flow cytometry-N isotope studies

 DSCF9503 FiltrationPN DSCF9277 P1010497
In the vast subtropical ocean (>50% of the global ocean surface area), intense upper ocean stratification limits the nitrate supply from below such that regenerated nitrogen (N) forms (e.g., ammonium) is assumed to fuel most phytoplankton growth. However, it is notoriously difficult to quantify N uptake in such nutrient-poor systems. One approach is to measure the naturally occurring ratio of N isotopes (δ15N) in suspended particles (PN), with high-δ15N PN implying phytoplankton growth on deep nitrate and low-δ15N PN indicating dependence on regenerated N. Past measurements reveal that the δ15N of bulk PN is low throughout the sunlit surface layer (the euphotic zone) of the North Atlantic subtropical gyre (the Sargasso Sea), which has been interpreted as indicating a system supported predominantly by regenerated N, in keeping with expectations for such a N-poor region of the ocean. However, in addition to phytoplankton, bulk PN includes heterotrophic organisms (e.g., bacteria, small zooplankton) and detritus, and thus cannot address potential differences in the N sources used by different phytoplankton. To overcome this problem, we use flow cytometry to separate the important Sargasso Sea phytoplankton populations (figure 1), and then measure the δ15N of these sorted populations using the high sensitivity persulfate-denitrifier method.

Assimilation of upwelled nitrate by small eukaryotes in the Sargasso Sea


figure 1: Cytograms (i.e., flow cytometry data) showing the phytoplankton populations sorted from bulk particle collections at BATS in the summer. A) Red fluorescence, which is roughly indicative of chlorophyll content, as a function of polarized forward scatter, an approximation of size. B) Phycoerythrin (PE), a light-harvesting accessory pigment characteristic of Synechococcus, as a function of chlorophyll content. Figure from Fawcett et al., 2011.

For particles collected in summer at the Bermuda Atlantic Time-series Study (BATS) site (31°40’N; 64°10’W), we find that prokaryotic cyanobacteria (Prochlorococcus and Synechococcus) and eukaryotic phytoplankton have distinct N isotope signatures, indicating that they rely on different N sources (figure 2A). The prokaryotes have a uniformly low δ15N, suggesting that they consume mostly regenerated N. However, the more complex (but not much larger) eukaryotic phytoplankton have a high δ15N, implying that they are predominantly supported by deep nitrate; a simple calculation reveals that these eukaryotes rely on nitrate for more than 50% of their N despite the extremely low nitrate concentrations that are typical of the Sargasso Sea euphotic zone in summer. We conclude that eukaryotic phytoplankton at BATS specialize in the assimilation of nitrate supplied from depth, thereby dominating new production in this region.

The clearly different δ15N signatures of prokaryotic versus eukaryotic phytoplankton imply that these taxa are physiologically distinct, despite fairly minimal differences in their cell size. Moreover, the δ15N of sorted eukaryotes is similar to both the subsurface nitrate supply and the organic matter sinking out of the surface ocean, suggesting that sinking material derives largely from eukaryotic, not prokaryotic, phytoplankton biomass. This implies that the Sargasso Sea’s biological pump is driven by eukaryotic phytoplankton, even though they are two orders of magnitude less numerically abundant than the prokaryotes. These data have implications for paleoceanographic studies as they indicate a disconnect between the prokaryote-dominated biomass in the low-latitude surface ocean and the organic matter sinking to the seafloor, which, at least in the Sargasso Sea, appears to be predominantly of eukaryotic origin.


figure 2: A) A summertime example of the δ15N of Prochlorococcus (blue diamonds), Synechococcus (red squares), and eukaryotic phytoplankton (green circles) at BATS, separated from the bulk suspended PN pool (black crosses) by fluorescence activated cell sorting. The δ15N of the nitrate supply is shown by the purple arrow, and the mixed layer depth (MLD) and deep chlorophyll maximum (DCM) are indicated by the dashed horizontal lines. B-D) Histograms showing the number of occurrences of binned δ15N for sorted phytoplankton taxa at BATS in the summer, fall, and early winter. Figure adapted from Fawcett et al., 2011; 2014.

