1 Bioremoval of copper and nickel on living and non-living Euglena gracilis 2 3 4 5 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master 6 of Science in the Faculty of Arts and Sciences 7 8 9 10 11 12 13 TRENT UNIVERSITY 14 Peterborough, Ontario, Canada 15 16 17 18 19 20 21 Environmental and Life Sciences MSc. Graduate Program 22 April 2016 23 © Cameron Winters 2016 24 i 25 Abstract 26 Bioremoval of Copper and Nickel on living and non-living Euglena gracilis 27 Cam Winters 2016 28 This study assesses the ability of a unicellular protist, Euglena gracilis, to remove 29 Cu and Ni from solution in mono- and bi-metallic systems. Living Euglena cells and 30 non-living Euglena biomass were examined for their capacity to sorb metal ions. 31 Adsorption isotherms were used in batch systems to describe the kinetic and equilibrium 32 characteristics of metal removal. In living systems results indicate that the sorption 33 reaction occurs quickly (<30 min) in both Cu (II) and Ni (II) mono-metallic systems and 34 adsorption follows a pseudo-second order kinetics model for both metals. Sorption 35 capacity and intensity was greater for Cu than Ni (p < 0.05) and were described by the 36 Freundlich model. In bi-metallic systems sorption of both metals appears equivalent. In 37 non-living systems sorption occurred quickly (10-30 min) and both Cu and Ni 38 equilibrium uptake increased with a concurrent increase of initial metal concentrations. 39 The pseudo-first-order model was applied to the kinetic data and the Langmuir and 40 Freundlich models effectively described single-metal systems. The biosorption capacity 41 of Cu (II) and) was 3x times greater than that of Ni (II). Sorption of one metal in the 42 presence of relatively high concentrations of the other metal was supressed. Generally, 43 it was found that living Euglena remove Cu and Ni more efficiently than non-living 44 Euglena biomass in both mono- and bi-metallic systems. It is anticipated that this work 45 should contribute to the identification of baseline uptake parameters and capacities for Cu 46 and Ni by Euglena as well as to the increasing amount of research investigating 47 sustainable bioremediation. ii 48 Keywords: biosorption, kinetics, Cu, Ni, accumulation, bioremediation, Euglena gracilis 49 Acknowledgements 50 I would like to offer my sincere thanks to my supervisor Dr. Céline Guéguen for 51 her guidance and support of this work. It has been my pleasure to work in her laboratory 52 and I am thankful for her clear perspective and valuable counsel. My thanks go also to 53 supervisory committee members Dr. Eric Sager and Dr. Neil Emery both for their time 54 and advice regarding the work. Additionally, I would like to thank Dr. Sager for his 55 friendship and guidance throughout my undergraduate and graduate studies. This work 56 would not have been possible without the assistance of Antoine Perroud whom I thank 57 for his ICPMS analysis and unfailingly good humour. I would also like to acknowledge 58 Jean- François Koprivnjak and the Water Quality Centre at Trent University for 59 assistance and expertise. My thanks also go to Noble Purification Inc. for their support 60 and encouragement of this project. Financial support from the Ontario Centre for 61 Excellence, NSERC, and the Canada Research Chair program is greatly appreciated. I 62 must also thank all my colleagues in the lab, especially Vaughn Mangal, Yong Xiang Shi, 63 and Chad Cuss for their valuable input and friendship. Finally, I thank my parents for 64 their abiding patience, love, and support over the course of this endeavour. It means 65 more than words can say, and I dedicate this work to you both. 66 Authors’ contributions 67 CW and CG developed study design and CG contributed revisions for all chapters. CW 68 collected and analyzed the data and wrote the first draft of all manuscripts. AN provided 69 technical and financial support for Chapter 2. All authors read and approved final drafts. iii 70 Table of Contents 71 72 Chapter 1: General Introduction 73 1 Metal pollution ............................................................................................................ v 74 2 Ecological and Human Health Effects of Metals ........................................................ 2 75 3 Conventional Methods of Metal Remediation ............................................................. 2 76 4 Bioremediation: an alternative method for metal removal .......................................... 5 77 5 Euglena gracilis: a potential biosorbent for metal remediation ................................ 13 78 6 Thesis objectives........................................................................................................ 