Dissolved organic phosphorus production and decomposition during open ocean diatom blooms in the subarctic Pacific

Dissolved organic phosphorus production and decomposition during open ocean diatom blooms in the subarctic Pacific

Marine Chemistry 165 (2014) 46–54 Contents lists available at ScienceDirect Marine Chemistry journal homepage: www.elsevier.com/locate/marchem Diss...

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Marine Chemistry 165 (2014) 46–54

Contents lists available at ScienceDirect

Marine Chemistry journal homepage: www.elsevier.com/locate/marchem

Dissolved organic phosphorus production and decomposition during open ocean diatom blooms in the subarctic Pacific Takeshi Yoshimura a,⁎, Jun Nishioka a,b, Hiroshi Ogawa c, Kenshi Kuma d, Hiroaki Saito c,e, Atsushi Tsuda c a

Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko, Chiba 270-1194, Japan Institute of Low Temperature Science, Hokkaido University, Kita-Ku, Sapporo 060-0819, Japan Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan d Graduate School of Fisheries Sciences and Faculty of Fisheries, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido 041-0861, Japan e Tohoku National Fisheries Research Institute, Fisheries Research Agency, Shiogama, Miyagi 985-0001, Japan b c

a r t i c l e

i n f o

Article history: Received 27 December 2013 Received in revised form 7 August 2014 Accepted 18 August 2014 Available online 27 August 2014 Keywords: Dissolved organic phosphorus Algal blooms Diatoms Subarctic Pacific

a b s t r a c t A significant part of phosphorus (P) in seawater is found in the dissolved organic matter (DOM) fraction as DOP, which plays a key role in the marine biogeochemical cycle of P. The DOM pool size changes with biological activity, but DOP production and decomposition processes, unlike carbon (C) and nitrogen, have been only infrequently studied during phytoplankton blooms when rapid production and accumulation of organic matter occurs. We observed the DOP dynamics during two phytoplankton blooms dominated by centric diatoms, the first induced by an in situ mesoscale iron enrichment experiment in the western subarctic Pacific in summer 2001 (SEEDS) and the second that occurred naturally in spring 2003 in the Oyashio region. DOP concentration increased with the buildup of phytoplankton biomass with DOP/chlorophyll-a production ratios (mol/g) of 0.0027 ± 0.0004 and 0.0044 ± 0.0010 for the SEEDS and Oyashio blooms, respectively. During the SEEDS and Oyashio blooms the amount of net DOP production corresponded to (4.9 ± 0.7) % and (4.5 ± 0.6) % of the consumed soluble reactive P, and (5.5 ± 0.8) % and (13 ± 3) % of the newly accumulated organic P was partitioned into DOP, respectively. Seawater culture bottle experiments showed that newly produced DOP during the bloom development was decomposed by free living bacteria over a time scale of a month even under soluble reactive P available conditions. C:P for the decomposed DOM (molar ratio of 78–88) showed a similar value to in situ net produced DOM (66) and POM (83) but much lower than that for the bulk DOM (395–706), suggesting that the composition of the freshly produced DOM with high lability differs significantly from the bulk DOM. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Phosphorus (P) is an essential element for organisms such as phytoplankton and bacteria and thus a major bioactive element as well as carbon (C) and nitrogen (N). In the biogeochemical cycle of bioactive elements, dissolved organic matter (DOM) plays important roles (Hansell and Carlson, 2002), and a significant part of P occurs in the dissolved organic P (DOP) fraction (Karl and Björkman, 2002). Although orthophosphate (PO4) is the most accessible form of P for planktonic microbes, DOP also plays an important role as a P source when the PO4 concentration is low (Björkman and Karl, 2003; Nausch and Nausch, 2006). Although DOP concentrations have not been routinely measured in some recent studies of marine environments probably due to the complex and difficult procedures for DOP analysis, DOP measurement

⁎ Corresponding author. Tel.: +81 47182 1181; fax: +81 47183 2966. E-mail address: [email protected] (T. Yoshimura).

http://dx.doi.org/10.1016/j.marchem.2014.08.003 0304-4203/© 2014 Elsevier B.V. All rights reserved.

is required for a better understanding of the marine biogeochemical cycle of P (Benitez-Nelson, 2000; Dyhrman et al., 2007; Karl and Björkman, 2002; Paytan and McLaughlin, 2007). DOM is variously produced via many potential processes including cytolysis by viral infection, sloppy feeding by crustacean zooplankton, release of ingested prey organic matter by protozoan grazers, and by direct extracellular release from phytoplankton (Nagata, 2000). Partitioning of newly produced organic matter into DOM and particulate organic matter (POM) has been focused on because they play different roles in the food web and biological pump (Passow and Carlson, 2012), which is the biologically mediated process responsible for transporting elements from the photic zone to the deep ocean. Organic matter production and partitioning have been studied from a standpoint of C (e.g. Carlson et al., 2000), but have been less examined for P. Similarities and dissimilarities between P and C and N should be examined for organic matter production and decomposition processes. Phytoplankton blooms are a good opportunity to study the dynamics of organic matter since rapid production and accumulation of organic matter occurs during the event. Past studies have focused on the

