Although the subsurface biosphere is now recognized as an important reservoir of life on our planet, until recently the microbial community beneath open-ocean oligotrophic gyres (making up the majority of the seafloor) has not been studied in detail (D’Hondt et al., 2004, 2009). IODP Expedition 329 has taken a first step at characterizing the microbial community beneath the South Pacific Gyre. This region has low biological surface productivity and therefore very low organic carbon burial rates (10-8 and 10-10 moles C cm-1 yr-1), deep oxygen penetration (sediments are oxidized to the basement), and low prokaryotic cell counts (106 cells cm-3 to <103 cells cm-3) (D’Hondt et al., 2009; Fischer et al., 2009, IODP Exp. 329 Preliminary Report, 2011). In these sediments, the dominant fraction of organic carbon may be aggregated or adsorbed to minerals (Arnarson & Kiel 2007). Thus the ability to colonize minerals should be an important ecological adaptation, with those microbes that are able to grow on the minerals creating potential “hotspots” of microbial activity within these oligotrophic sediments. Our project aims to determine whether there is stimulated microbial activity associated in long-term incubations with H13CO3- and 15NO3-. Specific mineral and clay fractions in the oligotrophic South Pacific Gyre sediment system were targeted using combination of magnetic and density separation and SEM-EDS. The bacterial and archaeal community were examined by CARD-FISH, CARD-FISH-nanoSIMS, and 16S rRNA tag sequencing. Overall results from this C-DEBI grant have shown the viability of magnetic separation and identification of single cells in subsurface sediments as a method for investigating mineral association in microbial communities. We have identified putatively viable cells attached to 7 Fe/Mn-rich minerals, potentially representing an unexplored strategy for low-carbon environments. We also have discovered a higher level of diversity in the paramagnetic (Fe/Mn-rich) mineral-associated bacteria and higher number of Marine Group I archaeal OTUs compared to the diamagnetic fraction in the oligotrophic subsurface sediment from the South Pacific Gyre.