14 79 References ......................................................................................................................... 18 80 Chapter 2: Equilibrium and kinetic studies of Cu (II) and Ni (II) sorption and 81 accumulation on living Euglena gracilis 82 Abstract ..................................................................................................................... 26 83 1 Introduction ............................................................................................................... 27 84 2 Materials and Methods .............................................................................................. 28 85 3 Results and Discussion .............................................................................................. 34 86 4 Conclusions ............................................................................................................... 39 87 References ......................................................................................................................... 41 88 Chapter 3: Equilibrium and kinetic studies of Cu (II) and Ni (II) biosorption on non- 89 living Euglena gracilis 90 Abstract ..................................................................................................................... 51 91 1 Introduction ............................................................................................................... 52 92 2 Materials and Methods .............................................................................................. 56 93 3 Results and Discussion .............................................................................................. 60 94 4 Conclusion ................................................................................................................. 64 95 Chapter 4: General Conclusion ......................................................................................... 78 96 1 Euglena biomass characterization and toxicity ......................................................... 78 97 2 Sorption kinetics ....................................................................................................... 79 98 3 Sorption Isotherms .................................................................................................... 80 99 4 Significance of the work ........................................................................................... 83 100 iv 101 List of Figures 102 Chapter 1: 103 Figure 1: Euglena gracilis at 400x magnification 104 Figure 2: Growth curve for Euglena gracilis 105 Chapter 2: 106 Figure 1: Experimental data for EC50 toxicity assay on living E. gracilis 107 for (a) Cu2+ and (b) Ni2+ 108 Figure 2: Experimental data for Cu2+ sorption kinetics on living E. gracilis 109 at initial concentrations of (a) 20 ug L-1 and (b) 50 ug L-1. Curves 110 represent pseudo-second-order kinetic model. 111 Figure 3: Experimental data for Ni2+ sorption kinetics on living E. gracilis 112 at initial concentrations of (a) 3 mg L-1 and (b) 100 mg L-1. Curves 113 represent pseudo-second-order kinetic model. 114 Figure 4: Experimental data for a) Cu2+ and b) Ni2+ sorption equilibrium 115 on living E. gracilis. Curve represents the Freundlich model. 116 Figure 5: Experimental data for Cu2+ and Ni2+ sorption equilibrium from 117 binary-metal solution on living E. gracilis. Error bars represent SE. 118 Chapter 3: 119 Figure 1: FTIR spectra of dried euglena biomass 120 Figure 2: Experimental data for Cu2+ sorption kinetics on non-living E. 121 gracilis at initial concentrations of (a) 20 ug L-1 and (b) 50 ug L-1 (c) 1 mg 122 L-1 (d) 25 mg L-1. Curves represent PFO (a, c) and PSO (b, d) kinetic 123 models. 124 Figure 3: Experimental data for Ni2+ sorption kinetics on non-living E. 125 gracilis at initial concentrations of (a) 1 mg L-1 and (b) 2 mg L-1 (c) 4 mg 126 L-1 (d) 20 mg L-1. Curves represent PFO (b, d) and PSO (a, c) kinetic 127 models. 128 Figure 4: Experimental data for Cu2+ sorption equilibrium on non-living E. 129 gracilis. Curves represent the Freundlich and Langmuir models. v 130 Figure 5: Experimental data for Ni2+ sorption equilibrium on non-living E. 131 gracilis. Curves represent the Freundlich and Langmuir models. 132 Figure 6: Experimental data for Cu2+ and Ni2+ sorption equilibrium from 133 binary-metal solution on living E. gracilis. Error bars represent SE. 134 Chapter 4: 135 Figure 1: Percentage metal removal of Cu and Ni in mono-metallic and bi- 136 metallic solutions by living and non-living E. gracilis. vi 137 138 139 140 141 142 143 144 145 146 147 148 vii 149 List of Tables 150 Chapter 2: 151 Table 1: Pseudo-second-order kinetics parameters for the sorption of 152 Cu2+and Ni2+ on living Euglena gracilis cells (±SE). 153 Table 2: Freundlich adsorption isotherm parameters