SCREENING, DOWN-SELECTION, CHARACTERIZATION, AND GENETIC TOOL DEVELOPMENT IN HIGH-PRODUCTIVITY MICROALGAE by Lukas Royce Dahlin A thesis submitted to the Faculty and the Board of Trustees of the Colorado School of Mines in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Applied Chemistry). Golden, Colorado Date: Signed: Lukas R. Dahlin Signed: Dr. Matthew C. Posewitz Thesis Advisor Signed: Dr. Michael T. Guarnieri Thesis Advisor Golden, Colorado Date: Signed: Dr. Thomas Gennett Professor and Head Department of Chemistry ii ABSTRACT Microalgae are a promising source of renewable biomass, and biocatalysts which can be utilized for the renewable production of fuel and chemical intermediates. During the time the work of this thesis was pursued microalgal productivity was recognized as a limiting factor to achieve competitive pricing with fossil fuel alternatives. To address this, a microalgal culture collection was screened, down-selecting for promising strains showing robust growth under relevant outdoor conditions. After isolating several promising strains, they were further characterized, ultimately leading to the identification of strains for robust winter and summer cultivation. Winter strains were grown outdoors in 1000 L raceway ponds in February of 2016 to confirm and characterize real world growth metrics. Two promising summer isolates were identified, Picochlorum renovo and Scenedesmus sp. These two strains both display high productivity, however, have remarkably different life cycles. Picochlorum renovo has a compact genome, grows rapidly, undergoes cell division during both day and night, and once nitrogen deprived photosynthesis ceases. In contrast, Scenedesmus sp. has a relatively large genome, regulates cell division to occur during dark periods, and has a remarkably high photosynthetic rate when nitrogen deprived. To further enhance growth and enable basic science inquiries genetic tools were developed for Picochlorum renovo. Tools necessary for the overexpression of proteins from either the nuclear or chloroplast genomes were developed. iii TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii LIST OF FIGURES .......................................................................................................... vii LIST OF TABLES ............................................................................................................. xi ACKNOWLEDGEMENTS .............................................................................................. xii CHAPTER 1 INTRODUCTION ........................................................................................ 1 1.1 Motivation ............................................................................................................... 1 1.2 Background and Previous Work ............................................................................. 3 1.3 Outline of Chapters ................................................................................................. 6 1.4 References ............................................................................................................... 7 CHAPTER 2 DOWN-SELECTION AND OUTDOOR EVALUATION OF NOVEL HALOTOLERANT ALGAL STRAINS FOR WINTER CULTIVATION ........................................................................................ 11 2.1 Abstract ................................................................................................................. 11 2.2 Introduction ........................................................................................................... 12 2.3 Results ................................................................................................................... 14 2.3.1 Indoor Screening .......................................................................................... 14 2.3.2 Outdoor Deployment ................................................................................... 15 2.4 Discussion ............................................................................................................. 21 2.4.1 Indoor Screening .......................................................................................... 21 2.4.2 Outdoor Deployment ................................................................................... 23 2.5 Methods................................................................................................................. 26 2.5.1 Culture Collection Screening ....................................................................... 26 2.5.2 18S Sequencing ............................................................................................ 28 2.5.3 Outdoor Growth ........................................................................................... 28 2.5.4 Compositional Analysis ............................................................................... 29 2.6 Acknowledgements ............................................................................................... 30 2.7 References ............................................................................................................. 31 iv CHAPTER 3 RECENT ADVANCES IN ALGAL GENETIC TOOL DEVELOPMENT ....................................................................................... 35 3.1 Abstract ................................................................................................................. 35 3.2 Introduction ........................................................................................................... 36 3.3 Random Gene Integration and Homologous Recombination ............................... 37 3.4 Multi-copy Gene Expression and Expression Control.......................................... 39 3.5 In vivo genome editing ......................................................................................... 41 3.6 Technical Hurdles and Considerations ................................................................. 43 3.7 Concluding Remarks ............................................................................................. 44 3.8 References ............................................................................................................. 46 CHAPTER 4 DEVELOPMENT OF A HIGH-PRODUCTIVITY, HALOPHILIC, THERMOTOLERANT MICROALGA, PICOCHLORUM RENOVO ...... 50 4.1 Abstract ................................................................................................................. 50 4.2 Introduction ........................................................................................................... 51 4.3 Results ................................................................................................................... 52 4.3.1 Down-Selection, Physiology, and Compositional Analysis ........................ 52 4.3.2 Genomic Analysis and Speciation ............................................................... 55 4.3.3 Transcriptome Response to Salinity............................................................. 59 4.3.4 Nuclear and Chloroplast Engineering .......................................................... 60 4.4 Discussion ............................................................................................................. 63 4.5 Conclusions ........................................................................................................... 67 4.6 Methods................................................................................................................. 68 4.6.1 Strain Screening and Characterization of Algal Growth.............................. 68 4.6.2 Compositional Analysis ............................................................................... 70 4.6.3 Genome Sequencing, Assembly and Annotation ......................................... 70 4.6.4 Transcriptome Response to Salinity............................................................. 71 4.6.5 Nuclear Engineering..................................................................................... 73 4.6.6 Chloroplast Engineering............................................................................... 74 4.6.7 Fluorescent Plate Reader Analysis ............................................................... 75 4.6.8 Statistics and Reproducibility ...................................................................... 76 4.6.9 Microscopy ................................................................................................... 76 4.7 Acknowledgements ............................................................................................... 77 v 4.8 Data Availability ................................................................................................... 77 4.9 References ............................................................................................................. 78 CHAPTER 5 CHARACTERIZATION OF A NOVEL SCENEDESMUS....................... 83 5.1 Abstract ................................................................................................................. 83 5.2 Introduction ........................................................................................................... 83 5.3 Results ................................................................................................................... 84 5.3.1 Strain Down-Selection, Physiology, and Compositional Analysis .............. 84 5.3.2 Preliminary Genetic Engineering ................................................................