Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Photobioreactor production of microalgae for potential fuel oils A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand Tiyaporn Luangpipat 2013 iii Abstract This work focussed on a detailed characterization of the freshwater microalga Chlorella vulgaris as a producer of potential fuel oils. Uniquely, growth and oil production of C. vulgaris were characterized in full strength seawater-based media, something that has not been previously reported. C. vulgaris was selected for a detailed study after a screening of six potential oil producing microalgae. For photoautotrophic growth, always under carbon sufficiency and at normal growth temperature, the characterization study covered: the biomass growth rate; lipid content in the biomass; productivities of the lipids and the biomass; the biomass loss in the dark; the lipid/biomass yields on macronutrients; and the energy content of the biomass. The above key production parameters were characterized in a purpose-built tubular photobioreactor (a80 L) and in stirred tank photobioreactors (a7.5 L) under conditions of nitrogen sufficiency and at various levels of nitrogen limitation. Production was evaluated in both batch and continuous cultures at various dilution rates using indoor light to mimic sunlight. The production temperature mimicked the relatively warm conditions that would be encountered in a potential production system located outdoors in a tropical climate. In seawater media at 25–27 qC, C. vulgaris was shown to have a crude oil productivity of >37 mg L1 d1 and the energy content of the biomass could exceed 25 kJ g1, depending on the culture conditions. Both these values were high compared with the reported data for this alga in freshwater media. Compared with continuously illuminated culture, day–night cycling of irradiance reduced oil productivity by a31%, but the energy content of the biomass were reduced by only about 8%. In seawater, the alga could be grown as rapidly and stably as in freshwater. The lipid content of the biomass commonly exceeded 30% by dry weight and in exceptional cases a lipid content of more than 50% (by weight) iv was achieved. Biomass calorific values of t27 kJ g1 could be attained in some cases. Nitrogen starvation enhanced the lipid contents of the biomass by >3-fold relative to the lipid contents for the nonstarved case. Steady-state continuous cultures were shown to be possible. Both batch and continuous operations were feasible, especially in stirred tanks, but the culture was more failure prone, or relatively less productive, in the tubular photobioreactor. v Acknowledgements My doctoral study and this thesis would not have been possible without the support of many individuals. My Thai friends and everyone I shared an office with always helped. The other students I shared the laboratory facilities with were immensely helpful. I would specially like to thank Tawan and his family and Pittiporn who were always supportive. I am thankful to the Laboratory Manager Ann-Marie Jackson, the other laboratory technicians and the Mechanical Workshop personnel who were consistently helpful. Encounters with Professors Clive Davies and Tony Paterson were always cheerful and motivating. A scholarship from my university, Nakhon Sawan Rajabhat University, facilitated my work and Massey University provided me with an excellent environment to work in. I am especially grateful to Neste Oil Corporation, Porvoo, Finland, for funding this research and allowing it to be published. My supervisor, Professor Yusuf Chisti, supported me academically as well as financially. You are an awesome supervisor ever! I very much enjoyed working with you and your kindness. I am greatly thankful to my family for everything. Papa, without your inspiration and love, my PhD could not have begun. Thank you for your love; you will always be in my heart. vii Table of Contents Abstract .................................................................................................................................... iii Acknowledgements................................................................................................................... v Table of Contents .................................................................................................................... vii List of Figures .......................................................................................................................... xi List of Tables ........................................................................................................................ xvii Abbreviations ......................................................................................................................... xix Chapter 1 ................................................................................................................................... 1 Introduction............................................................................................................................... 1 Chapter 2 ................................................................................................................................... 5 Literature Review ..................................................................................................................... 5 2.1 Microalgae and their culture ........................................................................................... 5 2.2 Attributes of a commercial alga for production of fuel oils ........................................... 5 2.3 Microalgae versus plants ................................................................................................ 