Immobilized cell hollow fiber membrane bioreactor for lignocellulosic bioethanol production

IMMOBILIZED-CELL HOLLOW FIBER MEMBRANE BIOREACTOR FOR LIGNOCELLULOSIC BIOETHANOL PRODUCTION NGUYEN THI THUY DUONG (B.Eng. (Hons.), Ho Chi Minh City University of Techonology, Vietnam M.Sc., Pukyong National University, Korea) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL & BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 i ACKNOWLEDGEMENTS The completion of my thesis and subsequent PhD has been a long journey with lots of ups and downs, hope and frustration. Along this journey, I have received tremendous help and support from many people, whom I would like to sincerely thank as I prepare to conclude my thesis. Firstly I would like to express my deepest gratitude to my thesis supervisor, Associate Professor Loh Kai Chee. I would like to thank him for the guidance, trust, independence, flexibility and finance he has given to me. At some critical points of time, especially in the first two years when my initial project did not work, he has given me so much encouragement and motivation to continue this path. Without his support, I may not have gotten to where I am today. I would like to sincerely thank National University of Singapore for the research scholarship. I thank Prof. Chung Tai-Shung Neal for giving me valuable opportunity to work on membrane fabrication and Ms. Ong Rui Chin for assisting in spinning technique. I thank the lab technologies: Ms. Tay Alyssa, Mr. Ang Wee Siong, Mr. Tan Evan Stephen for their continuous assistance in lab works. Special thanks are given to Dr. Cao Bin for assisting in the start-up of this bioethanol project. My gratitude is extended to Dr. Ji Liang Hui in Temasek Life Science Laboratory for his guidance on molecular engineering experiment. Though that project was not fruitful at the end, I have gained valuable experiences during those years working in his lab. I would also appreciate great support from my fellow lab members: Dr. Satyen Gautam, Dr. Karthiga Nagarajan, Dr. Vivek Vasudevan, Dr. Cheng Xiyu, Ms. Phay Jia Jia. Two other labmates I would like to mention specially ii are Dr. Prashant Praveen and Ms. Vu Tran Khanh Linh. Three of us were working on bioreactor development and have spent days and nights in the lab. During those long hours, we have not only run reactors and discussed about projects, we also have told stories, shared interest, made jokes, sometimes argued or yelled at each other. My heartfelt thanks go to Prashant and Linh for those beautiful memories. I am also grateful to Ms. Nguyen Thi Qui and Mr. Vu Viet Hung. They were the first friendly faces to greet me when I began this program and have always been a big help no matter what the task was. Other supporters were my childhood friends Ms. Pham Thi Thanh Truc and Mr. Le Nguyen Man. Though they were not physically in Singapore, but always mentally were beside me whenever I needed them. I must acknowledge with deep gratitude to my parents, my sister Nguyen Thi Lan Anh and my son Phan Nguyen Phuc Khang. It is their unconditional love, patience, support and unwavering belief in me has helped me to complete this long journey. Last but not least, special acknowledgements go to my devoted daughter Phan Nguyen Phuc An, who has been accompanying me for the past four years. She was a great help by growing to be an independent and responsible girl. After long hours working, I felt happy home as I knew there was always a loving girl greeting me with warm smile and fresh cakes she baked especially for me. With deepest gratitude, Nguyen Thi Thuy Duong iii TABLE OF CONTENTS SUMMARY . vii LIST OF TABLES xii LIST OF FIGURES . xiii NOMENCLATURE xv LIST OF SYMBOL . xvii CHAPTER 1. INTRODUCTION 1.1. Background and Research Motivations 1.2. Objectives . 15 1.3. Thesis Organization 17 CHAPTER 2. LITERATURE REVIEW 18 2.1. Lignocellulosic Bioethanol Production 18 2.2. Pretreatment of Lignocellulose Material and Inhibitors 20 2.3. Conversion of Glucose and Xylose to Bioethanol . 29 2.4. Lignocellulosic Ethanol Production at High-Solid Loading 36 2.5. Immobilized-Cell Reactor in fermentation of lignocellulosic bioethanol 40 2.6. Conclusion 45 CHAPTER 3. MATERIALS AND METHODS . 46 3.1. Bacterial Cultures . 46 3.2. Analysis methods . 47 3.2.1. Cell Concentration . 47 3.2.2. Ethanol . 47 3.2.3. Sugar Analysis . 