Dataset: Phytoplankton_Physiology_Fe_Temperature
Deployment: helicopter_Allen

Methodology:

Strains and growth conditions: Unialgal cultures of Pseudo-nitzschia subcurvata, Chaetoceros sp., Fragilariopsis cylindrus, and Phaeocystis antarctica were isolated from the ice edge in McMurdo Sound (77.62°S, 165.47°E) in the Ross Sea, Antarctica during January and February 2013. All stock cultures were maintained in 0.2 µM-filtered seawater that was collected using trace metal clean techniques from the same locale as the culture isolates (Hare et al. 2007, King et al. 2012). Cultures were grown at 0°C in a walk-in incubator under 24 hour (h) cold white fluorescence light (80 µmol photons m-2 s-1).

Experiments examined interactions between temperature and iron availability under four conditions: 0°C-Fe limited (+1nM Fe, abbreviated as 0C-Fe), 0°C-Fe replete (+500nM Fe, or 0C+Fe), 4°C-Fe limited (+1nM Fe, or 4C-Fe), and 4°C-Fe replete (+500nM Fe, or 4C+Fe). Fe concentration was amended by adding EDTA chelated FeCl3 (100: 1) into 0.2 µM-filtered trace metal-clean Ross Sea seawater. The seawater was collected late in the Antarctic summer, and so the concentrations of NO3 -, PO4 3- were relatively low for this region at 6.95 µmol L-1 and 0.66 µmol L-1 respectively. Si(OH)4 and dissolved Fe concentrations were 52.91 µmol L-1, and 0.2 nmol L-1 respectively (Feng et al. 2010). Chaetoceros sp. and one strain of Pseudo-nitzschia subcurvata (P. subcurvata) were grown in this seawater medium without any added nutrients. Another isolate of P. subcurvata, Fragilariopsis cylindrus, and Phaeocystis antarctica were maintained in the same four conditions, but the seawater was enriched with chelexed nutrient stocks to 50 µmol L-1 NO3 - and 10 µmol L-1 PO4 3- to examine growth effects of these two variables at higher nutrient levels. Cultures grown at high and low major nutrient levels will be identified as HN and LN treatments, respectively.

Experimental cultures were grown in triplicate 500 ml acid washed polycarbonate bottles under 24 h irradiance (80 µmol photons m-2 s-1). Semi-continuous culturing methods were used, whereby the cultures were diluted every 2 days with medium pre-acclimated to their respective temperatures. Dilution rates were based on the individually calculated growth rate of each replicate bottle (see ‘Growth rates’ section below), allowing each bottle to reach its own steady state exponential growth rate. All of the cultures were acclimated to their respective environmental conditions for 8 weeks before the commencement of the experiment. After the growth rates remained stable for at least three to five consecutive transfers, indicating steady state growth had been attained, the cultures were sampled 48 h after dilution.

Growth rates: Ten ml aliquot of culture samples were taken for visual cell counts directly before and after each treatment was diluted. Cell count samples were preserved with 0.5% glutaraldehyde (final concentration) and stored at 4°C for subsequent counting on a hemacytometer using an Olympus BX51 microscope (Olympus, Japan). Due to poor preservation, cell count samples of P. antarctica at 4°C for phosphorus cell quotas and Chl a per cell calculations were lost. Specific growth rates, expressed as h-1, were calculated as: µ = (ln N1 - ln N0)/t, where N0 and N1 are the cell density at the beginning and end of a dilution period, respectively, and t is the duration of the dilution period.

Elemental analysis: 50 ml and 20 ml culture samples of each treatment were filtered onto pre148 combusted GF/F filters (500°C for 2h) and dried in a 60°C oven overnight for particulate organic carbon/nitrogen (POC/PON) and particulate organic phosphorus (POP) analyses, respectively. POC/PON samples were analyzed using a 440 Elemental Analyzer (Costech Inc, Valencia, CA) following Fu et al. (2007) and Garcia et al. (2014). POP was analyzed using a molybdate colorimetric method according to Fu et al. (2007). A 20 ml aliquot of Chaetoceros sp. and P. subcurvata LN sample from each treatment was filtered onto 2 µm polycarbonate filters and dried in a 60°C oven overnight for biogenic silicate (BSi) analysis (Passche et al. 1973). Chlorophyll a analysis: 20 to 50 ml culture samples were filtered onto GF/F filters (Whatman) and extracted with 90% aqueous acetone for 24 h at -20°C, and measured using the non157 acidification method on a 10-AUTM fluorometer (Turner Designs, CA) (Fu et al. 2007). Cell volume and surface area: A minimum of 50 cells from each treatment were measured using an Olympus BX51 microscope (Olympus, Japan) with a coupled Excelis HD camera (ACCU8 SCOPE, NY). The length, height, or diameter of all cells were measured using ImageJ (NIH), and the volume and surface area of each cell was calculated following Hillebrand et al. (1999). Active fluorescence characteristics: A 6 ml aliquot of culture sample of P. subcurvata LN and Chaetoceros sp. LN from each treatment was dark-adapted for 5 min, and minimum fluorescence (F0) was measured using a 10-AUTM fluorometer (Turner Designs, CA). Next, maximum fluorescence (Fm) was recorded by adding 6 µl DCMU  (dichloromethylurea) to each sample followed by shaking for 30 seconds. The quantum efficiency of photosystem II Fv/Fm was calculated according to the equation Fv/Fm = (Fm – F0)/Fm (Schreiber 2004).

