Cultivation of Tetraselmis suecica Under Various Nutrient Concentrations and the Characterization of the Algal Biomass by Zoë Leigh Grabowski Submitted in partial fulfilment of the requirements for the degree of Master of Applied Science at Dalhousie University Halifax, Nova Scotia March 2017 © Copyright by Zoë Leigh Grabowski, 2017 To Grandpa Joe and Auntie Kathy Thank you for always believing in me ii Table of Contents LIST OF TABLES .............................................................................................................. v LIST OF FIGURES ........................................................................................................... vi ABSTRACT ..................................................................................................................... viii LIST OF ABBREVIATIONS AND SYMBOLS USED ................................................... ix ACKNOWLEDGEMENTS ................................................................................................ x Chapter 1 : Introduction ...................................................................................................... 1 1.1 Background ............................................................................................................... 1 1.2. Research Objectives ................................................................................................. 5 Chapter 2 : Literature Review ............................................................................................. 7 2.1 Microalgae:................................................................................................................ 7 2.2 Tetralsemis suecica: .................................................................................................. 8 2.3 KDO: ....................................................................................................................... 11 2.4 Effect of Cultivation Conditions ............................................................................. 16 2.4.1 Nitrogen and Phosphorous Stress: .................................................................... 16 2.4.2 Effect of Cultivation Condition on T. suecica .................................................. 19 2.5 Process Integration: ................................................................................................. 22 Chapter 3 : Materials and Methods ................................................................................... 24 3.1 Culture Growth ........................................................................................................ 29 3.1.1 Optical Density ................................................................................................. 29 3.1.2 Freeze Dried Mass ............................................................................................ 30 3.1.3 Cell Count ......................................................................................................... 30 3.2 Analysis of Cellular Content ................................................................................... 30 iii 3.2.1 Enzymatic Hydrolysis for total starch content ..................................................... 30 3.2.2 Derivatization of Sugars ................................................................................... 31 Chapter 4 : Results and Discussion ................................................................................... 34 4.1 Inoculum.................................................................................................................. 34 4.2 Experimental Cultures ............................................................................................. 35 4.2.1 Monitoring Biomass Growth by Optical Density Measurements: ................... 36 4.2.2 Cell Count: ........................................................................................................ 39 4.2.3 Freeze Dried Mass: ........................................................................................... 41 4.3 Carbohydrate Analysis ............................................................................................ 46 4.3.1 Enzymatic Hydrolysis: ..................................................................................... 46 4.3.2 KDO and Other Sugars: .................................................................................... 50 Chapter 5 : Conclusion...................................................................................................... 58 References ......................................................................................................................... 61 Appendix A: Culture Maintenance for Tetraselmis suecica ........................................ 69 Appendix B: Cultivation and Culture Monitoring ...................................................... 72 Appendix C: Starch Determination .............................................................................. 81 Appendix D: Methanolysis Method ............................................................................... 84 Appendix E: Photobioreactor Set-up ............................................................................ 86 Appendix F: Calibration Curves for Carbohydrate Analysis .................................... 87 iv LIST OF TABLES Table 2-1: Autotrophic T. suecica experiments investigating product accumulation and biomass ............................................................................................................................. 21 Table 3-1: Basic UTEX enriched seawater stock solution, and N- and P-limited stock solution concentrations. The dilution of these stock solution into the media is 20 mL/L [66] .................................................................................................................................... 27 Table 3-2: Final concentrations of sodium nitrate and glycerophosphate in 1L cultures . 28 Table 4-1: Analysis of variance for enzymatic hydrolysis measurements. SSw is the sum of squares within treatments, SSb is the sum of squares between treatments, F is the value used for the F-test and F0.05, 2, 6 is the critical value for a one tailed F-distribution. ......... 49 Table 4-2: Analysis of variance for the mg KDO per g dried mass as determined GC/MS results based on nutrient condition. SSw is the sum of squares within treatments, SSb is the sum of squares between treatments, F is the value used for the F-test and F0.05, 2, 6 is the critical value for a one tailed F-distribution. ..................................................................... 54 Table 4-3: Determination of value of T. suecica cultures based on mg of KDO per litre 56 Table 4-4: TMS derivatives of sugars found in T. suecica and their GC/MS characteristics (RT = Retention time) ....................................................................................................... 57 Table B-1: Enrichment N-free and P-free stock solution composition. Twenty milliliters of this solution will be added to 980 mL of autoclaved seawater to be added to the photobioreactor after sampling. ........................................................................................ 80 v LIST OF FIGURES Figure 2-1: Linear 3-deoxy-D-manno-2-octulosonic acid ................................................ 12 Figure 2-2: Inclusion of KDO in lipopolysaccharide as portion of the Lipid A-KDO2 component ......................................................................................................................... 13 Figure 2-3: Synthesis of KDO from D-arabinose ............................................................. 14 Figure 2-4: Biosynthesis of KDO in E. coli ...................................................................... 15 Figure 3-1: Experimental set-up for autotrophic batch cultures ....................................... 26 Figure 3-2: Methanolysis of glucose................................................................................. 33 Figure 3-3: Silylation of methylated glucose .................................................................... 33 Figure 4-1: Comparison of the inoculum cultures grown over the course of the experiment, with standard deviation calculated from simultaneously grown cultures within each replicate ......................................................................................................... 35 Figure 4-2: T. suecica biomass grown under a) Nutrient Replete, b) P-starved and ........ 36 Figure 4-3: Optical density at 750 nm versus time under nutrient-replete cultivation condition ........................................................................................................................... 37 Figure 4-4: Optical density at 750 nm versus time under P-starved cultivation condition38 Figure 4-5: Optical density at 750 nm versus time under N-starved cultivation condition ........................................................................................................................................... 38 Figure 4-6: Cell count per mL as over time under nutrient-replete condition .................. 39 Figure 4-7: Cell count per mL as over time under P-starved condition ............................ 40 Figure 4-8: Cell count per mL as over time under N-starved condition ........................... 40 Figure 4-9: Concentration of lyophilized algal biomass of Culture 2 versus time under nutrient-replete condition .................................................................................................. 42 Figure 4-10: