sources of alginates 14 F1.2 Molecular structure of alginates 14 iii Contents F2 F1.3 Gelation of alginates 17 F1.4 Limitations of alginates as bio-encapsulant 18 Gellan gum 21 F2.1 Sources of gellan gum 22 F2.2 Molecular structure of gellan gum 23 F2.2.1 Acetylated gellan gum 23 F2.2.2 Deacetylated gellan gum 25 F2.2.3 Commercial gellan gum 25 F2.3 Gelation of gellan gum 26 F2.4 Limitations of gellan gum 29 G Scaffold-coating 29 G1 Spray drying 30 G2 Coating material 32 G2.1 Ethylcellulose 33 G2.1.1 34 Aquacoat II HYPOTHESES AND OBJECTIVES 35 III EXPERIMENTAL 38 A Materials 38 A1 Model microorganism 38 A2 Cultivation and fermentation media 38 A3 Encapsulating polymers and chemicals 38 A4 Coating materials 39 A5 Chemicals for assay of ethanol by gas chromatography-mass 39 spectrometry iv Contents A6 B Chemicals for other studies 39 Methods 40 B1 Cultivation of yeast cells 40 B1.1 Saccharomyces cerevisiae ATCC 9763 40 B1.1.1 40 Determination of suitable solid media and incubation conditions for the growth of yeast B1.1.2 Optimisation of cultivation conditions 40 for mass production of yeast in broth B1.1.3 Determination of log phase of growth 41 curve B1.1.4 B1.2 Preparation of standardised inoculum 41 Turbo Extra Yeast 42 B1.2.1 42 Preparation of standardised inoculum B2 Study of temperature effect on yeast viability 42 B3 Study of concentration effect of polymer on congealation of 43 gellan gum B4 Optimisation of microspheres production B4.1 43 Investigation of process and formulation factors that 43 affect the properties of gellan gum microspheres B4.2 Investigation of process and formulation factors that 44 affect the properties of calcium alginate microspheres v Contents B5 Microencapsulation of yeast cells in gellan gum and alginate 46 microspheres B6 Physical characterisation of blank microspheres and 47 micro-bioreactors B7 Determination of viable yeast contents of yeast-calcium 48 alginate micro-bioreactors B7.1 Study of effects of sodium chloride solution 48 concentration on yeast viability B7.2 Liberation of yeast cells from micro-bioreactors for 48 viable count B8 B9 Study of emulsification process effect on yeast viability 48 B8.1 Production of gellan gum microspheres 48 B8.2 Production of calcium alginate microspheres 49 Method development for assay of ethanol by gas 49 chromatography-mass spectrometry (GC-MS) B9.1 Optimisation of operation conditions of GC-MS 49 B9.2 Optimisation of ethanol extraction from 50 fermentation medium B9.3 Construction of ethanol calibration plot 51 B9.4 Assay of ethanol produced in the fermentation 51 medium B10 Study of the fermentation process using free yeast cells 52 B10.1 Fermentation using Saccharomyces cerevisiae 52 ATCC 9763 vi Contents B10.1.1 Optimisation of fermentation conditions 52 B10.1.2 Influence of sucrose concentration 52 B10.2 Fermentation using Turbo Extra Yeast 53 B10.2.1 Influence of sucrose concentration 53 B10.2.2 Influence of malt extract broth 53 concentration B11 Mass production of blank calcium alginate microspheres for 54 scaffold-coating B12 Scaffold-coating of blank calcium alginate 54 (Macrocystis Kelp) microspheres B13 Mass production and scaffold-coating of yeast-calcium 55 alginate micro-bioreactors B14 Physical characterisation of yeast-calcium alginate 55 micro-bioreactors, with and without scaffold-coating B15 Spray drying of free yeast cells 55 B16 Fermentation using free yeast cells or micro-bioreactors 57 B17 Viable count of free yeast cells liberated from 59 micro-bioreactors into the fermentation medium B18 Fermentation using double and triple doses of gellan gum 59 micro-bioreactors with encapsulated TEY cells B19 Fermentation using recycled free yeast cells and 59 micro-bioreactors B20 Physical stability of blank beads and microspheres 60 B20.1 Preparation of beads 60 vii Contents B21 IV B20.1.1 Physical characterisation of beads 60 B20.1.2 Study on the stability of beads 60 B20.2 Study on stability of microspheres 62 Statistical analyses 62 RESULTS AND DISCUSSION 63 Part One: Production of gellan gum and calcium alginate 63 micro-bioreactors A Cultivation of yeast cells B 63 A1 Cultivation of Saccharomyces cerevisiae ATCC 9763 63 A2 Cultivation of Turbo Extra Yeast 67 Optimisation of microsphere production 71 B1 Factors affecting the production of gellan gum microspheres 71 B1.1 Temperature effect on yeast viability 71 B1.2 Concentration effect of polymer on congealation of 73 gellan gum B1.3 Effects of emulsification process and formulation 74 factors on the properties of gellan gum microspheres B2 Effects of the emulsification process and formulation factors 81 on the properties of calcium alginate 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Nussinovitch, A., 2003 Changes induced by DC electrical field in agar, agarose, alginate and gellan gum beads Food Hydrocolloids 17, 255-263 234 Publications and Papers VII PUBLICATIONS / PAPERS PRESENTED AT SCIENTIFIC MEETINGS Journal Publications: Tan, S.M., Chan, L.W., Heng, P.W.S., Novel method of immobilising yeast for potential application in continuous fermentation to produce bio-ethanol (under review) Tan, S.M., Chan, L.W., Heng, P.W.S., Influence of scaffold-coating on stability and fermentation efficiency of yeast-alginate micro-bioreactors (under review) Oral Presentations: S.M Tan, Fermentation efficiency of encapsulated yeast in gellan gum as reusable micro-bioreactors, ASEAN Scientific Conference in Pharmaceutical Technology 2008, 1st – 3rd June 2008, Penang, Malaysia S.M Tan, Stability of alginate and gellan gum micro-bioreactors in bio-ethanol production, Asian Association of Schools of Pharmacy (AASP) Conference 2007, 25th – 28th October 2007, Makati City, Philippines Poster Presentations: S.M Tan, L.W Chan and P.W.S Heng, Feasibility study of yeast-gellan gum microbioreactors for application in a continuous fermentation process, American Association of Pharmaceutical Scientists (AAPS) 2009, 8th – 12th November 2009, Los Angeles, California, USA L.W Chan, S.M Tan and P.W.S Heng, Influence of alginate and scaffold-coating on stability and fermentation efficiency of yeast-alginate micro-bioreactors, 4th Asian Association of Schools of Pharmacy – 9th Malaysian Pharmaceutical Society Pharmacy Scientific Conference (AASP-MPSPSC) 2009, 10th – 13th June 2009, Penang, Malaysia Best Poster Award S.M Tan, L.W Chan, P.W.S Heng, Alginate microencapsulation of yeast cells as reusable micro-bioreactors for bio-ethanol production, 16th International Symposium on Microencapsulation, 9th – 12th September 2007, Lexington, Kentucky, USA 235 Publications and Papers S.M Tan, L.W Chan, P.W.S Heng, Fermentation efficiency of gellan gum microbioreactors, PharmSci@Asia Symposium, 28th – 29th June 2007, Shanghai, China S.M Tan, L.W Chan, P.W.S Heng, Feasibility study of yeast encapsulation by the alginate-emulsification method, Asian Pharmaceutics Graduate Congress, 25th – 27th September 2006, Singapore 236