Table of Contents

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

Samples were collected from culture flasks of a Hemiaulus-Richelia diatom diazotroph association (DDA) over the course of 2.5 weeks. Sargasso Seawater collected on the R/V Atlantic Explorer AE1812 cruise in May 2018 (at 32.42°N, 63.48°W) was used for media and filtered via peristaltic pump over 0.2 micrometers (µm) 47-millimeter (mm) filters (Polyethersulfone filters Millipore Express PLUS #GPWP04700). All flasks were grown in f/5 media without nitrogen, one set of flasks had no added nitrogen (Only N2), one set 10 µM added ammonium treatment (+NH4), and one set 10 µM added nitrate treatment (+NO3). Flasks were grown at 22 degrees Celsius (°C), ~130 micromoles per square meter per second (µmol m-2 s-1) in a 12:12 light:dark cycle. Two time points were used, an initial (T=0) and a final (T=15,17,18,19, depending on flask growth). Samples from the culture innoculum (Innoc1, Innoc2, Innoc3) were collected before all three innoculum flasks were thoroughly mixed and added to the experiment flasks.

Total chlorophyll a fluorescence was sampled by filtering over GF/F (~0.7 µm Whatmanfilters) and immediately extracted using 90% acetone over 24 hours (Strickland & Parsons, 1968). Total chlorophyll a, phaeophytin, and relative fluorescence were measured daily using a 10 AU fluorometer (Turner). FRR (Fv_Fm and sigma) was measured using a FIRe Fluorometer System (Satlantic) (Kolber et al., 1998) with settings of 100 microseconds (µs) Single Turnover Flash (STF), 80 µs STRI, 20 µs MTF, 40 µs MTRP, 100 µs MTRI, and with gain adjusted based on the fluorescence yield. Samples were preserved for cell counts during the experiment by preserving with 0.125% gluteraldehyde, flash freezing, and storing at -80°C. Samples were then thawed and aliquoted onto a Sedgewick Rafter (PYSER-SGI) and counted. Cells were counted on an Eclipse E800 (Nikon) light microscope, with phycoerythrin emission (565 nanometers (nm) ± 40 nm) and excitation (530 nm ± 30 nm) wavelength filters to count the diazotroph symbionts. Cell size was averaged using at least 30 pictures each of the host and symbiont per flask, and measured using ImageJ and an image of a stage micrometer (OMAX A36CALM1 0.01 mm) at the same magnification of cell pictures. Cell volume and surface area were calculated using 29-H (Hemiaulus hauckii) and 1-H shaped cells (Richelia euintracellularis) equations from Sun and Liu (2003). Dissolved and particulate nutrients were sampled from the same ~100 milliliters (mL) aliqout (Part_vol_mL). Particulate nutrients were filtered out using pre-combusted GF/F (~0.7 µm) Whatman filters. Dissolved nutrients (NO3_uM, NO2_uM, NH4_uM, SiOH4_uM, and PO4_uM) were immediately frozen before measuring on a Seal Analytical AA3 nutrient autoanalyzer and total reduced nitrogen (TDN_uM) was analyzed by oxidizing all nitrogen to nitrate following a persulfate odixation method (Knapp et al., 2005). Particulate nutrients (PC and PN) were immediately frozen until processing, during which samples were dried at 60 °C for 24 hours, packed in 9x10mm tin capsules (Costech) and sent for analysis on the Carlo Erba NC 2500 Elemental Analyzer (with a Costech zero-blank autosampler) at the Central Appalachians Stable Isotope Facility (CASIF) at the University of Maryland.

Carbon and nitrogen fixation measurements were taken using ¹³C and ¹⁵N stable isotope incubations (Hama et al. 1983, Montoya et al. 1996) during the last 24 hours of the experiment (T=Final), following methods from White et al. (2020) and Klawonn et al. (2015). Sample aliqouts (~550 mL) were added to 630 mL polycarbonate bottles (Nalgene), with 214 mL of 0.05 grams per milliliter (g/mL) of H₁₃CO₃ (Sodium Bicarbonate, 13C 99% Cambridge Isotopes) for a final concentration of 200 µM and 63 mL ¹⁵N₂ solution using Cambridge Isotopes ¹⁵N₂, 98%+ (Lot No. I-24583/AR0483820) for a final ¹⁵N₂ dilution of 10% v/v. The ¹⁵N₂ (Cambridge Isotopes 15N2 98%+, Cat # NLM-363-1-LB, Lot # 1-24583/AR0483820) solution added to each flask was made using the dissolution method (Mohr et al. 2010; Klawonn et al. 2015) and letting serum bottles with dissolved gas sit for ~12 hours before the start of the incubation to allow more ¹⁵N₂ to move into solution. After 24 hours, 10 mL of incubation sample was collected through the bottle septum caps, added to helium-flushed 20 mL vials with 20 mm rubber butyl septa crimp caps (Sigma-Aldrich) for % ¹⁵N₂ dissolved gas analysis. A 50% solution of ZnCl₂ was added to each 20 mL vial for sample preservation and vials were stored upside down, submerged in DI water before sending for sample analysis at the UC Davis Stable Isotope Facility for analysis on the GasBench-Precon-IRMS for ¹⁵N₂/N₂ atom % measurements. After dissolved gas samples were collected from incubation bottles, 225-275 mL isotope incubation volume was filtered over pre-combusted GF/F (~0.7 µm) filters (Whatman), which were then immediately frozen and processed in the same way as particulate samples before sending to the CASIF at the University of Maryland (UMD) for isotope analysis (d¹⁵N and d¹³C) on a Thermo Fisher Delta V+ isotope ratio mass spectrometer interfaced with the Carlo Erba NC 2500 Elemental Analyzer. One sample (NegCon) was incubated with a diatom (Thalassiosira pseudonana - f/2 media) with no nitrogen fixers to test for contamination (¹⁵NO₃ or ¹⁵NH₄) in the ¹⁵N₂ gas stock, from which there was no indication of contamination.

