Table of Contents

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

Supplementary Table 3B: Overview of archaeal and bacterial lipid biomarkers and cell counts. Replicate cell counts for the 11 samples and alkaline phosphatase activity measurements available for any of the 11 samples. Samples were taken on board of the JOIDES Resolution  between November 30, 2015 and January 30, 2016 in the SW Indian Ridge.

Alkaline phosphatase (AP) activity was measured using the fluorogenic substrate 4-methylumbelliferyl phosphate (MUF-P) (Sigma-Aldrich, St. Louis, MO) and its reference standard, methylumbelliferone (MUF). Fluorescence was measured using black, flat bottom, 96-well microplates in a Spark 10M Multimode Microplate Reader (Tecan, Männedorf, Switzerland). Fluorescence of MUF is greatest at pH 10, therefore 25 μL of 0.4 M NaOH was added to the wells (final concentration 40 mM) to be read. 25 μL of 1M EDTA was added as well (100 mM final concentration) to prevent precipitation of c 439 arbonate from sampled veins.

Fluorescence was measured with an excitation wavelength of 380 nm and emission of 454 nm for all substrates and standards. One cm3 powdered rock was mixed with 5 cm3 of sterileartificial seawater (ASW) in a 8 mL serum vial with 90:5:5 N2:CO2:H2 headspace. 700 μL of each slurry was withdrawn with a sterile syringe to a 1.5 mL Eppendorf tube after setup but before sealing the vial; this sample served as T0, with triplicate 200 μL technical replicates. These 700 μL samples were briefly centrifuged (60 sec. at 2500 rpm) and the supernatant used for the T0 analyses.

Two additional samples were taken using the same methods as for T0 after at least 2 weeks and then again after 4-6 weeks to generate a slope of activity. Incubations were kept at 10˚C, the inferred in situ temperature, for the duration of each assay. Autoclaved, powderized rock from each of the samples was tested to determine the amount of fluorophore adsorbance to rock powder.

Adsorbance was found to behave in a systematic manner, resulting in a straight line when comparing fluorescence standards in artificial seawater (ASW) alone with fluorescence standards plus rock powder in ASW, although this relationship was found to be different when measured at 4 hours versus days later. Therefore, a correction factor for adsorbance was applied to the enzyme data for the initial measurement (t0, y=1.90x-676), taken <2 hours after experiment initiation, versus the second and third measurements (t1 and t2, y = 4.64x - 303), taken days to weeks later. Negative controls consisting of the same ASW used for the sample incubations plus substrate, but no sample, were consistently below detection. The limit of quantification for the AP assay, defined as 3X the standard deviation of the blank, was 0.0242 pmol cm-3 rock hour-1 based on analysis of eight blanks.

BCO-DMO processing notes:

• Added latitude and longitude of sample location
• Adjusted column headers to comply with database requirements

NSF abstract:

The lower ocean crust has remained largely unexplored and represents one of the last frontiers for biological exploration on Earth. Preliminary data indicate an active subsurface biosphere in samples of the lower oceanic crust collected from Atlantis Bank in the SW Indian Ocean as deep as 790 m below the seafloor. Even if life exists in only a fraction of the habitable volume where temperatures permit and fluid flow can deliver carbon and energy sources, an active lower oceanic crust biosphere would have implications for deep carbon budgets and yield insights into microbiota that may have existed on early Earth. This is all of great interest to other research disciplines, educators, and students alike. A K-12 education program will capitalize on groundwork laid by outreach collaborator, A. Martinez, a 7th grade teacher in Eagle Pass, TX, who sailed as outreach expert on Drilling Expedition 360. Martinez works at a Title 1 school with ~98% Hispanic and ~2% Native American students and a high number of English Language Learners and migrants. Annual school visits occur during which the project investigators present hands on-activities introducing students to microbiology, and talks on marine microbiology, the project, and how to pursue science related careers. In addition, monthly Skype meetings with students and PIs update them on project progress. Students travel to the University of Texas Marine Science Institute annually, where they get a campus tour and a 3-hour cruise on the R/V Katy, during which they learn about and help with different oceanographic sampling approaches. The project partially supports two graduate students, a Woods Hole undergraduate summer student, the participation of multiple Texas A+M undergraduate students, and 3 principal investigators at two institutions, including one early career researcher who has not previously received NSF support of his own.

Given the dearth of knowledge of the lower oceanic crust, this project is poised to transform our understanding of life in this vast environment. The project assesses metabolic functions within all three domains of life in this crustal biosphere, with a focus on nutrient cycling and evaluation of connections to other deep marine microbial habitats. The lower ocean crust represents a potentially vast biosphere whose microbial constituents and the biogeochemical cycles they mediate are likely linked to deep ocean processes through faulting and subsurface fluid flow. Atlantis Bank represents a tectonic window that exposes lower oceanic crust directly at the seafloor. This enables seafloor drilling and research on an environment that can transform our understanding of connections between the deep subseafloor biosphere and the rest of the ocean. Preliminary analysis of recovered rocks from Expedition 360 suggests the interaction of seawater with the lower oceanic crust creates varied geochemical conditions capable of supporting diverse microbial life by providing nutrients and chemical energy. This project is the first interdisciplinary investigation of the microbiology of all 3 domains of life in basement samples that combines diversity and "meta-omics" analyses, analysis of nutrient addition experiments, high-throughput culturing and physiological analyses of isolates, including evaluation of their ability to utilize specific carbon sources, Raman spectroscopy, and lipid biomarker analyses. Comparative genomics are used to compare genes and pathways relevant to carbon cycling in these samples to data from published studies of other deep-sea environments. The collected samples present a rare and time-sensitive opportunity to gain detailed insights into microbial life, available carbon and energy sources for this life, and of dispersal of microbiota and connections in biogeochemical processes between the lower oceanic crust and the overlying aphotic water column.

About the study area:

The International Ocean Discovery Program (IODP) Expedition 360 explored the lower crust at Atlantis Bank, a 12 Ma oceanic core complex on the ultraslow-spreading SW Indian Ridge. This oceanic core complex represents a tectonic window that exposes lower oceanic crust and mantle directly at the seafloor, and the expedition provided an unprecedented opportunity to access this habitat in the Indian Ocean.

The International Ocean Discovery Program (IODP) is an international marine research collaboration that explores Earth's history and dynamics using ocean-going research platforms to recover data recorded in seafloor sediments and rocks and to monitor subseafloor environments. IODP depends on facilities funded by three platform providers with financial contributions from five additional partner agencies. Together, these entities represent 26 nations whose scientists are selected to staff IODP research expeditions conducted throughout the world's oceans.

IODP expeditions are developed from hypothesis-driven science proposals aligned with the program's science plan Illuminating Earth's Past, Present, and Future. The science plan identifies 14 challenge questions in the four areas of climate change, deep life, planetary dynamics, and geohazards. 

IODP's three platform providers include:

• The U.S. National Science Foundation (NSF) 
• Japan's Ministry of Education, Culture, Sports, Science and Technology (MEXT) 
• The European Consortium for Ocean Research Drilling (ECORD)

More information on IODP, including the Science Plan and Policies/Procedures, can be found on their website at http://www.iodp.org/program-documents.