The sediments within South Pacific Gyre (SPG) represent the most biologically inactive sediments on the planet, despite dissolved oxygen, nitrate, phosphate and inorganic carbon being present throughout the entire sediment column. Detection and characterization of lineages associated with potential cryptic biogeochemical cycles (e.g., a microbial process that is not reflected in the geochemical signature due to populations utilizing the metabolic products from another population in the reverse redox reaction) within the subsurface biosphere may indicate that the SPG is more metabolically active than previously predicted through geochemical assessments. Presence of anaerobic respiring populations within aerobic, energy limited SPG sediments would expand our understanding of activity in the deep biosphere, extent and limits of life, and the evolution and survival of life. The overall objective of this proposal was to use culture independent and dependent techniques to provide the more complete assessment of the SPG subsurface microbial ecology. The central hypothesis was that anaerobic populations have remained viable and potentially metabolically active within these sediments despite the low energy availability and presence of oxygen. This project directly addressed the four fundamental research themes identified by C-DEBI. This work was also central to IODP’s mission in the deep subsurface: what is the diversity of microorganisms in this environment; and what are the strategies by which living systems obtain and use energy. Results provide the first highly detailed molecular characterization of the metabolically active fungal community structure and function within the extreme energy limited subsurface environments at the South Pacific Gyre. Molecular techniques, including qPCR, supported the culture based isolation of multiple fungal lineages distinctly unique from previously isolated fungal species as determined by functional gene characterization. These lineages will not only expand the potential for eukaryotic microbial population habitability zones, but also represents a significant potential for biotechnological advancement in the area of bioprospecting as these lineages are related to Penicilium. Results from this work have been reported at the post-cruise meeting, AGU, workshops in Brazil and Italy and multiple invited lectures within the US presented by both Mills and Reese.