C fixation and Fe uptake rates: To measure carbon fixation rates and iron uptake rates, a 30 ml aliquot of culture sample from each treatment was incubated with 37 kBq 14C -bicarbonate (MP Biomedicals), or ~2 kBq 55FeCl3 (PerkinElmer, 0.33 nM 55FeCl3 complexed to 120 µmol L-1 EDTA) under their respective treatment conditions. Samples were filtered onto GF/F filters after 24 h incubation. For Fe uptake rate samples, the filters were washed in oxalate reagent for 5 min to remove surface-adsorbed Fe (Tovar-Sanchez et al. 2003). To correct for filter absorption of both radiotracers, the same amount of stock solution was added to a 30 ml aliquot of sample and filtered immediately; these filter absorption count values were subtracted from reported activities. The radioactivities of 14C and 55Fe in each sample were counted in a Tri-Carb 2500TR (Packard, now Perkin Elmer). Carbon fixation rates and Fe uptake rates were calculated using the initial dissolved inorganic carbon (DIC) concentrations and initial total Fe concentrations of each bottle (1nM Fe and 500 nM for iron limited and iron replete cultures, respectively), and were normalized to cell density (Garcia et al. 2014). Because 55Fe additions were a large fraction of the total Fe present in the Fe-limited samples, these uptake values represent an upper rate estimate for this treatment.

Statistical analysis: All statistical analyses, including student t-tests, ANOVA, Tukey’s HSD test, and two-way ANOVA were conducted using the open source statistical software R version 3.1.2 (Systat Software, CA).

References:

Feng Y, Hare CE, Rose JM, Handy SM, DiTullio GR, Lee PA, Smith WO, Peloquin J, Tozzi S, Sun J, Zhang Y, Dunbar RB, Long MC, Sohst B, Hutchins, DA (2010) Interactive effects of iron, irradiance and CO2 on Ross Sea phytoplankton. Deep-Sea Res I 57: 368–383 doi:101016/jdsr200910013

Garcia NS, Fu F, Sedwick PN, Hutchins DA (2014) Iron deficiency increases growth and nitrogen-fixation rates of phosphorus-deficient marine cyanobacteria. ISME J 2015(9): 238–245

Hare CE, DiTullio GR, Riseman SF, Crossley AC, Popels LC, Sedwick PN, Hutchins DA (2007) Effects of changing continuous iron input rates on a Southern Ocean algal assemblage. Deep-Sea Res I 54: 732-746

Hillebrand H, Dürselen CD, Kirschtel D, Pollingher U, Zohary T (1999) Biovolume calculation for pelagic and benthic microalgae. J Phycol 35(2): 403-424

King AL, Sañudo-Wilhelmy SA, Boyd PW, Twining BS, Wilhelm SW, Breene C, Ellwood MJ, Hutchins DA (2012) A comparison of biogenic iron quotas during a diatom spring bloom using multiple approaches.  Biogeosciences 9: 667–687

Paasche E (1973) Silicon and the ecology of marine plankton diatoms. I. Thalassiosira pseudonana (Cyclotella nana) grown in a chemostat with silicate as limiting nutrient. Mar Bio 19(2): 117-126

Schreiber U (2004) Pulse-amplitude-modulation (PAM) fluorometry and saturation pulse method: an overview. In: Chlorophyll a Fluorescence, Springer Netherlands, p279-319

Tovar-Sanchez A, Sañudo-Wilhelmy SA, Garcia-Vargas M, Weaver RS, Popels LC, and others (2003) A trace metal clean reagent to remove surface-bound iron from marine phytoplankton. Marine Chemistry 82(1): 91-99.