T. Yoshimura et al. / Marine Chemistry 165 (2014) 46–54

direct measurement of C and N dynamics in POM and DOM pools during phytoplankton blooms (Biddanda and Benner, 1997; Carlson et al., 2000; Wetz and Wheeler, 2003). Despite its importance, DOP dynamics have been understudied. Seasonal variations in DOP concentration have been reported in coastal regions (Monaghan and Ruttenberg, 1999; Van Der Zee and Chou, 2005) and open subtropical regions (Karl and Tien, 1997; Lomas et al., 2010). We also reported multiyear DOP dynamics in Funka Bay, Hokkaido, Japan (Yoshimura and Kudo, 2011). However, the monthly sampling intervals used in previous studies were not frequent enough to enable examination of the DOP dynamics during spring phytoplankton blooms (Monaghan and Ruttenberg, 1999). Observations and incubation studies for understanding variations in DOP have been conducted during upwelling and subsequent relaxation events in the Oregon coastal area (Ruttenberg and Dyhrman, 2005, 2012) and Monterey Bay (Mackey et al., 2012), but details of the time course of DOP production during in situ phytoplankton bloom development still remains unclear. Yoshimura et al. (2009) measured DOP in fine time intervals during a phytoplankton bloom induced by an in situ iron enrichment (SEEDS-II experiment), but increases in DOP concentration were not observed because the buildup of phytoplankton biomass was small with a chlorophyll-a (Chl-a) concentration of less than 3 μg L–1 even at the peak of the bloom. Observations during a massive phytoplankton bloom are needed for a better understanding of in situ DOP dynamics. DOP dynamics have not been fully examined in the relatively productive subarctic regions compared with the comprehensive studies in subtropical oligotrophic regions such as stations ALOHA (Karl and Tien, 1997) and BATS (Lomas et al., 2010). In the Oyashio region, a western boundary current of the subarctic circulation in the North Pacific, phytoplankton growth is regulated by nitrate, silicic acid, and iron while a significant amount of soluble reactive P (SRP) remains throughout the year (Nakayama et al., 2010; Nishioka et al., 2007; Saito et al., 2002). P has not been considered to be the key nutrient to control phytoplankton growth, and thus P dynamics including DOP have not been thoroughly studied. Similar nutrient dynamics have been observed in Funka Bay, influenced by Oyashio-related waters, where the spring phytoplankton bloom depletes nitrate and then silicic acid (Kudo et al., 2000) but SRP of 0.3 μmol L–1 remained (Yoshimura and Kudo, 2011). In the bay, the remaining SRP seems to suppress the increase in alkaline phosphatase activity (APA) during the phytoplankton bloom (Yoshimura and Kudo, 2011). This suggests that utilization of DOP as P nutrition may be minor unlike with fast turnover of DOP in the subtropical regions (Björkman and Karl, 2003). This is supported by higher DOP concentrations in subarctic waters than in subtropical waters in the North Pacific (Yoshimura et al., 2007). Under such conditions, DOP dynamics can be illustrated in the subarctic regions through measurements of the temporal change in the DOP standing stock. The present study examined the DOP dynamics during two model phytoplankton blooms in the subarctic Pacific. One is a phytoplankton bloom induced by an in situ iron enrichment in the Subarctic Pacific Iron Experiment for Ecosystem Dynamics Study (SEEDS) conducted in 2001. The experiment observed a large phytoplankton bloom with a maximum Chl-a concentration of ca. 20 μg L−1 from the initial stage to the peak of the bloom during a 2 week study period (Tsuda et al., 2003). C budgets during SEEDS have been examined in Tsuda et al. (2003). The other bloom observed in this study was a naturally occurring spring phytoplankton bloom in 2003 in the Oyashio region. Spring phytoplankton blooms, dominated by diatoms such as Thalassiosira, Chaetoceros, and Fragilariopsis (Hattori-Saito et al., 2010), occur every year during April and May and maximum Chl-a concentrations of over 10 μg L−1 have been observed (Saito et al., 2002). C and N dynamics in POM and DOM pools during the Oyashio bloom in 2003 have been reported in Hasegawa et al. (2010), but the P dynamics have not yet been clarified. During the two phytoplankton blooms, field observations and seawater culture bottle experiments were conducted to understand the DOP production and decomposition processes.

