Resolving the apparent energy paradox for marine deep biosphere life how life exists on very limited amounts of energy is a grand challenge within the research community, as it is important to know for understanding how life evolved on Earth and if life might exist elsewhere in the Universe. Prior NSF and international funding supported the establishment of a subseafloor observatory system in the Pacific Northwest, on the flank of the Juan de Fuca Ridge, that our team thought would be an ideal goldilocks site to address this research challenge. That is because chemical and physical conditions at this site support some of the lowest energy ecosystems on Earth, and it has infrastructure to access these systems for experimentation and sampling. We proposed a series of high risk-high reward experiments to conduct on a research expedition with a remotely operated vehicle to measure the activity of individual microorganisms that exist in this habitat. These experiments were designed to understand how active the microbes are, and if they are all equally active or if only some members of the community are active. If successful, these would be among the first measurements of the activity of individual microbes in these low energy habitats. Despite setbacks from COVID and expedition delays, our team successfully conducted these experiments during a research expedition in 2023. Initial data analysis reveals that we can, in fact, measure the activity of individual microbes from this low-energy environment satisfying the major objective of this project. Meeting this objective was enabled by a new technological approach optimized in partnership with collaborators via parallel NSF funding (award OIA-1826734). After these data are quality checked, they will be made publicly available at this data repository (https://www.bco-dmo.org/project/700324). Proof of ability to measure individual microbial cell activity in low-biomass systems has application for a wide range of fields, from the search for life on other planets to biomedical research on rare and/or slow-growing but pathogenic microbes. During COVID interruptions, our team leveraged existing genomic datasets to reveal novel findings about life in the subsurface, such as evidence that the subsurface of the ocean crust is a reservoir for life that retains signatures of early evolutionary conditions and unique adaptations to thrive in the subsurface under low energy. This project also included several components to contribute to STEM education, STEM workforce, and inclusion broader impacts through training and professional development. Two postdoctoral scientists involved in the project gained training in conducting field research and new laboratory and bioinformatic skills, which they have leveraged to secure new positions in the STEM workforce. Multiple early career researchers, from students to technical staff to postdocs to early career faculty gained exposure to deep-sea research and how to approach research in a collaborative and inclusive manner. At the same time, the project also contributed to enhancing infrastructure for research by training a new generation of researchers in the use of subseafloor observatories. Some trainees also received detailed instruction in how to lead collaborative international deep-sea expeditions that involve stakeholder participation, which has already resulted in one of them being a co-chief scientist of an international expedition (with participants from Canada) and others submitting proposals to lead future research. Scientific staff received career promotions as a result of the professional growth and development resulting from involvement in this project, supporting retention in the STEM workforce. Several student trainees gained mentoring and skills during the project which they leveraged to secure positions in the STEM workforce and/or graduate school. Finally, the project supported building partnerships broader impacts through engagement with First Nations participants in field research and co-production of resources for community outreach about the value of deep-sea research. Last Modified: 04/30/2024 Submitted by: BethNOrcutt