OPTIMIZATION STUDY OF BIOMASS AND ASTAXANTHIN PRODUCTION BY HAEMATOCOCCUS PLUVIALIS UNDER MINKERY WASTEWATER CULTURES by Yu Liu Submitted in partial fulfilment of the requirements for the degree of Mater of Science at Dalhousie University Halifax, Nova Scotia March 2018 ©Copyright by Yu Liu, 2018 TABLE OF CONTENTS LIST OF TABLES .............................................................................................................. v LIST OF FIGURES ......................................................................................................... vii ABSTRACT ........................................................................................................................ x LIST OF ABBREVIATIONS USED ................................................................................ xi ACKNOWLEDGEMENTS .............................................................................................. xv CHAPTER I INTRODUCTION ...................................................................................... 1 1.1 Microalgae ................................................................................................................. 1 1.1.1 Haematococcus pluvialis .................................................................................... 3 1.2 Potential bioproducts from Haematococcus pluvialis ............................................... 4 1.3 Minkery wastewater as a nutrient resource ............................................................... 5 1.4 Objectives .................................................................................................................. 6 CHAPTER II LITERATURE REVIEW .......................................................................... 8 2.1 Biology of H. pluvialis .............................................................................................. 8 2.1.1 Classification, history, and distribution ............................................................... 8 2.1.2 Cellular morphology and life cycle ..................................................................... 8 2.1.3 Major biochemical compositions of H. pluvialis .............................................. 10 2.2 Introduction of astaxanthin ...................................................................................... 16 2.2.1 Structure, classification, and functions of astaxanthin ...................................... 17 2.2.2 Applications of astaxanthin ............................................................................... 18 2.2.3 Commercial market, safety, and challenges of astaxanthin .............................. 21 2.2.4 Biosynthesis pathway of astaxanthin in H. pluvialis ........................................ 23 2.3 Cultivation and processing of H. pluvialis for biomass and astaxanthin production ...................................................................................................................... 26 2.3.1 Culture conditions for vegetative stage ............................................................. 26 2.3.2 Culture conditions for cysts stage ..................................................................... 33 2.3.3 Production mechanisms .................................................................................... 37 ii 2.3.4 Cultivation systems ........................................................................................... 41 2.4. Microalgae production integrated in wastewater treatment system ....................... 43 2.4.1 Microalgae growth in different wastewaters ..................................................... 45 2.4.2 Minkery wastewater .......................................................................................... 48 2.4.3 H. pluvialis growth in wastewater .................................................................... 49 2.4.4 Mechanisms of nutrient removal ...................................................................... 50 2.5. Production process.................................................................................................. 54 2.5.1 Two-stage cultivation strategy for astaxanthin production ............................... 54 2.5.2 Harvesting techniques ....................................................................................... 56 2.5.3 Extraction and purification of astaxanthin ........................................................ 56 CHAPTER III BIOMASS AND ASTAXANTHIN PRODUCTION BY H. PLUVIALIS IN MINKERY WASTEWATER ................................................................... 58 3.1 Introduction ............................................................................................................. 58 3. 2. Methodology.......................................................................................................... 59 3.2.1 Microalgae strain and culture conditions .......................................................... 59 3.2.2 Preparation of minkery wastewater................................................................... 61 3.2.3 Cultivation system designed for the two-stage strategy ................................... 62 3.2.4 Experiment design ............................................................................................ 64 3.2.5 Analytical methods ............................................................................................ 67 3.3. Results and discussion ............................................................................................ 75 3.3.1 Effect of minkery wastewater concentration on microalgae growth ................ 75 3.3.2 Nutrient removal capacities from different cultivation mediums and biomass accumulation by H. pluvialis during cultivation stage ............................................... 81 3.3.3 Astaxanthin production during photoautotrophic induction ............................. 93 3.4 Conclusion ............................................................................................................... 97 CHAPTER IV EFFECTS OF ACETATE AND NACL CONCENTRATIONS ON ASTAXANTHIN PRODUCTION BY H. PLUVIALIS .................................................... 99 4.1 Introduction ............................................................................................................. 99 4.2 Methodology............................................................................................................ 99 iii 4.3 Results and discussion ........................................................................................... 100 4.3.1 Effects of acetate ............................................................................................. 100 4.3.2 Effects of salinity ............................................................................................ 102 4.4 Conclusion ............................................................................................................. 105 CHAPTER V OPTIMIZATION STUDY OF ASTAXANTHIN PRODUCTION BY H. PLUVIALIS IN MINKERY WASTEWATER CULTURES USING RESPONSE SURFACE METHODOLOGY ....................................................................................... 106 5.1 Introduction ........................................................................................................... 106 5.2 Methodology.......................................................................................................... 107 5.2.1 Cultivation and induction conditions .............................................................. 107 5.2.2 Experimental design and statistical analysis ................................................... 107 5.3 Results and discussion ........................................................................................... 109 5.3.1 Regression model and ANOVA analysis ......................................................... 109 5.3.2 Process optimization ....................................................................................... 115 5.3.3. RSM used for astaxanthin production by Haematococcus in other cultures . 116 5.4 Conclusion ............................................................................................................. 117 CHAPTER VI CONCLUSION ................................................................................... 118 6.1 Experimental conclusion ....................................................................................... 118 6.2 Future research ...................................................................................................... 119 BIBLIOGRAPHY ........................................................................................................... 121 iv LIST OF TABLES Table 2.1. Typical composition of H. pluvialis in vegetative and cyst stages. 11 Table 2.2. High value carotenoid products from microalgae and their current 14 use. Table 2.3. Comparison of the characteristics of different microalgae cultivation systems. 43 Table 2.4. Total nitrogen (TN) and total phosphorus (TP) contents of different wastewater sources. 45 Table 2.5. Nitrogen and phosphorus removal efficiency (%) by various microalgae species in different wastewater resources. 47 Table 3.1. Nutrient concentrations in Bold’s Basal Medium. 60 Table 3.2. The nutrient contents of the raw MW, 1%, 1.5% and 2% pre-treated 76 minkery wastewater. Table 3.3. The increased amount (mg L-1) and percent increase (%) of biomass production in BBM, 1% MW, and 1.5% MW mediums after 6-day cultivation 85 period. Table 3.4. The statistics analysis of specific growth rates (mg L-1 d-1), biomass productivities (mg L-1 d-1), increased