Table of Contents

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

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.

Data Processing
General linear mixed models (GLMM) were used to evaluate the physiological responses of Ulva lactuca to a range of pCO2 and NH4+ concentrations. Data analysis was done in R using the lmer4 and AICcmodavg packages (Mazerolle 2017).

BCO-DMO Processing
- Renamed fields to conform with BCO-DMO naming conventions.

Benthic macroalgae contribute to intensely productive near shore ecosystems and little is known about the potential effects of ocean acidification on non-calcifying macroalgae. Kübler and Dudgeon will test hypotheses about two macroalgae, Ulva spp. and Plocamium cartilagineum, which, for different reasons, are hypothesized to be more productive and undergo ecological expansions under predicted changes in ocean chemistry. They have designed laboratory culture-based experiments to quantify the scope for response to ocean acidification in Plocamium, which relies solely on diffusive uptake of CO2, and populations of Ulva spp., which have an inducible concentrating mechanism (CCM). The investigators will culture these algae in media equilibrated at 8 different pCO2 levels ranging from 380 to 940 ppm to address three key hypotheses. The first is that macroalgae (such as Plocamium cartilagineum) that are not able to acquire inorganic carbon in changed form will benefit, in terms of photosynthetic and growth rates, from ocean acidification. There is little existing data to support this common assumption. The second hypothesis is that enhanced growth of Ulva sp. under OA will result from the energetic savings from down regulating the CCM, rather than from enhanced photosynthesis per se. Their approach will detect existing genetic variation for adaptive plasticity. The third key hypothesis to be addressed in short-term culture experiments is that there will be a significant interaction between ocean acidification and nitrogen limited growth of Ulva spp., which are indicator species of eutrophication. Kübler and Dudgeon will be able to quantify the individual effects of ocean acidification and nitrogenous nutrient addition on Ulva spp. and also, the synergistic effects, which will inevitably apply in many highly productive, shallow coastal areas. The three hypotheses being addressed have been broadly identified as urgent needs in our growing understanding of the impacts of ocean acidification.

NSF Climate Research Investment (CRI) activities that were initiated in 2010 are now included under Science, Engineering and Education for Sustainability NSF-Wide Investment (SEES). SEES is a portfolio of activities that highlights NSF's unique role in helping society address the challenge(s) of achieving sustainability. Detailed information about the SEES program is available from NSF (https://www.nsf.gov/funding/pgm_summ.jsp?pims_id=504707).

In recognition of the need for basic research concerning the nature, extent and impact of ocean acidification on oceanic environments in the past, present and future, the goal of the SEES: OA program is to understand (a) the chemistry and physical chemistry of ocean acidification; (b) how ocean acidification interacts with processes at the organismal level; and (c) how the earth system history informs our understanding of the effects of ocean acidification on the present day and future ocean.

Solicitations issued under this program:
NSF 10-530, FY 2010-FY2011
NSF 12-500, FY 2012
NSF 12-600, FY 2013
NSF 13-586, FY 2014
NSF 13-586 was the final solicitation that will be released for this program.

PI Meetings:
1st U.S. Ocean Acidification PI Meeting(March 22-24, 2011, Woods Hole, MA)
2nd U.S. Ocean Acidification PI Meeting(Sept. 18-20, 2013, Washington, DC)
3rd U.S. Ocean Acidification PI Meeting (June 9-11, 2015, Woods Hole, MA – Tentative)

NSF media releases for the Ocean Acidification Program:

Press Release 10-186 NSF Awards Grants to Study Effects of Ocean Acidification

Discovery Blue Mussels "Hang On" Along Rocky Shores: For How Long?

Discovery nsf.gov - National Science Foundation (NSF) Discoveries - Trouble in Paradise: Ocean Acidification This Way Comes - US National Science Foundation (NSF)

Press Release 12-179 nsf.gov - National Science Foundation (NSF) News - Ocean Acidification: Finding New Answers Through National Science Foundation Research Grants - US National Science Foundation (NSF)

Press Release 13-102 World Oceans Month Brings Mixed News for Oysters

Press Release 13-108 nsf.gov - National Science Foundation (NSF) News - Natural Underwater Springs Show How Coral Reefs Respond to Ocean Acidification - US National Science Foundation (NSF)

Press Release 13-148 Ocean acidification: Making new discoveries through National Science Foundation research grants

Press Release 13-148 - Video nsf.gov - News - Video - NSF Ocean Sciences Division Director David Conover answers questions about ocean acidification. - US National Science Foundation (NSF)

Press Release 14-010 nsf.gov - National Science Foundation (NSF) News - Palau's coral reefs surprisingly resistant to ocean acidification - US National Science Foundation (NSF)

Press Release 14-116 nsf.gov - National Science Foundation (NSF) News - Ocean Acidification: NSF awards $11.4 million in new grants to study effects on marine ecosystems - US National Science Foundation (NSF)