Dataset: Results from a study of physiological responses of Ulva lactuca to ocean acidification and nutrient enrichment

The experiment took place in the laboratory at the Department of Biology, California State University, Northridge. The specimens used in the experiment were collected in Malibu, CA (34° 02' 29.0" N, 118° 34' 03.2" W) on May 26, 2016 for trial 1 and July 5, 2016 for trial 2. Each trial lasted 22 days.

Carbonate Chemistry
Carbonate chemistry parameters were calculated multiple times during each trial using measurements of pH and total alkalinity (AT). AT samples were collected in 50 mL Falcon tubes, stored wrapped in Parafilm at 4°C in the dark, and analyzed within two weeks by potentiometric titration coupled to a pH electrode (Mettler Toledo DGi-115-SC with T5 Rondolino) and thermometer. Most AT samples were measured on the day of sampling. The performance of the machine was checked with each measurement using certified reference material (CRM) from the Dickson laboratory at the Scripps Oceanographic Institute and the pH electrode was calibrated using TRIS buffer (Dickson et al., 2007). A spectrophotometric technique using m-cresol as an indicator dye was used to determine pHT (pH total scale). AT was calculated using potentiometric titration data and pHT using spectrophotometric data in the R-package Seacarb V 3.0.14 (Lavigne et al., 2011).

Nitrate reductase activity
An in vivo assay of nitrate reductase activity (NRA) was done according to the methods of Thompson & Valiela (1999) resulting in a colorimetric reaction with NO2- produced via NRA under dark, anoxic conditions. Absorbance was measured at 540 nm using a spectrophotometer.

Nutrient Analysis
NH4+ concentrations in the culture tanks were measured with a fluorometric method using OPA (Holmes et al., 1999) with the suggested modifications of Taylor et al. (2007), which included using an improved method for measuring background fluorescence. The raw fluorescence measurement of a sample was calibrated to a standard curve of an NH4+ stock solution using the standard additions protocol I of Taylor et al. (2007) which accounts for matrix effects that can alter fluorescence measurements.

NO3- concentrations in the culture tanks were determined from samples sent to the University of California, Santa Barbara Marine Science Institute Analytical Lab and were analyzed using a Lachat Instruments flow injection analysis instrument (QuikChem 8000).

Nutrient uptake rates
Uptake rates of NH4+ and NO3- were measured in situ on day twenty of the trials 8 – 10 hours into the light cycle for a period of one hour. The formula for chemostat nutrient uptake by Carmona et al. (1996) was used to determine nutrient uptake rates.

CN analyses and carbon stable isotope ratios
Tissue samples were dried for 24 hours at 60 degrees Celsisu. Dried samples were prepared for analysis by homogenizing samples using a metal laboratory scoop, cleaned with ethanol between each sample, which resulted in a fine power. Then, approximately 3 mg of the Ulva lactuca tissue powder was measured using an analytical balance (Mettler Toledo XP205) into a tin capsule and carefully enclosed with clean forceps. The tin capsules were put into 96-well tray plates and sent to the University of California, Davis Stable Isotope Facility (UCD-SIF). The samples were analyzed for δ15N and δ13C using the elemental analysis – isotope ratio mass spectrometry technique, which also provides results for tissue C and N content.

Seawater carbon stable isotopes
Seawater samples for delta13C of dissolved inorganic carbon (DIC) were stored in 20 mL glass vials with cone lids to exclude air from sample. Samples were stored at room temperature in low light until prepared for analysis using the exetainer gas evolution technique for DIC (Li et al., 2007). Then, the samples were sent to the UCD-SIF for analysis using the GasBench – isotope ratio mass spectrometry technique.

Internal soluble nitrogen pools
Internal NH4+ and NO3- pools were measured using the boiling water method (Hurd et al., 1996). One piece (0.04 ± 0.02 g FW, mean ± SEM) from each treatment was rinsed with deionized water to remove salt and nutrients on the surface. The pieces were placed in test tubes with 15 mL of deionized water and placed in a boiling water bath for 40 minutes. The water was decanted and analyzed for NH4+ and NO3-. This process was repeated on the same algal piece three times and the concentrations of internal soluble NH4+ and NO3- pools were calculated using the sum of the NH4+ and NO3- concentrations of the three water samples of each algal piece.

Soluble protein and carbohydrates
Pieces of Ulva lactuca tissue (0.04 ± 0.004 g FW, mean ± SEM) were ground in a mortar and pestle in 2 mL of a β-mercaptoethanol buffer, pH 7.5 and stored at 4°C for up to 72 hours. The extract was centrifuged at 16,000 g for 5 minutes. Soluble proteins and carbohydrates were determined spectrophotometrically (Milton Roy Spectronic Genesys 5) using the supernatant fraction. Soluble proteins were determined according to Bradford (1976) and soluble carbohydrates were determined using the phenol-sulfuric acid method according to Kochert (1978).

Chlorophyll a
Chlorophyll a was extracted in dimethylsulfoxide (DMSO) and methanol according to the methods of Duncan and Harrison 1982. Pieces of Ulva lactuca tissue (0.5 g FW) were placed in 1.25 mL of 80% DMSO for 10 minutes, and then suspended in two sequential 3 mL solutions of methanol for 10 min each to complete the extraction. The absorbance of the DMSO and methanol were measured using a spectrophotometer at the wavelengths indicated in the formulas below. The absorbance at each wavelength, volume of solvent, and the fresh weight of a fragment were used to calculate the concentration of Chl a from each solvent using the following formulas:

DMSO Solution Chl a (mg g⁻¹) = [ (A₆₆₅/(72.8)) * 1000 ] / g FW

Methanol solution Chl a (mg g⁻¹) = (13.8A₆₆₈ - 1.3A₆₃₅) / g FW

The Chl a concentration was the sum of the concentrations of the DMSO and methanol extracts.

Photosynthetic rates
Photosynthetic O2 evolution rates were measured using the Qubit systems O2 electrode in a water-jacketed cuvette connected to a laptop using a LabProTM interface. Small pieces of Ulva lactuca (1 – 2 cm2) were cut from thalli at least one hour prior to measurements. The pieces were placed in 20 mL of culture water at 16°C in a 2 cm2 mesh bag which held the pieces at a 90° angle to the Qubit LED light source. A photosynthesis-irradiance (P-I) curve was generated using various photon flux densities from 0 – 700 μM photons m-2 s-1 for 200 seconds each, following a 200 second dark period to measure dark respiration rate. The maximum photosynthetic rate (Pmax), light saturation point (Ik), and photosynthetic efficiency (the initial slope of the P-I curve) (α) were determined from the P-I curves.