Phosphate and adenosine-59-triphosphate uptake by cyanobacteria and heterotrophic bacteria in the Sargasso Sea

Transcription

1 Limnol. Oceanogr., 56(1), 2011, E 2011, by the American Society of Limnology and Oceanography, Inc. doi:10.319/lo Phosphate and adenosine-59-triphosphate uptake by cyanobacteria and heterotrophic bacteria in the Sargasso Sea Vanessa K. Michelou, a,1 Michael W. Lomas, b and David L. Kirchman a,* a School of Marine Science and Policy, University of Delaware, Lewes, Delaware b Bermuda Institute of Ocean Sciences, St. George s, Bermuda Abstract We used flow cytometry sorting to quantify assimilation of phosphate and adenosine-59-triphosphate (ATP) by heterotrophic bacteria and the cyanobacteria Prochlorococcus and Synechococcus during the fall of 2007 and 2008 and the spring of 2009 in the western Sargasso Sea. Phosphate and ATP uptake rates per cell were 50-fold and 80- fold higher, respectively, for Synechococcus than for the other two microbial groups, whereas there was no significant difference between ATP and phosphate uptake per cell by Prochlorococcus and heterotrophic bacteria. Both cyanobacterial groups had higher rates of phosphate uptake per surface area than did the heterotrophic bacteria; Synechococcus had the highest rates per cellular phosphorus (P) quota for ATP uptake, and Prochlorococcus usually had lower rates. Heterotrophic bacteria had the lowest uptake rates of ATP and phosphate per cellular P quota. In contrast, total uptake of phosphate and ATP per liter of seawater was dominated by heterotrophic bacteria, while uptake by Prochlorococcus and Synechococcus was less than 10% of the total, as a result of their low abundance (15% and 1.5% of total prokaryotes, respectively). Uptake rates of phosphate and ATP for heterotrophic bacteria and Prochlorococcus were less tightly coupled than for Synechococcus, and phosphate appeared to be favored over ATP for all three groups. Uptake rates of both compounds by Synechococcus were much higher than by the other microbial groups. Cyanobacteria are successful competitors for phosphate and ATP in the Sargasso Sea. Phosphorus (P) is now widely recognized as an important limiting nutrient for biomass production in the world s oligotrophic oceans and large seas as a result of low concentrations and supply rates of P-containing compounds. Phosphate concentrations in Sargasso Sea surface waters, for example, are low (, 5 nmol L 21 ) during the highly stratified summer months near Bermuda and can decline further southward through the North Atlantic (Wu et al. 2000). These low concentrations have been suggested to limit phytoplankton and heterotrophic bacterial production in the subtropical North Atlantic (Cotner and Wetzel 1992). In the absence of sufficient phosphate, phytoplankton and heterotrophic bacteria may assimilate substantial quantities of dissolved organic phosphorus (DOP) to satisfy cellular P quotas and support biomass production. Throughout most of the year in the Sargasso Sea, more than 80% of total dissolved P exists as DOP (Ammerman et al. 2003; Lomas et al. 2009). However, little is known about the uptake of phosphate and DOP by various microbial groups in these P-limited environments. Many studies have examined the role of heterotrophic bacteria vs. large phytoplankton in P cycling, mostly in freshwater environments, but much less is known about P uptake by cyanobacteria (Vadstein 2000). Heterotrophic bacteria are thought to be responsible for about 60% of the phosphate uptake in various aquatic ecosystems (Kirchman 2000), as determined by the phosphate taken up by the plankton fraction that passes through polycarbonate filters * Corresponding author: kirchman@udel.edu 1 Present address: Hopkins Marine Station, Stanford University, Pacific Grove, California 323 with a small pore size ( mm). However, some of this uptake might be due to cyanobacteria (Prochlorococcus and Synechococcus) or other small phytoplankton found in the same size fraction. A size fractionation study indicated that Synechococcus ( mm size fraction) had significantly higher phosphate uptake rates than heterotrophic bacteria (Moutin et al. 2002), but large heterotrophic bacteria and small eukaryotic microbes (picoeukaryotes) probably are also in the Synechococcus size fraction (Vaulot et al. 2008). Currently, the only approach that adequately separates heterotrophic bacteria, cyanobacteria, and picoeukaryotes is flow cytometric sorting (Czechowska et al. 2008). One of the few studies using this approach confirmed that heterotrophic bacteria are the main consumers of phosphate in the