Karen McLaughlin, Jill A. Sohm 1, Gregory A. Cutter 2, Michael W. Lomas 3 and Adina Paytan 4. University of Southern California, Los Angeles, CA

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Phosphorus cycling in the Sargasso Sea: Investigation using the oxygen isotopic composition of phosphate, enzyme labeled fluorescence, and turnover times Karen McLaughlin, Jill A. Sohm 1, Gregory A. Cutter
Phosphorus cycling in the Sargasso Sea: Investigation using the oxygen isotopic composition of phosphate, enzyme labeled fluorescence, and turnover times Karen McLaughlin, Jill A. Sohm 1, Gregory A. Cutter 2, Michael W. Lomas 3 and Adina Paytan 4 Abstract Dissolved inorganic phosphorus (DIP) concentrations in surface water of vast areas of the ocean are extremely low ( 1 nm) and phosphorus (P) availability could limit primary productivity in these regions. We utilized multiple techniques to investigate biogeochemical cycling of P in the Sargasso Sea, Atlantic Ocean. We found that dissolved organic phosphorus (DOP) is extensively utilized by phytoplankton and bacteria to supplement cellular requirements. Remineralization of the DOP pool was most extensive above the thermocline as indicated by expression of alkaline phosphatase, rapid P turnover (4-8 hours), and large δ 18 O p deviations from equilibrium. These data suggest that DOP remineralization by extracellular enzymes in the euphotic zone can account on average for 35% (range 1-5%) of P utilized. Below the thermocline, alkaline phosphatase expression is reduced, turnover times increase, and δ 18 O p values approach isotopic equilibrium. In the surface waters of the Sargasso Sea, C-fixation supported by regenerated DOP utilization may account for 4 x 1 9 mol C year -1. Introduction The oceanic phosphorus (P) cycle is closely coupled with the global carbon cycle through the role of P as a major nutrient supporting ocean primary productivity. The concentrations of dissolved inorganic phosphorus (DIP) in much of the open ocean surface waters are low and can limit or co-limit primary production (Wu et al. b, Vidal et al. 3, Lomas et al. 4, Mills et al. 4). In contrast, the dissolved organic phosphorus (DOP) pool is significantly larger than the DIP pool and thus, utilization of this chemically heterogeneous pool as a P source for living organisms can potentially influence carbon sequestration in the ocean via the biological pump (Dyhrman et al. 6, Torres-Valdes et al. 9, Lomas et al. 21). However, the complex biogeochemical cycling of P in the open ocean is poorly characterized and quantitative estimates of the bioavailability and utilization of DOP on large (ecologically relevant) spatial and temporal scales are lacking. In order to better understand P cycling in the open ocean and to estimate the degree of DOP utilization, we use a combination of techniques: δ 18 O p distribution in the water column (Blake et al. 1 University of Southern California, Los Angeles, CA 2 Old Dominion University, Norfolk, VA 3 Bermuda Institute of Ocean Sciences, St. George s, Bermuda 4 University of California, Institute of Marine Sciences, Santa Cruz, CA Sargasso Sea phosphorus cycling - 323 5, Colman et al. 5, McLaughlin et al. 6), enzyme-labeled fluorescence (ELF) analysis of alkaline phosphatase in single cells (Dyhrman and Palenik 1999, Ruttenberg and Dyhrman 5), and 33 P uptake derived P turnover times (Benitez-Nelson and Buesseler 1999, Sohm and Capone 21). Analyses were made on samples representing relatively high nutrients near-shore environment as well as open ocean oligotrophic nutrient depleted environments. Marine organisms cope with low levels of biologically available P in different ways. Some species have lower cellular P requirements (Christian 5, vanmooy et al. 9). Other species have adapted to utilize P from organic sources (DOP) and from polyphosphates (Dyhrman et al. 6, Dyhrman and Haley 6), evidenced by the abundance of alkaline phosphatase activity in seawater and phosphorus uptake measurements (Benitez-Nelson and Karl 2; Vidal et al. 3; Bjorkman and Karl 3, 5). A number of studies have shown that P is preferentially remineralized from dissolved organic matter relative to carbon, allowing efficient utilization of P in the euphotic zone (Clark et al. 1998, Hopkinson et al. 2, Aminot and Kerouel 4, Hopkinson and Vallino 5, Lomas et al. 21). Phosphorus turnover rates obtained using cosmogenic isotopes of P show that P recycling rates in the dissolved and particulate pools in surface waters are very rapid (less than a day to two weeks), suggesting that low P concentrations can support relatively high primary production (Benitez-Nelson and Buesseler 1999, Benitez-Nelson and Karl 2). Furthermore, P recycling rates vary spatially and temporally (Sohm and Capone 21) and picoplankton preferentially utilize certain DOP compounds to obtain P and other associated nutrients (Benitez-Nelson and Buesseler 1999). However, quantitative estimates of the contribution of DOP regeneration to carbon fixation in the open ocean is lacking, a data gap addressed by this study. Methods