9 2.4 Algae culture requirements ........................................................................................... 12 2.4.1 Culture media......................................................................................................... 13 2.4.2 Temperature ........................................................................................................... 15 2.4.3 pH .......................................................................................................................... 17 2.4.4 Light ....................................................................................................................... 17 2.5 Algal oils ....................................................................................................................... 21 2.6 Culture systems for microalgae .................................................................................... 33 2.6.1 Open culture systems ............................................................................................. 33 2.6.2 Closed photobioreactors ........................................................................................ 36 2.7 Objectives ..................................................................................................................... 43 viii Chapter 3 .................................................................................................................................45 Materials and Methods ............................................................................................................45 3.1 General methodology ....................................................................................................45 3.2 Microalgae ....................................................................................................................46 3.3 Growth media ................................................................................................................50 3.3.1 BG 11 culture medium ...........................................................................................50 3.3.2 Seawater .................................................................................................................52 3.4 Inoculum preparation and Duran bottle cultures...........................................................53 3.5 Tubular photobioreactor ................................................................................................54 3.6 Stirred tank photobioreactor..........................................................................................59 3.6.1 Batch culture ..........................................................................................................59 3.6.2 Continuous culture .................................................................................................60 3.7 A 10 L stirred photobioreactor (Corning photobioreactor) ...........................................62 3.8 Biomass separation by centrifugation ...........................................................................63 3.8.1 Batch centrifugation ...............................................................................................63 3.8.2 Continuous centrifugation ......................................................................................64 3.9 Biomass freeze drying ...................................................................................................65 3.10 Measurements .............................................................................................................66 3.10.1 Biomass concentration .........................................................................................66 3.10.2 Irradiance .............................................................................................................72 3.10.3 Nitrate...................................................................................................................74 3.10.4 Phosphate .............................................................................................................76 3.10.5 Total lipids ...........................................................................................................78 3.10.6 Neutral lipids ........................................................................................................79 3.10.7 Triglyceride in neutral lipids ................................................................................79 ix 3.10.8 Calorific values .................................................................................................... 80 3.10.9 Nile red staining of cells (modified from Elsey et al., 2007) .............................. 80 3.10.10 Gram staining (Mudili, 2007; Cappuccino & Natalie, 2011) ............................ 81 3.10.11 Algae morphology ............................................................................................. 81 3.10.12 Calculations of kinetic parameters (Doran, 1995; Shuler & Kargi, 2002) ........ 81 Chapter 4 ................................................................................................................................. 85 Results and Discussion ........................................................................................................... 85 4.1 Studies in Duran bottles ................................................................................................ 85 4.1.1 Salinity effects on growth ...................................................................................... 85 4.1.2 Light/dark cycle effects on Chlorella vulgaris ...................................................... 