INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. ProQuest Information and Learning 300 North Zeeb Road. Ann Arbor, Ml 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DETECTION, RECOVERY, ISOLATION AND CHARACTERIZATION OF BACTERIA IN GLACIAL ICE AND LAKE VOSTOK ACCRETION ICE DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brent C. Christner, M.S. The Ohio State University 2002 Dissertation Committee: Dr. John N. Reeve, Adviser Dr. Ellen Mosley-Thompson Approved by Dr. Lonnie G. Thompson Dr. Olli H. Tuovinen Dr. Charles J. Daniels A d v i s e r Department of Microbiology Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number 3039458 UMI_ __ (fi) UMI Microform 3039458 Copyright 2002 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT An extraction system has been constructed that melts ice from the interior of ice cores and collects the resulting melt water aseptically. Using this system, bacteria entrapped in modem and ancient glacial ice from worldwide locations and in an ice core extending into accreted Lake Vostok ice have been isolated using enrichment culture and identified by amplification and sequencing of DNA-encoding 16S rRNA genes. In general, ice cores from non-polar locations contained larger numbers and species of cultivable bacteria than samples from polar ices, presumably due to the closer proximity of terrestrial biological ecosystems and exposed landscape. When compared with other polar locations, higher numbers of isolates were obtained from ices adjacent to the Dry Valley complex of Antarctica, consistent with the influx of airborne biological particles from local environments serving as the primary factor controlling the numbers of microorganisms present. The numbers of recoverable bacteria did not correlate directly with the age of the ice, and isolates ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were recovered from the oldest samples examined (>500K years old) . The 16S rDNA sequences from bacterial isolates and amplicons obtained directly from, samples position within 6 different bacterial lines of descent (a— , (3—, and y-proteobacteria, high and low G+C gram positive bacteria, a n d the Cytophaga/Flavobacterium/Bacteroides) . Some of the isolated bacteria have close phylogenetic relationships with species originating from permanently cold environments, and other ice core sites or different portions (time periods) of the same core. Macromolecular synthesis was demonstrated in bacteria frozen under conditions analogous to those in glacial ice, and the possibility exists that metabolic activity and repair may occur during extended periods of glacial entrapment. Several of the species identified in Lake Vostok accretion ice are also related to glacial isolates and species from other cold environments. These ice core studies have provided a glimpse of the microorganisms likely to inhabitant this potentially unique subsurface ecosystem. Investigating microbial survival in ice and exploring potential habitats for activity within the glacial and subglacial environment has confirmed that these could have served as refuge environments for life during periods of iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. global glaciation (Snowball Earth) , and has provided data for extrapolations to the likelihood of microorganisms surviving frozen in extraterrestrial habitats or during interplanetary transport. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS This study was made possible by financial support from the National Science Foundation and the scientific guidance, time and resources supplied by my advisers, John Reeve, Ellen Mosley-Thompson and Lonnie Thompson. I feel very fortunate to have worked amongst this fine group. Their scientific dedication and encouragement have served as a constant source of motivation. I am also grateful to my thesis committee members Olli Tuovinen and Charles Daniels, who provided an interesting subject for my general exam, and have offered useful criticisms and comments on project design. Many thanks are also due to Victor Zagorodnov, who designed and constructed the automated ice core sampler. I am indebted to the knowledge and friendship of past and present members of the Reeve laboratory, including Kathryn Bailey, Trevor Darcy, Brian Hanzelka, Wen-tyng Li, Frederic Marc, Rod Morgan, Suzette Pereira, Kathleen v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sandman, Rachel Samson, Divya Soares, Mark Xie, and Li Yu, a former technician on this project. I would also like to thank Andrea Wolfe, Laurie Achenbach, Tom Schmidt, and Joel Klappenbach for assistance with phylogenetic analysis, Ahmed Yousef for help with the disinfection procedures, Scott Rogers for recommendations on PCR amplification from low biomass material, Wade Jeffrey for advice on macromolecular synthesis, and Dorota Porazinska and Allison Murray for obtaining ice core samples from the Canada Glacier. I am also appreciative of funding supplied by NSF to participate in the Antarctic Biology Course (2001, McMurdo Station, Antarctica) and by OSU to participate in the 1999 Woods Hole Microbial Diversity course. vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VITA July 31, 1970 ..................... B o m - Mt.Pleasant, Pennsylvania 1988-1992 ......................... B.S. Molecular Biology, Westminster College, New Wilmington, Pennsylvania 1992-1993 ......................... Research Technician, Eye and Ear Institute (UPMC) , Pittsburgh, Pennsylvania 1993-1996 ......................... M.S. Microbiology, University of Dayton, Ohio 1997-present ...................... Graduate Student, Department of Microbiology, The Ohio State University PUBLICATIONS Research Publications 1. Christner, B.C., E. Mosley-Thompson, L.G. Thompson. V. Zagorodnov, K. Sandman, and J.N. Reeve. 2000. Recovery and identification of viable bacteria immured in glacial ice. Icarus 144:479-485. 2. Christner, B.C., E. Mosley-Thompson, L.G. Thompson, and J.N. Reeve. 2001. Isolation of bacteria and 16S rDNAs from Lake Vostok accretion ice. Environ. Microbiol. 3:570- 577. FIELDS OF STUDY Major Field: Microbiology vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE 07 CONTENTS Page A b s t r a c t ...............................................ii Acknowledgments ........................................ v V i t a .................................................. vii List of Tables ......................................... x List of Figures ....................................... xi C h a p t e r s : 1. General introduction ................................ 1 Glacial ice, paleoclimatology, and the cold biosphere ................................ 1 Microorganisms immured in glacial i c e .......... 10 Astrobiology implications of microbiological investigations of terrestrial glacial i c e ......................... 24 Objectives of this study ........................ 35 2. Procedures used to prevent contamination during sampling and analysis ..............................37 Introduction ..................................... 37 Materials and methods ........................... 38 R e s u l t s .......................................... 52 Discussion ....................................... 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3. Isolation and characterization of bacteria and 16S rDNA