surface waters of the central North Atlantic Ocean, even though the per-cell rates for one group of heterotrophic bacteria (SAR11) were lower at two of three stations than the per-cell rates for Prochlorococcus and Synechococcus (Zubkov et al. 2007); the per-cell rates for the entire heterotrophic bacterial community were not examined. Prochlorococcus accounted for a large fraction of phosphate uptake, while Synechococcus and picoeukaryotic phytoplankton played only a minor role in the uptake of this compound in these P-limited waters (Zubkov et al. 2007; Larsen et al. 2008). More work is needed to determine the contribution of these bacterial groups to P cycling and the relative competitive abilities for P uptake among microbial groups. Size fractionation studies indicate that heterotrophic bacteria also dominate DOP uptake in lakes (Berman 1988), but less is known about oceanic microbes. One of the few marine studies using size fractionation found that uptake of adenosine-59-triphosphate (ATP) per liter by

2 32 Michelou et al. Fig. 1. Locations of sampling stations in the Sargasso Sea: Cruise Bval 39 (open circles; October 2007), X0808 (open squares; September 2008), and BATS2 (cross; March 2009; referred to as B2). The contour is soluble reactive phosphate concentrations at 25 m; data to the west of 3uW are from this study, while data to the east of 3uW are from the study of Mather et al. (2008). All of the stations from Casey et al. (2009) (filled circles) and Zubkov et al. (2007) (filled triangles) where taxon-specific uptake was measured. Bermuda is located at 32u189N, 6u79W. heterotrophic bacteria was much higher than by phytoplankton in Baltic Sea mesocosms (Lovdal et al. 2007), but uptake by cyanobacteria was not examined. The proportion of heterotrophic bacteria and Prochlorococcus with alkaline phosphatase activity was very low compared to larger phytoplankton in the Mediterranean and Arabian Seas (Duhamel et al. 2008). In contrast, Casey at al. (2009) found that cyanobacteria are physiologically superior to larger eukaryotes in scavenging organic as well as inorganic P in the Sargasso Sea. However, that study did not compare cyanobacteria to heterotrophic bacteria, and we are not aware of any other study using flow cytometry to examine DOP uptake in the oceans. Results from culture experiments indicate that Synechococcus populations must rely on the DOP pool to support growth in P-limited waters (Fu et al. 2006), but it is unclear whether cyanobacteria can effectively compete with heterotrophic bacteria for DOP in oligotrophic regions of the oceans. The goal of this study was to use flow cytometry to examine measures of uptake of 33 P-ATP and 33 P-PO 3{ by heterotrophic bacteria, Synechococcus, and Prochlorococcus in the Sargasso Sea. We expected heterotrophic bacteria to have the highest per-cell uptake rates and to dominate total assimilation of phosphate and ATP because their high surface area to-volume ratio makes them superior competitors for both of these P compounds and because they are the most abundant prokaryotes in the Sargasso Sea (Carlson et al. 1996). We found that cyanobacteria were capable of assimilating organic and inorganic P at significant rates, even though their contribution to total uptake was low as a result of their low abundance. Methods Samples were collected from cruises in the North Atlantic subtropical gyre during October 2007, September 2008, and March The uptake rates for cyanobacteria during the fall of 2007 were reported by Casey et al. (2009), but here we add new data from the other two cruises, in addition to estimates for heterotrophic bacteria. Transects from Bermuda extended as far north as 38uN and as far south as 19uN and from 55uW to 66uW (Fig. 1). Eight stations were sampled in October, four in September, and five in March. Water for taxon-specific experiments was collected from 5 m, 0 m, and the deep chlorophyll maximum (ranging from 80 to 120 m) in acid-cleaned Niskin bottles and kept in subdued lighting until incubation. We quantified assimilation of 15-pmol L 21 (final concentration) additions of 33 PO 3{ ( 6 Ci mmol 21 ) and c-at 33 P (3000 Ci mmol 21 ). Assimilation was measured in triplicate incubations in the dark at the in situ temperature

