Table of Contents

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

Three species of marine phytoplankton – Micromonas commoda, Emiliania huxleyi, and Phaeodactylum tricornutum – were grown under four phosphorus (P) conditions. These include phosphate (Pi) replete and deplete conditions and the phosphonate conditions where cultures received either methylphosphonate (MPN) or 2-aminoethylphosphonate (2-AEPN) as the sole source of phosphorus at replete levels. Samples for cell abundance were collected throughout the experiment to monitor growth. In addition, particulate P, to calculate cellular P quota, as well as alkaline phosphatase activity was measured at a single time point when cells were in exponential growth.

Axenic cultures of the pico-prasinophyte Micromonas commoda (CCMP 2709), the coccolithophore Emiliania huxleyi (CCMP 2090), and the diatom Phaeodactylum tricornutum (CCMP 2561) were obtained from the National Center for Marine Algae and Microbiota (Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine). The cultures remained axenic throughout the experiments as determined by SYTO-staining and flow cytometric counting on a BD FACSJazz cell sorter; all cultures were free of bacteria during these experiments. Phytoplankton were grown in artificial sea water amended with L1 media with (P. tricornutum) or without (M. commoda and E. huxleyi) silica. The P source was added separately to achieve the desired growth conditions; Pi-replete media contained 36 µM PO₄³⁻, the Pi-deficient condition received 0.1 µM PO₄³⁻, and the phosphonate treatments received either 36 µM MPN or 2-AEPN. The Pi-deficient treatment (0.1 µM) represents a control for the low level of contaminating Pi measured in the phosphonate media; thus, an increase in growth in the MPN and 2-AEPN conditions above that measured in the Pi-deficient condition is due to phosphonate utilization. The potential for abiotic breakdown of phosphonate to Pi was investigated in media-only tubes exposed to the experimental temperature and light conditions for 10 days. Pi levels did not change throughout the experimental period (MPN average Pi = 0.11 µM ± 0.02; 2-AEPN average Pi = 0.10 µM ± 0.02), strongly supporting the notion of active enzymatic breakdown of phosphonates for growth. Natural P concentrations are much lower than those used in this study; the replete nutrient concentrations were used to support high cell yields necessary for analytical measurements. Cultures were acclimated to the four growth conditions described above as they had been maintained in each P treatment for a minimum of two transfers (20 days). Cultures were grown at 20°C in a 14h light/10h dark cycle at ~100 µE m⁻² s⁻¹ with a starting concentration of ~1x10⁴ cells mL⁻¹ in 25 mL culture volumes. Phytoplankton growth was monitored by fluorescence measurements using a Turner TD-700 fluorometer and cell counts analyzed by flow cytometry. Specific growth rates (μ) were calculated from the linear regression of the natural log of cell counts during the exponential growth phase of cultures. Quadruplicate cultures were setup for each treatment; three replicates were harvested in the late exponential phase of growth for physiological measurements, while the fourth was used to monitor cell abundances later in the growth cycle.

Physiological measurements:
Cell samples (5 mL culture volume) for particulate P were collected onto precombusted 25 mm Whatman glass fiber filters, rinsed with 0.17 M Na₂SO₄, and stored frozen at -20°C until analysis. Determinations were made as previously described (Lomas et al. 2010). Briefly, filters were rinsed with 0.017 M MgSO₄, dried at 90°C, and combusted at 500°C for 2 h. Upon cooling, 0.2 M HCl was added and hydrolyzed at 80°C for 30 min. After cooling, mixed reagent was added, the samples were centrifuged, and absorbance was read at 885 nm using a Genesys 10 spectrophotometer.

Alkaline phosphatase content (APA) measurements were made by quantifying the hydrolysis of 6,8-difluoro-4-methylumbelliferyl phosphate using a Molecular Devices FilterMax F5 microplate reader. Abiotic substrate hydrolysis was accounted for in killed controls that were boiled and cooled prior to substrate addition, as well as in media-only controls. The fluorescent reference standard, 6,8-difluoro-4-methylcoumarin was used to calculate the rate of hydrolysis, which was then normalized to cell abundance to determine APA per cell.

Cell abundance data were processed from flow cytometry files by gating logic using Sortware software. Nutrient concentrations, both dissolved and particulate, were calculated based upon the instrument absorbance response to known concentration standards. Alkaline phosphatase activity was calculated based on fluorescence response of a known standard.

Any samples that were not collected are represented by 'nd' in the dataset.

BCO-DMO Processing:
- renamed fields;
- replaced 'NaN' with 'nd' (no data).

NSF Award Abstract:
Phosphorus (P) is an essential nutrient for all living cells. It is a central component of genetic material and cellular membranes and is integral to energy production and regulating enzyme activity. In the marine environment, P occurs as inorganic (Pi) and dissolved organic (DOP) forms; the availability and concentration of these different forms of P is an important control on marine phytoplankton growth. Marine phytoplankton are single-celled photosynthetic organisms and can be both prokaryotic bacteria and eukaryotic plants. While Pi is the preferred form of P for marine phytoplankton, in large regions of the oceans it is at such low levels that it restricts phytoplankton growth. In these regions, DOP is the most important P source. The composition of the DOP pool can generally be divided into two major groups: P esters and phosphonates. All marine phytoplankton are capable of using P esters to support growth; in contrast, phosphonates have only been shown to be an important source of P in the nutrition of bacteria to date. This project will determine the ability of marine eukaryotic phytoplankton to use phosphonates as a source of P for growth. Genomic analyses will determine the metabolic response of eukaryotic phytoplankton species to growth on phosphonates as well as the relevance of phosphonate use by natural populations. It is critical to understand the metabolic capabilities of phytoplankton which control marine nutrient cycling. In addition, the project is of great value in understanding the potential impacts of a changing ocean on phytoplankton growth. The project supports reseach opportunities for undergraduates from a local community college as well as hands-on enrichment programs for an afterschool program that serves a diverse student population.

Comprising up to 10% of the marine DOP pool, phosphonates have been shown to be a dynamic P pool both being assimilated and produced by marine photosynthetic bacteria. The ability of eukaryotic phytoplankton to supplement their growth with phosphonates remains vastly unexplored. Several eukaryotic phytoplankton species have been shown to use glyphosate, a chemically synthesized herbicidal phosphonate, as a P source; it remains unknown if open ocean eukaryotic phytoplankton can utilize phosphonates found naturally in the marine environment. Preliminary experiments suggest at least some eukaryotic phytoplankton are able to directly utilize extracellular phosphonates. This project characterizes the pervasiveness of phosphonate utilization within eukaryotic phytoplankton lineages and identifies the cellular underpinnings that support the acquisition of and growth on naturally occuring phosphonates. The project uses whole-cell transcriptomics and functional gene complementation assays, in addition to phylogenetic analyses, to understand the bioavailability of phosphonates and relevance of phosphonate utilization by natural eukaryotic phytoplankton populations. It is critical to understand the metabolic capabilities of phytoplankton which control marine biogeochemical cycles. This is especially important given the prediction that future oceans may become more stratified which could increase the importance of DOP, including phosphonates, in supporting phytoplankton growth.