96 4.1.3 Chlorella vulgaris biomass loss in the dark ........................................................ 100 4.2 Culture profiles in tubular photobioreactor ................................................................ 103 4.2.1 Batch cultures ...................................................................................................... 112 4.2.1.1 Effect of nitrate concentration ...................................................................... 112 4.2.1.2 Effect of light ................................................................................................ 117 4.2.1.3 Effect of copper ............................................................................................ 118 4.2.2 Continuous cultures ............................................................................................. 121 4.3 Culture profiles in stirred-tank bioreactor .................................................................. 124 4.3.1 Batch cultures ...................................................................................................... 137 4.3.2 Continuous cultures ............................................................................................. 146 4.4 Investigation of MGA-1-NZ cultures ......................................................................... 156 4.4.1 MGA-1-NZ culture in Duran bottles ................................................................... 156 4.4.2 MGA-1-NZ culture in 10 L Corning stirred photobioreactor .............................. 157 4.5 Characteristics of the algal lipids ................................................................................ 165 4.5.1 Chlorella vulgaris ................................................................................................ 166 x 4.5.1.1 Concentrations of elements in Chlorella vulgaris total lipid extract .............166 4.5.1.2 Fractionation of Chlorella vulgaris total lipid extract ...................................167 4.5.1.3 Fatty acid profiles of Chlorella vulgaris total lipid extract ...........................169 4.5.2 The alga MGA-1-NZ ...........................................................................................175 4.5.2.1 Concentrations of elements in MGA-1-NZ total lipid extract ......................175 4.5.2.2 Fractionation of MGA-1-NZ total lipid extract ............................................175 4.5.2.3 Fatty acid profiles of MGA-1-NZ total lipid extract.....................................175 4.6 Comparison of the lipid productivity of C. vulgaris ...................................................179 Chapter 5 ...............................................................................................................................185 Summary and Conclusions....................................................................................................185 5.1 Summary .....................................................................................................................185 5.2 Conclusions .................................................................................................................190 References .............................................................................................................................193 Appendix 1 ............................................................................................................................225 Standard deviation calculations ............................................................................................225 Appendix 2 ............................................................................................................................227 Experimental data .................................................................................................................227 xi List of Figures Figure 2.1 A microalga (Chlorella vulgaris) with a dispersed cell morphology. ..................... 6 Figure 2.2 Microalgae with filamentous morphologies. ........................................................... 7 Figure 2.3 Oil productivity of various sources. ...................................................................... 10 Figure 2.4 A typical photosynthesis response curve. ............................................................. 19 Figure 2.5 Some components of algal crude oil. .................................................................... 23 Figure 2.6 Open systems for growing microalgae.. ................................................................ 34 Figure 2.7 Closed systems for growing microalgae.. ............................................................. 39 Figure 2.8 A tubular photobioreactor with a single continuous looped tube. ......................... 41 Figure 3.1 Chlorella vulgaris in BG-11 made in freshwater. ................................................. 47 Figure 3.2 Choricystis minor in BG-11 made in freshwater. ................................................. 47 Figure 3.3 Neochloris sp. in BG-11 made in freshwater. ....................................................... 48 Figure 3.4 Pseudococcomyxa simplex in BG-11 made in freshwater. ................................... 48 Figure 3.5 Scenedesmus sp. in BG-11 made in freshwater..................................................... 49 Figure 3.6 MGA-1-NZ in BG-11 made in seawater. .............................................................. 49 Figure 3.7 A typical set up for algae growth in gas sparged culture bottles........................... 54 Figure 3.8 Tubular photobioreactor. ....................................................................................... 55 Figure 3.9 Broth recirculation in the tubular loop photobioreactor. ....................................... 