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2. Materials and methods 2.1. Field samplings Field observations were conducted during the SEEDS experiment and during a naturally occurring diatom bloom in the Oyashio region. SEEDS was conducted on the R/V Kaiyo-Maru (KY0103 cruise) from 18 July to 1 August 2001 in the western North Pacific (48.5°N, 165°E; Fig. 1) (Tsuda et al., 2003). In the SEEDS experiment, an 8 km by 10 km patch was fertilized with 350 kg of iron as FeSO4 · 7H2O (Tsumune et al., 2005). The patch was tracked for 13 days after the iron enrichment, and seawater samples were taken both inside and outside of the patch at 1- or 2-day intervals. In the second set of observations, the Oyashio phytoplankton bloom was observed once or twice a month on four cruises of T/S Oshoro-Maru in February and March 2003 (OS0302 and OS0303), and R/V Wakataka-Maru in April and May 2003 (WK0304 and WK0305) in a project, the Study for Plankton and Iron Dynamics in the Northwestern Subarctic Pacific (SPINUP). During the SPINUP observations, sampling was conducted at stations on the Aline (Stns A4, A7, A11, and A17), which is a monitoring line used by the Fisheries Research Agency (Saito et al., 2002), and station H (Stn H; 41.50°N, 145.78°E) in the Oyashio region (Fig. 1). Seawater samples were taken vertically from 5 m to 100 m depth with acid washed Niskin-X bottles on a Kevlar wire. The samples for SRP and total dissolved phosphorus (TDP) analysis were drawn from the Niskin-X bottles by gravity filtration through an in-line precombusted (450 °C for 4 h) GF/F filter, attached directly to the spigot of the Niskin bottle. The filtrates were collected in acid cleaned polypropylene bottles. For particulate P (PP) analysis, subsamples (1–2 L aliquots) were filtered through a precombusted and acid washed 25 mm GF/F filter under gentle vacuum (b 0.01 MPa). The filtrate and filter samples were stored at −20 °C until analysis on land. For Chl-a analysis, subsamples were filtered through GF/F filter under a gentle vacuum, and Chl-a was extracted immediately with N,N-dimethylformamide under − 20 °C in the dark over 24 h (Suzuki and Ishimaru, 1990). The Chl-a data shown in this paper have already been reported for SEEDS (Kudo et al., 2005; Ramaiah et al., 2005) and partly for SPINUP (Nishioka et al., 2007). 2.2. DOM decomposition experiments To monitor the DOM decomposition processes in bottles, seawater culture experiments were examined using seawater samples collected at Stn A4 during the SPINUP observations. Seawater samples were collected at 10 m depth in three different stages of the phytoplankton bloom (23 April for Exp-1, 9 May for Exp-2, and 17 May 2003 for Exp3) which had Chl-a concentrations of 6.1, 13.3, and 13.9 μg L−1, respectively. Each seawater sample was gravity filtered through a 0.22 μm

60˚N

SEEDS Site H

40˚N

A-line (Stns A4, A7, A11, A17) 20˚N 120˚E

140˚E

160˚E

180˚

160˚W

Fig. 1. Locations of sampling stations during the SEEDS experiment in summer 2001 and the SPINUP observations along the A-line and Stn H in the Oyashio region in spring 2003.

Decomposed fraction

– – – 88 ± 61 78 ± 50 177 39 107 47 34 ± ± ± ± ± 1.1 0.8 1.0 0.9

Initial

706 500 590 395 479 UDL 4.1 ± 3.6 ± 5.3 ± 3.9 ±

DOC DOP

UDL UDL UDL 0.06 ± 0.04 0.05 ± 0.03 56.5 60.9 61.3 61.8 63.2 0.06 0.11 0.09 0.11 0.09 1.34 0.29 0.29 0.50 0.51 1.40 0.40 0.38 0.61 0.60 56.5 65.0 64.9 67.1 67.1 0.08 0.13 0.11 0.17 0.14 1.31 0.28 0.28 0.47 0.48 17 May Exp-3

22.7

1.39 0.41 0.39 0.64 0.62 22.9 18.2 23 April 9 May Exp-1 Exp-2

TDP

± ± ± ± ±

0.01 0.00 0.01 0.01 0.00

PO4

± ± ± ± ±

0.01 0.01 0.01 0.01 0.01

DOP

± ± ± ± ±

0.02 0.01 0.02 0.02 0.01

DOC

± ± ± ± ±

0.3 0.4 0.4 0.5 0.5

TDP

± ± ± ± ±

0.00 0.00 0.00 0.01 0.01

PO4

± ± ± ± ±

0.00 0.01 0.01 0.01 0.01

DOP

± ± ± ± ±

0.00 0.01 0.01 0.02 0.02

DOC

± ± ± ± ±

0.4 0.7 0.4 0.5 0.4

Decomposed fraction (μmol L−1) Final (μmol L−1) Initial (μmol L−1) Incubation period (days)

P concentrations were measured for three operationally defined pools, DOP, SRP, and PP. The concentration of DOP was estimated as the difference between TDP and SRP concentrations. SRP was measured manually by the molybdenum blue method (Hansen and Koroleff, 1999) using a 50 mm path length quartz cell and a spectrophotometer (U-2001, Hitachi). The calibration was performed using KH2PO4 (Suprapur, Merck). Samples for TDP analysis were autoclaved in an acid potassium persulfate (N and P analysis grade, Wako) solution at 123 °C for 120 min (Hansen and Koroleff, 1999; Ridal and Moore, 1990). TDP concentrations were measured as SRP after removing excess free chlorine by placing the samples in a hot water bath for 2 h. For the SEEDS experiment, each sample analysis for SRP and TDP was done singularly and in duplicate, respectively, and the mean ± range is reported for DOP. For the SPINUP observations, analyses for SRP and TDP were done in triplicate for each sample, and the mean ± 1 standard deviation (SD) was reported for SRP and DOP. The detection limit for SRP was 0.01 μmol L−1. The precision of the DOP concentration for a single sample analysis was typically ±0.02 μmol L−1 (ranging ±0.00 μmol L−1 to

Sampling date

2.3. Chemical analyses

Exp.

