NER: Molecular-To-Microscale Investigation Of Microbial Habitats: Exploratory Studies Using Molecular Probes and Microcomputed Tomography and Fluorescence

O‘DayNER-0210819本提案是对NSF 01-157类纳米科学与工程倡议的响应，由地球观测组织和生物理事会共同资助。将微生物和地球化学系统的研究结合起来的一个重大挑战是在其自然环境中以适当的长度和时间尺度可视化、识别和测量化学反应和反馈。这一建议旨在探索一种新的分子生物学探针和同步辐射X射线微型计算机断层扫描(m-CT)和荧光的组合，以获得关于自然系统中物理和化学微生境以及微生物在二维和三维分布的补充信息。微生物群落存在于孔隙空间和矿物表面，其长度尺度与单个细菌和细菌群落(约1-100毫米)相对应。微生物与水溶液、胶体和矿物的化学反应是在分子水平上进行的，但微生物群落的相互作用体积可能在~5-1000mm3或更多。传统的检查技术，如电子或荧光显微镜，需要破坏性的样品制备方法，这种方法可能会严重改变生物和物理结构。这项探索性研究的目的是探索一种在原位和三维上检查微生物群落分布的方法，并开发耦合元素特定光谱探针和分子生物学探针的互补技术。我们建议使用针对rRNA靶标的核酸生物探针，并用重元素(碘、金和银)标记，使用密度对比度同步加速器m-CT绘制天然多孔材料中微生物群落的空间分布。此外，我们将研究荧光m-CT测量使用这些目标元素和矿物底物的元素绘制特定元素在二维分布的能力。在密度对比和荧光填图的基础上，探索微生物群落和矿物底物的空间分布，并尝试特定元素的XANES光谱，以获取化学信息。二维和三维的物理和化学数据的耦合，信息量从分子到微米级，将使未来对涉及地质流体、矿物和微生物之间的元素循环的基本过程的创新研究成为可能。

O'DayNER- 0210819This proposal was received in response to the Nanoscale Science and Engineering initiative NSF 01-157, category NER, and is co-funded by the GEO and BIO Directorates. A significant challenge to integrating the study of microbiological and geochemical systems lies in visualizing, identifying, and measuring chemical reactions and feedbacks at appropriate length and time scales within their natural environments. This proposal seeks to explore a novel combination of molecular biological probes and synchrotron X-ray micro-computed tomography (m-CT) and fluorescence to obtain complementary information about physical and chemical microhabitats and microbial distribution in natural systems in two and three dimensions. Microbial communities are found in pore spaces and on mineral surfaces at length scales corresponding to individual bacteria and bacterial communities (~1-100 mm). Chemical reactions involving microorganisms and aqueous solutions, colloids, and minerals take place at the molecular level, but the interaction volumes of microbial communities may range from ~5-1000 mm3 or more. Conventional examination techniques such as electron or fluorescence microscopy require destructive sample preparation methods that can severely alter biological and physical structures. The aim of this exploratory study is to investigate a method for examining the distribution of microbial communities in situ and in three dimensions, and to develop complementary techniques for coupling element-specific spectroscopic probes and molecular biological probes. We propose to use nucleic acid biological probes directed against rRNA targets and tagged with heavy elements (iodine, gold, and silver) to map the spatial distribution of microbial communities within natural porous material using density-contrast synchrotron m-CT. In addition, we will investigate the ability of fluorescence m-CT measurements to map specific element distribution in two dimensions using these target elements and those of the mineral substrates. Based on density contrast and fluorescence mapping, we will explore the spatial distribution of microbial communities and mineral substrates, and attempt XANES spectroscopy of specific elements to gain chemical information. The coupling of physical and chemical data in two and three dimensions, with information content ranging from molecular to micron scale, will enable innovative future studies of fundamental processes involving element cycling among geologic fluids, minerals, and microbes.