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

摘要本研究探讨了微生物在地质材料中长期生存的基本问题。虽然Vreeland等人（2000年）表明，生物体可以在岩盐晶体内的流体包裹体中存活250 Ma，但关于地质材料中捕获的微生物数量以及这些生物体存活104至106年的信息很少。对加州死亡谷、智利阿塔卡马盐沼和玻利维亚乌尤尼盐沼的第四纪盐岩进行跨学科研究，将为研究盐矿物流体包裹体中微生物的分布提供丰富的资料。拟议研究的目标是获得关于在地下存在了几十万年的微生物的分布、生存和多样性的数据。在岩盐晶体中的流体包裹体中的微生物的存在或不存在将被解释的背景下，原始的表面环境（盐湖，盐池，含盐地下水）和物理化学条件（温度，主要离子化学）的盐沉淀。从这项研究中获得的知识将有助于改进在地球上和可能的火星上的古代碳酸盐岩中的流体包裹体中寻找微生物生命的工作。将对岩心样品的薄切片进行检查，以确定岩盐沉淀的原始环境。用ESEM-X射线能谱技术对岩盐中的流体包裹体进行化学分析，将记录含有古代微生物的盐水的主要元素化学。盐水包合物的化学知识将指导培养基的制备，以模拟古代盐细菌生活的化学环境。将测量岩心中来自岩盐的流体包裹体的均一化温度，以获得盐沉淀的盐水的实际古温度。孵育温度将设定为接近这些古生物温度。用于微生物工作的岩盐样品将在宾厄姆顿大学（BU）进行岩相学筛选。将切割单个岩盐晶体以分离原生流体包裹体群，并检查离子微粒。（可能的微生物）在可见光和紫外光下。石盐晶体将在BU和西切斯特大学（WCU）进行表面灭菌，并溶解在无菌蒸馏水中。从岩盐晶体中提取的DNA将通过针对16 S rRNA基因部分的PCR测定进行分析，然后通过变性梯度凝胶电泳（DGGE）来表征微生物（BU和WCU）的多样性。将对PCR产物和从DGGE凝胶中切下的单个条带进行克隆和测序，以确定微生物的系统发生亲和力（BU）。WCU实验室将使用培养方法尝试从岩盐晶体中的流体包裹体中分离微生物，并将使用生理学、化学分类学和分子参数对嗜盐和耐盐菌和细菌进行分类学表征。拟议的项目涉及两个机构的地球科学和微生物学跨学科研究，涉及四名PI，由一名博士后研究员，一名博士，一名博士后研究员和一名博士后研究员协助。候选人，和几个付费的本科生。来自BU的研究小组将参观WCU的微生物实验室，以交流小组之间的想法，并接受进一步的微生物实验室方法培训。在BU，地球生物学研讨会强调这一研究项目的结果将每年提供给研究生和本科生。BU集团计划参加为期一周的夏季实地和实验室充实计划，为小学生和组织1-2天的夏季研讨会，为15名当地教师从5-9年级提供培训和课程开发。将为研究人员和K-12学生创建一个关于古老微生物生命的网站，介绍BU和WCU目前的研究。WCU本科生将在研究的各个方面就业。Vreeland博士的微生物生态学课程的材料将来自该项目期间开发的数据。

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.