3 P and cyano- and heterotrophic bacteria 325 for 1 h. Killed controls consisted of samples to which paraformaldehyde (PFA; 2% final concentration) was added before the addition of isotopes. At the end of the incubation, samples were fixed with 2% PFA and stored frozen in liquid nitrogen before analysis by flow cytometry and sorting in the lab. Flow cytometry analysis Samples for picoplankton enumeration by flow cytometry were collected from the same depths as the uptake measurements and fixed with PFA (2% final concentration) at uc for 2 h before being frozen. Samples were analyzed on a Becton Dickinson (formerly Cytopeia) high-speed jet-in-air InFlux Cell Sorter at an average flow rate of 0 ml min 21. Samples were analyzed for Prochlorococcus, Synechococcus, and heterotrophic (non-pigmented) bacteria. A few cells counted as heterotrophic bacteria may be archaea, but their abundance is negligible in the surface layer of the Sargasso Sea (Herndl et al. 2005). After exclusion of laser noise gated on pulse width and forward light scatter (FSC), cyanobacterial cells were discriminated by chlorophyll (to separate autotrophs from similarly sized heterotrophs) and phycoerythrin fluorescence (to separate Prochlorococcus from Synechococcus), as well as FSC (Olson et al. 1993; Gasol and Del Giorgio 2000). Samples for total prokaryote enumeration were stained with a green fluorescent nucleic acid stain (SYTO 13) (5-mmol L 21 final concentration) for min at room temperature in the dark (Troussellier et al. 1999). A70-mm nozzle tip was used with a sample pressure of 196,500 Pa (689 Pa higher than the sheath fluid pressure) to optimize speed while maintaining high fluorescent signal resolution. Sheath fluid was made fresh daily from deionized water and molecular-grade NaCl and was filtered through a 0.2-mm capsule filter (Pall Life Sciences). A 100- mw blue (88-nm) excitation laser operating at 100% power was used in conjunction with three color and two scatter detectors. Analog signals from red (. 650-nm), orange (585-nm), and green (530-nm) band pass filters as well as direct laser light from FSC and side scatter (SSC) detectors (Hamamatsu C6270 photomultiplier tubes) were log amplified and converted to digital input and output. Mean coincident abort rates were, 1% and mean recovery from secondary sorts (n 5 25) was 97.5% 6 1.1% (data not shown). Spigot (Becton Dickinson) was used for data acquisition and FCS Express version 3 (DeNovo Software) was used for post-acquisition analysis. Estimates of surface area from flow cytometry and epifluorescence microscopy We directly related forward light scattering to cell size of cyanobacteria and heterotrophic bacteria using empirically determined calibrations (Olson et al. 1993). The FSC geometric mean area for a specific target cell gate was compared to the total FSC area of all autotrophic and heterotrophic cells, as described in Casey et al. (2009). Cell size and forward light scattering are influenced by the cell s refractive index and shape (Green et al. 2003). However, for spherically shaped cells, there is a strong correlation between forward light scatter (FLS) and cell size, despite small changes in refractive index (Durand et al. 2001). For this study, the relationships between light scatter and particle diameter were determined daily on the cytometer used for analysis. Particle diameter and FSC were always found to follow a positive linear relation (r , Model 1 linear regression) on a log log scale as determined by using 0.53-mm, 3.0-mm, and 6.0-mm polystyrene calibration beads (Spherotech). Cell surface area was calculated assuming all particles were spheres, thus: SA~ p ( a 2 mzb)2 where SA is the mean surface area (mm 2 ); a is the logtransformed geometric mean FSC of the target gate; and m and b are the slope and intercept, respectively, of the Model I linear regression of forward angle light scatter and calibration bead diameter (Casey et al. 2009). The relationship between heterotrophic bacterial surface areas estimated by flow cytometry and by epifluorescence microscopy was assessed in samples from two cruises (October 2007 and September 2008). Surface area and cell abundance by epifluorescence microscopy were estimated for cells stained with 9,6-diamidino-2-phenylindole, and then size was determined by image analysis, as described previously (Sieracki et al. 1989). Surface area estimates from both methods were significantly related (r , p, 0.001, n 5 20), and the slope of a linear regression analysis (epifluorescence vs. flow cytometry) was not significantly different from 1 (y X, R ; p, 0.001), indicating that the two methods produce similar estimates of surface area (data not shown). Other studies also found that cell size for heterotrophic bacteria estimated by flow cytometry was statistically the same as that estimated by epifluorescence microscopy (Felip et al. 2007). Flow cytometric counts of bacteria stained with SYTO 13 are