Dataset: Ulva pCO2 - NH4 enrichment

Ulva australis was collected from Blackmans Bay, Tasmania, Australia (approx. 42°59'56"S 147°19'8"E) in July 2015 (Austral winter). Three Ulva australis thalli were placed in chambers filled with sterile seawater and circulated with fresh seawater. The pHT of seawater pumped to each tank was maintained using an automated pH control system. Seawater was equilibrated using a membrane contactor where the appropriate mix of N2 and CO2 gas was achieved using three pairs of mass flow controllers (MFCs) set to pHTs of 8.05, 7.85, and 7.65. Each of the three MFCs were randomly assigned to four ambient NH4+ and four enriched NH4+growth chambers for a total of 24 chambers.

Growth rates: Ulva australis thalli were blotted with a tissue to remove excess water and weighed before the start of the experiment and after seven days. The total weight of the three thalli from each chamber was used for the analysis. The RGR, expressed as % day-1, was calculated as RGR = ln(FWf/FWi) x t-1 x 100 where FWi is the initial fresh weight, and FWf is the final fresh weight after t days.

NH4+ uptake rates: At the end of the seven-day incubation period, one of the three Ulva australis thalli (0.43 ± 0.03 g of FW) was removed from each chamber to an Erlenmeyer flask containing 200 mL of filtered seawater with overhead light of 200 µmol photons m-2 s-1. The seawater in each flask was obtained from the automated pH control system shortly before the start of the experiment so the seawater pHT in the flasks was representative of the seawater in the chambers the algae came from. The initial NH4+ concentration of 20 µM was obtained with the addition of NH4Cl to ambient seawater. Flasks were placed on an orbital shaker (RATEK OM7, Victoria, Australia) set to 80 rpm and continuously stirred to induce water motion and reduce boundary layer effects. A 10 mL sample of the water was taken at 0 and 30 minutes, and frozen at -20°C, until defrosted and analyzed for NH4+ concentration using a QuickChem 8500 series 2 Automated Ion Analyzer (Lachat Instrument, Loveland, USA). The uptake rate (V) was determined according to Pedersen [42]] using the formula V = [(Si × voli)-(Sf × volf)]/(t × FW) where Si and Sf are the initial and final NH4+ concentrations (µM) over a period of time (t), vol is the seawater volume in the flask and FW is the fresh weight (g) of the algae.

Internal soluble NH4+ pools: The boiling water extraction method was used to determine the internal soluble NH4+ pool. Ulva australis tissue (0.18 ± 0.01 g FW) was put in a boiling tube with 20 mL of deionized water then placed in a boiling water bath for 40 minutes. The liquid was cooled, decanted, and then filtered through a 0.45 µm Whatman filter (GF/C). This process was repeated on the same algal piece three times and the concentration of internal soluble NH4+ pools was calculated using the sum of the NH4+ concentrations of the three water samples of each algal piece. NH4+ concentrations were measured as stated above.

Photosynthetic pigments: Following the experiment, a 0.04 ± 0.001 g FW piece of Ulva australis from each experimental chamber was kept at -20°C pending analysis. Each sample was then ground in 5 mL of 100% ethanol with a ceramic mortar and pestle in dim light and with the samples shaded. The extract was poured into 10 mL centrifuge tubes and placed in the dark at 4°C for six hours. Samples were then centrifuged for 10 min at 4000 rpm at 4°C. Total Chl a and b concentrations in the supernatant were determined according to the quadrichroic formula from Ritchie [2008] using a spectrophotometer (S-22 UV/Vis, Boeco, Germany).

A complete description of methods for implementation and monitoring of experimental treatments, and sampling methods to estimate response variables provided in the following publication:

Related Reference:

Reidenbach LB, Fernandez PA, Leal PP, Noisette F, McGraw CM, Revill AT, et al. (2017). Growth, ammonium metabolism, and photosynthetic properties of Ulva australis (Chlorophyta) under decreasing pH and ammonium enrichment. PLoS ONE 12(11): e0188389. https://doi.org/10.1371/journal.pone.0188389