Assessing the influence of environmental pH on algal physiology using a novel culture system By Rachel L. Golda A DISSERTATION Presented to the Division of Environmental and Biomolecular Systems and the Oregon Health & Science University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Environmental Science and Engineering August, 2017 i School of Medicine Oregon Health & Science University CERTIFICATE OF APPROVAL _________________________________ This is to certify that the PhD dissertation of Rachel L. Golda has been approved ________________________________________________ Mentor/Advisor: Tawnya Peterson (Oregon Health & Science University) ________________________________________________ Mentor/Advisor: Joseph Needoba (Oregon Health & Science University) ________________________________________________ Committee Chair: Paul Tratnyek (Oregon Health & Science University) ________________________________________________ Member: Anne Thompson (Portland State University) i TABLE OF CONTENTS CERTIFICATE OF APPROVAL ........................................................... Error! Bookmark not defined. TABLE OF CONTENTS ........................................................................................................................... ii LIST OF FIGURES .................................................................................................................................... v LIST OF EQUATIONS ............................................................................................................................ vii ACKNOWLEDGMENTS ....................................................................................................................... viii ABSTRACT ................................................................................................................................................. x CHAPTER 1 ................................................................................................................................................ 1 Introduction ................................................................................................................................................. 1 1.1 Ocean acidification and aquatic carbon chemistry .............................................................. 1 1.2 pH as an independent, ecologically significant variable ...................................................... 3 1.3 Homeostasis of intracellular pH ............................................................................................ 6 1.4 Esterase activity and cellular energetics ............................................................................... 8 1.5 Ecological impact: potential effects of ambient pH on phytoplankton ecology ................. 8 1.6 Summary and research needs .............................................................................................. 11 1.7 Dissertation overview ............................................................................................................ 12 CHAPTER 2 .............................................................................................................................................. 15 2.1 Abstract .......................................................................................................................................... 15 2.2 Introduction ................................................................................................................................... 16 2.3 Materials and Methods ................................................................................................................. 19 2.4 Major System Components .......................................................................................................... 20 2.4.1 System Overview ..................................................................................................................... 20 2.4.2 Computer Program ................................................................................................................. 21 2.4.3 pH Sensor ................................................................................................................................ 22 2.4.4 Input/Output (I/O) Device ....................................................................................................... 23 2.4.5 GEORG – Conditioning Electronically Operated Relay Grouping ....................................... 23 2.4.6 Reagent Manifold .................................................................................................................... 24 2.4.7 Gas manifold ........................................................................................................................... 25 2.4.8 Culture Vessel ......................................................................................................................... 26 2.4.9 Pump ....................................................................................................................................... 27 2.4.10 Culture Conditions .................................................................................................................. 28 2.4.11 Measurements ......................................................................................................................... 28 2.4.12 Performance Testing ............................................................................................................... 29 2.6 Discussion....................................................................................................................................... 38 2.7 Conclusions .................................................................................................................................... 44 CHAPTER 3 .............................................................................................................................................. 45 ii 3.1 Abstract .................................................................................................................................. 45 3.2 Introduction ........................................................................................................................... 46 3.3 Methods .................................................................................................................................. 49 3.3.1 Phytoplankton isolates and culture conditions ................................................................... 49 3.3.2 Fluorophore loading ........................................................................................................... 50 3.3.3 Fluorescence measurements ............................................................................................... 52 3.4 Confocal Laser Scanning Microscopy ................................................................................. 52 3.5 Fluorescence spectroscopy ................................................................................................... 53 3.6 Flow Cytometry ..................................................................................................................... 54 3.6.1 SNARF calibration curves .................................................................................................. 54 3.6.2 Radioisotopic pHi measurements ........................................................................................ 55 3.6.3 Esterase activity .................................................................................................................. 57 3.6.4 Statistics .............................................................................................................................. 58 3.8 Discussion ............................................................................................................................... 68 CHAPTER 4 .............................................................................................................................................. 74 4.1 Abstract ............................................................................................................................................ 74 4.2 Introduction ........................................................................................................................... 75 4.3 Methods .................................................................................................................................. 81 4.3.1 Cell culture .......................................................................................................................... 81 4.3.2 pH environments ................................................................................................................. 81 4.3.3 Determination of biomass ................................................................................................... 82 4.3.4 Measurement of photosynthetic efficiency .......................................................................... 83 4.3.5 Intracellular pH measurements .......................................................................................... 83 4.3.6 Determination of esterase activity ...................................................................................... 84 4.3.7 Fluorescence measurements ............................................................................................... 84 4.3.8 Gibbs calculations............................................................................................................... 85 4.4 Results