Fast repetition rate fluorometer (FRRF) study of sea ice algae in low iron conditions. Algae were grown in an ice tank and the measurements were made at the end with a Chelsea Insrtuments FRRF.
Materials and Methods (see the download document for original formatting and formulas)
1. Ice tank incubation
The polar pennate diatom Fragilariopsis cylindrus, isolated from Antarctic pack ice in 2015 (Davis station, East Antarctica) was incubated in a purpose designed ice tank (Island Research, Tasmania). The ice tank, which was contructed of titanium to minimise dissolved Fe, was placed into a freezer (–20 degrees C), and the ice thickness and temperature gradient controlled by interaction between a basal heater and the adjustable ambient freezer temperature (see Kennedy et al., 2012). This enabled an ice thickness of approximately 5.5 cm to be maintained during the experiment. The diatom F. cylindrus was incubated in Aquil media (Price et al. 1989) buffered with ethylenediaminetetraacetic acid (EDTA) at 150 micro mol photons m−2 s−1 (PAR), a salinity of 35, and a Fe concentration of 400 nM, where the concentration of total inorganic forms of Fe (Fe') was 1.54 nM, this being continuously supplied to the medium and the exact value calculated using the software Visual MINTEQ, ver. 3.1 (https://vminteq.lwr.kth.se). Before a freezing cycle started, the seawater temperature was maintained at 2.5 degrees C, and a sample was obtained to assess the original physiological state of the algae (Day−5, hereafter). After obtaining the sample, the seawater temperature was set to −1.8 degrees C to initiate ice formation in the ice tank. Once ice had formed at Day−2, the under-ice seawater was partially replaced with ultrapure water to reduce the salinity down to 35, because the salinity had increased (to approximately 38) as a result of brine rejection from the ice. After a 2-day acclimation to the new salinity, ice samples were obtained every 5 days for 20 days (i.e., Days 0, 5, 10, 15, and 20). To minimize the heterogeneity among ice cores, ice samples were randomly collected from the tank chamber with a trace metal-free hand drill (2 cm in diameter) from randomly annotated grids on the ice surface, following normal random sampling numbers generated by the software R (https://www.r-project.org/). To assess the effects of melting and high light exposure, the ice was melted at 2.5 degrees C for 2 days. After the ice had completely melted, the seawater was exposed to a high light level, which was adjusted to represent the likely summer light intensity at the surface in ice-edge regions (800 micro mol photons m−2 s−1; MODIS Aqua), Seawater samples were obtained both after the melting and light exposure events (Melt and Light, respectively, hereafter).
2. Fast repetition rate (FRR) fluorometry
To monitor the photophysiology of F. cylindrus during the freezing and melting processes, variable chlorophyll a fluorescence (ChlF) measurements were conducted using a bench-top Fast Repetition Rate fluorometer (FRRf) (FastOcean Act2Run Systems, Chelsea Technologies) with Act2Run software (Chelsea Technologies). Ice samples were directly thawed at 2 degrees C in the dark for 30 min, and the slushily melted ice samples were placed in a quartz tube and their flouresence (ChlF) was measured. A single turnover protocol was applied for the ChlF measurements; 100 flashlets with 1 micro second duration at a wavelength 450 nm and 2 micro second intervals for excitation of reaction centres of photosystem II (PSII, hereafter), and 20 flahlets with 1 μs duration and 100 micro second intervals for relaxation. Eighteen light steps were applied to generate a rapid light curve (RLC) from 0 to 1800 μmol photons m−2 s−1, taking less than 5 min to complete one RLC. At each light step (~15 s), at least five induction and relaxation curves were averaged to obtain ChlF yields, described in Table, after calibrating the ChlF yields with filtered seawater. According to the models proposed by Kolber et al. (1998), photosynthetic parameters of chlorophyll a (chl a) induction and relaxation curves were calculated based on the ChlF yields as shown in Table. Electron transport rate though the reaction centres of PSII (RCII) (ETRRCII) was calculated as per the equation detailed in the download document.