Study Site We collected depth profiles at 6 stations along a transect from the shelf break off the coast of Virginia (USA) through the Sargasso Sea (Figure 1) on March 4. Profiles ranged in depth from m (at the shelfbreak) to 4, m at Station 6. Station 2 ( N, W) approximately coincides 4º 2º º Station 1 Station 2 Station 3 Station 4-8º -6º -4º km 5 Figure 1. Map of sampling stations. Station 5 Station 6 with the existing Bermuda Atlantic Time Series (BATS) Station ( N, W). Sampling Approach Water samples were collected from Niskin bottles mounted to a 24-place rosette. Water samples at each depth were analyzed for δ 18 O p and soluble reactive phosphate (SRP) concentration (SRP operationally defined and is primarily (87%) DIP, but may also include some easily hydrolyzable inorganic and organic forms of P). Surface water samples were also analyzed for chlorophyll a and ELF. Phosphorus turnover time was assessed at Stations 3 through 6. Chlorophyll a (Chl a) concentration (mg chl m -3 ) was measured by a modified fluorometric procedure in which water is filtered through 25 mm Whatmann GF/F filters, filters were extracted in 9% acetone in a freezer overnight, and Chl a is measured using a Turner Designs Model-1 fluorometer calibrated with a commercial Chl a standard. Approximately 5 ml of water was collected for SRP concentration analysis. These samples were concentrated by the MagIC method of Karl and Tien (Karl and Tien 1992) and analyzed on an Alpkem autoanalyzer. Surface waters (1 L) were collected for alkaline phosphatase (AP) enzyme- labeled fluorescence (Dyhrman and Palenik 1999). This involved collecting plankton samples on a.22 µm filter (by low-vacuum filtration), resuspending the sample in an ethanol solution, adding the label (ELF-97), and transferring Sargasso Sea phosphorus cycling - 324 the sample to an Epitube. Samples are stored in the dark at 4 C until analysis. Cell counts are performed using a Nikon epifluorescent microscope using a -W mercury lamp (DAPI filter set, excitation at 35 nm, and maximum ELF emission at 52 nm) for ELF activity as well as with standard illumination. Slides are scanned, and each identifiable cell is tallied as either positive or negative for ELF labeling, indicating AP activity. A positive tally is given to any cell that had a considerable amount ( 1% of cell area) of visible fluorescent ELF labeling. At least 3 individual cells were tallied for each sample. We note that the ELF procedure is not a quantitative measure of enzyme activity, but rather an account of the percentage of cells that have expressed the enzyme over the past week or two (Dyhrman and Palenik 1999, Lomas et al. 4). Approximately 4 L of water was collected from each depth for oxygen isotope analysis of phosphate (δ 18 O p ) in high density polyethylene (HDPE) acid washed Jerrycans (two 25 L containers per depth). DIP was stripped from seawater by adding 1 M sodium hydroxide to each sample immediately after collection and rigorously shaking (Karl and Tien 1992, Thomson-Bulldis and Karl 1998). Because two Jerrycans were collected per depth, each container was treated as a separate sample for the initial precipitation. Magnesium hydroxide floc was allowed to settle in each container for 2 hours in a cold room (4 C) before supernatant was siphoned off, leaving approximately 5 L of floc in seawater. The floc from both Jerrycans was then combined into a single container in which the floc was allowed to continue to settle. After another hour additional supernatant was siphoned off leaving approximately 1-2 L of floc in seawater. This was then stored in a 2 L HDPE bottle and frozen until analysis for δ 18 O p (McLaughlin, et al. 4). Isotopic analyses were conducted on a Eurovector Elemental Analyzer coupled to a mass spectrometer at the US Geological Survey in Menlo Park, California. Two calibrated internal silver phosphate standards, STDH (δ 18 O p = 2. ) and STDL (δ 18 O p = 11.3 ) were analyzed throughout each mass spectrometer run for calibration and drift correction. Results from field replicate analyses of water samples (collected at the same depth and time thus including natural variability and processing reproducibility) fell within acceptance limits of 8-12% relative percent difference. Water samples were also collected in 2 ml HDPE-depressed cap scintillation vials with no headspace for δ 18 O w analysis. δ 18 O w was determined using a Finnigan MAT 251 mass spectrometer also at the US Geological Survey in Menlo Park. All oxygen isotopic composition measurements are reported in standard delta notation (δ 18 O) relative to Vienna Standard Mean Ocean Water (VSMOW). To measure phosphate turnover times, duplicate 5 ml samples of seawater were placed in 6 ml acid washed polycarbonate bottles with µci of H 3 PO -3 4 and incubated in 25% light on deck (euphotic zone samples), in a dark incubator on deck (samples m) or in the dark at 4 C incubator (samples m) for 6-9 minutes. To calculate an instantaneous uptake rate, samples were collected within the linear range of increase of isotope in the cell, rather