pore size durapore filter (Millipak 100, Millipore) into an acid washed polycarbonate bottle or through a precombusted GF/F filter into an acid washed glass bottle. The 0.22 μm filtrate (2 L) was inoculated with 5% v/v of GF/F filtrate in which ambient bacteria should be retained, and incubated in the dark under 4 °C. We note that a substantial abundance of the total bacterial biomass including attached bacteria was removed by the filtration through GF/F filter and the initial abundance of bacteria being diluted to ~5% in the incubation bottle. Therefore, the experiments could lead to an underestimation of the ambient activity of DOM decomposition by bacteria. However, the bacterial abundance usually increases rapidly under grazer-free conditions as reported in Carlson et al. (2004), thus we measured the potential DOM decomposition by free living bacteria in these experiments. Similarly, since phytoplankton-derived alkaline phosphatase was removed, the experiments could underestimate the ambient activity of DOP decomposition by APA. Although Yoshimura and Kudo (2011) reported that relatively low APA (maximal potential rates of b3 nmol L−1 h−1) has been observed during the spring phytoplankton bloom in Oyashiorelated waters (SRP N 0.3 μmol L−1) compared with SRP depleted periods in summer in Funka Bay (a different station to the present study), the APA is equivalent to the background level of up to several nmol L− 1 h− 1 observed in the Pacific (Dyhrman and Ruttenberg, 2006; Sato et al., 2013; Suzumura et al., 2012) and other open seas (Lomas et al., 2010; Sebastián et al., 2004). Since the APA level can potentially play a role to hydrolyze DOP during the 3-week culture periods, the underestimation of DOP decomposition relative to in situ conditions in the present experiments may have been significant. The experiments were conducted in single bottles for Exp-1 and in duplicate bottles for Exp-2 and Exp-3. Subsamples for SRP, TDP, and dissolved organic C (DOC) analyses were collected over time, filtered through 0.2 μm pore size polycarbonate filter (Nuclepore), and stored in acid cleaned polypropylene bottles and precombusted (550 °C for 4 h) glass ampoules, respectively, at − 20 °C until analysis on land. Since the decomposed DOM fraction was estimated from the decrease in DOP and DOC concentrations in 0.2 μm filtrate during the seawater culture experiments, possible coagulation of DOM into particulate fractions such as abiotic formation of transparent exopolymer particles (Passow, 2012) and adsorption of them onto the bottle wall, if it occurs can potentially lead to an overestimation of the decomposed fractions. Similarly, adsorption of SRP onto newly produced particles and bottle wall can potentially lead to an underestimation of the DOP decomposition. Impacts of these mechanisms on the estimate of DOP and DOC decomposition are currently unknown, but would be minimal for DOP because TDP concentrations were almost stable during the experiments (Table 1).

C:P of DOM (molar)

T. Yoshimura et al. / Marine Chemistry 165 (2014) 46–54 Table 1 DOM decomposition experiments using seawater samples collected at Stn A4 during the phytoplankton bloom in the Oyashio region. The experiments were conducted in duplicate bottles (values in upper for bottle #1 and lower for #2) except in Exp-1 of the single bottle experiment. Values are mean ± 1 standard deviation of triplicate analyses, and the errors are propagated in calculating the decomposed fraction and C:P of DOM. UDL, under detection limit.

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T. Yoshimura et al. / Marine Chemistry 165 (2014) 46–54

0.04 μmol L−1). We measured reference materials (RMs) for nutrients in seawater (Aoyama et al., 2012) and confirmed that our SRP results were consistent with the consensus value for SRP. Although we cannot evaluate the accuracy of our DOP measurement due to a lack of appropriate RMs for DOP analysis (Yoshimura, 2013), a good comparability in our analytical results was confirmed with stable DOP results (±0.01 μmol L−1) of the successive measurements of a batch of aged surface seawater sample (Yoshimura and Sharp, 2010). PP was measured as SRP after high-temperature combustion and acid hydrolysis of the filter samples as described by Solórzano and Sharp (1980). Analyses for PP were done on single samples that were analyzed singularly for SEEDS and in duplicate for SPINUP, and the mean ± range is reported. A higher precision (± b 1 nmol L−1) was obtained for PP analyses due to the ca. 30 fold concentration factor used in the analytical procedure. DOC was measured by a high-temperature combustion method (TOC-V, Shimadzu). The analyzer was calibrated with the 5 point standard solutions of glucose (analytical grade, Merck). The analyses were done on single samples with multiple (typically four) replicate injections per sample and the mean ± 1 SD is reported. For an accuracy check, we measured at least one consensus RM for DOC (Lot #12-07), distributed by Prof. D.A. Hansell's Laboratory (University of Miami), with the samples during every analysis run. Our measurements of (42.2 ± 0.4) μmol L−1 (mean ± 1 SD, n = 4) for the RMs were within the consensus values of 41–44 μmol L− 1. The precision of replicate

