skip to main content

This content will become publicly available on May 23, 2023

Title: Quantifying Per-Cell Chlorophyll a in Natural Picophytoplankton Populations Using Fluorescence-Activated Cell Sorting
Marine phytoplankton play a central role in global biogeochemical cycling, carbon export, and the overall functioning of marine ecosystems. While chlorophyll a (Chl a ) is widely used as a proxy for phytoplankton biomass, identifying the proportion of Chl a attributable to different phytoplankton groups remains a major challenge in oceanography, especially for the picophytoplankton groups that often represent the majority of phytoplankton biomass in the open ocean. We describe a method for measuring picophytoplankton per-cell Chl a in field samples using fluorescence-activated cell sorting followed by solvent-based Chl a extraction and fluorescence quantification. Applying this method to surface samples from the Gulf of Mexico, we determined per-cell Chl a to be 0.24 ± 0.07, 0.6 ± 0.33, and 26.36 ± 20.9 fg Chl a cell -1 for Prochlorococcus , Synechococcus , and PPE, respectively (mean ± SD). Measurements of per-cell Chl a using this method are precise to within 1.7, 2.1, and 3.1% for Prochlorococcus , Synechococcus , and PPE, respectively. We demonstrate that this approach can be used to obtain estimates of group-specific Chl a for Prochlorococcus , Synechococcus , and picophytoeukaryotes, the latter two of which cannot be captured by existing methods. We also demonstrate that measurements more » of per-cell Chl a made using this method in field samples are sufficiently precise to capture relationships between per-cell Chl a and cytometer red fluorescence, providing a bridge between biomass estimates from cell counts and bulk measurements of total Chl a . « less
Authors:
; ; ; ;
Award ID(s):
1458070 1737240
Publication Date:
NSF-PAR ID:
10331660
Journal Name:
Frontiers in Marine Science
Volume:
9
ISSN:
2296-7745
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Nitrogen (N) is a limiting nutrient in vast regions of the world’s oceans, yet the sources of N available to various phytoplankton groups remain poorly understood. In this study, we investigated inorganic carbon (C) fixation rates and nitrate (NO3), ammonium (NH4+) and urea uptake rates at the single cell level in photosynthetic pico-eukaryotes (PPE) and the cyanobacteriaProchlorococcusandSynechococcus. To that end, we used dual15N and13C-labeled incubation assays coupled to flow cytometry cell sorting and nanoSIMS analysis on samples collected in the North Pacific Subtropical Gyre (NPSG) and in the California Current System (CCS). Based on these analyses, we found thatmore »photosynthetic growth rates (based on C fixation) of PPE were higher in the CCS than in the NSPG, while the opposite was observed forProchlorococcus. Reduced forms of N (NH4+and urea) accounted for the majority of N acquisition for all the groups studied. NO3represented a reduced fraction of total N uptake in all groups but was higher in PPE (17.4 ± 11.2% on average) than inProchlorococcusandSynechococcus(4.5 ± 6.5 and 2.9 ± 2.1% on average, respectively). This may in part explain the contrasting biogeography of these picoplankton groups. Moreover, single cell analyses reveal that cell-to-cell heterogeneity within picoplankton groups was significantly greater for NO3uptake than for C fixation and NH4+uptake. We hypothesize that cellular heterogeneity in NO3uptake within groups facilitates adaptation to the fluctuating availability of NO3in the environment.

    « less
  2. Abstract. Oligotrophic regions play a central role in global biogeochemical cycles, with microbial communities in these areas representing an important term in global carbon budgets. While the general structure of microbial communities has been well documented in the global ocean, some remote regions such as the western tropical South Pacific (WTSP) remain fundamentally unexplored. Moreover, the biotic and abiotic factors constraining microbial abundances and distribution remain not well resolved. In this study, we quantified the spatial (vertical and horizontal) distribution of major microbial plankton groups along a transect through the WTSP during the austral summer of 2015, capturing important autotrophicmore »and heterotrophic assemblages including cytometrically determined abundances of non-pigmented protists (also called flagellates). Using environmental parameters (e.g., nutrients and light availability) as well as statistical analyses, we estimated the role of bottom–up and top–down controls in constraining the structure of the WTSP microbial communities in biogeochemically distinct regions. At the most general level, we found a typical tropical structure, characterized by a shallow mixed layer, a clear deep chlorophyll maximum at all sampling sites, and a deep nitracline. Prochlorococcus was especially abundant along the transect, accounting for 68±10.6% of depth-integrated phytoplankton biomass. Despite their relatively low abundances, picophytoeukaryotes (PPE) accounted for up to 26±11.6% of depth-integrated phytoplankton biomass, while Synechococcus accounted for only 6±6.9%. Our results show that the microbial community structure of the WTSP is typical of highly stratified regions, and underline the significant contribution to total biomass by PPE populations. Strong relationships between N2 fixation rates and plankton abundances demonstrate the central role of N2 fixation in regulating ecosystem processes in the WTSP, while comparative analyses of abundance data suggest microbial community structure to be increasingly regulated by bottom–up processes under nutrient limitation, possibly in response to shifts in abundances of high nucleic acid bacteria (HNA).