48 3.2.4. Inhibitors Analysis 49 3.2.5. Activity of Cellulase Activity 49 3.2.6. Activity of β-glucosidase 49 3.2.7. Scanning Electron Microscope 50 3.3. Processing Lignocellulosic Material 50 3.3.1. Biomass preparation 50 3.3.2. Cellulose and hemicellulose determination . 51 3.3.3. Acid Hydrolysis and Sugar Composition Analysis . 51 3.3.4. Pretreatment . 52 3.3.5. Enzymatic Hydrolysis . 53 iv 3.3.6. Separate Hydrolysis and Fermentation 53 3.3.7. Simultaneous Saccharification and Fermentation . 54 3.4. Membrane Bioreactor . 55 3.4.1. Membrane Fabrication 55 3.4.2. Sterility 56 3.4.3. Bioreactor Setup 56 CHAPTER 4. IMMOBILIZED-CELL HOLLOW FIBER MEMBRANE BIOREACTOR TO ALLEVIATE INHIBITORS FOR BIOETHANOL PRODUCTION . 63 4.1. Introduction 63 4.2. Results & Discussion . 68 4.2.1. Effect of Inhibitors on Suspended Cells 68 4.2.2. Abiotic Absorption and Desorption of Inhibitors 74 4.2.3. Immobilized Cell and Morphological Characteristics . 76 4.2.4. Fermentation of Glucose with Inhibitors by Immobilized Cells . 78 4.3. Conclusions 84 CHAPTER 5. IMMOBILIZED-CELL HOLLOW FIBER MEMBRANE FOR FERMENTATION OF HIGH SUGAR CONCENTRATION . 86 5.1. Introduction 86 5.2. Results and Discussion . 89 5.2.1. Fermentation in Suspension 89 5.2.2. Effect of glucose concentration on performance of IHFMB . 92 5.2.3. Effect of packing density . 96 5.2.4. Effect of Flow Rates 97 5.2.5. Bioreactor Stability 98 5.3. Conclusions 100 CHAPTER 6. IMMOBILIZED-CELL HOLLOW FIBER MEMBRANE BIOREACTOR FOR CO-CULTURE ON GLUCOSE AND XYLOSE . 101 6.1. Introduction 101 6.2. Results 102 6.2.1. Co-culture in suspension . 102 6.2.2. Sequential Fermentation without Cell Removal . 105 6.2.3. Sequential Co-culture with Cell Removal . 108 6.2.4. Co-culture in Submerged Immobilized-Cell Hollow Fiber Membrane Bioreactor (SIHFMB) . 114 6.2.4. Effect of Aeration 115 v 6.2.5. Effect of Cell Ratio 115 6.2.6. Effect of Initial Sugar Concentration 116 6.3. Conclusions 121 CHAPTER 7. SIMULTANEOUS SACCHARIFICATION AND COFERMENTATION WITH HIGH SOLID LOADING FOR LIGNOCELLULOSIC BIOETHANOL PRODUCTION 123 7.1. Introduction 123 7.2. Results & Discussion . 124 7.2.1. Composition of Jatropha curcas fruit hulls 124 7.2.2. Simultaneous saccharification and co-fermentation in SHFMB . 131 7.2.3. Effect of Aeration 134 7.2.4. Effect of Cell Ratio 134 7.2.5. Long-term operation 135 7.3. Conclusions 136 CHAPTER 8. CONCLUSIONS AND RECOMMENDATIONS . 138 8.1. Conclusion 138 8.2. Recommendations 140 REFERENCES 143 vi SUMMARY Production of bioethanol from lignocellulosic residues has attracted considerable research interest in the past decade, as lignocellulosic residues are the most abundant renewable material and they have the potential to serve as a sustainable feedstock for biofuel production. In the fermentation of lignocellulosic biomass, the microorganisms must be robust to the growth inhibitors resulting from the pretreatment of the biomass, that they can effectively convert high sugars concentrations, while they can concomitantly tolerate high ethanol concentration. Furthermore, for economical feasibility of lignocellulosic bioethanol, both glucose and xylose in the pretreatment hydrolysate must to be converted to bioethanol. These challenges to lignocellulosic biomass fermentation are more severe when fermentation is carried out at high-loading solid and cells are exposed to much higher stresses from the inhibitory present in the fermentation broth. In this study, an immobilized-cell hollow fiber membrane bioreactor (IHFMB) was developed to mitigate these challenges and facilitate high throughput fermentation of lignocellulosic biomass to bioethanol. In the first part of this research, an IHFMB resembling a shell and tube dialysis module was designed and operated to mitigate the effect of various inhibitors present in lignocellulosic hydrolysate. In this configuration, the hollow fiber membrane served as a barrier to shield the actively growing Zymomonas mobilis ATCC 31821 immobilized within the porous matrix from the toxic inhibitors. Four common inhibitors including furfural (1- g/L), 5vii hydroxymethylfurfural (2- g/L), vanillin (1-2 g/L) and syrinaldehyde (0.5- g/L) were used in the fermentation medium and their effects on growth and ethanol production in IHFMB were investigated. In the suspension, individual compound had negative effects on cell growth and ethanol production as growth rate of Z. mobilis decreased by 20-50%, cell concentration declined by 10-70%, and ethanol concentration lowered by 10-60%. In the medium with the mixture of low concentration of inhibitors (1 g/L furfural, g/L hydroxymethylfurfural, g/L