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DOC measurements for a single sample typically was 0.6 μmol L–1 (ranging 0.3 μmol L−1 to 1.2 μmol L−1). Chl-a concentrations were measured with a fluorometer (Turner Design, Model 10-AU) onboard within 48 h for the SEEDS experiment and on land within 1 month for the SPINUP observations using the nonacidification method of Welschmeyer (1994). The Chl-a data for the SEEDS experiment have already been published in Kudo et al. (2005) and Ramaiah et al. (2005). A part of the Chl-a data (averaged within the surface mixed layer) for the SPINUP observations have already been published in Nishioka et al. (2007). 3. Results 3.1. SEEDS experiment The single in situ iron enrichment induced a phytoplankton bloom. During the experiment, seawater temperature at 10 m depth in the iron patch gradually increased from 6.4 °C to 8.4 °C. In the iron enriched patch, Chl-a concentration increased from less than 1 μg L−1 to nearly 20 μg L− 1 within the surface mixed layer of upper 20 m depth (Fig. 2a). Chl-a concentration at 5 m depth started to increase exponentially on day 7 and reached a maximum of 19.2 μg L− 1 on day 13 (Fig. 2c). Similarly, PP concentration increased from 0.14 μmol L−1 to 1.17 μmol L−1. SRP was consumed concurrently with the increase in Chl-a and PP, and the concentration at 5 m depth decreased from

Fig. 2. Vertical profiles of (a) chlorophyll-a and (b) dissolved organic phosphorus concentration inside of the iron enriched patch, and temporal variations in (c) chlorophyll a, (d) soluble reactive phosphorus, and (e) dissolved organic phosphorus concentration at 5 m depth of inside and outside of the patch during the SEEDS experiment. Error bars show the range of duplicate analysis for DOP. Chlorophyll-a data were obtained from the dataset presented in Kudo et al. (2005) and Ramaiah et al. (2005).

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1.49 μmol L−1 at the beginning to 0.35 μmol L−1 by the end of the experiment (Fig. 2d). Increase in DOP was observed in the surface mixed layer (Fig. 2b) and the concentration increased significantly (one-way ANOVA, P b 0.05) from (0.15 ± 0.02) μmol L−1 to (0.21 ± 0.00) μmol L−1 at 5 m depth (Fig. 2e). Thus, a net DOP production of (0.06 ± 0.02) μmol L−1 occurred during the phytoplankton bloom development. On the other hand, phytoplankton did not increase outside of the iron enriched patch, so stable Chl-a, SRP, and DOP concentrations were observed throughout the experiment (Fig. 2c e). DOP at 5 m depth of iron enriched patch composed 9% of TDP pool on day 0, and the proportion increased to 37% on day 13. Correlation analyses revealed that DOP concentration increased with the increase in Chl-a concentration with a slope of 0.0027 ± 0.0004 (Fig. 3a; F-test, P b 0.001). DOP concentration showed a significant negative correlation with SRP concentration with a slope of 0.049 ± 0.007 (Fig. 3b; F-test, P b 0.001). In addition, the correlation between DOP and PP plus DOP showed a significant slope of 0.055 ± 0.008.

3.2. SPINUP observations A natural phytoplankton bloom developed along the A-line during April and May. During our observations, surface water temperature ranged from 1.0 °C in February to 2.9 °C on 17 May at Stn A4, a representative station of the Oyashio current. Southern stations (A11 and A17) showed a surface water temperature of 5 6 °C in April. A gradual increase in Chl-a was observed in the upper 25 m depth at Stn A4 within a month from April to May (Fig. 4a). Chl-a concentration at 10 m depth along the A-line was 0.2 0.3 μg L−1 during February and March, and increased from 0.5 μg L−1 in the middle of April to 14 μg L−1 in the middle of May (Fig. 4c). PP concentration at 10 m depth at Stn A4 increased from (0.067 ± 0.001) μmol L−1 on 18 March to (0.481 ± 0.002) μmol L−1 on 9 May and (0.489 ± 0.000) μmol L−1 on 17 May. SRP concentration at 10 m depth along the A-line decreased from the pre-bloom conditions of (1.87 ± 0.01) μmol L−1 in March at Stn A4 to (0.34 ± 0.01) μmol L− 1 in May at Stn A4 and (0.09 ± 0.01) μmol L− 1 at Stn A17 (Fig. 4d). Obvious increase in DOP concentration was observed in the surface mixed layer at Stn A4 (Fig. 4b). The DOP concentration at 10 m depth at the A-line stations increased significantly (one-way ANOVA, P b 0.005) from (0.08 ± 0.01) μmol L− 1 in March at Stn A4 to (0.19 ± 0.02) μmol L− 1 in May at Stn A4 (Fig. 4e). Thus, net DOP production of (0.11 ± 0.03) μmol L− 1 occurred during the phytoplankton bloom development. DOP at 10 m depth composed only 4% of TDP pool in March at Stn A4, and the proportion increased to 30% in May. Correlation analyses revealed that DOP concentration increased with the increase in Chl-a concentration with a slope of 0.0044 ± 0.0010 (Fig. 5a; F-test, P b 0.001). DOP concentration showed a significant negative correlation with SRP concentration with a slope of 0.045 ± 0.006 (Fig. 5b; F-test, P b 0.001). In addition, the correlation between DOP and PP plus DOP showed a significant slope of 0.13 ± 0.03.

3.3. DOM decomposition experiments during the Oyashio bloom Decomposed DOP concentrations during ca. 3-week incubations were under the detection limit in Exp-1 and Exp-2, and 0.05 0.06 μmol L−1 in Exp-3 (Table 1). TDP concentration was almost stable during the experiments and the decomposed DOP concentrations matched with the increased SRP concentrations within analytical uncertainty. DOC decomposition of 4 5 μmol L−1 occurred in Exp-2 and Exp-3, while it was under the detection limit in Exp-1. Whereas C:P molar ratios were 395 706 for the initial bulk DOM in Exp-1 3, ratios were 78 88 for the decomposed fraction of DOM in Exp-3 . The DOP and DOC decompositions in Exp-3 occurred under SRP available (0.5 μmol L− 1) conditions.