R v4.3.0 was used for all isotope incubation calculations for carbon (pp_rate_ugCperLperday) and nitrogen fixation (NFR_nmolNperLperday).

- Imported original file "Meta-DDA_NitAdd_2022.csv" into the BCO-DMO system.
- Marked "NA" as a missing data value; note that missing data are empty/blank in the final CSV file.
- Saved the final file as "921924_v1_dda_nutrient_acquisition.csv".

Diatom diazotroph associations (DDAs) have broad geographic distributions, provide bioavailable nitrogen to the biosphere via nitrogen fixation, affect ecosystem functioning and influence biogeochemical cycling. Despite the importance of these symbioses, little is known about their basic physiology and metabolism because DDAs are rarely brought into and kept in culture. Without cultured strains, it is difficult to study experimentally how the partners interact, share and potentially compete for resources. Here we evaluate and model key physiological characteristics of a DDA (Hemiaulus hauckii-Richelia intracellularis)  isolated from the Sargasso Sea to investigate how their physiology is altered by nitrogen sources and temperature. The isolated strain is growing well, fixing nitrogen and can be manipulated in the laboratory, allowing for an unprecedented view into the physiology and metabolism of a biogeochemically important ocean symbiosis. Empirical data informs the development of quantitative cell flux model of DDAs (CFM-DDA) embedded into a simple ecosystem model to test how different nitrogen sources and temperatures shape the niche of DDAs. The physiology of DDAs is compared to asymbiotic diatoms to examine the conditions where diazotrophic symbionts benefit the host diatoms and allow them to expand their ecological niche. This research addresses fundamental knowledge gaps that will lead to an enhanced understanding of DDA distributions and activities both in today's ocean and in a future ocean with altered temperature and nutrient fields. 

NSF Award Abstract:
Phytoplankton are photosynthetic microbes that inhabit the surface ocean, form the base of marine food webs, and drive the global cycling of elements like carbon and nitrogen. To survive in regions with limiting nutrients for growth, some phytoplankton have evolved symbiotic relationships. In many cases, it remains unknown how the symbioses influence the survival of each partner or their impacts on ecosystem function and cycling of nutrients. This project focuses on the symbiotic relationship between two phytoplankton -- a single-celled eukaryote diatom and a single-celled nitrogen fixing cyanobacteria called a diazotroph. These diatom-diazotroph associations (DDAs) have broad geographic distributions, provide bioavailable nitrogen to the biosphere via the fixation of nitrogen gas, affect marine food webs, and influence the cycling of carbon and nitrogen. Despite the importance of these symbioses, little is known about their basic physiology and metabolism because it has been difficult for researchers to grow DDAs in the laboratory. In this project, a team of investigators from the University of Rhode Island is applying a method they developed to grow DDAs in the laboratory and conducting experiments on the effects of temperature and nutrients on DDA cellular metabolism. The newly-generated laboratory data is informing the development of a computer model of DDA cellular functioning that embedded in a simple ecosystem model to test how different nitrogen sources and temperatures influence DDA ecology and ecosystem function. The project is addressing fundamental knowledge gaps, leading to an enhanced understanding of DDA geographic distributions and activities both in today's ocean and in a future ocean with altered temperature and nutrient fields. Broader impacts of this study include the provision of DDA cultures to the oceanographic community, graduate student training, computational model distribution, and outreach to the broader community. Graduate students supported by the project are being cross-trained in experimental and modeling approaches. Outreach to the broader community includes hosting a high school student intern in the lab each year and the development of educational videos for the general public and K-12 students. Collectively, these activities are designed to broaden the public understanding of DDAs, a globally significant symbiosis.

This project is examining the cellular metabolism and physiology of diatom-diazotroph associations (DDAs) and evaluating their ecosystem and biogeochemical impacts by addressing the following critical, longstanding questions: 1) What is the cellular response of the Hemiaulus DDA to different nitrogen sources? 2) How does the thermal niche of the DDA influence nitrogen fixation, nutrient stoichiometries, and geographic distribution? 3) How does DDA physiology and metabolism differ from asymbiotic diatoms, and what are the ecosystem-level impacts of the symbiosis? Due to a scarcity of culture data, major ecological models assume DDAs gain 100% of their nitrogen from N2, although there is intriguing experimental evidence suggesting otherwise. If DDA physiology is affected by different N sources, key assumptions in modern ecosystem models will be altered, refining our understanding of the role DDAs play in ecosystem and biogeochemical functioning. In addition to N sources, temperature is an important regulator of cellular metabolism and a key variable in ecosystem models. The team of researchers is examining the roles of each partner in the symbiosis in setting the DDA thermal niche and examining ecosystem-level impacts via modeling both in the present day and future oceans. Finally, the impacts of the endosymbiont on the host genome, transcriptome, and resulting physiology are practically unknown. Comparison of DDAs with asymbiotic diatoms is providing new insights into the metabolic modifications of the host and providing new understanding of DDAs as a symbiosis. Addressing these three questions advances fundamental understanding of the impact of this widely-distributed symbiosis.