also highly correlated with epifluorescence microscopic counts in seawater samples (Troussellier et al. 1999). Sorting after uptake of phosphate and ATP After the incubations with 33 PO 3{ or AT 33 P, duplicate samples containing 50, ,000 Synechococcus, Prochlorococcus, and stained heterotrophic bacterial cells were sorted, filtered, and assayed by liquid scintillation counting. Sorted cells were gently filtered onto 0.2-mm Nuclepore polycarbonate filters and rinsed with 10 ml of 0.2-mm filtered seawater. The filters were placed in scintillation cocktail (Ultima Gold, Analytical Sciences) and radioassayed. Cellspecific uptake rates were calculated as follows: r Pi ~ b ð ln 2 DT l Þ sort R ð2þ n b TA T inc where r Pi is the cell-specific utilization rate (amol 33 Pi or AT 33 P cell 21 h 21 ); b sort and b TA are the radioactivity (Bq min 21 ) of the sorted sample and the total radioactivity added, respectively; n is the number of cells sorted; DT is the elapsed time from 33 P isotopic tracer addition to counting; T inc is the incubation time; l is the decay constant of 33P (half life d); and P is the ambient concentration of the P source (nmol L 21 ). Soluble reactive ð1þ

4 326 Michelou et al. Fig PO 3{ uptake rates of three flow-sorted microbial groups and the sum of the three vs. total rates. The three groups were heterotrophic bacteria (hbac), Prochlorococcus (Pro), and Synechococcus (Syn). The lines are linear regressions, which were significant for each group and for the sum (slope for the sum , p, ). phosphate concentrations were measured for all samples using standard methods, except for those samples collected in September For that cruise, we assumed concentrations were equal to values measured on another cruise to our stations 3 d after our cruise. An ATP concentration of 1 nmol L 21 was assumed based upon chemical data from the North Pacific (Karl and Bossard 1985) and bioassaymeasured ATP in the North Atlantic (Zubkov et al. 2007). This concentration was also used by Casey et al. (2009). The average uptake was determined from the slope of the linear regression of radioactivity vs. the number of sorted cells. Uptake rates based upon a single sorted sample were not significantly different from utilization rates using the slope of radioactivity vs. cell number on the same sample (data not shown). Sorting purity was assessed routinely by sorting microbes from a sample and re-analyzing the sorted cells by flow cytometry. This test confirmed that the sorted samples contained nearly only the targeted microbial group. We also compared total P uptake and the sum of the three sorted microbial groups. Uptake by a group was calculated by multiplying the mean per-cell uptake of a group by the cell abundance of that group. There was little difference between the measured total and the calculated sum of the groups (98.% 6 1.1%) (Fig. 2). Fixation of cells prior to analysis may lead to some leakage of the isotope (Larsen et al. 2008). In several tests, we found that 25% 6 5% of the isotope in phosphate incubations leaks from cells within 2 h (data not shown). The quantity lost from Synechococcus and Prochlorococcus did not differ significantly (data not shown). These results agree with those of others testing under similar conditions (Larsen et al. 2008). Leakage from cells in ATP incubations is lower (13% 6 7%) within 2 h. The uptake rates presented in this article have not been corrected for this leakage. Fig. 3. Average abundance of Prochlorococcus (Pro), Synechococcus (Syn), and heterotrophic bacteria (hbac) during October 2007, September 2008, and March Error bars represent standard errors of six stations. Results Microbial abundance and uptake rates Uptake of phosphate and ATP and microbial abundance were determined in the Sargasso Sea in October 2007, September 2008, and March Total prokaryotic abundance varied with depth more than with location during all months (Fig. 3). Abundance of heterotrophic bacteria ranged from cells ml 21 to cells ml 21 at the surface on the three sampling dates (Fig. 3). Prochlorococcus abundance at the surface ranged from cells ml 21 in September 2008 to cells ml 21 in October This group reached maximum numbers above the bottom of the euphotic zone between 80 and 120 m and was least abundant at the surface. In contrast, Synechococcus abundance decreased steadily with depth and was lowest around 120 m ( cells ml 21 ). Overall, the average abundances were cells ml 21 for heterotrophic bacteria, cells ml 21 for Prochlorococcus, and cells ml 21 for Synechococcus for all cruises. Phosphate and ATP uptake per cell Cyanobacteria and bacteria without chlorophyll a ( heterotrophic bacteria)

