Seawater for short 15N incubations was collected from three depths throughout the euphotic zone - the shallow euphotic zone (0-40m) where irradiances are high and nutrients are depleted, the DCM which is in the main NO3- gradient, and ~20m below the DCM where light is very low (<<1%) but NO3- concentrations are high - using clean methods employed by BATS for collecting samples for primary production. Prior to initiating the incubation we measured nutrient concentrations using high-sensitivity colorimetric methods to calculate true tracer (10% ambient) 15N additions. All incubations were carried out under simulated in-situ conditions of irradiance and temperature, which were maintained in screened deckboard incubators. Other water-column samples and data were collected using established protocols (Knap et al. 1997, Sedwick et al. 2005, Steinberg et al. 2001). As much as possible, we collected the seawater for our experiments and corresponding water-column samples and data at the same time of day, in order to minimize the possible confounding effects of diel variations.
15N-uptake measurements: Nitrogen (NH4+, urea, NO2-, and NO3-) uptake rates were measured following the isotopic-tracer procedures described previously (Lipschultz, 2001; Lomas et al., 2002; Slawyk et al., 1977). Incubations were short (4h) and run as a time course (1, 2 and 4h), with replicate bottles sacrificed at each time point. At the final time point from each incubation, seawater samples were taken for isotopic dilution of the added substrate and used in the calculation of uptake rates. Each incubation included control samples with no added 15N, but are taken through the entire incubation/sorting protocol to serve as our ‘initial’ isotopic value for rate estimations (Lipschultz, 2008). Whole population and cell-specific uptake rates were estimated sensu Dugdale and Goering (1967).
Following incubations, samples were filtered under gentle vacuum (<50 mm Hg), transferred to a vial with filtered seawater and paraformaldehyde (0.5% final concentration), kept at 4°C for ~1 hour, and stored in liquid nitrogen until analysis. Then, we split each sample into two subsamples immediately prior to sorting with one subsample sorted for Prochlorococcus and the other for total autotrophs. Next, we flow sorted the isotopically labeled cells using a previously published method (Casey et al., 2007). All samples were sorted with a Cytopeia Influx Cell Sorter (see facilities) using 0.2 µm filtered 3.6% NaCl solution as the sheath fluid and 488 nm laser excitation (we also have 355nm and 635nm solid state lasers). Sorting was optimized for maximum purity, rather than yield, by triggering on forward scatter, using single drop sorting and 2/16th drop criterion to ensure cells are away from drop boundaries. Purity of sorted samples was routinely assessed by post-sort re-analysis.
Once sorted, cells were filtered (0.2um silver filter) and analyzed on a Europa 20/20 isotope ratio mass spectrometer at the UC Davis isotope facility.
Prochlorococcus enumeration: Separate subsamples for total Prochlorococcus counts - by flow cytometry - were fixed in para-formaldehyde (0.5% final concentration), and stored in liquid nitrogen (Sieracki et al., 1995). Red fluorescence histogram of the total Prochlorococcus population, normalized to 0.53 µm bead fluorescence, will be used as a qualitative criteria to distinguish between ‘low-light’ and ‘high-light’ sub-populations (Campbell and Vaulot, 1993).
High sensitivity nutrient analysis and isotopic composition of dissolved substrates: Samples for NH4+, urea, NO2-, and NO3- were filtered through 0.8 µm polycarbonate filters in duplicate and analyzed immediately prior to the incubations in order to calibrate the 15N tracer addition. All analyses were colorimetric and run on an Alpkem Flow Solution IV autoanalyzer that will be modified as part of this project with fiber-optic flow cells to improve methodological sensitivity to the nanomolar level. Relevant methods are: NO3- and NO2- (Lomas et al., 2009), NH4+ (Li et al., 2005), urea (Cozzi, 2004) and PO4- (Li and Hansell, 2008; Li et al., 2008).
