ANALYSIS OF MICROBES IN GREENLAND ICE CORES FROM PERIODS OF HIGH AND LOW ATMOSPHERIC CARBON DIOXIDE LEVELS Caitlin Nicole Knowlton A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE May 2013 Committee: Scott O. Rogers, Advisor Paul Morris Vipaporn Phuntumart 1 ii ABSTRACT Scott O. Rogers, Advisor Glacial ice entraps microbial cells and nucleic acids that have been deposited from the atmosphere by precipitation or on dust particles carried by the wind. The ice is deposited in sequential layers representing distinct past periods of time. Metagenomics and metatranscriptomics offer means to perform comprehensive analyses of microbial genes and taxa within the ice. During the past 400,000 years, fluctuations in atmospheric carbon dioxide levels have directly coincided with temperature fluctuations. Three ice core sections from Byrd Station, Antarctica (80° 1’ S, 119° 31’ W) and three ice core sections from GISP 2D (Greenland Ice Sheet Project, core 2D; 72o 36' N, 38o 30'W) were aseptically melted. Two of the ice core sections from GISP 2D (at 1600 m = 10,500 ybp [years before present] and at 3014 m = 157,000 ybp) were analyzed using metagenomic and metatranscriptomic analyses. The 157,000 year old ice core section was deposited during low atmospheric CO2 levels that would have led to low temperatures, low precipitation rates, and high dust levels, while the 10,500 year old core section was deposited during high CO2 levels that would have led to higher temperatures, higher precipitation rates, and lower dust levels. Nucleic acids from ultracentrifuge- concentrated meltwater were subjected to 454 pyrosequencing. Taxonomic analyses were based on rRNA and mRNA sequences. Taxa in the 10,500 ybp ice primarily included bacterial species of Proteobacteria, Firmicutes, and Cyanobacteria. This sample also contained black particulate matter indicative of volcanic activity. Major volcanic activity was reported in Iceland, Alaska, and from a super volcano in Italy ii iii during the 10,500 ybp time period. The 157,000 ybp sample also contained a large number of Proteobacteria and Firmicute species, a few Actinobacteria and Cyanobacteria, but most were common soil bacteria, as well as animal pathogens. The number and frequency of cyanobacterial species was much higher. While some species were in common to both samples, a large number of unique taxa and genes were present in each of the samples. These unique taxa and genes suggest different atmospheric conditions may influence the deposition of microbes in ice. iii iv ACKNOWLEDGEMENTS It is important to recognize that this research was partially funded by BGSU through FRC-RIG (Faculty Research Committee Research Incentive Grants). The ice in this study was provided by NICL (in Colorado) and NICL-SMO (in New Hampshire). I acknowledge my advisor, Dr. Scott Rogers, who has always pushed me towards excellence. I am thankful for his gentle guidance and patient teaching in my years at Bowling Green State University. I am grateful for all he has done to make this a successful growing experience. Many thanks to the educators that have fueled my curiosity to learn: Mr. Mike Sutter-who introduced me to the mystery of science in high school, Dr. Andrew Whipple-my undergraduate advisor, Dr. Anita Hopper-for giving me a chance to understand the joy of lab work, Dr. Ray Larsen-for his stimulating classes and enthusiasm to bring me to Bowling Green State University and Dr. Carmen Fioravanti– who provided insight to my research in the beginning stages. I also thank my committee members-Dr. Vipa Phuntumart and Dr. Paul Morris. I appreciate all their friendly advice. I am indebted to Dr. Morris for his generous funding through scholarships during my graduate studies. I owe a huge thank you to my lab mates, Sammy Juma, Jen Hofacker and especially Yury Shtarkman, who generously conversed and shared ideas with me. It was a pleasure to work alongside such kind individuals. I would also like to thank Seung-Geuk Shin for his assistance with parts of my lab work. I am grateful for Dr. Ram iv v Veerapaneni and Dr. Tom D’Elia’s comradery and previous work in our lab, that when combine with my work, was invaluable in producing our publication. I am beyond thankful to family, my father and mother-Rob and Mary Knowlton, and my siblings- Allison and Nate Knowlton, for their unwavering faith in my ability and the support that they have been. I must also thank my friends for the moments of laughter that kept me sane. My friends and family have been a joy and comfort throughout this process. I particularly thank Elizabeth Metz for the rock that she is in my life, and Alexandra Leguire for sharing in my every success throughout my research. Additionally, I am ever grateful to the love of my life, Samuel Shepard, for his constant affirmation. His understanding and encouragement was so important in this journey. Most importantly, I thank my heavenly Father for the grace He shows to me through life in Jesus Christ. I am honored to study His hand in the intricacy, beauty, and complexity of biology. I would have never made it through this time without continual renewal, peace, and strength from above. v vi TABLE OF CONTENTS Page CHAPTER I. INTRODUCTION TO POLAR MICROBIOLOGY AND METAGENOMICS/METATRANSCRIPTOMICS ..................................................... 1 1.1 Why study environmental ice deposits? ....................................................... 2 1.1.1 Background......................................................................................... 2 Application of ice: atmosphere and microbes ............................ 2 Atmospheric changes and genome recycling ............................. 2 Glacial formation............................................................................. 4 Dating ice cores ............................................................................... 5 Adaptations of microbes living in ice .......................................... 6 1.1.2 Evolutionary and practical application of ice study..................... 9 1.2 How to study the ice ........................................................................................ 9 1.2.1 Obtaining ice ...................................................................................... 9 The ice cores..................................................................................... 9 The approach ................................................................................... 11 1.2.2 Eliminating Contamination ............................................................. 12 1.2.3 Methods .............................................................................................. 14 Retrieving nucleic acids from ice ................................................. 14 Sample preparation for sequencing ............................................. 15 Sequencing ....................................................................................... 18 Data Analysis................................................................................... 20 vi vii Assembling Data................................................................... 20 Phylogenetics......................................................................... 22 Microscopy of black particulate matter ............................. 24 REFERENCES......................................................................................................... 27 CHAPTER II. RESEARCH .............................................................................................. 34 2.1 Introduction ....................................................................................................... 35 2.2 Materials and Methods .................................................................................... 37 2.2.1 Ice core selection and samples ........................................................ 37 2.2.2 Surface sterilization and melting..................................................... 37 2.2.3 Culturing ............................................................................................. 38 2.2.4 Ultracentrifugation ........................................................................... 39 2.2.5 Isolation of RNA ................................................................................ 39 2.2.6 Isolation of DNA ................................................................................ 40 2.2.7 Preparation of samples for sequencing........................................... 41 2.2.8 Sequencing ......................................................................................... 47 2.2.9 Analysis of sequences........................................................................ 48 2.2.10 Scanning Electron Microscope (SEM) ........................................... 49 2.2.11 Sequencing of Cultures ................................................................... 49 2.3 Results……......................................................................................................... 50 2.3.1 Taxa...................................................................................................... 54 2.3.2 Phylogenetics...................................................................................... 56 2.3.3 Culturing and sequencing ................................................................ 63 vii viii 2.3.4 Elemental analysis ............................................................................. 66 2.4 Discussion