5 P and cyano- and heterotrophic bacteria 327 Fig.. Uptake rates per cell of (A) ATP and (B) phosphate (PO 3{ ) by Prochlorococcus (Pro), Synechococcus (Syn), and heterotrophic bacteria (hbac) for October 2007, September 2008, and March Error bars indicate standard errors of all dates. Notice the log scale of the y-axis. Statistically significant differences (Student s t-test: t , df 5 21, p, 0.001) are indicated by the different letters; means with the same letter are not significantly different. were sorted by flow cytometry to determine phosphate and ATP assimilation per cell. ATP uptake rates for Prochlorococcus and heterotrophic bacteria were not significantly different (Student s t-test: t , df 5 21, p ), while ATP uptake rates by Synechococcus were significantly higher than by the other groups (Student s t-test: t , df 5 21, p ; Fig. A). The rates for Synechococcus averaged amol P cell 21 h 21 over all depths, while heterotrophic bacteria and Prochlorococcus had lower per-cell ATP uptake rates of amol P cell 21 h 21 and amol P cell 21 h 21, respectively. Synechococcus also had the highest per-cell uptake rates of phosphate, although rates varied with depth differently than ATP uptake (Fig. B). The per-cell rate for Synechococcus averaged over all depths was 2.3 amol P cell 21 h 21, a measure that is approximately two- to fivefold higher than rates for the other two microbial groups. Per-cell uptake rates of phosphate by Prochlorococcus and heterotrophic bacteria were not significantly different (p. 0.05, n 5 68), averaging and 0.12 amol P cell 21 h 21, respectively. Phosphate uptake was highest at the surface for all three groups (Fig. B). Uptake of phosphate and ATP per surface area and per cellular P quota P uptake normalized to average cell surface area was examined because of the difference in cell size for the three bacterial groups. For simplicity, the averages for the entire water column are given for each station (Table 1); the differences among the three groups did not vary substantially with depth (data not shown). Surface area normalized uptake rates of phosphate by Synechococcus were higher than for heterotrophic bacteria, which in turn were higher than the rates for Prochlorococcus (Table 1) (p, 0.001, n 5 68), except in October 2007, when rates for the heterotrophic bacteria were too variable. Overall, Synechococcus had higher surface area normalized uptake rates for phosphate and ATP than did heterotrophic bacteria and Prochlorococcus. We also examined P uptake normalized to the cellular P quota of each microbial group analyzed (Table 2). The assumed P quotas for Prochlorococcus and Synechococcus ( fg P cell 21 and fg P cell 21, respectively) (Bertilsson et al. 2003) were lower than those for heterotrophic bacteria ( fg P cell 21 ) (Gundersen et al. 2002). Uptake rates of phosphate by Synechococcus per cellular P were higher than for Prochlorococcus, which in turn were higher than the rates for heterotrophic bacteria (Table 2). Synechococcus had the highest ATP uptake rates per cellular P during all cruises, while uptake by heterotrophic bacteria was lowest (October 2007 and September 2008) or equal to rates by Prochlorococcus (March 2009). Overall, heterotrophic bacteria had the lowest ATP uptake rates, normalized to cellular P content, during our study. Total uptake of ATP and phosphate The contribution of each microbial group to total uptake of ATP and phosphate was calculated by multiplying the per-cell rates by the cell abundance for each group. Heterotrophic bacteria dominated total ATP uptake throughout the water column, with rates highest at 120 m (Fig. 5A). There was no difference between total ATP uptake by Prochlorococcus and Synechococcus between 0 and 80 m, even though there were more Prochlorococcus cells than Synechococcus cells at 0 and 80 m, because cell-specific uptake compensated for cell numbers to some degree. At

