Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

For XRMA analysis, ~ 0.4 mL of Synechococcus culture cells were transferred to a 1.5 mL microtube and washed with 0.8 mL of cold buffered ultrapure water (pH 8) to remove salts. After centrifugation (1 min at 7000 rpm at 15 ºC), 1.1 mL supernatant was replaced by cold buffered ultrapure water and centrifuged under the same conditions. This last step was repeated twice, and 1.1 mL was carefully discarded. The concentrated cells were homogenized before transferring 6 µL onto a 3 mm TEM grid coated with formvar and evaporated carbon (Ted Pella, Redding, CA, USA). The grid was air-dried and stored in a vacuum cabinet until analysis. Cells were analyzed for elemental composition on a Hitachi SU8600 Field-Emission Scanning Electron Microscope (FE-SEM) equipped with two high-resolution (123 eV) Bruker X-Flash 7|30 Energy Dispersive Spectrometers. To avoid interference with the holder, the grids were attached to the edge of the SEM pins. XRMA spectra were obtained at 20 kV of incident energy during 90 seconds of live time. The absolute beam current was measured using a Faraday cup before each analysis session, which allowed for correcting for beam current drift during the session, and ensured an accurate reference to the calibration. Polystyrene latex spheres with mean particle size of 0.6 and 1.1 µm  (Sigma LB6 and LB11) were used to calibrate carbon, and ATP (Sigma, A2383) was used to obtain the calibration constants for N and P, following Norland et al. (1995). 

Cell dimensions were measured from SEM micrographs using ImageJ (version 1.54g, Abràmoff et al. 2004). Synechococcus cells were assumed to exhibit a rod-shaped morphology and were therefore approximated geometrically as a cylinder with two hemispherical ends. Based on these measurements, cellular biovolumes were calculated using standard geometric approximations for this morphology.

Latex spheres with a 0.6 µm mean particle size were used as internal standards and were analyzed at the start and end of each sample analysis session. Detection limits were calculated as 3 times the standard deviation of the blank (formvar film) measured in the energy region of each target element's spectral peak and were 26.3, 0.54, and 0.45 fg cell^-1 for C, N, and P, respectively. In the present study, only elemental quotas above the detection limit, which account for more than 98% of the quotas measured, are shown and used to calculate ratios. 

- Loaded Sheet1 from xray_syn_wh8102_dataset_bco.xlsx into table named xrma_single_cell_stoichiometry; configured empty string and "nd" as missing values; applied floating point error adjustment and preserved formatting during load
- Renamed column "Exp" to "exp" to match the lowercase column name used in the README, since the actual data file used "Exp" as the treatment column header
- Output as 1005186_v1_xrma_single_cell_stoichiometry.csv

NSF Award Abstract:
Phosphorus (P) is a vital nutrient required by all forms of life. In the ocean, which sustains half of global photosynthesis and oxygen supply, P can be scarce enough to constrain biological productivity, carbon dioxide uptake, and therefore climate. Human activity is accelerating the delivery of nutrients like P to the ocean, but rates of nitrogen inputs are even greater. This imbalance may increase levels of P stress in marine ecosystems, placing ocean productivity more and more under the control of P supply. Given the critical role of P in sustaining ocean health and ecosystem services now and into the future, a comprehensive understanding of its utilization and fate in the marine environment is necessary. In this project, the research team investigates marine polyphosphate (polyP), a ubiquitous yet poorly understood form of P made by all living organisms. To close major knowledge gaps on marine polyP, the investigators are overcoming major technical barriers to produce the first quantitative measurements of polyP in marine microorganisms. These measurements are being conducted on laboratory microbial cultures, as well as field samples from environments with high P supply, such as the California Current Ecosystem, or very low P supply, such as the Mediterranean Sea. In addition, the research team is resolving the cellular function of marine polyP across these different organisms and environments in order to clarify its role in biological P nutrition. This work helps advance polyP research across disciplines, including terrestrial science and even cancer research, and has broad application to P bioremediation. This project supports a postdoctoral researcher, a graduate student, and several undergraduate students in the labs of two female scientists. New educational tools to teach the public about marine polyP are being produced and disseminated through this project. A K12 teacher is participating in the work and communicating the findings to their classrooms and to broad audiences online.

PolyP is ubiquitous in marine systems, where it plays critical roles in microbial P nutrition and P mineral formation and sequestration. In these ways, polyP has the potential to shape long- and short-term marine P cycling, primary productivity, microbial ecology, and global climate. However, major knowledge gaps still exist. Due to technical limitations, the scientific community currently lacks a quantitative understanding of marine polyP pools, their chain lengths, and biological origins. Furthermore, given the view of polyP as a P storage molecule, recent observations pointing to the preferential retention of particulate polyP in low phosphate (Pi) environments raise new questions about its ecophysiological functions. To close these knowledge gaps, two research questions are addressed: Q1: What is the total content and chain length distribution of polyP across different microbial groups and environmental conditions? Q2: How do the production of particulate polyP contribute to microbial P demand and stoichiometry across a broad range in Pi availability? The following hypotheses are tested: H1 (Q1): Functionally and environmentally diverse plankton produce a broad range of polyP chain lengths and concentrations. H2 (Q2): The preferential retention of polyP in low Pi environments can be reconciled with its role as a P storage molecule by a combination of taxonomic and physiological factors. These hypotheses are tested in the laboratory using representative cultures of marine plankton and in the field using observational approaches along natural Pi gradients in the Pacific Ocean and Mediterranean Sea. Applying a new P-targeted method using mass spectrometry, the team is resolving an unprecedented level of detail in marine polyP content and speciation. By combining cell sorting with elemental, biochemical, and radiotracer analyses, the team is gaining a mechanistic understanding of polyP physiology and its cycling in the ocean.

This project is supported by the Biological Oceanography and Chemical Oceanography Programs. 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.