DETECTION AND CHARACTERIZATION OF A UNIQUE AMMONIA OXIDIZING ARCHAEA; CULTURED FROM LAKE SUPERIOR Michael J. Schlais A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2014 Committee: George S. Bullerjahn, Advisor John Snyder Graduate Faculty Representative Robert M. McKay Scott O. Rogers Paul Morris © 2014 Michael J. Schlais All Rights Reserved iii ABSTRACT George Bullerjahn, Advisor In the past century Lake Superior has seen a 5-fold increase in nitrate levels. Previous research has shown this increase to be due to as of yet undescribed in-lake oxidation processes. It has recently been shown that ammonia oxidizing archaea (AOA) in marine and freshwater environments belonging to the phylum Thaumarchaeota have the ability to oxidize ammonium, and are specifically suited to the low NH /oligotrophic environment of Lake Superior. In this study isolation and enrichment of these unique₄⁺ freshwater ammonia oxidizing archaea from Lake Superior has enabled the direct measurement of NO ¯ and N O production and NH metabolism. In the search for in-lake nitrifying organisms₂ we have₂ identified and described₄⁺ a novel group of freshwater ammonia oxidizing archaea using the ammonia monooxygenase alpha (amoA) subunit gene as a diagnostic tool for microbes capable of ammonia oxidation. Flow cytometry was used to determine cell abundances and ideal incubation times and temperatures for these AOA taken from these Lake Superior. These data along with nitrification measurements enabled us to determine per cell nitrification rates for our AOA enrichment cultures, which have shown that they do indeed account for a major component of in-lake nitrification processes. With the exception of the marine archaeon Nitrosopumilus maritimus, most research has been focused on culture-independent methods for the characterization of nitrifying microbes. This study is among the first of these culture dependent studies to describe methods for culturing a freshwater ammonia oxidizing archaea. iv Dedicated to my father who has been an effortless teacher and philosopher and who always encouraged my curiosity for life; to my uncle Ed who has been my lifelong mentor and who taught me one of the key components of being a scientist, whether he knew it or not, how to “improvise”; to my wife and to my mother who were both limitless sources of love and encouragement along this journey; and to my two children who were a constant reminder that nothing good is accomplished without hard work and determination. v ACKNOWLEDGMENTS I would like to thank my advisor Dr. George Bullerjahn for his patience and guidance throughout my research. He has been an amazing source of knowledge that not all graduate students are fortunate enough to get in an advisor. He provided the kind of freedom in his research lab that enabled all of his graduate students to follow their own curiosity. I would also like to thank him for his understanding and support with some of the personal life challenges that came my way during my work at Bowling Green. I would also like to thank Dr. Michael McKay who was always a great sounding board for ideas and who often guided me through problems when my advisor was not available. Dr. McKay has been an invaluable co-advisor on my research journey. It is rare to have one great advisor during ones graduate experience, but in my case I believe I had not one but two. I would also like to thank my committee members Dr. Paul Morris and Dr. Scott Rogers who both brought their unique ways of seeing the world to my studies and to my research at Bowling Green State University. I must also thank my fellow lab members for their help and support and most of all their friendship throughout my PhD journey. Finally I cannot thank my parents enough for the sacrifices they both made for me during this journey. Without their support I could never have achieved this great accomplishment. Thanks to my wife who supported me through thick and thin and who believed in me even when I did not. vi TABLE OF CONTENTS Page CHAPTER I INTRODUCTION ……………………………………………………. 1 1.1A Description of Laurentian Great Lakes as Freshwater ecosystems ……………………………………………. 1 1.1B Lake Superior ………………………………………………………… 2 1.1C The Nitrogen Cycle …………………………………………………… 3 1.1D Nitrate Buildup ………………………………………………………… 4 1.2A Characterization and Diversity of AOB ………………………………. 5 1.2B Culture Characteristics of Nitrosomonas europaea …………………... 6 1.3A Characterization of ammonia oxidizing microbes: Archaea ………….. 7 1.3B Archaeal Cell Membrane Structure …………………………………… 9 1.3C Archaeal Ammonia Oxidation Pathways ……………………………... 10 1.3D Anthropogenic Influences on Lake Superior …………………………. 15 1.4A Identification of AOA and AOB Communities in Lake Superior …….. 