6 328 Michelou et al. Table 1. Uptake of PO 3{ and ATP normalized to surface area for each sorted microbial group. Uptake rates were averaged over the entire water column. Standard error values are presented. hbac, heterotrophic bacteria; Pro, Prochlorococcus; Syn, Synechococcus; BVAL 39, October 2007 cruise; X0808, September 2008 cruise; B2, March 2009 cruise; HS, hydrostation; Bats, Bermuda time-series station. Surface area normalized uptake rates (fmol P cm 22 h 21 ) PO 3{ ATP Cruise hbac SE Pro SE Syn SE hbac SE Pro SE Syn SE BVAL Mean (SE) (13.67) (1.69) (25.68) (1.17) (0.6) (6.22) X0808 HS Bats HS Mean (SE) (5.) (0.56) (18.82) (0.3) (0.28) (2.73) B2 HS Bats Mean (SE) (2.18) (1.26) (16.2) (0.56) (0.05) (.9) 120 m, ATP uptake per liter by Prochlorococcus was higher than the Synechococcus per-liter rates. In contrast, ATP uptake per liter at the surface was higher for Synechococcus than for Prochlorococcus. Synechococcus uptake rates per liter decreased significantly with depth (p, 0.01), while uptake rates for Prochlorococcus were not significantly different throughout the water column (p. 0.07). Heterotrophic bacteria also dominated phosphate uptake at all depths, with rates that were eight- to 10-fold higher than those of the cyanobacterial groups (Fig. 5B). Prochlorococcus followed, with highest rates at 80 m, where they were most abundant. There was no significant difference in the uptake of phosphate by Prochlorococcus and Synechococcus at the surface. However, Prochlorococcus had significantly higher uptake rates than Synechococcus below the surface. Synechococcus had the lowest overall rates of phosphate uptake, which decreased with depth (Fig. 5B) as a result of abundance (Fig. 3). Relationship between phosphate and ATP uptake rates We compared uptake of phosphate and ATP for each microbial group using correlation and Model II regression analyses (Fig. 6). Phosphate uptake rates were highly correlated with ATP uptake for Prochlorococcus, with a correlation coefficient of The regression analysis indicated that inorganic and organic P uptake varied differently for heterotrophic bacteria (slope ) (Fig. 6A), Synechococcus ( ) (Fig. 6B), and Prochlorococcus ( ) (Fig. 6C). In all samples, ATP uptake per cell was lower than phosphate uptake Table 2. Uptake of PO 3{ and ATP normalized to cellular P quota, averaged over the entire water column for the three bacterial groups. Prochlorococcus and Synechococcus P quotas were assumed to be fg P cell 21 and fg P cell 21, respectively (Bertilsson et al. 2003). The cellular P quota for heterotrophic bacteria was fg P cell 21 (Gundersen et al. 2002). Heterotrophs refer to heterotrophic bacteria. The means (and standard errors [SEs]) are from n number of samples. PO 3{ uptake (fmol P fg P 21 h 21 ) ATP (fmol P fg P 21 h 21 ) Date Heterotrophs Prochlorococcus Synechococcus Heterotrophs Prochlorococcus Synechococcus n October (27.23) (56.60) (289.16) 51.0(13.16) 10.51(2.36) 7.3(105.19) 28 September (33.32) 19.87(21.91) (289.23) 15.39(3.26) 2.57(.25) (33.50) 16 March (20.12) (0.9) (237.30) 5.(.23) 22.01(16.02) 90.73(27.06) 20

7 P and cyano- and heterotrophic bacteria 329 Fig. 5. Total uptake of (A) ATP and (B) phosphate (PO 3{ ) by Prochlorococcus (Pro), Synechococcus (Syn), and heterotrophic bacteria (hbac) for October 2007, September 2008, and March Notice the log scale of the y-axis. Error bars indicate standard errors of all dates. Statistically significant differences (p, 0.01) are indicated by lowercase letters; means with the same letter are not significantly different. (Fig. 6) for all of the microbial groups. The magnitude of uptake of both compounds was much higher for Synechococcus than for Prochlorococcus and heterotrophic bacteria (Fig. 6C). Discussion Previous studies indicate that bacteria are responsible for over half of phosphate uptake in various aquatic ecosystems (Kirchman 2000; Vadstein 2000). However, there is uncertainty about which group, cyanobacteria or heterotrophic bacteria, dominates P uptake in the oceans, in part because of methodological problems and because much less is known about DOP uptake. Most studies have used size fractionation to determine the contribution of various microbial groups to phosphate and DOP uptake. The problem with this method is that Synechococcus and Prochlorococcus are in the same size fraction as heterotrophic bacteria. Our study, using flow cytometry, examined per-cell rates and specific contributions of autotrophic and heterotrophic bacteria to P uptake. It was necessary to fix and preserve samples until the flow cytometric analyses could be completed back at our shore-based facilities. The radioactivity lost during the formaldehyde fixation step seems unlikely to affect our conclusions, because the lost fraction was small compared to the large differences observed among microbial groups. In an analogous study, formaldehyde was found to act like Fig. 6. ATP uptake vs. phosphate (PO 3{ ) uptake in the Sargasso Sea by (A) heterotrophic bacteria, (B) Synechococcus, and (C) Prochlorococcus. Data are from October 2007, September 2008, and March Each point is a mean of triplicates. The slope is from Model II linear regression analyses. The line is the one-to-one line.

