LEXEN: Paleobiology of Ancient Salt Formations: Examination of Primary Crystals for Biological Materials

这个建议是基于一个假设，即极端嗜盐细菌和/或大分子（生物材料）可以在NaCl晶体中保存很长一段时间。该项目有三个目标：1)对各种古代地质盐层进行取样，并清楚地记录样品的年龄；2)从物镜#1中获得的晶体中轻轻提取和研究生物材料；3)实验考察盐晶体内部各种生物材料的稳定性。这项研究将通过增加对极端环境中原始生物的本质的科学理解，以及它们赖以生存的早期极端环境的本质，以及微生物对地球早期环境的影响，来推进NSF的几个LExEN项目。本项目研究将在最初的假设下进行，即发现现存生物材料的最佳地点将是在至少200平方微米的原始流体填充包裹体内。目前，已经为这个项目选择了四个样本站点。这些包括：现代沉积盐（130万年或更少），一个包含二叠纪盐（约250万年）的地层；含泥盆世盐（约360-400 Ma）的地层；第四处是志留纪时期（430 Ma）。本项目最关键的问题是明确确定用于分离生物材料的每个晶体样品的年龄和质量。对单个样本进行年龄测定基本上包括在两个层面上努力收集数据。这些级别是：周围晶体的微化石分析，将在阿尔伯塔省德拉姆海勒皇家泰瑞尔博物馆的丹尼斯·布拉曼博士的指导下进行；以及提取的包裹体盐水的化学分析，这些将在橡树岭国家实验室的堀田Juske博士的指导下完成。生物取样将在项目主任（Dr. Russell H. Vreeland）的实验室进行。生物材料的取样涉及使用表面灭菌的原生晶体，将使用无菌技术穿透。包裹体将使用显微操作器和灭菌的毛细管取样，这样可以在不破坏晶体的情况下提取样品。该项目将在以下领域提供重要数据：1)证明生物材料可以在晶体中存活很长一段时间；2)开发并测试了从矿物包裹体中分离古代脆弱生物材料的程序；3)提供了可用于确定2.5 ~ 4.3亿年B.P.E微生物进化状态的生物材料；4)提供数据和抽样方法，可用于在长期死亡的行星上寻找外星生命（DeVincenzi, 1992, DeVincenzi et al. 1990）；5)将已证实的完整生物材料的存活时间推后10倍（1.5-2.0 x 107） （Cano和Borucki， 1995）至2.5-4.3 x 108年。项目主任的实验室一直在积极进行几项相关实验，为这个项目做准备。弗里兰博士研究嗜盐细菌的生态学、分类学和生理学已有近25年。在过去的十年里，他一直从事地下盐层的取样工作。他发表了25篇主要研究出版物，并拥有一项涉及嗜盐细菌的专利。

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.