DIVERSITY AND POTENTIAL GEOCHEMICAL FUNCTIONS OF PROKARYOTES IN HOT SPRINGS OF THE UZON CALDERA, KAMCHATKA by WEIDONG ZHAO (Under the Direction of Chuanlun L Zhang and Christopher S Romanek) ABSTRACT Hot springs are modern analogs of ancient hydrothermal systems where life may have emerged and evolved. Autotrophic microorganisms play a key role in regulating the structure of microbial assemblage and associated biochemical processes in hot springs. This dissertation aims to elucidate the diversity, abundance and ecological functions of multiple groups of chemoautotrophs that use diverse energy sources including CO, NH3, and H2 in terrestrial hot springs of the Uzon Caldera, Kamchatka (Far East Russia). This dissertation consists of seven chapters. Chapter 1 reviews studies in geochemistry and microbiology of studied area. 13 Chapter 2 reports H2, CO2, CH4 and CO contents and the δ C values in vent gas samples. The gases were determined to be thermogenic with small temporal but large spatial variations among the springs investigated. Chemical and partial isotope equilibria between CO2 and CH4 may be attained in the subsurface at elevated temperature. Chapter 3 shows the distribution of bacteria was spatially heterogeneous whereas that of archaea was related to geographic features. Cluster analyses group bacterial and archaeal communities according to their similarities of lipid compositions. Hydrogen-utilizing Aquificales appeared to be dominant in two of the four bacterial groups, but was outnumbered by presumably Cyanobacteria-Thermotogae or Proteobacteria-Desulfurobacterium types in the other two groups. The lipid data also suggest the existence of possibly three types of archaea with each type producing one of GDGT-0, GDGT-1, and GDGT-4 as the main membrane lipids, respectively. Chapters 4 and 5 describe a novel aerobic, thermophilic and alkalitolerant bacterium Caldalkalibacillus uzonensis, which tolerated >90% headspace CO but cannot utilize CO as carbon and energy sources. Chapter 6 focuses on the study of carbon isotope fractionation associated with carbon fixation by an anaerobic CO oxidizing hydrogenogen Carboxydothermus hydrogenoformans. It reveals that a feedback 13 system tends to hold the δ C value constant among CO, CO2 and biomass. A steady state point and associated carbon isotope fractionations (εA-B = δA-δB) were hypothesized to be 18‰ for the CO2-CO system, 2‰ for the bulk biomass-CO2 system, and -20‰ for the CO-biomass system. Chapter 7 reports the diversity and distribution of ammonia-oxidizing archaea (AOA) in hot springs of the Uzon Caldera. A patchy presence of AOA and the absence of AOB (ammonia-oxidizing bacteria) were observed through PCR based surveys. AOA diversity correlated to abundances of archaea or crenarchaeota but was not directly correlated to temperature or chemical variables. INDEX WORDS: Kamchatka, hot springs, PLFA, GDGT, hydrogen-oxidizing bacterium, Aquificales, CO-oxidizing bacterium, ammonia-oxidizing archaea, AOA, CO2 fixation, carbon stable isotopes, isotope fractionation, pathways, Carboxydothermus hydrogenoformans, Caldalkalibacillus uzonensis DIVERSITY AND POTENTIAL GEOCHEMICAL FUNCTIONS OF PROKARYOTES IN HOT SPRINGS OF THE UZON CALDERA, KAMCHATKA by WEIDONG ZHAO B.S. Peking University, P. R. China, 1997 M.S. The Institute of Oceanology, Chinese Academy of Sciences, P. R. China, 2000 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2008 © 2008 Weidong Zhao All Rights Reserved DIVERSITY AND POTENTIAL ECOLOGICAL FUNCTIONS OF PROKARYOTES IN HOT SPRINGS OF THE UZON CALDERA, KAMCHATKA by WEIDONG ZHAO Major Professors: Chuanlun L Zhang Christopher S Romanek Committee: Juergen Wiegel Gary Mills Ming-Yi Sun Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2008 DEDICATION This work is dedicated to my wife Xiaozhen, who has been a constant source of unwavering love, concern, support and strength through all these years. iv ACKNOWLEDGEMENTS I would like to thank my major advisors Dr. Chuanlun Zhang and Dr. Christopher Romanek for the guidance and instilling in me the qualities of being a good scientist. Their insightful comments and constructive criticisms at different stages of my research have been the major driving force to a high standard research. A special thank goes to my committee member Dr. Juergen Wiegel. He has been an excellent mentor who taught me many concepts and techniques of microbiology. It has been a wonderful experience working in his lab during the first half term of my graduate studies. I would like to thank my other committee members Dr. Gary Mills and Dr. Ming-Yi Sun for supporting my research by providing timely advices and access to instrumentation available in their