Fig. 3. Correlations between dissolved organic phosphorus and (a) chlorophyll-a, (b) soluble reactive phosphorus, and (c) total organic phosphorus (particulate phosphorus + dissolved organic phosphorus) concentration at 5 m and 10 m depths of inside of the iron enriched patch during the SEEDS experiment. For DOP and PP data, mean ± range for duplicate analyses are presented. Chlorophyll-a data were obtained from the dataset presented in Kudo et al. (2005) and Ramaiah et al. (2005).

4. Discussion The present study clearly demonstrated that DOP accumulated during phytoplankton blooms in the SEEDS experiment and in the Oyashio region. Phytoplankton community compositions in the SEEDS and Oyashio blooms were dominated by centric diatoms Chaetoceros debilis (Tsuda et al., 2005) and Chaetoceros and Thalassiosira species (Suzuki et al., 2011), respectively. So our data showed that DOP was produced during the diatom dominated blooming conditions at DOP/Chl-a (mol/g) of 0.0027 ± 0.0004 and 0.0044 ± 0.0010 for the SEEDS and Oyashio blooms, respectively (Figs. 3a and 5a). A comparable dataset of time

T. Yoshimura et al. / Marine Chemistry 165 (2014) 46–54

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Fig. 4. As in Fig. 2, but at 10 m depth at A-line stations and station H for the SPINUP observations during the Oyashio phytoplankton bloom. Error bars show 1 standard deviation for the triplicate analysis for soluble reactive phosphorus and dissolved organic phosphorus. Surface mixed layer-averaged chlorophyll-a concentrations have already been reported in Nishioka et al. (2007).

course DOP data during the phytoplankton bloom has only been reported in the North Sea using a moored automated sampling buoy, showing DOP/Chl-a of 0.33 (Suratman et al., 2010). On the other hand, van Beusekom and de Jonge (2012) found a good correlation between the spatial data of Chl-a and DOP in the productive summer season among the sites located in the coastal Dutch Wadden Sea (DOP/Chl-a of 0.011). In our previous bottle incubation experiments using open water plankton communities in the subarctic Pacific and Bering Sea, DOP/Chl-a of 0.037 0.16 has been observed for iron limited conditions (Yoshimura et al., 2013) and 0.0038 0.017 for iron enriched conditions (Yoshimura et al., 2014), suggesting that iron is a factor controlling the DOP/Chl-a. Although Ruttenberg and Dyhrman (2012) have reported that NO3 vs. PO4 availability controls net DOP production in simulated phytoplankton blooms, other mechanisms also are required to explain the surprisingly wide range of DOP/Chl-a. Site specific factors such as phytoplankton growth limiting factors, plankton community structures, ecosystem functions, and physical conditions will be driving phytoplankton production and DOP release. Our observations provided quantitative data of P flows during the diatom bloom developing phase. We found that a significant part of P, ranging from (4.9 ± 0.7) % in SEEDS and (4.5 ± 0.6) % in Oyashio, consumed as SRP was transferred into the DOP pool by surface plankton organisms (Figs. 3b and 5b). We also found that (5.5 ± 0.8%) and (13 ± 3%) of newly accumulated organic P (accumulated PP + DOP) at 5 m and 10 m depths were partitioned into the DOP pool in SEEDS and Oyashio, respectively (Figs. 3c and 5c). Because the time scale of the bloom development was longer in the Oyashio observations than SEEDS (ca. 5 weeks vs. 2 weeks), a part of the newly accumulated PP in the surface of the Oyashio bloom may have been lost by sinking to the depths, resulting

in a higher value of DOP/(PP + DOP) in the Oyashio bloom than SEEDS. The P partitioning values were similar to the C partitioning data described in past studies, showing that 9% (Tsuda et al., 2003) and 20% (Hasegawa et al., 2010) of newly produced organic C was partitioned into DOC in SEEDS and the Oyashio bloom, respectively. Among the many potential processes for DOP production as reported for DOC (Nagata, 2000), microzooplankton grazing may be a possible process, because microzooplankton played an important role to graze down the phytoplankton biomass during the SEEDS experiment (Saito et al., 2005, 2006) and spring phytoplankton bloom in the Oyashio region (Shinada et al., 2001). In addition, extracellular release of DOC by actively growing phytoplankton represents 2 10% of the total primary production (Nagata, 2000), so this process also should be considered as potential DOP production. As a result of SRP consumption and DOP production, DOP composed a large part of the TDP pool (37% for SEEDS and 30% for the Oyashio bloom) around the peak of the phytoplankton blooms, indicating that DOP can play a role as an important P source for microorganisms in the declining phase and post bloom periods. When our observations on DOP are combined with previously reported data on DOC and DON in other research, the dataset provided new insights into the elemental composition of newly produced DOM during the developing phase of diatom blooms. Hasegawa et al. (2010) and the present study observed the same phytoplankton bloom that occurred in the Oyashio in spring 2003 although sampling was done using a different research vessel. Our observation dates for the A-line stations corresponded to those in Hasegawa et al. (2010) who conducted surveys on 15 16 April and 17 22 May 2003. Our measurements of the DOC concentration for the DOM decomposition experiments which increased from 57 μmol L−1 in April to 67 μmol L−1 in

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Fig. 5. As in Fig. 3, but at the A-line stations for the SPINUP observations during the Oyashio phytoplankton bloom. Mean ± 1 standard deviation of triplicate analyses for DOP and SRP, and mean ± range for duplicate analyses for PP are presented.

