Table of Contents

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

The following results paper has been submitted to Scientific Reports. A pre-print is available at https://doi.org/10.21203/rs.3.rs-6149474/v1

Armstrong D. A., McNicholl, C., and Bahr, K. C. (n.d.) Species-specific proton and oxygen flux in Hawaiian corals under ocean acidification—a microsensor analysis of the concentration boundary layer. Submitted to Scientific Reports. 

Coral Collection and Acclimation

Six large colonies each of Montipora capitata and Pocillopora acuta were collected approximately 300 m offshore of the Hawai‘i Institute of Marine Biology (HIMB) in Kāne‘ohe Bay, Hawai‘i (21.4337 N, 157.7893 W). Colonies were transported to HIMB in ambient seawater and placed into a flow-through mesocosm (area 1.37 m2; volume ~50 m3). Mesocosms received natural seawater from Kāne‘ohe Bay (<1 km from collection site) with a residence time of about one hour. Temperature, dissolved oxygen, salinity, and carbonate chemistry were not manipulated and followed natural diel patterns. Light was natural but shaded to approximately 50 percent (midday PAR ~800 µmol photons m-2 s-1). Colonies acclimated for 10 days, were then halved and tagged by genotype, and acclimated for an additional 3 days before experiments began. Initial buoyant weights were recorded prior to treatment placement.

Experimental Treatments

Two carbonate chemistry conditions were established: an ambient control and an elevated pCO2 treatment (target pH offset of approximately -0.3 units relative to the control). Treatment seawater carbonate chemistry was manipulated by bubbling pure CO2 or CO2-scrubbed air into mesocosm mixing pumps using a pH-stat approach. All other seawater variables (temperature, salinity, nutrients) were unmodified. Colonies were placed in similar positions within each mesocosm to minimize spatial variation during the 19-day exposure.

Seawater Chemistry Measurements

Temperature, salinity, dissolved oxygen, and pHNBS were measured with a YSI ProDSS multiparameter instrument. pHNBS values were corrected to total scale (pHT) using Tris buffer calibrations following Dickson et al. (2007).
Total alkalinity (AT) was measured via open-cell titration using a Metrohm Titrino 877 Plus with 0.1 M HCl. All AT values were corrected using certified reference materials from A. Dickson (batch 205). Carbonate system parameters (DIC, HCO3-, CO32-, pCO2, and Omega aragonite) were calculated using the R package seacarb.

Seawater Sample Collection, Filtration, and Storage

For AT and DIC measurements, seawater samples were collected in 250 mL borosilicate glass bottles with no headspace. Samples were poisoned immediately with 100 µL saturated HgCl2 to halt biological activity. Bottles were sealed with ground-glass stoppers greased with Apiezon L and stored in the dark at room temperature for no more than 7 days prior to analysis.  No nutrients or chlorophyll samples were collected for this study.

Flume System

A custom acrylic flume was used for microsensor profiling. The system consisted of a concentrator section (2.09 m2) supplied by a 24 V Hygger pump (6511 L h-1), a flow-reducing baffle, and a hexagonal flow straightener leading into a test section (88.9 cm by 22.3 cm; 1.8 m2). Flow velocity was approximately 1250 µm s-1 (residence time 44 minutes). Lighting was provided by a 180 W full-spectrum LED aquarium light delivering approximately 800 µmol photons m-2 s-1.
The flume received water from the control mesocosm each morning. For elevated pCO2 flume conditions, CO2 was bubbled into 1 L of seawater until pH 4.00, which was then mixed into the flume reservoir and equilibrated with circulation for one hour, achieving a pH offset similar to the mesocosm treatment.

Microsensor Measurements

Oxygen was measured using a PreSens PSt7 micro-optode (230 µm tip diameter), and pH was measured using a Unisense glass microelectrode (tip diameter 100 µm). Measurements were recorded every 6 seconds.
Sensors were mounted on a dual-probe automated micromanipulator (Zaber T-LSR075A). Movement followed scripted profiles with 100 µm vertical steps. At each height, sensors paused for 60 seconds before recording. Profiles extended from the coral surface (0 µm) upward until bulk seawater values were reached, followed by an additional 2000 µm at 500 µm intervals.
Sensor placement targeted branch tips oriented into flow and positioned between polyps. Sensors were lowered in fine increments (1–10 µm) until maximum steady values of oxygen and pH were observed at the tissue surface. Bulk seawater values were measured with the YSI instrument and used to standardize microsensor readings.