Methods References:
Campbell, L., Vaulot, D., 1993. Photosynthetic picoplankton community structure in the subtropical north Pacific ocean near Hawaii (station ALOHA). Deep-Sea Research (Part 1, Oceanographic Research Papers) 40, 2043-2060.
Casey, J.R., Lomas, M.W., Mandecki, J., Walker, D.E., 2007. Prochlorococcus contributes to new production in the Sargasso Sea deep chlorophyll maximum. Geophysical Research Letters 34 (10), L10604, doi:10.1029/2006GL028725.
Cozzi, S., 2004. A new application of the diacetyl monoxime method to the automated determination of dissolved urea in seawater. Marine Biology 145 (4), 843-848.
Dugdale, R.C., Goering, J.J., 1967. Uptake of new and regenerated forms of nitrogen in primary productivity. Limnology and Oceanography 12, 196-206.
Knap, A., A. Michaels, D. Steinberg, et al. 1997. BATS Methods Manual Version 4. U.S. JGOFS Planning Office.
Li, Q.P., Hansell, D.A., 2008. Intercomparison and coupling of magnesium-induced co-precipitation and long-path liquid-waveguide capillary cell techniques for trace analysis of phosphate in seawater. Analytica Chimica Acta 611 (1), 68-72.
Li, Q.P., Hansell, D.A., Zhang, J.Z., 2008. Underway monitoring of nanomolar nitrate plus nitrite and phosphate in oligotrophic seawater. Limnology and Oceanography-Methods 6, 319-326.
Li, Q.P., Zhang, J.Z., Millero, F.J., Hansell, D.A., 2005. Continuous colorimetric determination of trace ammonium in seawater with a long-path liquid waveguide capillary cell. Marine Chemistry 96 (1-2), 73-85.
Lipschultz, F., 2001. A time-series assessment of the nitrogen cycle at BATS. Deep-Sea Research 48 (8-9), 1897-1924.
Lipschultz, F., 2008. Isotope Tracer Methods for Studies of the Marine Nitrogen Cycle. In: Mulholland, M.R., Bronk, D.A., Capone, D.G., Carpenter, E.J. (Eds.), Nitrogen in the marine environment. Academic Press, New York, pp. 1345-1384.
Lomas, M.W., Lipschultz, F., Nelson, D.M., Bates, N.R., 2009. Biogeochemical responses to late-winter storms in the Sargasso Sea. I. Pulses of new and primary production. Deep Sea Research 56:843-860.
Lomas, M.W., Trice, T.M., Glibert, P.M., Bronk, D.A., McCarthy, J.J., 2002. Temporal and spatial dynamics of urea uptake and regeneration rates and concentrations in Chesapeake Bay. Estuaries 25 (3), 469-482.
Sedwick, P. N., T. M. Church, A. R. Bowie, et al. 2005. Iron in the Sargasso Sea (Bermuda Atlantic Time-series Study region) during summer: Eolian imprint, spatiotemporal variability, and ecological implications. Global Biogeochemical Cycles 19: GB4006, doi:10.1029/2004GB002445.
Sieracki, M.E., Haugen, E.M., Cucci, T.L., 1995. Overestimation of Heterotrophic Bacteria in the Sargasso-Sea - Direct Evidence by Flow and Imaging Cytometry. Deep-Sea Research Part I-Oceanographic Research Papers 42 (8), 1399.
Slawyk, G., Collos, Y., Auclair, J.C., 1977. Use of C-13 and N-15 Isotopes for Simultaneous Measurement of Carbon and Nitrogen Turnover Rates in Marine-Phytoplankton. Limnology and Oceanography 22 (5), 925-932.
Steinberg, D. K., C. A. Carlson, N. R. Bates, et al. 2001. Overview of the US JGOFS Bermuda Atlantic Time-series Study (BATS): a decade-scale look at ocean biology and biogeochemistry. Deep Sea Research Part II, 48:1405-1447.
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