MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF HYDROCARBON PRODUCTION IN THE GREEN MICROALGA Botryococcus braunii A Dissertation by TAYLOR LEIGH WEISS Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2012 Major Subject: Biochemistry Molecular and Biochemical Characterization of Hydrocarbon Production in the Green Microalga Botryococcus braunii Copyright 2012 Taylor Leigh Weiss MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF HYDROCARBON PRODUCTION IN THE GREEN MICROALGA Botryococcus braunii A Dissertation by TAYLOR LEIGH WEISS Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Timothy P. Devarenne Committee Members, Pingwei Li Andreas Holzenburg Comer O. Patterson Head of Department, Gregory D. Reinhart August 2012 Major Subject: Biochemistry iii ABSTRACT Molecular and Biochemical Characterization of Hydrocarbon Production in the Green Microalga Botryococcus braunii. (August 2012) Taylor Leigh Weiss, B.S., Rochester University Chair of Advisory Committee: Dr. Timothy P. Devarenne Botryococcus braunii (Chlorophyta, Botryococcaceae) is a colony-forming green microalga that produces large amounts of liquid hydrocarbons, which can be converted into transportation fuels. While B. braunii has been well studied for the chemistry of the hydrocarbon production, very little is known about the molecular biology of B. braunii. As such, this study developed both apparatus and techniques to culture B. braunii for use in the genetic and biochemical characterization. During genetic studies, the genome size was determined of a representative strain of each of the three races of B. braunii, A, B, and L, that are distinguished based on the type of hydrocarbon each produces. Flow cytometry analysis indicates that the A race, Yamanaka strain, of B. braunii has a genome size of 166.0 ± 0.4 Mb, which is similar to the B race, Berkeley strain, with a genome size of 166 ± 2.2 Mb, while the L race, Songkla Nakarin strain, has a substantially larger genome size at 211.3 ± 1.7 Mb. Phylogenetic analysis with the nuclear small subunit (18S) rRNA and actin genes were used to classify multiple strains of A, B, and L races. These analyses suggest that the iv evolutionary relationship between B. braunii races is correlated with the type of liquid hydrocarbon they produce. Biochemical studies of B. braunii primarily focused on the B race, because it uniquely produces large amounts of botryococcenes that can be used as a fuel for internal combustion engines. C30 botryococcene is metabolized by methylation to generate intermediates of C31, C32, C33, and C34. Raman spectroscopy was used to characterize the structure of botryococcenes. The spectral region from 1600–1700 cm-1 showed v(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm-1 were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm-1 was assigned to the backbone C=C bond stretching. Finally, confocal Raman microspectroscopy was used to map the presence and location of methylated botryococcenes within a living colony of B. braunii cells. v DEDICATION This work is dedicated to my grandmother. Though she did not live to see its completion, this work would not have been possible without her love and support. vi ACKNOWLEDGEMENTS I would foremost like to thank my advisor, Tim Devarenne. I came to him without a lab, a project, nor direction. He gave me all these things plus the freedom and confidence to pursue my most ambitious intellectual pursuits. I am forever grateful for his mentoring, guidance, and friendship. I also thank our collaborator and good friend, Shigeru Okada. Without his aid, great patience, deep wisdom, and vast knowledge none of my studies would have been possible. I would especially like to thank both Arum Han and Christian Hilty for allowing me to collaborate and work with my friends Hyun Soo Kim and Giridhar Sekar. I look forward to many years of friendship and science together. I would like to thank all of the members of my committee, Andreas Holzenburg, Pingwei Li, James Manhart, C.O. Patterson, and Michael Polymenis, for all their service, suggestions, comments, and helpful conversations during the course of my studies. I would similarly like to thank Stanislav Vitha, Jaan Laane, Hye Jin Chun, Amanda Young, Margret Glasner, Wei-chuan Shih, Ji Qi, Larry Dangott, Eunah Lee, Patrick Killough, for all their collaborations, technical expertise, and fruitful conversation. Finally, I would like to thank all the members of the Devarenne lab, past, present, temporary, transient, and otherwise honorary for their help and conversation. In particular, I am grateful to have both worked and laughed beside classmates Anna Nelson, Joel Gray, and Julian Avila. vii TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi TABLE OF CONTENTS .......................................................................................... vii LIST OF FIGURES ................................................................................................... xi LIST OF TABLES .................................................................................................... xiv CHAPTER I INTRODUCTION ................................................................................ 1 Algae biofuels ................................................................................ 1 Algae .............................................................................................. 2 Botryococcus braunii ..................................................................... 4 General biology ........................................................................ 4 Biofuel potential ....................................................................... 9 Hydrocarbon synthesis ................................................................... 11 Isoprenoids ............................................................................... 11 B Race ...................................................................................... 17 L Race ...................................................................................... 22 A Race ...................................................................................... 25 Hydrocarbon accumulation ............................................................ 27 Rationale ......................................................................................... 30 II MATERIALS AND METHODS ......................................................... 31 Growth apparatus design ................................................................ 31 Mixed-gas system ..................................................................... 31 Lighting system ........................................................................ 34 Liquid media growth ...................................................................... 34 Flasks ........................................................................................ 34 Carboy ...................................................................................... 37 Solid media growth ........................................................................ 39 viii CHAPTER Page Culturing of algae ........................................................................... 42 Cyrogenic storage ........................................................................... 43 Gene sequencing ............................................................................ 44 Berkeley strain .......................................................................... 44 18S rRNA ......................................................................... 44 β-actin ............................................................................... 45 Additional strains ..................................................................... 46 18S rRNA ......................................................................... 46 β-actin ............................................................................... 47 Phylogenetic analysis ..................................................................... 48 Genome size estimation ................................................................. 50 Sample preparation ................................................................... 50 Flow cytometry ........................................................................ 50 Berkeley strain GC-content estimation .......................................... 51 Histochemical staining ................................................................... 52 Microscopy ..................................................................................... 53 Shell preparation ............................................................................ 54 Carbohydrate gas chromatography/mass spectrometry .................. 55 Purification and identification of botryococcenes .......................... 57 Raman spectroscopy ......................................................................