The counterintuitive effect of seasonal mixed layer deepening on nitrate assimilation by eukaryotes

Extending our coupled flow cytometry-N isotope approach to the fall and early winter reveals that Prochlorococcus and Synechococcus rely uniformly on recycled N (figure 2B and C). By contrast, eukaryotic phytoplankton appear to shift from summertime nitrate dependence to more complete reliance on recycled N in the fall and winter. This finding is counterintuitive given that the summer is considered the most stratified and nutrient-poor season at BATS; due to high heat fluxes and low wind stresses, the low-density, wind-mixed surface layer shoals (to <20 m at times) and the density gradient at its base strengthens, a state that is presumed to impede the upward mixing of subsurface nitrate. As the surface ocean cools into the fall, the mixed layer deepens, which is expected to correspond with an increased supply of nutrients to phytoplankton. However, our sorted δ15N data do not support this, and instead suggest that nitrate assimilation is greatest when the mixed layer is shallowest (i.e., in the summer).


figure 3: The hypothesized relationship between seasonal changes in the density structure of the upper water column and the nitrate supply to the euphotic zone. The x-axis indicates water density (top axis, red (summer) and blue (fall) lines) and the concentration of nitrate (bottom axis, solid gray lines). A) During the summer, the low-density mixed layer is very shallow, making it easier for nitrate to be imported into the lower euphotic zone below the mixed layer, where it is consumed by eukaryotic phytoplankton. B) In the fall and winter, the mixed layer deepens, reducing the potential for nitrate to be imported across the base of the euphotic zone and thus decreasing nitrate-supported phytoplankton growth. It has been hypothesized that, with global warming, the density structure of the upper tropical and subtropical ocean will resemble that shown in panel A for longer periods of the year, which might increase (rather than decrease) the total quantity of nitrate imported into the euphotic zone annually. Figure adapted from Fawcett et al., 2014.

To explain this, we turn to the stratification of the euphotic zone as a whole: In the summer, the mixed layer is so shallow that it does not contribute to stratification at the base of the euphotic zone. There is thus little impediment to low levels of subsurface nitrate being supplied into the photosynthetically-active lower euphotic zone (figure 3A) to be consumed by eukaryotic phytoplankton. Indeed, our seasonal δ15N data suggest that if nitrate is available, it will be consumed exclusively by eukaryotic phytoplankton (figure 2D). While this does not rule out the possibility of nitrate assimilation by the prokaryotes, which may occur during times of prolonged nitrate availability (e.g., following spring mixing), in the summer and fall, Prochlorococcus and Synechococcus rely predominantly on N recycled, thus contributing little to new production. The progressive deepening of the mixed layer from summer to fall winter, although reducing the surface-to-deep density contrast, increases the density difference of the euphotic zone as a whole from underlying nutrient-rich waters (figure 3B). Under these conditions, the subsurface nitrate supply to the euphotic zone decreases, and all phytoplankton must rely on recycled N forms.

Implications for subtropical ocean productivity under global warming: Over the coming decades, surface ocean density stratification characteristic of the subtropics and tropics is expected to strengthen and expand pole-ward. Low-latitude productivity is predicted to decline with increased stratification, rendering the ocean less efficient at sequestering atmospheric CO2; however, our work at BATS suggests that depending on the depth structure of this increase (figure 3), productivity throughout the sunlit surface ocean may actually be stable or even rise. Investigating this suggestion will be a focus of  future research, with a view to developing expectations for phytoplankton productivity and ecology, the carbon cycle, and low- and mid-latitude fisheries in response to increased stratification.

Relevant publications:

Treibergs, L.A., Fawcett, S.E., Lomas, M.W., Sigman, D.M. Nitrogen isotopic response of prokaryotic and eukaryotic phytoplankton to nitrate availability in Sargasso Sea surface waters. Limnology and Oceanography 59: 972-985 (2014). Treibergs L&O 2014

Fawcett, S.E., Lomas, M.W., Ward, B.B., Sigman, D.M. The counterintuitive effect of summer-to-fall mixed layer deepening on eukaryotic new production in the Sargasso Sea. Global Biogeochemical Cycles 28: 86-102 (2014). Fawcett GBC 2014

Newell, S.E., Fawcett, S.E., Ward, B.B. Depth distribution of ammonia oxidation rates and ammonia-oxidizer community composition in the Sargasso Sea. Limnology and Oceanography 58: 1491-1500 (2013). Newell L&O 2013

Fawcett, S.E., Lomas, M.W., Casey, J.R., Ward, B.B., Sigman, D.M. Assimilation of upwelled nitrate by small eukaryotes in the Sargasso Sea. Nature Geoscience 4: 717-722 (2011). Fawcett NatGeo 2011 (See also: news article)

Collaborators: John Casey, University of Hawaii; Michael Lomas, Bigelow Laboratory for Ocean Sciences; Daniel Sigman, Princeton University; Lija Treibergs, University of Connecticut; Bess Ward, Princeton University

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