15 1.4B Community Diversity of Lake Superior AOAs ………………………. 16 1.4C Characterization of the AOA Culture ………………………………… 17 1.4D Nitrifier Denitrification Processes ……………………………………. 17 CHAPTER 2 MATERIALS AND METHODS …………………………………… 19 2.1A Sample Collection; Water Column …………………………………… 19 2.1B Sample Collection; Sediment …………………………………………. 19 2.2A DNA Extraction from Environmental Samples; Water Column ……...... 22 2.2B DNA Extraction from Environmental Samples; Sediment …………… 22 2.2C DNA Extraction from Plate Cultures …………………………………. 22 vii 2.2D DNA Extraction from Flask Cultures ………………………………… 22 2.3A Amplification of Bacterial and Archaeal amoA Gene Sequences ……… 23 2.3B Amplification of Archaeal 16SrDNA and amoA Gene Sequences from Cultures …………………………………………………………. 24 2.3C Amplification of Archaeal nirK Sequences ……………………………. 24 2.4 Construction of Clone Libraries ……………………………………….. 24 2.5 DNA Sequencing ………………………………………………………. 25 2.6A Culture Establishment …………………………………………………. 25 2.6B Media Development for AOA Culture ………………………………… 26 2.6C Methods for Maintenance of Culture Purity; 15N- NH Assimilaion Assay ………………………………………………………………….₄⁺ 27 2.7A Colorimetric Nitrite Detection Assay ………………………………….. 28 2.7B Colorimetric Ammonium Detection Assay …………………………….. 28 2.7C. Nitrous Oxide Detection using Gas Chromatography …………………. 29 2.7D AOA Culture Negative Control for all N Measurement Assays ………. 30 2.7E Copper Utilization and Dependence using Cyclam ……………………. 30 2.8. Flow Cytometric Analysis ……………………………………………… 31 CHAPTER 3 RESULTS …………………………………………………………….. 32 3.1A Bacterial and Archaeal amoA Detection ……………………………….. 32 3.1B Alternative Archaeal amoA Primers ……………………………………. 33 3.1C Bacterial and Archaeal amoA Detection from Sediment ………………. 34 3.2 Bacterial amoA Community Analysis ………………………………….. 35 3.3 Archaeal amoA Detection in the Water Column ……………………..... 37 3.4A Archaeal nirK-like Sequence Detection from Environment viii and Cultures ……………………………………………………….. 39 3.4B Archaeal nirK-like Phylogenetic Analysis …………………………..... 40 3.5A Culture Establishment ………………………………………………… 42 3.5B Culture Morphology …………………………………………………. 44 3.5C Culture Growth Conditions ………………………………………….. 44 3.5D Viability of AOA and AOB Cultures ………………………………... 45 3.5E Establishment of Cultures Containing a Sole Ammonium Oxidizing Microbe ………………………………………………….. 46 3.5F AOA Culture Characterization ………………………………………. 47 3.6 nirK-like Sequences from Lake Superior ……………………………. 49 3.7 Description of 3 Experimental Groups ………………………………. 51 3.7A Preliminary Growth Assay: Experiment Group 1 …………………… 51 3.7B Nitrification Based on Culture Morphology: Experiment Group 1 …………………………………………………………….. 52 3.7C Preliminary Growth Assay Flow Cytometry ………………………… 54 3.8A Flow Cytometry to Determine Culture Purity: Experiment Group 2 …………………………………………………………….. 55 3.8B Nitrification and Ammonium Depletion: Experiment Group 2 ……..... 58 3.9A Experiment Group 3: Detection of Nitrite, Nitrous Oxide and Ammonium Depletion …………………………………………. 59 3.9B Experiment Group 3: AOA Culture Nitrogen Stoichiometry values ……………………………………………………………….. 60 3.9C Experiment Group 3: Extended ……………………………………… 62 3.10 Natural Ammonium Conversion Measurement ……………………… 63 ix 3.11 Analysis of Copper Availability/Recruitment on Nitrification ……….. 64 CHAPTER 4 DISCUSSION ……………………………………………………….. 66 4.1 Identification of a Novel Freshwater AOA and AOB from Lake Superior ………………………………………………………... 66 4.2 Lake Superior AOA Detection and Phylogeny: amoA sequences ……... 67 4.3 Lake Superior AOA Enrichment Culture ……………………………… 67 4.4A Nitrogen Stoichiometry in AOA Cultures …………………………….. 68 4.4B nirK Activity …………………………………………………………… 69 4.4C Comparing AOA Culture Growth and Nitrification Rates …………….. 69 4.5 Copper Recruitment and Requirements for Oxidative Pathways ………. 70 4.6 The Role of AOA Activity with Regard to the Existing Nitrogen Budget for Lake Superior ……………………………………………… 71 4.7A Further Study …………………………………………………………….. 72 4.7B Examination of Lake Superior AOAs for Mixotrophic/Heterotrophic Growth ………………………………………………………………… 72 4.7C Presence and Activity of nirK-like Sequences and nirK Activities …….. 73 4.7D Continued Culture Work ………………………………………………... 74 4.7E Metagenomic Analysis …………………………………………………... 75 REFERENCES ………………………………………………………………………… 76 - 90 APPENDIX A. …………………………………………………………………………. 91 x LIST OF FIGURES Figure Page 1.0 Plastocyanin-like proteins in AOA ETC 12 2.0 Comparison of AOB/AOA nitrification pathways 14 2.1a Sampling sites throughout Lake Superior 20 3.1a Detection of AOB/AOA amoA sequences from stations WM and CD-1 32 3.1b Detection of AOA amoA sequences from station WM 33 3.1c Detections of archaeal amoA sequences from sediment