Intellectual Merit: The intellectual merit of this activity relates to continuing the search for ancient biomaterials trapped in primary halite crystals of significant geological age. These live organisms, DNA and/or viruses represent the state of evolution during each of the ages represented by the salts. They are also significant study materials for issues of long-term survival of microbial species and the possible movement of microorganisms from planet to planet across space. Recent research has shown that halophilic microorganisms can be isolated from brine inclusions in salt crystals of various ages (Norton and Grant 1988, Vreeland et al. 2000) and that fragments of bacterial and haloarcheal DNA can be recovered from fluid inclusions in ancient halite (Fish et al. 2002, Park et al. 2008 PNAS submitted). These reports are only the latest in a series of announcements made over the last century (Namyslowski 1919, Dombrowski 1961, Tasch 1963, Reiser and Tasch 1960). One study (Vreeland et al. 2000) has received significant publicity because of the extreme sterilization techniques used to avoid contamination by modern microorganisms. The 250-million-year-old Virgibacillus (strain 2-9-3) reported by Vreeland et al. (2000) has profound implications for long-term survival on Earth and for finding life on planets such as Mars. This project will continue to advance these fascinating discoveries. The research will be conducted in two different Permian-aged salt formations in an attempt to duplicate the original NSF funded discovery. Further, the project will gather additional samples from even older formations (Devonian 320 million years of age; Silurian 412 MYA) in an attempt to document sound evidence for even longer term survival in primary halite minerals. Due to the excellent preservation of the sites, vertical samples will also be obtained from an entire evaporation cycle in order to examine population differences that might have occurred as the saline chemistry of the ancient lakes changed over time. In addition, the project will examine these same layers in different horizontal samples of each of the mines in order to develop data regarding the spatial distribution of biological materials in each of the formations. The microbial populations in these various formations will be studied using both culture dependent and independent assays. Consequently, this project will provide data on the types of microbes that are able to survive within the salts while concurrently providing the first systematic DNA-based survey of microbial diversity within the formations and that may have been present during the original precipitation of the salt formations. The project also includes the first examination of these primary salt brines for the presence of bacterial viruses trapped within the ancient brines that formed the fluid inclusions within the crystals.
Broader Impact: The project has many broad areas of impact including the support and training of several research interns, supporting training for several K-12 teachers in a joint educational effort between the PI and the Kansas Underground salt museum, and providing training in geobiology to a current NSF minority Post-Doctoral fellow who has expressed a sincere desire to become involved in the rapidly developing field of geological microbiology with a specific interest in the study of ancient biomaterials. In addition, the research focuses on an area of great public interest as it seeks to break the current record for the oldest live organism known while at the same time attempting to duplicate previous work that isolated the current record, a Permian aged microbe (ca. 250 MYA).
