Table of Contents

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

Sediment was collected with an MC-800 multicorer aborad the R/V Oceanus (expedition 1703A) approximately 0-300 km off the coast of San Francisco, CA, USA. Cores were stored at 4C until extrusion and sectioning within 24h of collection. Cores were sectioned into 2.5-5cm vertical horizons, and approximately 2g of sediment were sampled from each horizon with a cut-off syringe, flash frozen in liquid nitrogen, and stored at -80C until extraction of nucleic acids. DNA was extracted with an RNeasy PowerSoil DNA elution kit (Qiagen, cat. no 12867-25) in combination with an RNeasy PowerSoil Total RNA kit (Qiagen, cat. no 12866-25). The manufacturer's protocol was modified to include a bead-beating step of 5.5 m/s for 2x45s with a FastPrep-24 instrument. DNA and RNA were eluted in 100 microliters of DNAse and RNAse-free water or 1xTE and stored at -80C. Total RNA was treated with DNAse (Thermo Fisher Scientific,cat. no AM1907). Three DNA samples were additionally processed for long-read sequencing by size-selection with the BluePippin instrument targeting the size range of 10-50kb and a library was prepared for sequencing with the 10x Genomics Chromium system according to manufacturer's protocol and described in Bishara et al. (2018, https://doi.org/10.1038/nbt.4266). Chromium libraries were sequenced with 2 x 151bp sequencing on an HiSeq 4000 instrument (Illumina). All other samples were processed by the Joint Genome Institute (Department of Energy). DNA was sheared to approximately 300bp using a Covaris LE220 ultrasonicator and size selected with SPRI beads and libraries prepared and barcoded using Kapa Biosystems library preparation kit. Total RNA was treated with Ribo-Zero rRNA removal kit (Illumina) and cDNA libraries generated using the Illumina Truseq Stranded mRNA Library Prep kit. The rRNA depleted RNA was fragmented and reversed transcribed using random hexamers and SSII (Invitrogen) followed by second strand synthesis. The fragmented cDNA was treated with end-pair, A-tailing, adapter ligation, and 10 cycles of PCR. DNA and cDNA were sequenced with 2 x 151bp sequencing on a Nova Seq S4 (Illumina) instrument.

BCO-DMO Processing:
- replaced "na" with "nd" (no data)

NSF Award Abstract:
Life requires nitrogen for growth. Atmospheric nitrogen (N2) is the most abundant form of nitrogen on the surface of the planet, but most organisms cannot assimilate N2 directly. Habitats can therefore be nitrogen limited, meaning the demand for "bioavailable" nitrogen exceeds the supply, and its availability controls the overall growth and productivity of the community. A small subset of microorganisms, termed diazotrophs, convert N2 to bioavailable forms of nitrogen, including ammonium and nitrogenous organic matter, in a process known as N2 fixation. Diazotrophs are the largest natural source of bioavailable nitrogen on the planet, and the rate at which they fix N2 can control the rates at which other important microbial processes occur, such as the production and consumption of greenhouse gases. Understanding diazotrophs in the environment - their identity, distribution, activity levels, and biogeochemical controls - is therefore essential to understanding overall microbial community activity and biogeochemical cycling. The goal of this project is to characterize N2 fixation in deep-sea sediments, a generally understudied but expansive habitat, covering nearly two thirds of our planet. The project will have broader impacts via educational outreach, support and training of early career scientists, and scientific impact: since rates of marine methane, carbon dioxide, and nitrous oxide cycling are affected by nitrogen availability, the results will inform our understanding of greenhouse gas cycling in the marine environment, and therefore climate stability, a topic central to global security.

N2 fixation is a critical and intensely studied metabolism in the marine photic zone. Much less is known about N2 fixation in deep-sea sediments, but it could be an important factor in both benthic productivity and ocean-scale elemental cycling. Several observations have suggested or directly detected N2 fixation at localized areas of enhanced productivity on the seafloor (e.g., methane seeps and hydrothermal vents), raising the possibility that deep-sea N2 fixation is widespread. However, few measurements of N2 fixation have been made outside of these anomalous areas, and thus little is known about N2 fixation in the vast majority of the deep ocean floor. Preliminary data suggest N2 fixation does occur in typical deep marine sediment, and is mediated by a diverse set of yet unidentified microorganisms. This project will combine techniques from molecular biology and geochemistry to systematically investigate N2 fixation in representative deep-sea sediments collected along a depth profile (500 to 4500 m water depth) offshore California. The project will determine the (1) rates and distribution of N2 fixation (2) abundance, diversity, and distribution of genes and transcripts associated with N2 fixation (nif) (3) phylogenetic identity of the biological mediators (diazotrophs) and (4) physiochemical controls on diazotrophic community structure and activity. For context, the activity of the non-diazotrophic bacterial community will also be characterized. The results may lead to upward revisions of the estimates of new nitrogen production in the seafloor, and therefore change our understanding of the current balance of the marine nitrogen cycle. Together, this hypothesis-driven characterization of N2 fixation in deep-sea sediments will shed light on an expansive, climatically important, and traditionally understudied habitat, and facilitate more accurate extrapolation of the rates and distribution of N2 fixation on the whole seafloor as well as the metabolic response of the seafloor community to environmental change.