56 Figure 3.10 Main control panel of the tubular photobioreactor. ............................................. 56 Figure 3.11 A stirred tank photobioreactor illuminated using LED lamps. ........................... 61 Figure 3.12 Continuous culture in a stirred tank photobioreactor. ......................................... 61 Figure 3.13 The 10 L Corning stirred photobioreactor. .......................................................... 63 Figure 3.14 The cell paste after centrifugation. ...................................................................... 64 Figure 3.15 Continuous centrifugation. .................................................................................. 65 Figure 3.16 Freeze-dried biomass........................................................................................... 66 xii Figure 3.17 The calibration curve for Chlorella vulgaris in BG-11 made in freshwater. ......68 Figure 3.18 The calibration curve for Chlorella vulgaris in BG-11 made in seawater. .........69 Figure 3.19 The calibration curve for Choricystis minor in BG-11 made in freshwater. .......69 Figure 3.20 The calibration curve for Neochloris sp. in BG-11 made in freshwater. .............70 Figure 3.21 The calibration curve for Pseudococcomyxa simplex in BG-11 made in freshwater. ...............................................................................................................................70 Figure 3.22 The calibration curve for Scenedesmus sp. in BG-11 made in freshwater. .........71 Figure 3.23 The calibration curve for MGA-1-NZ in BG-11 made in seawater. ...................71 Figure 3.24 Relationship between the percentage light output and irradiance (QSL-2101 sensor) for LEDs of tubular photobioreactor.. ........................................................................72 Figure 3.25 Relationship between the percentage light output and irradiance (QSL-2101 sensor) for LEDs units A and B of the stirred tank photobioreactor unit 2.. ..........................73 Figure 3.26 Relationship between the percentage light output and irradiance (QSL-2101 sensor) for LEDs units C and D of the stirred tank photobioreactor unit 1.. ..........................73 Figure 3.27 A nitrate calibration curve for BG-11 made with freshwater. .............................75 Figure 3.28 A nitrate calibration curve for BG-11 made with seawater. ................................75 Figure 3.29 A phosphate calibration curve. ............................................................................77 Figure 4.1 Growth curves of Chlorella vulgaris in different BG11 formulations in 1 L culture bottles. .........................................................................................................................86 Figure 4.2 Growth curves of Choricystis minor in different BG11 formulations in 1 L culture bottles. .....................................................................................................................................86 Figure 4.3 Growth curves of Neochloris sp. in different BG11 formulations in 1 L culture bottles. .....................................................................................................................................87 Figure 4.4 Growth curves of Pseudococcomyxa simplex in different formulations of BG11 in 1 L culture bottles. ..................................................................................................................87 xiii Figure 4.5 Growth curves of Chlorella vulgaris in different BG11 formulations in 2 L culture bottles.......................................................................................................................... 89 Figure 4.6 Growth curves of Choricystis minor in different BG11 formulations in 2 L culture bottles. ..................................................................................................................................... 90 Figure 4.7 Growth curves of Neochloris sp. in different BG11 formulations in 2 L culture bottles. ..................................................................................................................................... 90 Figure 4.8 Growth curves of Psuedococcomyxa simplex in different formulations of BG11 in 2 L culture bottles. .................................................................................................................. 91 Figure 4.9 Growth curves of Scenedesmus sp. in different formulations of BG11 in 2 L culture bottles.......................................................................................................................... 91 Figure 4.10 Growth curves of Chlorella vulgaris in BG11 made with seawater, illuminated continuously (24/0) and every 12 h (12/12) by fluorescent lamps. ........................................ 97 Figure 4.11 Chlorella vulgaris biomass loss in the dark during incubation at various temperatures. At 25 qC, the data sets marked (1) and (2) are from two parallel flasks. ....... 100 Figure 4.12 Plots of ln(C/C0) versus time for C. vulgaris at various temperatures in the dark. At 25 qC, the data sets marked (1) and (2) are from two parallel flasks. ............................. 102 Figure 4.13 Culture profile of C. vulgaris in the run PBR 1 with 1133 mg L1 of initial nitrate concentration. ............................................................................................................ 112 Figure 4.14 Culture profile of C. vulgaris in the run PBR 9 with 537 mg L1 of initial nitrate concentration and 0.02 mg L1 of copper concentration. ..................................................... 