Table of Contents

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

For this alkaline phosphatase activity dataset, G. huxleyi CCMP371 was grown under low N and low P conditions by modulating dissolved inorganic nitrogen (DIN) to dissolved inorganic phosphorus (DIP) ratios. This experiment began as a single batch culture until a maximum in vivo chlorophyll fluorescence was reached (based on a previous growth experiment; data contributed to BCO-DMO in this same project), which initiated a semi-continuous phase of the experiment. Briefly, CCMP371 (in L1/5-Sargasso seawater base media) was used to inoculate L1/5-artificial seawater base media (ASW) in each of six 2 L Erlenmeyer flasks. The cultures were kept at 18ºC (the optimum in terms of its thermal range; see Krinos et al. 2025) on a 14 h:10 h light-dark cycle with a light intensity of 100 µE m-2 s-1. Growth was monitored daily by in vivo fluorescence and by microscopic cell counts (reported elsewhere). A total of 3 flasks were randomly selected as low N treatment flasks, and the remaining 3 were selected as low P treatment flasks for the semi-continuous phase of the experiment, with daily removal of 0.4 L of culture and replacement with 0.4 L modified L1/5-ASW, with an N:P of 0.5:1 (0.5 µM N and 1 µM P) for low N and an N:P of 125:1 (25 µM N and 0.2 µM P) for low P until steady-state growth, as determined by in vivo fluorescence and microscopic cell counts (reported elsewhere), was maintained. Dilutions were made at the same time each day, roughly 5 h into the light cycle, to avoid diel effects. Daily dilutions continued until the cultures reached a steady state that was maintained for at least 4 days after the daily removal/dilution began. Once the low N and low P triplicate cultures reached steady state, samples were collected for a suite of physicochemical parameters, including alkaline phosphatase activity (reported here).

For APA analysis, samples (5 mL) from each experimental treatment (low N and low P) and replicate were filtered onto 47-mm polycarbonate membranes (0.2 μm) and stored at −20°C until analysis. APA was assayed after Dyhrman and Ruttenberg (2006) on a Biotek Synergy H1 hybrid microplate reader in fluorescence mode (with an excitation wavelength of 360 nm and an emission wavelength of 460 nm) using the fluorogenic phosphatase substrate 6,8-difluoro-4-methylumbelliferyl phosphate. Subsamples (0.2 mL) from each sample were transferred to a black, flat-bottom, untreated 96-well microplate and assayed immediately and then at intervals adjusted to the activity of the sample, such that all readings fell within the linear range of the assay (at least four measurements within 1 h or less). A standard curve from 0 to 600 nM DiFMU in artificial seawater was generated and used to calculate the rate of DiFMUP hydrolysis. Values were normalized per G. huxleyi cell (cell counts reported elsewhere).

Details for the reagents used are as follows:

6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP; Invitrogen/Thermo Scientific D6567); Abs/Em = 358/455

6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU; Invitrogen/Thermo Scientific D-6566); reference standard

A standard curve from 0 to 600 nM DiFMU in artificial seawater was generated and used to calculate the rate of DiFMUP hydrolysis.  Rates were divided by the number of G. huxleyi cells collected on the filter to obtain a rate per cell for each treatment and replicate.

- Loaded data from "APA_CCMP371_BCO_DMO.xlsx" (sheet 1), treating empty strings and "nd" as missing values
- Renamed column "G. huxleyi_ID" to "G_huxleyi_ID"
- Set column types: "APA_nmolP_cell_hour" as number with scientific notation output format, "Experimental_Treatment" as string, "G_huxleyi_ID" as string, and "Replicate" as integer
- Output written to "998945_v1_G_huxleyi_CCMP371_alkaline_phosphatase_activity.csv"

NSF Award Abstract:
Emiliania huxleyi is a numerically and ecologically important phytoplankton species in the ocean known for its cosmopolitan distribution and ability to form large blooms in coastal and open ocean regions. Studies of E. huxleyi variants in culture have found differences in growth, function and activity potential among them. The E. huxleyi variants also differ in some of the genes they carry. It has been hypothesized that this genomic variability may underlie the global success of this phytoplankton species by allowing adaptation of variants to diverse environments. Yet, the direct connection between genomic content and ecological success remains unclear. This project investigates how the conserved and variable portions of E. huxleyi genome may be connected to its success and its dynamics under varied environmental conditions. This work is critical to our understanding of how this important phytoplankton species may shift and respond to future changes. This project also supports the development of a series of hands-on activities designed to teach middle school students advance computational data analysis in ocean science. These activities are in collaboration with the Girls Who Code Club at the Our Sisters School, a tuition-free, non-sectarian, independent school for girls from low-income families, located in New Bedford, MA.

Understanding how phytoplankton diversity and phenotype are driven by changes in the environment is crucial for better predicting carbon cycle dynamics in the future ocean. While much work has investigated competition among phytoplankton species, intraspecific diversity and dynamics remain largely unknown for many eukaryotic phytoplankton. The overarching goal of this project is to define the role of the pan genome (set of variable genes) in E. huxleyi ecology and biogeography through a series of genomic analyses, computational field surveys, and laboratory-based experiments. This project is sequencing the genomes of several E. huxleyi isolates from across the global ocean and combining them with existing genome sequences to constrain the core and variable portions of the pan genome. Using this new pan genome reference database and leveraging global scale metagenomics and metatranscriptomic surveys, this project is estimating ecotype diversity of E. huxleyi across ocean regions to identify patterns of environmental selection. This project additionally focuses on identifying the physiological and transcriptional responses of a selection of sequenced strains and their responses to shifts in their nutrient environments in controlled laboratory studies. As E. huxleyi plays such a significant role in marine ecosystems and the global carbon cycle it is important that its pan genome and its impact on the biogeography and ecology of E. huxleyi is taken into consideration. It is likely that these dynamics are acutely important to predicting how this genus, and perhaps others, respond to changing environmental conditions in the future ocean.

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.