labs. I am indebted to many US and Russian colleagues. I thank Dr. Ann Pearson and Yundan Pi (Harvard University) for helping with the archaeal lipid analysis, Dr. Tatyana Sokolova and Dr. Elizaveta Osmolovskaya of Institute of Microbiology, Russia, and Dr. Frank Robb and Steve Techtmann of University of Maryland for providing me strains of bacteria. I thank Dr. Douglas Crowe (Geology, UGA) for providing me the site maps, Dr. Mary Ann Moran for allowing me to use her lab space in one of my projects and helps throughout my graduate study, and Dr. Paul Schroeder (Geology, UGA) for sharing the sampling logs. Special thanks are due to all people who participated in the field expeditions to Uzon Caldera, Kamchatka from 2003 to 2006, which made available the samples for my dissertation. I thank Qi (Ellen) Ye, Zhiyong Huang, Yiliang Li, Heather Brant, Julie Fiser, Noelle Garvin, Morris Jones, Yong-Jing Lee, Isaac Wagner, Noha Mesbah and Elizabeth Burgess for their supports and friendships. Finally, I appreciate the financial support from the National Science Foundation and the Department of Energy of the United States. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.......................................................................................................................... v LIST OF TABLES......................................................................................................................................vii LIST OF FIGURES ..................................................................................................................................... ix INTRODUCTION ........................................................................................................................................ 1 CHAPTER 1. LITERATURE REVIEW: GEOCHEMISTRY AND MICROBIOLOGY OF HOT SPRINGS IN KAMCHATKA, RUSSIA .....................................................................................................3 2. GEOCHEMISTRY OF REDUCED GASES AND CARBON DIOXIDE IN KAMCHATKA HOT SPRINGS.........................................................................................................................20 3. LIPIDS AND STABLE CARBON ISOTOPES IN HOT SPRINGS OF THE UZON CALDERA, KAMCHATKA: IMPLICATIONS FOR MICROBIAL COMMUNITY DYNAMICS .............................................................................................................................38 4. THERMALKALIBACILLUS UZONENSIS GEN. NOV. SP. NOV., A NOVEL AEROBIC ALKALITOLERANT THERMOPHILIC BACTERIUM ISOLATED FROM A HOT SPRING IN THE UZON CALDERA, KAMCHATKA...........................................................86 5. CALDALKALIBACILLUS UZONENSIS SP. NOV., AND EMENDED DESCRIPTION OF THE GENUS CALDALKALIBACILLUS................................................................................109 6. STABLE CARBON ISOTOPE FRACTIONATION ASSOCIATED WITH CARBON FIXATION BY CHEMOLITHOAUTOTROPHIC ANAEROBIC CO-OXIDIZING BACTERIUM CARBOXYDOTHERMUS HYDROGENOFORMANS ...................................116 7. AMMONIA-OXIDIZING ARCHAEA IN KAMCHATKA HOT SPRINGS........................138 CONCLUSIONS....................................................................................................................................... 171 vi LIST OF TABLES Page Table 1.1. Gas composition, surface water temperature and dominant minerals of hot springs in Kamchatka ................................................................................................................................11 Table 1.2. Thermophilic bacteria isolated from Kamchatka hot springs ....................................................12 Table 1.3. Archaea isolated from Kamchatka hot springs. .........................................................................13 Table 2.1. Gas composition in vent gas samples collected in 2005 and 2006 ............................................27 Table 2.2. Carbon isotope values of gas samples collected in 2005 and 2006 ...........................................28 Table 3.1. Sample identification and description........................................................................................58 Table 3.2. Mole% of 13 major phospholipid fatty acids from Kamchatka samples ...................................59 Table 3.3. Results of SIMPER analysis showing similarity of PLFA between samples in each group identified by the cluster analysis ...............................................................................................60 Table 3.4. Results of SIMPER analysis showing primary contributors (cumulative contribution >90%) to dissimilarities between groups ..................................................................................................61