Dataset: Perturbation experiments: pigments

Sampling and Analytical Methodology: 

Experiments were conducted between July 2011 and April 2013 during five research cruises to Station ALOHA (22.75°N, 158°W), the well-characterized study site of the Hawaii Ocean Time-series (HOT) program.  Sampling occurred during four HOT cruises and one Center for Microbial Oceanography: Research and Education (C-MORE) cruise (termed HOE-DYLAN 5) aboard the R/V Kilo Moana. Seawater was collected in 12 L polyvinylchloride bottles affixed to a 24-bottle rosette sampler equipped with a Sea-Bird 911+ conductivity, temperature, and depth profiler. Nine 20-L polycarbonate carboys were filled with 25 m Station ALOHA seawater pre-filtered off the rosette sampler through a Nitex mesh (pore size ~202 μm) to exclude larger zooplankton. Of these, 3 carboys received additions of nitrate (target ~2.8 μM N final concentration as NaNO₃) and three carboys received additions of ammonium (target ~2.8 μM N final concentration as NH₄Cl). All carboys, including three ‘Control’ carboys, received additions of phosphate (target ~0.2 μM P final concentration as KH₂PO₄) and silicic acid (target ~2.8 μM Si final concentration as Na₂SiO₃) to achieve a final N:P:Si stoichiometric ratio (14:1:14). Carboys were incubated for 120 to 144 hours and subsampled at approximately daily scales throughout the experiment (Table 1). All sampling was conducted before sunrise in order to allow productivity rate measurements to span the full photoperiod.

For photosynthetic pigment analyses using high performance liquid chromatography (HPLC), seawater (2 L) was collected into brown, narrow-mouthed HDPE bottles and subsequently filtered using a peristaltic pump onto 25 mm diameter, GF/F filters. Filters were immediately flash-frozen in liquid nitrogen and stored at -80°C until analyzed. Photosynthetic pigments were extracted from the filters in 3 mL 100% acetone (HPLC grade) in culture tubes along with 50 µL canthaxanthin, an internal standard, and placed at 4oC for 24 hours. Chlorophyll and carotenoid pigments were separated on a Varian 9012 HPLC system and analyzed using SpectraSYSTEM Thermo Separation Products dual wavelength UV/VIS UV2000 and fluorescence FL2000 detectors. Pigment identifications were based on absorbance spectra, co-chromatography with standards, and relative retention time with a monovinyl Chl a standard and representative culture extracts, and Spectra-Physics WOW® software was used to calculate peak area.