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

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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.