May (Table 1) matched closely with the values reported in Hasegawa et al. (2010). Therefore, the estimated newly produced DOC (7.3 μmol L−1) and dissolved organic N (DON) (1.0 μmol L−1) based on field observations in Hasegawa et al. (2010) can be combined with our field observation result for DOP (0.11 μmol L–1), resulting in a C:N: P ratio of 66:9:1 for the newly produced DOM in the surface waters. The estimated newly produced DOM may have already been dephosphorylated because P can be more rapidly remineralized than C during DOM decomposition (Lønborg et al., 2009). This value deviates from the elemental composition of the bulk DOM in the pre-bloom conditions for C:N of 15.5 (Hasegawa et al., 2010) and C:P of 706 derived from the decomposition Exp-1 of the present study, resulting in a C:N:P of 706:46:1 for the bulk DOM. Rather, the elemental ratio of newly produced DOM was close to that of POM of 83:14:1, estimated by the same procedure to the DOM using data in Hasegawa et al. (2010) and the present study. The P and N rich POM composition is consistent with Martiny et al. (2013) which shows that the POM elemental composition varies systematically by ecosystem from oligotrophic low-latitude

to productive high-latitude regions. These results observed in the Oyashio spring bloom are consistent with past studies demonstrating lower C:N ratio of newly produced DOM than that of bulk DOM in high latitude regions (e.g. 6.2 reported in Carlson et al. (2000) for the Ross Sea). Similarly in SEEDS, since net produced DOC/Chl-a (mol/g) is reported to be 0.34 (Yoshimura and Ogawa, 2006), we estimated the C:P of newly produced DOM to be 126. We conclude that the stoichiometry of the newly produced DOM during the subarctic diatom blooms is quite different from that of the bulk DOM and, more importantly, is similar to or relatively P-rich compared to the Redfield ratio (Redfield et al., 1963) with a C:N:P of 106:16:1. The newly produced DOP during the developing phase of the diatom bloom was speculated to have a high lability. Our DOP decomposition experiments demonstrated that the elevated DOP concentrations decreased to the background level by ambient bacterial activity within a ca. 3-week incubation period. DOP can be decomposed more rapid under in situ conditions because we may underestimate the DOP decomposition in the bottle experiments due to the removal of phytoplankton APA as mentioned in Section 2.2. This is consistent with rapid cycling of P component including DOP in the surface ocean revealed in the P radioisotope study of Benitez-Nelson and Buesseler (1999). Interestingly, since our results showed that the DOP decomposition occurred under SRP replete (0.5 μmol L−1) conditions (Table 1), the decomposition of DOP by bacteria would not be for acquiring P from the DOP but may be the result of acquiring C or other nutrients. Another explanation may be enzymatic hydrolysis of DOP via dissolved alkaline phosphatase (Boge et al., 2013; Duhamel et al., 2011). Our results were comparable to past studies that showed that most of the DOM decomposition occurs in a few days (Wetz et al., 2008) or within the first 20 days (Lønborg et al., 2009). A recent study by White et al. (2012) has revealed that remineralization of model organic Pmonoesters occurs within hours at 24 °C. However, they showed that the decomposition rate of the labile model DOP is low at 4 °C at which our decomposition experiments were conducted. The decomposed DOP fraction during the 3-week incubation periods represented 35 36% of the initial bulk DOP, and may be partly composed of alkaline phosphatase-hydrolyzable labile DOP (L-DOP) previously reported in Suzumura et al. (1998) and Suzumura et al. (2012). L-DOP has been found ubiquitously in the Pacific (Hashihama et al., 2013; Sato et al., 2013) and represents 22 39% of the bulk DOP in the offshore and 85% in the nearshore surface waters (Suzumura et al., 2012). C:P of decomposed DOM around the peak of the bloom was 78 88 (Table 1; decomposition Exp-3), and was quite different from that of the bulk DOM at the start of the decomposition Exp-3 (C:P = 395 479), indicating preferential remineralization of P over C in the ambient bulk DOM. This is consistent with the results of Kolowith et al. (2001) and Lønborg et al. (2009) that show the difference in lability of elements in DOM (higher lability for DOP N DON N DOC). The relatively rapid production and decomposition of DOM with low C:P and deviation of the composition from the bulk DOM may explain the nearly invariant composition of DOP throughout the ocean as revealed by Sannigrahi et al. (2006) and Young and Ingall (2010). While our data were obtained in the developing phase of the bloom, it is unknown whether the DOM produced in the declining phase shows similar characteristics with the DOM discussed here. Norrman et al. (1995) and Wetz and Wheeler (2003) reported that DOC production mainly occurs in the declining phase. Hasegawa et al. (2010) indeed observed higher DOC concentration in the post bloom period in June 2003 than that in the blooming periods in the Oyashio region in May 2003. Since some parts of DOC produced during the diatom bloom are resistant to microbial decomposition for periods of up to several years (Fry et al., 1996), the accumulated DOC can contribute to horizontal and vertical C transport (Hopkinson and Vallino, 2005). This is the mechanism of the microbial carbon pump proposed in Jiao et al. (2010). In contrast, since a different trend of selective decomposition of DOC over DON is also reported in Wetz et al. (2008), lability of each constituting element

