9714203 Vreeland This proposal is based on the hypothesis that extremely halophilic bacteria and/or macromolecules (biological materials) can be preserved for long periods of time inside of NaCl crystals. This project has three objectives: 1) To sample a variety of ancient geologic salt formations and to clearly document the ages of the samples; 2) To gently extract and study biological materials from the crystals obtained in objective #1; 3) To experimentally examine the stability of various biological materials inside of salt crystals. This research will advance several of the NSF's LExEN program by increasing the scientific understanding of the nature of primitive organisms from extreme environments, the nature of the early extreme environment in which they thrived and the influence of microorganisms on the Earth's early environment. This project research will be conducted under the initial assumption that the best place to find extant biological materials will be inside primary fluid filled inclusions that are at least 200 microns square. At this time, four sample sites have been selected for this project. These include: depositional salts from the modern era 1.3 Million years of age (Ma) or less, a formation which contains Permian salt (ca 250 Ma); a formation containing Devonian age salt (ca. 360-400 Ma); and a fourth site made of Silurian age (430 Ma). The most critical issue for this project is to clearly establish the age and quality of each crystal sample used for the isolation of biological materials. Age dating the individual samples will basically involve collecting data at two levels of effort. These levels are: microfossil analyses of surrounding crystals, which will be performed under the direction of Dr. Dennis Braman at the Royal Tyrrel Museum in Drumheller, Alberta; and chemical analysis of the extracted inclusion brines which will be accomplished under the direction of Dr. Juske Horita at Oak Ridge National Laboratories. The biological sampling will be performed in the laboratory of the project director (Dr. Russell H. Vreeland). The sampling for biological material involves the use of surface sterilized primary crystals, which will be penetrated using aseptic techniques. The inclusions will be sampled using a micromanipulator and sterilized capillary tubes that will allow the sample to be withdrawn without destroying the crystal. This project will provide significant data in the following areas: 1) Demonstrated that biological materials can survive inside crystals for very long periods of time; 2) Developed and tested procedures for isolating ancient, fragile biological materials from mineral inclusions; 3) Provided biological material that can be used to determine the evolutionary state of microorganisms 250 to 430 million years B.P.E; 4) Provided data and sampling methods that can be used in a search for extraterrestrial life on long dead planets (DeVincenzi, 1992, DeVincenzi et al. 1990); 5) Pushed back the envelope for proven survival of intact biological materials by a factor of 10 1.5-2.0 x 107 (Cano and Borucki, 1995) to 2.5-4.3 x 108 years . The project director's laboratory has been actively conducting several relevant experiments in order to prepare for this project. Dr. Vreeland has studied the ecology, taxonomy and physiology of halophilic bacteria for nearly twenty-five years. He has been involved in sampling underground salt formations for the last ten years. He has published twenty-five primary research publications and has one patent dealing with halophilic bacteria.