113 Figure 4.15 Culture profile of C. vulgaris in the run PBR 13 with 291 mg L1 of initial nitrate concentration. See Table 4.6 for further details. ....................................................... 113 Figure 4.16 Broth foaming in the degassing column. ........................................................... 114 Figure 4.17 Culture profile of C. vulgaris in the run PBR 10 with 1372 mg L1 of initial nitrate concentration under a light level of 314-458 Pmol m2s1. ....................................... 116 xiv Figure 4.18 Culture profile of C. vulgaris in the run PBR 14 with 236 mg L1 of initial nitrate concentration under a light level of 314-458 Pmol m2s1. .......................................116 Figure 4.19 Culture profile of C. vulgaris in the run PBR 3 with 1088 mg L1 of initial nitrate concentration under a light level of 251-361 Pmol m2s1. .......................................119 Figure 4.20 Culture profile of C. vulgaris in the run PBR 6 without copper under a light level of 115-171 Pmol m2s1. .......................................................................................................121 Figure 4.21 Continuous growth profile of C. vulgaris in PBR 8 at a dilution rate of 0.3 d1. ...............................................................................................................................................123 Figure 4.22 Continuous growth profile of C. vulgaris in PBR 9 at a dilution rate of 0.12 d1. ...............................................................................................................................................123 Figure 4.23 Growth profile of C. vulgaris in STR 1 with normal nitrate (BG-11 seawater) under illumination of 292 Pmol m2s1 by fluorescent lights. ..............................................138 Figure 4.24 Growth profile of C. vulgaris in STR 2 with half nitrate under illumination of 292 Pmol m2s1 by fluorescent lights. .................................................................................138 Figure 4.25 Growth profile of C. vulgaris in STR 4 with half nitrate under illumination of 292 Pmol m2s1 by fluorescent lights. .................................................................................141 Figure 4.26 Growth profile of C. vulgaris in STR 5 with half nitrate under illumination of 292 Pmol m2s1 by fluorescent lights. .................................................................................143 Figure 4.27 Growth profile of C. vulgaris in STR 6 with half nitrate under illumination of 292 Pmol m2s1 by fluorescent lights. .................................................................................144 Figure 4.28 Growth profile of C. vulgaris in STR 10 with normal nitrate under illumination of 200 Pmol m2s1 by light-emitting diodes. .......................................................................145 Figure 4.29 Culture profile of C. vulgaris in STR 7. On day 31, the dilution rate was set at 0.157 d1. Irradiance level was 292 Pmol m2s1 (fluorescent light). ...................................146 xv Figure 4.30 Culture profile of C. vulgaris in STR 8. The dilution rate was 0.22 d1 (day 30) and 0.079 d1 (day 55). Irradiance level was 1123 Pmol m2s1 (light emitting diodes). .... 147 Figure 4.31 Culture profile of C. vulgaris in STR 9. On day 41, the dilution rate was set at 0.079 d1. Irradiance level was 292 Pmol m2s1 (fluorescent light). ................................... 147 Figure 4.32 Culture profile of C. vulgaris in STR 11 at various dilution rates (Table 4.8). The incident irradiance levels were 600 and 862 Pmol m2s1 (light-emitting diodes) as noted in Table 4.8. .......................................................................................................................... 148 Figure 4.33 Culture profile of C. vulgaris in STR 12 at various dilution rates (Table 4.8). The incident irradiance level were 177 and 76 Pmol m2s1 (light-emitting diodes) as noted in Table 4.8. .......................................................................................................................... 149 Figure 4.34 Relationship between dilution rate and the steady state biomass productivity. 155 Figure 4.35 Relationship between dilution rate and the steady state lipid productivity. ...... 156 Figure 4.36 Culture profiles of MGA-1-NZ in BG11 seawater medium in 2 L Duran bottle. .............................................................................................................................................. 157 Figure 4.37 Culture profile of MGA-1-NZ in BR 1. ............................................................ 160 Figure 4.38 Culture profile of MGA-1-NZ in BR 2. ............................................................ 160 Figure 4.39 Culture profile of MGA-1-NZ in BR 3. ............................................................ 161 xvii List of Tables Table 1.1 Some companies engaged in commercializing algal fuels ....................................... 3 Table 2.1 Various factors that affect lipid content and composition ...................................... 29 Table 3.1 BG11 stock 1 .......................................................................................................... 51 Table 3.2 BG11 stock 2 .......................................................................................................... 51 Table 3.3 BG11 stock 3 .......................................................................................................... 51 Table 3.4 BG11 stock 4 .......................................................................................................... 52 Table 3.5 Vitamins solution .................................................................................................... 