Chlorophyll a

Chlorophyll samples (50–250 mL) were filtered onto either a GF/F or a 20μm polycarbonate filter using low vacuum pressure. Samples were extracted for at least 24 h in the dark at −20 deg C in 90% acetone and read on a Turner Designs fluorometer (Welschmeyer, 1994).

Welschmeyer, N.A. 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and phaeopigments. Limnology and Oceanography 39: 1985-1992.

Nanoplankton Abundance - Flow cytometry

Nanophytoplankton abundance was determined using flow cytometry. Two milliliters of sample were preserved in 1% seawater-buffered, 0.2 um-filtered formalin (final concentration) and frozen at −80 deg C until analysis. Samples were run on a FACSCalibur flow cytometer for 5 min on the high flow rate setting (Campbell, 2001). Nanophytoplankton were identified on two dimensional cytograms based on forward scatter (FSC) and red fluorescence (FL3).

Campbell, L. 2001. Flow cytometric analysis of autotrophic picoplankton. In Methods in Microbiology, 317-341: Academic Press.

Taxon-specific pigments - High Performance Liquid Chromatography

Samples for taxon-specific pigments (600–1000 mL) were filtered under low vacuum onto GF/F filters and frozen in liquid nitrogen until analysis using high performance liquid chromatography (HPLC). An automated Hewlett Packard 1100 HPLC system was used to separate pigments with a reverse-phase Waters Symmetry C-8 column and a solvent gradient containing methanol, aqueous pyridine, acetone, and acetonitrile (DiTullio and Geesey, 2002). A diode array detector was used to record pigment spectra between the wavelengths 350 and 600 nm, as well as continuous chromatograms at 410, 440, and 455 nm every 5 s, and Chl a and c were quantified with an HP 1046A fluorescence detector (excitation 421 nm, emission 666 nm). Unialgal laboratory cultures with the appropriate pigments were used to generate purified pigment standards for system calibration (DiTullio and Geesey, 2002).

DiTullio, G.R., and M.E. Geesey. 2002. Photosynthetic pigments in marine algae and bacteria. In Encyclopedia of Environmental Microbiology, ed. G. Bitton, 2453-2470. NY, NY: J. Wiley and Sons.

Maximum Quantum Yield Efficiency for PSII (Fv/Fm)

Photochemical efficiency of PSII was measured using a MBARI 4th generation bench-top fast repetition rate fluorometer (FRRF) (Kolber et al., 1994). Samples were collected each day from experimental bottles, immediately placed on ice and kept in low light conditions (5– 10 mol photons m−2 s−1) for 30–40 min prior to analysis. The light and cuvette chamber were constantly flushed with dry nitrogen gas to avoid condensation on the exterior of the cuvette due to the temperature difference between the cold seawater and the laboratory air. Minimal (F0) and maximal (Fm) fluorescence and the effective absorption cross section (ơPSII) were calculated from each single turnover (ST) saturation curve. The maximum quantum yield efficiency for PSII (Fv/Fm) was calculated (Genty et al., 1989) by normalizing Fm by the difference between the fluorescence at saturation (Fm) and the minimum fluorescence (F0):

Genty, B., J.M. Briantais, and N.R. Baker. 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990: 87-92.

Kolber, Z.S., R.T. Barber, K.H. Coale, S.E. Fitzwater, R.M. Greene, K.S. Johnson, S. Lindley, and G.P. Falkowski. 1994. Iron limitation of the phytoplankton photosynthesis in the equatorial Pacific Ocean. Nature 371: 145-149.