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may change with the compositions of the supplied DOM and remaining nutrients. Additional data for DOP production as well as DON and DOC, and their lability in the declining phase will strengthen our understanding of whole DOM dynamics for the subarctic ecosystems. 5. Conclusion Although DOP has not been fully examined in the relatively productive subarctic regions, the present study found DOP to be a dynamic P pool in the biogeochemical cycle of P in this region. Our study further showed that the elemental composition of newly produced and decomposed DOM during the diatom bloom in the subarctic regions differs from that in the bulk DOM and shows similar values to newly produced POM being relatively P and N rich compared to the canonical Redfield ratio. POM elemental composition shows latitudinal variation with different ecosystem types (Martiny et al., 2013), so newly produced DOM also may vary with POM compositions. Concurrent measurements of P, N, and C in the POM and DOM pools and analyses of their detailed dynamics are required for a complete understanding of marine biogeochemical cycle of C and nutrients. Acknowledgments This work was conducted in the framework of the SEEDS and SPINUP projects, and we thank the scientists who took part in the projects for their contributions to the success of the projects. We acknowledge the captain and crew of R/V Kaiyo-Maru, T/S Oshoro-Maru, and R/V Wakataka-Maru for their field assistance. We also thank C. Norman for his help to improve the English of the manuscript. We acknowledge the associate editor and three anonymous reviewers for providing valuable comments that improved the manuscript significantly. This work was supported by a grant from CRIEPI. References Aoyama, M., et al., 2012. Current status of homogeneity and stability of the reference materials for nutrients in seawater. Anal. Sci. 28 (9), 911–916. Benitez-Nelson, C.R., 2000. The biogeochemical cycling of phosphorus in marine systems. Earth Sci. Rev. 51 (1–4), 109–135. Benitez-Nelson, C.R., Buesseler, K.O., 1999. Variability of inorganic and organic phosphorus turnover rates in the coastal ocean. Nature 398 (6727), 502–505. Biddanda, B., Benner, R., 1997. Carbon, nitrogen, and carbohydrate fluxes during the production of particulate and dissolved organic matter by marine phytoplankton. Limnol. Oceanogr. 42 (3), 506–518. Björkman, K.M., Karl, D.M., 2003. Bioavailability of dissolved organic phosphorus in the euphotic zone at Station ALOHA, North Pacific Subtropical Gyre. Limnol. Oceanogr. 48 (3), 1049–1057. Boge, G., Lespilette, M., Jamet, D., Jamet, J.L., 2013. The relationships between particulate and soluble alkaline phosphatase activities and the concentration of phosphorus dissolved in the seawater of Toulon Bay (NW Mediterranean). Mar. Pollut. Bull. 74 (1), 413–419. Carlson, C.A., Hansell, D.A., Peltzer, E.T., Smith Jr., W.O., 2000. Stocks and dynamics of dissolved and particulate organic matter in the southern Ross Sea, Antarctica. Deep-Sea Res. II 47 (15–16), 3201–3225. Carlson, C.A., et al., 2004. Interactions among dissolved organic carbon, microbial processes, and community structure in the mesopelagic zone of the northwestern Sargasso Sea. Limnol. Oceanogr. 49 (4), 1073–1083. Duhamel, S., Björkman, K.M., Van Wambeke, F., Moutin, T., Karl, D.M., 2011. Characterization of alkaline phosphatase activity in the North and South Pacific subtropical gyres: implications for phosphorus cycling. Limnol. Oceanogr. 56 (4), 1244–1254. Dyhrman, S.T., Ruttenberg, K.C., 2006. Presence and regulation of alkaline phosphatase activity in eukaryotic phytoplankton from the coastal ocean: implications for dissolved organic phosphorus remineralization. Limnol. Oceanogr. 51 (3), 1381–1390. Dyhrman, S., Ammerman, J. Van, Mooy, B., 2007. Microbes and the marine phosphorus cycle. Oceanography 20 (2), 110–116. Fry, B., Hopkinson Jr., C.S., Nolin, A., 1996. Long-term decomposition of DOC from experimental diatom blooms. Limnol. Oceanogr. 41 (6), 1344–1347. Hansell, D.A., Carlson, C.A., 2002. Biogeochemistry of Marine Dissolved Organic Matter. Academic Press, San Diego. Hansen, H.P., Koroleff, F., 1999. Determination of nutrients, In: Grasshoff, K., Kremling, K., Ehrhardt, M. (Eds.), Methods of Seawater Analysis, Third ed. Wiley-VCH Verlag GmbH, Weinheim, Germany, pp. 159–228. Hasegawa, T., Kasai, H., Ono, T., Tsuda, A., Ogawa, H., 2010. Dynamics of dissolved and particulate organic matter during the spring bloom in the Oyashio region of the western subarctic Pacific Ocean. Aquat. Microb. Ecol. 60 (2), 127–138.

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