Award: OCE-1658031

The lower oceanic crust is one of the last biological frontiers for exploration of life on Earth. Samples of lower oceanic crustal rocks were obtained during IODP Expedition 360 at Atlantis Bank from Hole U1473A down to ~800 meters below the seafloor (mbsf) (Figure 1). Analyses of enzyme activities, marker genes, lipid biomarkers, and microscopy counts of cells revealed heterogeneously-distributed, ultra-low cell densities (<2000 cells/cm3), some fraction of which are active. Messenger RNA recovered from these rocks provides evidence for heterotrophic activities of bacteria, archaea and fungi, including use of polyhydroxyalkanoates as carbon storage molecules (likely under long periods of austerity), degradation of polyaromatic hydrocarbons, and recycling of different amino acids for use in redox reactions, energy production and storage. These findings all provide insights into how microorganisms are able to survive in such an inhospitable environment where sources of carbon and energy are limited. Active carbon cycling in this extensive realm is likely unevenly distributed and occurs at extremely slow rates, Nonetheless, subsurface fluid circulation connects activities in the lower crust with the deep ocean, so detected activities have implications for carbon and other nutrient cycling in the deep sea. The results of this study extend what we know as the habitable biosphere on Earth. They also have implications for our understanding of the evolution of life on Earth. Since this realm provides an analog environment for certain extraterrestrial environments, our results also provide possible biomarkers to use in future searches for life elsewhere. Analyses of lipid biomarkers reveal little accumulation of fossil material in these samples, archaeal intact polar lipids that are distinct from those observed in typical deep biosphere settings, and the presence of bacterial diether glycerol lipids. Raman spectroscopy of a sample from 182 mbsf shows filamentous inclusions of organic compounds consistent with those found in microbial cells, inter-grown with the calcite host rock, evidence of their in situ origin. Marker genes reveal low diversity microbial communities including heterotrophs and chemoautotrophic taxa, many described from dee-sea and polyextreme habitats. Significant correlations were found between detection of biomarkers/cells and the presence of veins and fractures in the host rock. Local faulting and fractures in the rocks provide conduits for the circulation of fluids through the crust at this site, providing sources of carbon and energy to sustain life, and likely cells entrained in those fluids from the seafloor. In addition to recovery of expressed genes from microbial fungi, further evidence for viable (and some fraction active) fungi comes from a collection of fungal isolates cultured from these samples, most of which are closely-related to ubiquitous terrestrial and deep-sea taxa. This project provided opportunities for participation and training of twelve individuals, including undergraduate and graduate students, postdocs, and two visiting sabbatical professors. Talks at (inter)national meetings and posters were delivered by team members, and an education and outreach program involving primary school students from under-represented communities in Texas was conducted. The outreach program provided an immersive full-day experience for these students in coastal oceanography, hands-on experience with basic oceanography sampling gear on a coastal research ship, and classroom/school visits to engage students in our research. Last Modified: 03/22/2021 Submitted by: Virginia P Edgcomb