8 330 Michelou et al. trichloroacetic acid in releasing radioactivity from lowmolecular-weight intracellular pools (Kiene and Linn 1999). If so, the uptake measured by this study probably reflects incorporation into macromolecules such as deoxyribonucleic acid, ribonucleic acid, and lipids. Inclusion of the lost radioactivity would lead to more accurate total uptake rates, but the overall picture of differences among microbes would be similar. Using methods similar to those of this study, previous studies determined that bacteria in the SAR11 clade and Prochlorococcus were the two major groups using phosphate in the central and eastern North Atlantic Ocean (Zubkov et al. 2008). These two groups were each found to be responsible for about 5% of total uptake, while Synechococcus contributed only 7%. In contrast, our results indicate that Prochlorococcus and Synechococcus both contribute less than 10% to total phosphate assimilation. This difference between the eastern and western North Atlantic Ocean is due to differences in cyanobacterial abundance because cell-specific phosphate assimilation rates (expressed as moles per cell per hour) in the two regions were similar (Zubkov et al. 2007; this study). Zubkov et al. (2007) observed Prochlorococcus abundances of over 10 5 cells ml 21 in May and October, while we observed lower abundances, between and cells ml 21, in the fall. Our study sampled the part of the seasonal cycle during which Prochlorococcus abundance is historically low (Durand et al. 2001). During spring and summer Prochlorococcus abundance at our stations in the Sargasso Sea reaches similar numbers (Durand et al. 2001) to those in the eastern North Atlantic (Zubkov et al. 2007). Total phosphate uptake rates during our cruises in the western North Atlantic were several orders of magnitude lower than those observed in the eastern North Atlantic (Zubkov et al. 2007). The difference is due in part to the lower phosphate concentrations in the western North Atlantic waters that we sampled (Fig. 1), as measured by standard chemical assays, although the concentrations at the stations where Zubkov and colleagues sampled were similar to those at some of the stations we sampled. In contrast, DOP hydrolysis rates appear to be higher in the western than in the eastern North Atlantic (Mather et al. 2008; Casey et al. 2009). The substantial rates of ATP utilization by cyanobacteria indicate that DOP plays a critically important role in supporting primary production in the western North Atlantic, which may not be the case in the eastern North Atlantic (Zubkov et al. 2007). These differences are among the many large differences in biogeochemical variables and physical forcing between the two regions of the North Atlantic Ocean (Cianca et al. 2007). Surprisingly, DOP uptake by heterotrophic bacteria had not been examined using flow cytometry prior to this study, although size fractionation studies indicated that heterotrophic bacteria dominate ATP uptake in lakes (Berman 1988). To our knowledge, there is only one size fractionation study (Lovdal et al. 2007) that found that heterotrophic bacteria dominate ATP uptake in a marine environment. Uptake of ATP does not necessarily reflect utilization rates of the entire DOP pool, which consists of a mixture of many compounds that differ in lability. Unfortunately, the only commercially available radiolabeled organic phosphorus compounds are nucleotides. Other components of the DOP pool include phosphonates, which may be bioavailable to some diazotrophs (Dyhrman et al. 2002) as well as to other marine microorganisms, including picocyanobacteria and heterotrophic bacteria (Ilikchyan et al. 2009). All marine cyanobacterial genomes sequenced so far have genes for putative ATP-binding cassette (ABC-type) phosphonate transporters (phnd) (Ilikchyan et al. 2009; Martinez et al. 2009). In addition, Synechococcus sp. strain WH8102 has been shown to utilize phosphonates for growth and to induce the phnd gene during P stress (Ilikchyan et al. 2009). In any case, ATP is likely to be a good model compound for highly labile DOP components, since 75% of the natural DOP pool consists of P esters such as ATP (Kolowith et al. 2001). All of the sorted microbial groups were capable of assimilating ATP as well as phosphate, but there were clear physiological differences among heterotrophic bacteria, Prochlorococcus, and Synechococcus. We found that, similar to phosphate uptake, heterotrophic bacteria accounted for most of the total ATP uptake, again as a result of their high abundance. In the western Sargasso Sea, DOP accounted for. 70% of the total measured P uptake, as cyanobacteria (Prochlorococcus and Synechococcus) are