than after the isotopic equilibration outside the cells. Incubated samples were filtered onto.2 µm polycarbonate filters placed into 7 ml plastic scintillation vials which were rinsed with filtered seawater. To control for abiological adsorption, control samples amended with glutaraldehyde were also incubated. Activity of 33 P was measured in a scintillation counter after addition of 5 ml of scintillation cocktail. The turnover time of the phosphate pool in a sample is calculated as T = R t *t/(r f -R k ), where t is the incubation time and R t, R f and R k are the radioactivity (in counts per minute) of the total pool added, the filter and the killed control, respectively. The data obtained indicates the length of time that would be required for uptake and utilization of all the SRP in each respective sample. The average coefficient of variation associated with the turnover time calculations was 27%. For more detail see Sohm and Capone (Sohm and Capone 6). Results and Discussion Our results indicate that the Sargasso Sea is deficient in DIP such that the biological communities are utilizing extracellular enzymes to access the DOP pool. The first line of evidence is observed δ 18 O p values in the mixed layer (above the thermocline) at most of the stations are significantly lower than values expected for equilibrium (paired t-test; P .1), suggesting large scale recycling of the DIP pool (Figure 2). A shift in the isotopic composition towards values less than equilibrium implies isotopic fractionation associated with extracellular enzyme hydrolysis such as alkaline phosphatase or 5 -nucleotidase, or possibly some other uncharacterized enzyme that imparts a negative Sargasso Sea phosphorus cycling - 325 d 18 O p ( ) d 18 O p ( ) d 18 O p ( ) Depth (m) Stn Stn Temperature (ºC) Stn 2 Temperature (ºC) Temperature (ºC) d 18 O p ( ) d 18 O p ( ) d 18 O p ( ) Depth (m) Stn 4 5 Stn Temperature (ºC) Temperature (ºC) 3 Stn 6 d 18 O p observed d 18 O p equilibrium Temperature Temperature (ºC) Figure 2. Depth profiles of d 18 O p along with the calculated equilibrium values and the temperature. The solid line represents the expected equilibrium d 18 O p calculate based on the oxygen isotope value of seawater and the temperature at the respective depth using the equation for equilibrium (Longinelli and Nuti 1968). Observed d 18 O p values are significantly lower than equilibrium d 18 O p (p .1) and increasing turnover times are correlated with increasing d 18 O p (p =.4). fractionation at the P-O bond site, is shifting the product (phosphate) isotopic composition towards lower than equilibrium values. While several enzymes and substrates are involved in P cycling in the ocean and the precise isotopic fractionation associated with some of these enzymes is not known (Liang and Blake 6, 9), the enzymes and substrates (phosphoesters) for which the activity is characterized and these conclusions are based, are the most abundant in the water column (Kolowith et al. 1, Cotner and Biddanda 2, Paytan and McLaughlin 7, Young and Ingall 9). The second line of evidence is the high percentage of eukaryotes expressing alkaline phosphatase activity in surface waters at all stations (up to 8%). This is within the range reported by Lomas et al. (4); though the expression is variable among sites and decreases with depth (Figure 3; Table 1). Furthermore, bulk alkaline phosphatase activities in the euphotic zone were also high reaching 8 nmol μg chl a 1 h 1 (Sohm and Capone 6; Table 1). High levels of alkaline phosphtase activity are indicative of P-deficiency and the utilization of DOP (Vidal et al. 3). The surface waters where ELF is observed are also characterized by lower δ 18 O p values, suggesting that the disequilibrium in δ 18 O p can largely be attributed to utilization of DOP and likely involves the abundant Sargasso Sea phosphorus cycling - 326 SRP (um) SRP (um) SRP (um) Depth (m) Stn 1 Stn 2 Stn ELF (% cells) ELF (% cells) ELF (% cells) SRP (um) SRP (um) SRP (um) Depth (m) Stn 4 Stn 5 Stn ELF (% cells) ELF (% cells) ELF (% cells) SRP (um) DOP (um) ELF (% Cells) Figure 3. Depth profiles of soluble reactive phosphate concentration and percent cells labeled for alkaline phosphatase expression. DOP data is also shown for some stations (M. Lomas, unpublished). Surface water DOP data for additional stations are shown in Table 1. Table 1. Data for representative samples collected in the upper water column during this cruise. Additional data for other depths is shown in the figures and given in Table SI-1. Here we present the data for select depths for which additional measures of P dynamics were measured (e.g., turnover time, uptake rates, or Al-P activity) for the same samples. Bulk APA = bulk alkaline phosphatase activity from Sohm and Capone 6 collected on the same cruise within the euphotic zone. PO 4 uptake rate data from Sohm and Capone 6 collected on the same cruise within the euphotic zone. PO 4 uptake at station 6 was measured twice d 18 O EQ = d 18 O p expected for equilibrium with temperature and d 18 O w (Longinelli