Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • BCO-DMO Processing Description
• Parameters
• Deployments
• Project Information
• Funding

Samples were collected from the Northeast Pacific Ocean along the Juan de Fuca ridge flank, off of the continent of North America. CORK observatories are on the seafloor at a water column depth of approximately 2660 meters. CORKs are located in the general vicinity of 47.75 lat, -127.76 lon. Samples were collected during the following R/V Atlantis cruises:

2008 cruise: AT15-35, James Cowen (Chief Sci)
2009 cruise: AT15-51, Andrew Fisher (Chief Sci)
2010 cruise: AT15-66, James Cowen (Chief Sci)
2011 cruise: AT18-07, Andrew Fisher (Chief Sci)
2013 cruise: AT26-03, Andrew Fisher (Chief Sci)
2014 cruise: AT26-18, Geoff Wheat (Chief Sci)

Deep subseafloor basement crustal fluids were collected from boreholes fitted with CORKs (subseafloor borehole observatory systems) along the Juan de Fuca Ridge flank, which are located at a water column depth of approximately 2,650 meters (m) and penetrate through sediment and into the igneous basement. The CORKs fitted to these boreholes feature fluid delivery lines that access fluids at different depth horizons. Custom sampling equipment enabled the pumping of borehole fluids directly through 0.22 micrometer (µm) pore-size polyethersulfone filters or filter cartridges at the seafloor. DNA was extracted directly from the filters using either phenol-chloroform or commercially available kits, as specified in the sample log. Genomic DNA was submitted to the Joint Genome Institute for sequencing.

Sampling used custom-designed equipment: the Medium Volume Bag Sampler (MVBS), Large Volume Bag Sampler (LVBS), and In Situ Passive Viral Filter.

- Imported original file "rappe_deepSS_BCODMO.csv" into the BCO-DMO system.
- Created the Creation_Date column in YYYY-MM-DD format.
- Renamed fields to comply with BCO-DMO naming conventions.
- Saved final file as "945915_v1_sequencing_samples.csv".

NSF Award Abstract:
Our planet's seafloor consists primarily of sediment layered over a basement of basalt rock. Every 50,000 to 100,000 years, a volume of seawater equivalent to the entire global ocean circulates through cracks and fissures of this basement beneath the seafloor, forming one of the largest reservoirs for microscopic life on Earth. While high temperature fluids discharging at iconic hydrothermal vents at mid-ocean ridges are visually striking, the fluid flowing in and out of the flanks of these ridges is around three orders of magnitude greater and rivals the discharge of all rivers to the ocean. As it travels through the deep subseafloor, this fluid is significantly altered by water and rock interaction and the metabolic activity of microorganisms that are thought to ultimately help shape nutrient and energy budgets of the global ocean. However, our knowledge and understanding of this system suffer greatly from logistical difficulties in accessing it for scientific inquiry. Initial evidence suggests uncharacterized microbes that possess ancient homologs of enzymes involved in key metabolic pathways thought to be important to Earth's early microbial inhabitants populate this biome and are infected by novel viruses. In this study, the investigators are performing an integrated set of observations, experiments, and analyses aimed to advance our understanding of deep subseafloor microbes and their viruses by providing new, fundamental insights into which organisms and metabolisms are active in this environment, their evolutionary history and genetic characteristics, and their interactions. This project contributes to the development of a diverse STEM-educated workforce, and incorporate the training of one postdoctoral scientist, two graduate students, and ~29 undergraduate students in field-based research, wet-lab experimentation, and bioinformatics. This project also fosters a unique collaboration between scientists and the University of Hawaii Academy for Creative Media that supports undergraduate interns from the Academy to work with project personnel and produce creative videos and graphics to communicate aspects of our research to diverse audiences. Finally, this project supports two early career female scientists who started faculty positions in 2017.

This project leverages existing sampling infrastructure, DNA sequencing by the Department of Energy Joint Genome Institute, and a research expedition to the Juan de Fuca Ridge (JdFR) flank off the coast of Washington, USA, that is already supported by NSF. Here, subseafloor observatories have been previously installed to aid in exploring actively flowing subseafloor crustal fluids. These fluids will be collected from different sampling depths for an integrated set of geochemical, genomic, and cultivation studies. The project's specific objectives are to (i) use genomics to characterize microbial and viral populations inhabiting crustal fluids of the JdFR flank, (ii) use transcriptomics to identify the active metabolic pathways that are performing transformations relevant to elemental cycling within microorganisms of the JdFR flank, as well as identify active viral infections in these microbes, and (iii) generate microbial and viral pure cultures or limited diversity enrichments from crustal fluids of the JdFR flank. It combines bioinformatic analyses, controlled laboratory experiments, and field sampling to pursue both hypothesis-driven and discovery-based cultivation experiments, viral assays, and strain-level ecogenomic and metatranscriptomic analyses. Importantly, the investigators intend to generate new cultivated microbes and viruses to serve as model systems for investigating the characteristics of life in the deep ocean crust.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.