physiologically superior to larger phytoplankton in taking up ATP (Casey et al. 2009). Competitive ability among osmotrophs is often linked to cell size, and the traditional view is that small cells with a large surface-to-volume ratio take up substrates more efficiently than do larger organisms. Despite having the largest surface area, however, Synechococcus had the highest uptake of organic and inorganic P per surface area in our study, while Prochlorococcus had some of the lowest uptake rates of ATP. This indicates that DOP may play a bigger role in the support of Synechococcus communities than for Prochlorococcus communities in P-limited environments. Despite having the smallest size, heterotrophic bacteria had some of the lowest phosphate uptake rates per surface area. As another approach for exploring differences among the three microbial groups, we examined ATP and phosphate uptake normalized to cellular P quota for each microbial group. We had hypothesized that Prochlorococcus, given its lower P quota ( fg P cell 21 ), would have low uptake rates, and heterotrophic bacteria would have the highest rates as a result of their high cellular demand for P. Our results show that surprisingly, heterotrophic bacteria had the lowest uptake rates normalized to P quotas for both phosphate and ATP. In contrast, Synechococcus had significantly higher P quota normalized uptake rates, similar to what was observed for the surface area normalized uptake rates for both compounds. One explanation is that cyanobacteria have evolved more efficient systems for nutrient acquisition in oligotrophic systems, compared to heterotrophic bacteria (Van Mooy et al. 2009). Prochlorococcus and Synechococcus substitute non-phosphorus membrane lipids for phospholipids under P limitation (Van Mooy et al. 2009). In laboratory cultures Van Mooy et al. (2009) observed higher ratios of these substitute lipids to phospholipids in Synechococcus than in

9 P and cyano- and heterotrophic bacteria 331 Prochlorococcus. Results from several studies (Martiny et al. 2006) indicate that different species and even different strains of the same species are likely to react quite differently to P stress. Phosphate and ATP uptake were highly correlated for all three microbial groups, reflecting the connection between metabolic needs for P and uptake of these two P sources in a P-limited environment. However, slopes from regression analyses suggest differences in organic and inorganic P processing by the three groups. These data also suggest that Prochlorococcus favors phosphate over ATP, more so than the case for Synechococcus and heterotrophic bacteria. Still, phosphate uptake was substantially higher than ATP uptake for all microbial groups. This is consistent with results from previous studies in the North Atlantic Ocean (Zubkov et al. 2007; Casey et al. 2009). The estimates for ATP uptake depend on the assumed ATP concentration, but since the same concentration is assumed for all three microbial groups, the differences among the three groups would remain regardless of the ATP concentrations. Heterotrophic bacteria compete with cyanobacteria and other phytoplankton for phosphate and other inorganic nutrients. The outcome of this competition potentially influences the carbon cycle, both through heterotrophic microbes indirectly limiting primary production by depriving phytoplankton of nutrients and through phytoplankton indirectly limiting heterotrophic degradation of organic material (Havskum et al. 2003). The small size of bacteria should give them a competitive advantage over large phytoplankton, as was shown to be the case for cyanobacteria in the Sargasso Sea (Casey et al. 2009). However, our data and those of other studies clearly indicate that size alone is not an adequate predictor of uptake rates by a microbial group. In the Sargasso Sea, the small cell size of heterotrophic bacteria was not enough to ensure they had the highest uptake rates per cell or per surface area of phosphate or of ATP. Prochlorococcus appears to have a different strategy than heterotrophic bacteria and Synechococcus for using ATP and potentially other DOP components. These results indicate differences in potential P limitation among the three bacterial groups in the Sargasso Sea and potentially other P-limited environments. Acknowledgments We thank John Casey for his help with the flow cytometry analysis and the captain and crew of the R/V Atlantic Explorer for their support during sample collection. This research was supported by National Science Foundation (NSF) grants Molecular and Cellular Biosciences (to D.L.K.) and Ocean Sciences (to M.W.L.). 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