University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2020-01-24 Towards the optimization of lipid and antioxidant production in microalgae: A metabolic study Ujan, Safina Naz Ujan, S. N. (2020). Towards the optimization of lipid and antioxidant production in microalgae: A metabolic study (Unpublished master's thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/111572 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Towards the optimization of lipid and antioxidant production in microalgae: A metabolic study by Safina Naz Ujan A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN CHEMICAL ENGINEERING CALGARY, ALBERTA JANUARY, 2020 © Safina Naz Ujan 2020 Abstract This research explores the design of cultivation strategies for a microalgal system, which will enable production of selected products based on economic value and market size. The microalgae are cultivated under heterotrophic conditions, which involves a nutrient feed of organic carbon substrate, to prevent light dependency. Glucose yields high cell density and lipid content for biofuel production from Auxenochlorella protothecoides under nitrogen-deplete conditions but leads to chloroplast bleaching. Hence, waste streams from the biodiesel industry, in the form of glycerol, are incorporated in the batch level system in order to modulate the pigment content. The glycerol rich conditions enabled production of significantly high levels of lutein and beta-carotene at about 52.43 mg.L-1 and 6.2 mg.L-1 respectively. High biomass growth of 11.53 g.L-1 was reported for C:N ratio of 10:1 in glucose rich conditions. Since distinct nutrients promote accumulation of specific products, a mathematical model is used to simulate and predict micro-algal response to varying cultivation conditions in the batch level operation. For a reliable model, the biochemical reactions within the micro-algae are reconstructed and calibrated to predict the performance results for varying nutrient conditions. The mathematical model enables scale-up and control of process conditions within the bioreactor system. A sustainable and feasible solution for a micro-algal production system strengthens the bio-economy and renewable energy sector. ii Preface This thesis is original, unpublished, independent work by the author, S. Ujan. iii Acknowledgements ﷽ First and foremost, I am thankful to Allah for blessing me with opportunities and challenges that have expanded my knowledge and understanding of the world. Secondly, I would like to convey my deepest appreciation and gratitude to my research project supervisor, Dr. Hector De La Hoz Siegler for his constant guidance and support for the entirety of my research project. Dr. Hector promoted a great deal of independent learning by pushing me to resolve faults in research and the greater thought process. I would like to thank National Sciences and Engineering Research Council (NSERC) for providing financial support. I am appreciative of Schulich School of Engineering and Alberta Graduate Excellence Scholarship for recognizing my academic achievement with scholarships. I am gratified to be part of a remarkably supportive and resourceful research group ‘IamBio’ that created a positive environment for achieving high caliber research work. Particularly, I feel obliged to mention Maria Camila Caceres Falla and Cigdem Demirkaya for providing emotional support through stressful times. I am appreciative for the persistence demonstrated by summer student Thinh Tuan Tong at the preliminary stages of the research project. Lastly and most importantly, I would like to thank my parents and siblings for cheering me on through the difficult days. You hold a special place in my heart for your unwavering belief in my abilities. iv Dedication I would like to dedicate this thesis to my loving parents for believing in my potential especially when the world seemed leaps and bounds ahead. Thank you for celebrating my achievements no matter how small they seemed! v Table of Contents Abstract ...................................................................................................................................... ii Preface ....................................................................................................................................... iii Acknowledgements ..................................................................................................................... iv Dedication .................................................................................................................................... v List of Tables ............................................................................................................................ viii List of Figures and Illustrations ................................................................................................... ix List of Symbols ............................................................................................................................ x List of Abbreviations.................................................................................................................. xii Chapter 1: Introduction............................................................................................................. 1 1.1 Problem definition ...................................................................................................... 1 1.2 Research objectives .................................................................................................... 4 1.3 Research Scope .......................................................................................................... 4 Chapter 2: Literature Review .................................................................................................... 6 2.1 Microalgal biotechnology ........................................................................................... 6 2.2 Metabolic model ....................................................................................................... 16 2.3 Metabolic regulation and biosynthesis pathways ....................................................... 25 Chapter 3: Materials and Methods .......................................................................................... 31 3.1 Microalgal strain ...................................................................................................... 31 3.2 Chemical Reagents ................................................................................................... 31 3.3 Microalgal cultivation............................................................................................... 32 3.4 Experimental analysis ............................................................................................... 33 3.5 Batch reactor setup ................................................................................................... 42 Chapter 4: Study effect of nutrients on product composition ................................................... 44 4.1 Modulation of photo-bleaching effect with addition of glycerol ................................ 44 4.2 Assimilation of glucose and glycerol as organic carbon source ................................. 45 4.3 Assimilation of glycine as source of nitrogen ............................................................ 49 4.4 Influence of nutrient variation on biomass growth .................................................... 51 4.5 Influence of nutrient variation on antioxidants .......................................................... 53 4.6 Influence of nutrient variation on lipid content.......................................................... 56 4.7 Yield and productivity of heterotrophic growth conditions........................................ 58 4.8 Optimal cultivation strategy based on batch operation .............................................. 59 Chapter 5: Metabolic reconstruction and calibration ............................................................... 61 5.1 Reconstruction of metabolic model ........................................................................... 61 5.2 Localization of intracellular components .................................................................. 68 5.3 Cellular growth ......................................................................................................... 69 5.4 Mathematical modelling (Mechanistic expression) ................................................... 69 5.5 Kinetic and parameter estimation .............................................................................. 70 5.6 Model refinement ..................................................................................................... 73 Chapter 6: Dynamic metabolic modelling ............................................................................... 78 6.1 Framework of the dynamic model............................................................................. 78 6.2 ODE solver ..............................................................................................................