52 Table 4.1 Kinetic parameters for the algae cultured in 1 L bottles (Figures 4.1-4.4)............. 88 Table 4.2 Total lipid contents and calorific values for the algae cultured in 2 L bottles ....... 92 Table 4.3 Kinetic parameters for the algae cultured in 2 L bottles......................................... 93 Table 4.4 Comparison of continuous illumination and light-dark cycling (12 h/12 h) on culture kinetics and biomass properties of Chlorella vulgaris ............................................... 99 Table 4.5 C. vulgaris biomass consumption rate constant k in the dark at various temperatures .......................................................................................................................... 103 Table 4.6 Summary of C. vulgaris production in the photobioreactor runs ......................... 104 Table 4.7 Total lipid content and calorific value of C. vulgaris biomass produced in the photobioreactor ..................................................................................................................... 109 Table 4.8 Kinetic parameters of C. vulgaris production in tubular photobioreactor ............ 110 Table 4.9 Summary of C. vulgaris cultures in stirred-tank bioreactors................................ 124 Table 4.10 Total lipid contents and calorific values of C. vulgaris biomass from stirred-tank bioreactors............................................................................................................................. 131 Table 4.11 Culture kinetic parameters for C. vulgaris from stirred-tank bioreactors .......... 133 Table 4.12 Lipid production by C. vulgaris in stirred-tank bioreactors ............................... 135 Table 4.13 Summary of biomass concentrations at various steady states ............................ 150 xviii Table 4.14 Summary of MGA-1-NZ cultures in 10 L Corning stirred photobioreactor ......159 Table 4.15 Total lipid contents and calorific values of MGA-1-NZ biomass ......................162 Table 4.16 Growth kinetic parameters of MGA-1-NZ .........................................................163 Table 4.17 Lipid production parameters of MGA-1-NZ ......................................................164 Table 4.18 Concentrations of various elements in C. vulgaris total lipid extract from stirred- tank photobioreactor runs STR 1 and STR 2 ........................................................................166 Table 4.19 Neutral lipid content and triglyceride content of C. vulgaris oils .......................168 Table 4.20 Fatty acid profiles of C. vulgaris total lipid extract from photobioreactor runs .170 Table 4.21 Fatty acid profiles of C. vulgaris total lipid extract from stirred-tank photobioreactor runs .............................................................................................................172 Table 4.22 Proportions of the various classes of fatty acids in lipids from some tubular photobioreactor and stirred-tank photobioreactor runs .........................................................174 Table 4.23 Concentrations of elements in crude oil of MGA-1-NZ .....................................175 Table 4.24 Fatty acid profiles of MGA-1-NZ total lipids .....................................................177 Table 4.25 Proportions of the various classes of fatty acids in lipids of MGA-1-NZ ..........179 Table 4.26 The lipid productivity of C. vulgaris in Duran bottles, tubular photobioreactors and stirred-tank bioreactors ...................................................................................................180 xix Abbreviations A Area illuminated by light (m2) Axxx Spectrophotometric absorbance at a wavelength of xxx nm BR n Corning stirred photobioreactor run n C Biomass concentration at time t (g L1) C0 Biomass concentration at time zero (g L 1) D Dilution rate (d1) DCW Dry cell weight (g L1) DHA Docosahexaenoic acid DNA Deoxyribonucleic acid DO Dissolved oxygen d Diameter (m) EPA Eicosapentaenoic acid F Flow rate of the feed (mL d1) I Irradiance (Pmol m2 s1) k First-order rate constant for biomass loss (h1) LED Light emission diodes ND Not determined Nf Final concentration of nitrate (mg L 1) Ni Initial concentration of nitrate (mg L 1) PAR Photosynthetically active radiation PBR n Tubular photobioreactor run n Pf Final concentration of phosphate (mg L 1) Pi Initial concentration of phosphate (mg L 1) xx PTFE Poly (tetrafluoroethylene), or Teflon QL Final lipid productivity (g L 1d1) QX Final biomass productivity (g L 1d1) qL Specific lipid production rate (d 1) qN Specific nitrate consumption rate (mg g 1d1) qP Specific phosphate consumption rate (mg g 1d1) RNA Ribonucleic acid RTD Resistance temperature detector, a type of temperature sensor RUBISCO Ribulose-1,5-bisphosphate carboxylase/oxygenase RuBPCase Ribulose-1,5-bisphosphate carboxylase/oxygenase SARDI South Australia Research and Development Institute STR n Stirred tank bioreactor (BioFlo) run n TL Total lipids t Duration of a batch (d) tf Time at the end of a batch (d) ti Time at the beginning of a batch (d) V Working volume of the reactor, or volume (L) Xf Final concentration of biomass (g L 1) Xi Initial concentration of biomass (g L 1) YL/Light Lipid yield on light (g Pmol1) YL/N Lipid yield coefficients on nitrate (g mg 1) YL/P Lipid yield coefficients on phosphate (g mg 1) YX/Light Biomass yield coefficient on light (g Pmol1) YX/N Biomass yield coefficient on nitrate (g mg 1) YX/P Biomass yield coefficient on phosphate (g mg 1) xxi y Weight fraction of lipids in the biomass ICP-MS Inductively coupled plasma mass spectrometry ICP-OES Inductively coupled plasma optical emission spectrometry