vanillin and 0.5 g/L syrinaldehyde), suspended cells was unable to survive. However, the Z. mobilis immobilized in IHFMB showed success in fermenting 20 g/L of glucose into bioethanol in the presence of high concentration of inhibitors (2 g/L furfural, g/L hydroxymethylfurfural, g/L vanillin and g/L syrinaldehyde). Glucose was consumed within 15 hours and 95% of the theoretical ethanol yield was achieved. By doubling the packing density from 0.13 to 0.26, a 71% increase in ethanol productivity could be achieved. Likewise, doubling feed flow rate from 10 to 20 mL/min gave a 28% increase in ethanol productivity. The IHFMB was operated for 20 consecutive batch operations for 240 h at identical conditions and the bioreactor performance remained stable. The results indicate that the use of IHFMB can simplify bioethanol production process by doing away with any pre-fermentation treatment for removal of inhibitors from the hydrolysate and it can then save time and energy. In the second part of this research, the performance of the IHFMB was investigated in mitigating substrate inhibition at high glucose concentration. viii Prior to IHFMB operation, the inhibitory concentration of glucose for suspended cells of Z. mobilis determined was 140 g/L. At this concentration microorganism exhibits a long lag phase (6 h), low growth rate and low ethanol yield (65% theoretical ethanol yield). However, using IHFMB microorganism could successfully ferment 200 g/L of glucose with high ethanol yield (76% theoretical ethanol yield). By optimization of operating parameters such as packing density at 0.26, and flow rate at 20 mL/min, IHFMB performance could be further increased and the ethanol yield achieved was near the max theoretical yield (92%). The reusability results demonstrated that the IHFMB was stable for batches over 252 h. To further improve the efficiency of lignocellulosic bioethanol fermented in the IHFMB, the IHFMB was modified to submerged immobilized-cell hollow fiber membrane bioreactor (SHFMB) to simultaneously convert glucose and xylose to ethanol through co-culture. 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Science 267(5195): 240-243. 156 BIBLIOGRAPHY EDUCATION 1992-1997 Bachelor of Engineering (Honour) Faculty of Food & Chemical Engineering, Ho Chi Minh City University of Technology, Vietnam 2003-2005 Master of Science Faculty of Microbiology Pukyong National University, Korea 2008-2014 Research Scholar Department of Chemical & Biomolecular Engineering National University of Singapore, Singapore EXPERIENCE 1997-1999 Assistant Lecturer Faculty of Food & Chemical Engineering Ho Chi Minh City University of Technology, Vietnam 1999-2000 Research Scholar Osaka University, Japan 2000-2003 Lecturer Faculty of Environmental Engineering Ho Chi Minh City University of Technology, Vietnam 2005-2008 Lecturer Department of Biotechnology Ho Chi Minh City University of Technology, Vietnam 2011-2014 Research Engineer Department of Chemical & Biomolecular Engineering, Singapore 157 LIST OF PUBLICATIONS & PRESENTATIONS Duong TT Nguyen, Kai-Chee Loh. Immobilized-Cells Hollow Fiber Membrane Reactor for Fermentation of Inhibitors in Lignocellulosic Hydrolysates. AIChE 2012 - 2012 AIChE Annual Meeting, Conference Proceedings Duong TT Nguyen, Kai-Chee Loh. Immobilized-Cell Hollow Fiber Membrane Bioreactor to Overcome Inhibitors in Lignocellulose Hydrolysate for Bioethanol Production. World Biotechnology Congress 2013, Conference Proceedings Duong TT Nguyen, Kai-Chee Loh. Co-Culture in Submerged Hollow Fiber Membrane Bioreactor for Bioethanol Production on Glucose and Xylose. AIChE 2013 – 2013 AIChE Annual Meeting, Conference Proceedings Duong TT Nguyen, Kai-Chee Loh (2014). Immobilized-Cell Hollow Fiber Membrane Bioreactor to Overcome Inhibitors in Lignocellulose Hydrolysate for Ethanol Production (to be submitted) Duong TT Nguyen, Kai-Chee Loh (2014). Co-culture in Submerged Immobilized-Cell Hollow Fiber Membrane Bioreactor for Efficient Ethanol Production on Glucose and Xylose (to be submitted) Duong TT Nguyen, Kai-Chee Loh (2014). Submerged Immobilized-Cell Hollow Fiber Membrane Bioreactor for Co-culture on Glucose and Xylose (to be submitted) Duong TT Nguyen, Kai-Chee Loh (2014). Immobilized-Cell Hollow Fiber Membrane Bioreactor for High Sugar Fermentation (to be submitted) 158 [...]