This study was conducted at the University of Rhode Island (URI) Phytoplankton Culture Laboratory and Ocean Ecogeochemistry Isotope Laboratory.

A library of three species from each of four major eukaryotic microalgae classes - diatoms (Class Bacillariophyceae), dinoflagellates (Class Dinophyceae), raphidophytes (Class Raphidophyceae), and prasinophytes (Class Mamiellophyceae) - was generated from established laboratory culture lines in the URI microalgal libraries and the National Center for Marine Algae and Microbiota (NCMA; formerly CCMP). Cultures were grown in triplicate in either f/2 or L1 media created using 0.22-micrometer (µm) filtered, autoclaved Narragansett Bay, Rhode Island seawater. All seawater was collected at the same time and location to ensure consistent water conditions for all cultures. Cultures were grown in climate-controlled incubators under a light intensity of 55 ± 10 micromoles photons per square meter per second (µmol photons m-2 s-1) on a 12-hours:12-hours light:dark cycle. One species from each of the four Classes was grown in triplicate under three temperature treatments: 15° Celsius (C), 20°C, and 25°C. A microplate reader was used to obtain growth rates to target biomass collection. The Supplemental File "Stahl_et_al_2023_L&O_BCO-DMO_Supplemental_DataSet.pdf" contains the identification and laboratory culture conditions for the four major groups of eukaryotic microalgae.

Once cultures reached sufficient density to obtain ~5 milligrams (mg) of dry weight needed for amino acid isotope analysis, cultures were gently vacuum filtered onto either 5 µm PETE membrane filters (Sterlitech), 2 µm PETE membrane filters (Sterlitech), or 0.22 µm PES membrane filters (Millipore Express PLUS) depending on the size of the species being filtered. Filtered biomass was frozen at -20°C, freeze dried for 72 hours, and homogenized prior to isotope analysis. Dried, homogenized samples were acid hydrolyzed in 6 N hydrochloric acid at 110°C for 20 hours, filtered through a 0.45 µm nylon syringe filter (Restek), and evaporated to dryness under a gentle stream of N₂. Five µl of nor-leucine (Sigma-Aldrich) with a known δ13C value was added to each sample and standard as an internal calibration. Acid hydrolyzed samples were derivatized to N-trifluoroacetic acid isopropyl esters and the carbon isotope values of 13 individual amino acids were separated and analyzed on a BPX5 column (60 meters length, 0.32 millimeters internal diameter (ID), 1 µm film thickness) in a Thermo Trace 1310 gas chromatograph (GC) and analyzed on a Finnegan MAT Delta V Plus Isotope Ratio Mass Spectrometer (IRMS) interfaced to the GC through a GC-IsoLink II and reduction furnace (1000°C) at the University of Rhode Island, Graduate School of Oceanography. Standardization of runs was achieved using intermittent pulses of a CO₂ reference gas of known isotopic value. Amino acid standards of known isotopic value were derivatized concurrently with samples and analyzed bracketing each sample. All samples were analyzed minimally in triplicate along with the amino acid mixed standard and a cyanobacteria working lab standard. Normalized δ13CAAnorm values were calculated using the following equation: δ13CAAnorm = δ13CAA - δ13CAAmean

905161_v1_pp_amino_acid_c_isotopes.csv