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

摘要本研究旨在探讨微生物在地质物质中长期生存的基本问题。尽管弗里兰等人（2000年）表明，生物可以在岩盐晶体内的流体包裹体中存活250 Ma，但关于被困在地质物质内的微生物数量以及这些生物存活104至106年的信息却很少。对三个盆地（加利福尼亚死亡谷、智利阿塔卡马盐湖和玻利维亚乌尤尼盐湖）第四纪盐岩心的跨学科研究将为研究含盐矿物流体包裹体中微生物的分布提供丰富的资料。这项拟议研究的目标是获得几十万年来地下微生物的分布、生存和多样性的数据。盐岩晶体中流体包裹体中是否存在微生物，将根据盐沉淀的原始地表环境（盐湖、盐田、含盐地下水）和物理化学条件（温度、主要离子化学）来解释。从这项研究中获得的知识将有助于改进对地球上甚至可能是火星上古代蒸发岩中流体包裹体中微生物生命的研究。将检查岩心样品的薄片，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，将记录下孕育古代微生物的盐水的主要元素化学成分。盐水包裹体化学的知识将指导培养基的制备，以模拟古代盐杆菌生活的化学环境。通过测量岩心中岩盐体流体包裹体的均一温度，可以得到析出盐的卤水的实际古温度。孵化温度将设定在接近这些古盐水温度的水平。用于微生物学研究的岩盐样品将在宾厄姆顿大学（BU）进行岩石学筛选。单个岩盐晶体将被切割以分离原生流体包裹体种群，并检查是否有微颗粒。（可能是微生物）在可见光和紫外线下。盐晶体将在波士顿大学和西切斯特大学（WCU）进行表面消毒，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16S rRNA基因部分的PCR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DGGE凝胶的PCR产物和单个条带进行克隆和测序，以确定微生物（BU）的系统发育亲和性。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、化学分类和分子参数对嗜盐和耐盐古生菌和细菌进行分类表征。拟议的项目涉及两个机构在地球科学和微生物学方面的跨学科研究，涉及四名PI，由一名博士后研究员，一名博士候选人和几名付费本科生协助。来自BU的研究小组将访问WCU的微生物实验室，进行小组间的交流，并接受进一步的微生物实验室方法培训。在波士顿大学，每年将为研究生和本科生举办一次突出该研究项目成果的地理生物学研讨会。BU小组计划参加为期一周的小学儿童暑期实地考察和实验室丰富项目，并组织1-2天的暑期讲习班，为当地5-9年级的15名教师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代微生物生命的网站，介绍波士顿大学和威斯康星大学的最新研究。WCU的本科生将参与研究的各个方面。弗里兰博士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.