and Nuti 1968): Equilibrium d 18 O p = ((Temperature )/(-4.3)) + d 18 O w. % P Utilized from DOP based on best estimate see text and Table SI-2. d d ( ) ( ) Sargasso Sea phosphorus cycling - 327 enzyme alkaline phosphatase (and possibly other extracellular enzymes, see below). This is consistent with the observations of Mather et al. (8), who found widespread alkaline phosphatase activity in the North Atlantic. They concluded this activity was due to enhanced utilization of the DOP pool in response to high levels of nitrogen fixation, forcing the system towards P-limitation. The third line of evidence is rapid turnover times above the thermocline (4-8 hours) that increases with depth to ~17 hours just below the thermocline, and up to 6 days at greater depth (Figure 4; Table 1 and Supplemental Table SI-1 in Supplemental Information). Phosphate uptake rates were also high with rates of over 15 nmol μg chl a 1 h 1 (Sohm and Capone 6; Table 1). These data are consistent with similar measurements in this region obtained at different times (Sohm and Capone 21). Comparison of phosphate turnover times and nutrient addition bioassays in the P limited Mediterranean Sea suggest that turnover times of less than 7 hours indicate P-deficiency (Zohary and Roberts 1998). The near surface turnover times seen in this study are comparable to the values seen in the Mediterranean and suggest low phosphate availability compared to demand in the surface waters of the Sargasso Sea (Ammerman et al. 3, vanmooy et al. 9). These conditions are ideal for promoting the utilization of the DOP pool, consistent with the high surface ELF-expression and δ 18 O p values below equilibrium in surface waters that are indicative of the activity of extracellular enzymes. DOP concentrations were not measured on all samples in this cruise; however, unpublished data at Station 2 (M. Lomas, Bermuda Atlantic Time Series Station), data from Sohm and Capone (6, 21) at these and many other stations in the area, as well as additional literature data, indicate that DOP values from this area are consistently highest in surface waters and decrease with depth (Wu et al. a,b; Hopkinson and Vallino 5; Karl 9; vanmooy et al. 9). In general, in the open ocean sites within the thermocline there is a tendency for lower δ 18 O p values to coincide with lower DIP (p .1), higher DOP concentrations (p .1), faster turnover rates (p =.4) and the greatest amount of cells exhibiting ELF (p =.72; Table 1). This is consistent with the hypothesis that in the absence of sufficient inorganic orthophosphate, the biological communities utilize labile DOP. Below the thermocline, the situation was reversed. The δ 18 O p values are close to equilibrium, DIP concentrations are higher, DOP concentrations are lower than in the surface, and turnover times are longer. These results indicate utilization of DOP is slower below the euphotic zone, though still present. This is also noted by Colman et al. (5). They suggest that the observed trend at depth was due to the continued slow metabolism of sinking particulate and dissolved organic matter (POM and DOM respectively) by heterotrophic bacteria in the deep ocean likely using hydrolytic phosphoenzymes such as alkaline phosphatase to obtained C (or N) and not P for metabolic needs. Additional processes, Turnover Time (hr) Turnover Time (hr) Turnover Time (hr) Turnover Time (hr) Stn 3 Stn 4 Stn 5 Stn 6 Depth (m) Figure 4. Depth profiles of turnover times for Stations 3 through Sargasso Sea phosphorus cycling - 328 including the contribution of preformed DIP derived from surface waters, and oxygen inheritance effects on POM/DOM formed in warmer surface waters but hydrolyzed at depth, also contribute to the observed trend. The δ 18 O p values of the deep water DIP pool may shed light on the relative contribution of different sources and cycling of DIP. Specifically, three processes can be considered: 1) remineralization of sinking particulate organic matter from the surface waters using extracellular enzymes that would most likely tend to shift δ 18 O p towards lower than equilibrium values; 2) DIP processed intracellularly by deep heterotrophic organisms and released to seawater, shifting δ 18 O p values towards equilibrium; and 3) preformed phosphate which will carry δ 18 O p signatures of the DIP pool at the site of deep water formation (e.g., high latitudes). Since deep ocean values approach the equilibrium isotopic composition, we can infer that extracellular remineralization rates (non-equilibrium processes) are less important compared to those in surface waters or to intracellular cycling at depth (which imparts an equilibrium isotopic signature). Our turnover time data indicates phosphate is consistently recycled even at depth (e.g., turnover time 5 - hours; Table SI-1). However, it is recycled more slowly than in surface waters, indicating that the equilibrium values are at least in part achieved by intracellular P cycling. We used an isotope mass balance model to estimate the fraction
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