    « less
  3. Abstract
    Excessive phosphorus (P) applications to croplands can contribute to eutrophication of surface waters through surface runoff and subsurface (leaching) losses. We analyzed leaching losses of total dissolved P (TDP) from no-till corn, hybrid poplar (Populus nigra X P. maximowiczii), switchgrass (Panicum virgatum), miscanthus (Miscanthus giganteus), native grasses, and restored prairie, all planted in 2008 on former cropland in Michigan, USA. All crops except corn (13 kg P ha−1 year−1) were grown without P fertilization. Biomass was harvested at the end of each growing season except for poplar. Soil water at 1.2 m depth was sampled weekly to biweekly for TDP determination during March–November 2009–2016More>>
  4. Abstract. We assessed the influence of the marine diazotrophic cyanobacterium Trichodesmium on the bio-optical properties of western tropical South Pacific (WTSP) waters (18–22°S, 160°E–160°W) during the February–March 2015 OUTPACE cruise. We performed measurements of backscattering and absorption coefficients, irradiance, and radiance in the euphotic zone with a Satlantic MicroPro free-fall profiler and took Underwater Vision Profiler 5 (UPV5) pictures for counting the largest Trichodesmium spp. colonies. Pigment concentrations were determined by fluorimetry and high-performance liquid chromatography and picoplankton abundance by flow cytometry. Trichome concentration was estimated from pigment algorithms and validated by surface visual counts. The abundancemore »of large colonies counted by the UVP5 (maximum 7093coloniesm−3) was well correlated to the trichome concentrations (maximum 2093trichomesL−1) with an aggregation factor of 600. In the Melanesian archipelago, a maximum of 4715trichomesL−1 was enumerated in pump samples (3.2m) at 20°S,16730°E. High Trichodesmium abundance was always associated with absorption peaks of mycosporine-like amino acids (330, 360nm) and high particulate backscattering, but not with high Chl a fluorescence or blue particulate absorption (440nm). Along the west-to-east transect, Trichodesmium together with Prochlorococcus represented the major part of total chlorophyll concentration; the contribution of other groups were relatively small or negligible. The Trichodesmium contribution to total chlorophyll concentration was the highest in the Melanesian archipelago around New Caledonia and Vanuatu (60%), progressively decreased to the vicinity of the islands of Fiji (30%), and reached a minimum in the South Pacific Gyre where Prochlorococcus dominated chlorophyll concentration. The contribution of Trichodesmium to zeaxanthin was respectively 50, 40 and 20% for these regions. During the OUTPACE cruise, the relationship between normalized water-leaving radiance (nLw) in the ultraviolet and visible and chlorophyll concentration was similar to that found during the BIOSOPE cruise in the eastern tropical Pacific. Principal component analysis (PCA) of OUTPACE data showed that nLw at 305, 325, 340, 380, 412 and 440nm was strongly correlated to chlorophyll and zeaxanthin, while nLw at 490 and 565nm exhibited lower correlations. These results, as well as differences in the PCA of BIOSOPE data, indicated that nLw variability in the greenish blue and yellowish green during OUTPACE was influenced by other variables associated with Trichodesmium presence, such as backscattering coefficient, phycoerythrin fluorescence and/or zeaxanthin absorption, suggesting that Trichodesmium detection should involve examination of nLw in this spectral domain.

    « less
  5. Johnson, Karyn N. (Ed.)
    ABSTRACT Coral reefs are possible sinks for microbes; however, the removal mechanisms at play are not well understood. Here, we characterize pelagic microbial groups at the CARMABI reef (Curaçao) and examine microbial consumption by three coral species: Madracis mirabilis , Porites astreoides , and Stephanocoenia intersepta . Flow cytometry analyses of water samples collected from a depth of 10 m identified 6 microbial groups: Prochlorococcus , three groups of Synechococcus , photosynthetic eukaryotes, and heterotrophic bacteria. Minimum growth rates (μ) for Prochlorococcus , all Synechococcus groups, and photosynthetic eukaryotes were 0.55, 0.29, and 0.45 μ day −1 , respectively, and suggestmore »relatively high rates of productivity despite low nutrient conditions on the reef. During a series of 5-h incubations with reef corals performed just after sunset or prior to sunrise, reductions in the abundance of photosynthetic picoeukaryotes, Prochlorococcus and Synechococcus cells, were observed. Of the three Synechococcus groups, one decreased significantly during incubations with each coral and the other two only with M. mirabilis. Removal of carbon from the water column is based on coral consumption rates of phytoplankton and averaged between 138 ng h −1 and 387 ng h −1 , depending on the coral species. A lack of coral-dependent reduction in heterotrophic bacteria, differences in Synechococcus reductions, and diurnal variation in reductions of Synechococcus and Prochlorococcus , coinciding with peak cell division, point to selective feeding by corals. Our study indicates that bentho-pelagic coupling via selective grazing of microbial groups influences carbon flow and supports heterogeneity of microbial communities overlying coral reefs. IMPORTANCE We identify interactions between coral grazing behavior and the growth rates and cell abundances of pelagic microbial groups found surrounding a Caribbean reef. During incubation experiments with three reef corals, reductions in microbial cell abundance differed according to coral species and suggest specific coral or microbial mechanisms are at play. Peaks in removal rates of Prochlorococcus and Synechococcus cyanobacteria appear highest during postsunset incubations and coincide with microbial cell division. Grazing rates and effort vary across coral species and picoplankton groups, possibly influencing overall microbial composition and abundance over coral reefs. For reef corals, use of such a numerically abundant source of nutrition may be advantageous, especially under environmentally stressful conditions when symbioses with dinoflagellate algae break down.« less