... hollow fiber membrane bioreactor to alleviate inhibitors in lignocellulosic hydrolysate 2 To investigate the performance of immobilized- cell hollow fiber membrane bioreactor in mitigating substrate inhibition 3 To co-coculture Zymomonas mobilis and Pichia stipitis in immobilized- cell hollow fiber membrane for simultaneous fermentation on glucose and xylose 4 To investigate immobilized- cell membrane bioreactor. .. 20 Figure 2-2 Illustration of lignocellulosic biomass structure 22 Figure 2-3 Inhibitors formed as degradation products from hydrolysis of lignocellulose 24 Figure 3-1 Schematic diagram of Immobilized- Cell Hollow Fiber Membrane Bioreactor 58 Figure 3-2 Schematic diagram of Submerged Immobilized- Cell Hollow Fiber Membrane Bioreactor 61 Figure 4-1... and Loh 2006) There are several attractive properties of hollow fiber membrane for the purpose of microbial immobilization Firstly, hollow fiber immobilized cells, or immobilized cells in general, have numerous advantages compared to free cell systems Immobilization allows easy recycling of cells for subsequent batch of production whereas in free cell system, every fresh batch must be inoculated with... processes in lignocellulosic bioethanol production, current problems and motivation for this research An in-depth literature review is presented in Chapter two The third chapter covers all materials and methods used in the research Chapter four presents the results on development of Immobilized- Cell Hollow Fiber Membrane Bioreactor (IHFMB) to alleviate inhibitors in lignocellulosic bioethanol production. .. 7-4 Effect of ratio of cell 135 Figure 7-5 Long-term operation 136 xiv NOMENCLATURE Symbol Description SSF Simultaneous saccharification and fermentation SHF Separate hydrolysis and fermentation IHFMB Immobilized- Cell Hollow Fiber Membrane Bioreactor SHFMB Submerged Immobilized- Cell Hollow Fiber Membrane Bioreactor 5-HMF 5-hydrolxymethylfurfural AFEX Ammonia fiber explosion AIL Acid... membrane bioreactor for simultaneous saccharification and co-fermentation of high-loading of solid The schematic layout of research is shown in Figure 1-1 This research demonstrates the application of Immobilized- Cell Hollow Fiber Membrane Bioreactor to alleviate most of the critical problems encountered in lignocellulosic bioethanol production The membranes provided a barrier for cells to alleviate... compromising cell viability and productivity In addition, reusability is also convenient 14 1.2 Objectives The overall objective of this thesis is to develop an immobilized- cell hollow fiber membrane bioreactor to alleviate the challenges in current lignocellulosic bioethanol production, and improve the efficiency of fermentation The specific research objectives include: 1 To develop an immobilized- cell hollow. .. shorter time The immobilized microorganisms could biodegrade phenol concentrations as high as 3500 mg/L (Li and Loh 2005; Li and Loh 2006) A hollow fiber is a cylindrical membrane structure with a hollow tubular centre, termed as the lumen The hollow fiber membrane can be either symmetric or asymmetric The structure of a symmetric hollow fiber 12 membrane is uniform whereas an asymmetric membrane typically... stable over three runs during 252 h with high yield and productivity The results from this research demonstrated the strengths and potential of the Immobilized- Cell Hollow Fiber Membrane Bioreactor in lignocellulosic bioethanol production The membranes barrier for cells could alleviate inhibitory effects of the toxic compounds in the hydrolysate, high concentration of ethanol and glucose The IHFMB could... of immobilized- cell in fermenting real hydrolysate at high-solid loading producing ethanol at high concentration and high yield 11 In biotechnology, the hollow fiber membrane has been widely used as a tool for immobilization of microorganism to carry biotransformation processes Inloes and co-workers designed a hollow- fiber membrane bioreactor to immobilize recombinant Escherichia coli C600 for the production . the strengths and potential of the Immobilized- Cell Hollow Fiber Membrane Bioreactor in lignocellulosic bioethanol production. The membranes barrier for cells could alleviate inhibitory effects. lignocellulose 24 Figure 3-1 Schematic diagram of Immobilized- Cell Hollow Fiber Membrane Bioreactor 58 Figure 3-2 Schematic diagram of Submerged Immobilized- Cell Hollow Fiber Membrane Bioreactor. fermentation IHFMB Immobilized- Cell Hollow Fiber Membrane Bioreactor SHFMB Submerged Immobilized- Cell Hollow Fiber Membrane Bioreactor 5-HMF 5-hydrolxymethylfurfural AFEX Ammonia fiber explosion