Sampling note -  The measurements were conducted at staggered intervals in the month of August. The days by species and genotype:
08-13 (m.cap 1)
08-14 (p. acuta 1)
08-15 (m.cap 2)
08-16 (p.acuta 2)
08-17 (m.cap 3)
08-18 (p.acuta 3)

All raw data and source code used in the analysis are publicly available and found here at https://github.com/CROH-Lab/Proton_and_oxygen_flux_concentration_boundary_layer.git 

This dataset was used to make the following calculations were reported in the results publication Armstrong D. A., McNicholl, C., and Bahr, K. C. (n.d.) Species-specific proton and oxygen flux in Hawaiian corals under ocean acidification—a microsensor analysis of the concentration boundary layer. Submitted to Scientific Reports. Pre-print https://doi.org/10.21203/rs.3.rs-6149474/v1

Calculation of Diffusive Boundary Layer Thickness

Diffusive boundary layer (DBL) thickness was calculated from log-transformed concentration profiles. A linear model of log10(concentration) versus distance was used to estimate the distance at which the concentration equaled the bulk seawater concentration.

Proton and Oxygen Flux Calculations

Fluxes were calculated using Fick’s first law of diffusion. The linear portion of each concentration gradient was used to determine the slope (m). Proton flux was calculated using a diffusion coefficient of 9.31 × 10-5 cm2 s-1, and oxygen flux using a diffusion coefficient of 2.20 × 10-5 cm2 s-1 at 26.5 C and salinity 35. These methods follow approaches described by Martins et al. (2020, 2021) and Pacherres et al. (2023).

- Loaded main data table from profiles_calculated.csv as resource 995180_v1_microsensor-profiles, with missing values interpreted as "" and "NA"
- Loaded supplemental table from calcification.csv with missing values interpreted as "" and "NA"
- Loaded supplemental table from sw_chem.csv with missing values interpreted as "" and "NA"

Main Data Table:
- Set types on 995180_v1_microsensor-profiles: bulk and hplus and value as number, distance_um as integer, genotype as integer, condition/measure/species/treatment as string
- Split "value" column in "995180_v1_microsensor-profiles" into two separate columns "pH" and "O2" based on whether the "measure" column indicated it was a pH or O2 measurement, as best practice is to have different measurement types in distinct columns; "pH" populated where measure == 'pH', "O2" populated where measure == 'o2'
- Deleted column "measure" and "value" from "995180_v1_microsensor-profiles" after splitting
- Added constant computed field experiment_year with value "2023" to 995180_v1_microsensor-profiles

Supplemental Tables:
- Set types on calcification: genotype as integer, value as number, measure/species/treatment as string
- Set types on sw_chem: crm_205, o2_mg_l, pH_nbs, salinity, ta_umol_kg, temp_c, tris_buffer_41 as number; date as date type with format %Y-%m-%d; tank, tod, treatment as string
- Converted date field in sw_chem from format %m/%d/%Y to ISO date format %Y-%m-%d
- Renamed column "value" to calcification_value in calcification table, as the "measure" col contained only the value "cal" indicating all values were calcification measurements, making an explicit field name clearer
- Deleted column "measure" from calcification table (redundant after col rename)

- Output three final resources: 995180_v1_microsensor-profiles.csv, calcification.csv, and sw_chem.csv

NSF Award Abstract:
Corals build calcium carbonate skeletons to maintain the three-dimensional structure of a coral reef, which provides habitat for many organisms and protects shorelines from bioerosion and storm damage. However, changes in ocean chemistry threaten the ability of corals to build and sustain these ecological important structures. To further the understanding of how climate change impacts coral reefs, this project investigates how changes in ocean carbonate chemistry directly influence coral calcification. The researchers are conducting a series of experiments on corals grown in seawater tanks to study corals responses to seawater chemistry in a changing ocean. Broader impacts of the project include student research opportunities, science-inquiry labs, and virtual learning. This project supports the training of several early career researchers, Ph.D. students, undergraduates, and high school students in the disciplines of chemistry, engineering, and marine ecology. Researchers partner with the Texas State Aquarium to communicate with the general public through a virtual research expedition series that will focus on coral reef health. This series includes interviews, behind the scene tours, and virtual dives on coral reefs in Hawaii.

This project examines the fundamental connections between seawater chemistry and coral physiology by investigating the modulation of seawater chemistry in the microenvironment surrounding corals. Specifically, this project 1) examines the response of corals to differing carbonate chemistry and 2) characterizes the proton gradient across the corals' boundary layers under differing ocean acidification conditions. Results of this work isolate whether carbonate ions or hydrogen ions have a stronger influence on calcification rates. This work utilizes a state-of-the-art experimental mesocosm facility that combines an automated systems to simultaneously and independently control both total alkalinity and carbon dioxide in the tanks to examine coral response under different carbon chemistry scenarios. Small-scale gradients in carbon chemistry surrounding the corals are being characterized using an innovative solid-state, reagentless sensor capable of making simultaneous measurements of two critical carbon system parameters. Coral biological response variables are quantified during short-term incubations and long-term mesocosm manipulations to understand physiological implications across multiple scales (i.e., individual and community scales) and across boundary layers.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.