Collaborative Research: Preservation and Long-Term Bacterial Survival in Quaternary Age Salt from Death Valley, Chile and Bolivia

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AbstractThe proposed research explores the fundamental problem of long-term survival of microorganisms in geological materials. Although Vreeland et al. (2000) showed that an organism can survive in a fluid inclusion within a halite crystal for 250 Ma, there is little information about the number of microorganisms trapped inside geological materials and the survival of those organisms for periods of 104 to 106 years. The proposed interdisciplinary research on Quaternary age salt cores from three basins (Death Valley, California, Salar de Atacama, Chile, and Salar de Uyuni, Bolivia) will provide a rich archive for study of the distribution of microorganisms in fluid inclusions in saline minerals. The goal of the proposed research is to obtain data on the distribution, survival, and diversity of microorganisms that have been in the subsurface for periods of several hundred thousand years. The presence or absence of microorganisms in fluid inclusions in halite crystals will be interpreted in the context of the original surface environments (saline lakes, saline pans, saline groundwaters) and physico-chemical conditions (temperatures, major ion chemistries) in which the salts precipitated. The knowledge gained from this study will help refine the search for microbial life in fluid inclusions in ancient evaporites on Earth and possibly Mars.Thin sections of core samples will be examined to determine the original environments in which halite precipitated. Chemical analyses of fluid inclusions in halite by the ESEM-X-ray EDS technique will document the major element chemistry of the brines that harbored ancient microbes. Knowledge of brine inclusion chemistries will guide preparation of culture media to mimic the chemical environment in which the ancient halobacteria lived. Homogenization temperatures of fluid inclusions from halites in the cores will be measured to obtain the actual paleotemperatures of the brines from which the salt precipitated. Incubation temperatures will be set close to these paleobrine temperatures. Samples of halite for microbiological work will be petrographically screened at Binghamton University (BU). Individual halite crystals will be cleaved to isolate primary fluid inclusion populations and inspected for iomicroparticlesl. (possible microorganisms) under visible and ultraviolet light. Halite crystals will be surface sterilized at BU and West Chester University (WCU) and dissolved in sterile distilled water. DNA extracted from halite crystals will be analyzed by PCR assays targeting portions of 16S rRNA genes, followed by denaturing gradient gel electrophoresis (DGGE) to characterize the diversity of microorganisms (BU and WCU). Cloning and sequencing of PCR products and individual bands excised from DGGE gels will be done to establish the phylogenetic affinities of the microorganisms (BU). The WCU lab will use culturing methods to attempt to isolate microbes from fluid inclusions in halite crystals and will perform taxonomic characterization of halophilic and halotolerant Archaea and Bacteria, using physiological, chemotaxonomic and molecular parameters.The proposed project involves interdisciplinary research in geoscience and microbiology at two institutions, involving four PI's, aided by one post-doctoral researcher, a Ph.D. candidate, and several paid undergraduate students. The research group from BU will visit the microbiology labs at WCU to exchange ideas between groups and receive further training in microbiological lab methods. At BU, a geobiology seminar highlighting the results of this research project will be offered each year to graduate and undergraduate students. The BU group plans participation in a weeklong summer field and laboratory enrichment program for elementary school children and organized 1-2 day summer workshops to provide training and curriculum development for 15 local teachers from grades 5-9. A website for researchers and K-12 students will be created on ancient microbial life featuring current research at BU and WCU. WCU undergraduates will be employed in all aspects of the research. Material for Dr. Vreeland's Microbial Ecology course will come from data developed during this project.