The proposed work intends to specifically characterize the temperature and pressure as microbial physiological variables and explore quantitatively and qualitatively, the metabolic capacities of single microbial cells in deeply buried habitats. We hypothesize that the microbial physiological responses to ambient temperatures may be used to characterize the nature, in terms of the geographical origin, of the microorganisms present in deeply buried habitats. A high pressure thermal gradient system will be used to study the pressure and temperature relationships of microbial metabolism in basaltic fluids. Pulse-chase incubation experiments using radio- as well as stable isotope labeled substrates will be performed in order to quantify relevant metabolic processes rates under energy limiting conditions and identify potential isotopic effects during specific metabolic steps. In addition, voltammetric measurements will be conducted to potentially quantify real-time changes on manganese, iron and sulfur species of intermediate oxidation state in samples incubated using the high pressure thermal gradient system. Nanometer-scale secondary ion mass spectrometry (NanoSIMS) will also be used in combination with halogen in situ hybridization (HISH-SIMS) for simultaneous quantification of cell-specific rates and phylogenetic identification under different temperature and pressure regimes. We expect that the physiological characterization of microorganisms as a function of temperature and pressure in the basement fluids will help to elucidate dispersal mechanisms that structure microbial diversity.