Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Sediments samples dedicated to nutrient analyses recovered from 8 drilling sites from up to 427 meter below seafloor. Two ~40ml samples of sediment were placed in 50 ml Falcon tubes and were centrifuged at 3000 rpm for 15 minutes to separate porewater from the sediment. The porewater was collected into vials for dissolved inorganic carbon (DIC), dissolved organic carbon (DOC), NOx, and total nitrogen (TN). Samples were stored at 4oC.

Colorimetric determinations of ammonium, nitrate, were performed using a OI Analytical Flow Solutions IV auto analyzer. Total nitrogen and carbon (weight %) are measured with a Costech 1040 CHNOS Elemental Combustion system. Filtered porewater samples were analyzed for dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) using a Shimadzu TOC/TN analyzer. Dissolved organic nitrogen (DON) was calculated by subtracting TDN from the sum of the inorganic nitrogen species.   

All nutrient analyses were performed at Wetland Biogeochemistry Analytical Services, LSU, LA.

NSF Award Abstract:
The marine subsurface is one of the least explored habitats on Earth. International Ocean Discovery Program (IODP) Expedition 385 drilled into the seafloor in Guaymas Basin, Mexico, and was the first to drill directly into subsurface sediments and sediment-hosted basalt sill intrusions of an active hydrothermal basin. This expedition provides a direct microbiological window into a deep hydrothermal biosphere across an active plate spreading center where complex hydrocarbons are generated by heating of buried organic matter under high temperature and pressure. Mounting evidence suggests that Fungi constitute an active and ecologically important fraction of the subsurface biosphere community. This is especially true in organic-rich continental margin sediments that are ideal for colonization by aerobic and anaerobic Fungi, where fungal activities may contribute significantly to nutrient cycling. Major knowledge gaps in our knowledge of subsurface microbiota preclude our ability to estimate their full impact: how active Fungi are distributed along temperature and depth gradients, the range of substrates utilized by active cells, and how Fungi may cooperate with bacteria in degradation of complex organic matter, including hydrocarbons. Fungi are known to participate in degradation of refractory organics and cycling of metals and to produce novel metabolites with interesting properties. This project informs us on origins of different lineages of microbial life on Earth, the extent of marine subsurface carbon cycling, limits of life, how life adapts to environmental change, and the potential for Fungi to accelerate the biodegradation of complex hydrocarbons. Given the extent of the potential subsurface biosphere, Fungi likely play an important role in global nutrient cycling. The culture collection of fungal isolates created by this project will be available for exploration of their ecology and novel properties by other interested researchers, and may also yield insights into basal fungal lineages. These biogeochemical and potential evolutionary outcomes are of great interest to other research disciplines, educators, and students alike. The project’s K-16 education program capitalizes on programs aimed at increasing involvement of under-represented undergraduate populations in research. High school students, undergraduates, a graduate student, and postdoc are involved in the research. An art-in-science project with a local high school is being displayed at the community library along with education materials on marine Fungi and their ecological roles.

This project examines how abundance, diversity and distribution of Fungi and co-inhabiting bacteria and archaea changes in subsurface sediment samples exhibiting a wide range of in situ temperatures and pressure, what the active fraction of cells is along these gradients, and whether/how Fungi impact carbon cycling in this biosphere by interacting metabolically with bacteria to break down hydrocarbon substrates. The project is assessing the activities of in situ microorganisms in this active hydrothermal subsurface biosphere using a cutting-edge combination of molecular approaches and culture-based studies of enrichments and microbial isolates applied to an extensive collection of samples from 8 sites in Guaymas Basin varying in temperature profile, presence of old, buried magmatic sills, and geochemical conditions. The investigators are examining 1) marker genes and metagenomes of sorted active cells using new bioorthogonal non-canonical amino acid tagging (BONCAT) approaches, 2) the distribution of bacterial, archaeal, and fungal cells and their marker genes along depth and geochemical gradients using microscopy, ‘meta-omics’ and lipid biomarker analyses, 3) substrate usage by fungal isolates, 4) metabolite pools, nutrients, and hydrocarbons with depth, and 5) fungal metabolism of complex organics (and syntrophies between Fungi and Bacteria) using time-course stable isotope probing of RNA from culture-based studies coupled with analyses of expressed genes and pools of metabolites.

Broader Impacts:  The proposed project can transform our understanding of microbial life in the sedimented marine subsurface biosphere because active mycobiota would have implications for deep carbon budgets. Our culture collection is estimated to generate hundreds of new strains of fungi that will be available for exploration of their ecology and novel properties by interested researchers, and may also yield insights into basal fungal lineages. These biogeochemical and potential evolutionary outcomes are of great interest to other research disciplines, educators, and students alike. Our proposed K-16 education program capitalizes on programs aimed at increasing involvement of under-represented undergraduate populations in research. High school students, undergraduates (4 per year), a graduate student, and postdoc will be involved. The PI and an art teacher at a local high school will teach an art-in-science unit. The product (a large quilt of art inspired by fungal cultures) will be displayed at the community library along with education materials on marine fungi and their ecological roles.  The project involves two international collaborators, and will partially support 4 principal investigators at three institutions, including two early career researchers, Roland Hatzenpichler and Paraskevi Mara.