Impacts of elevated CO2 on organic carbon dynamics in nutrient depleted Okhotsk Sea surface waters

Journal of Experimental Marine Biology and Ecology 395 (2010) 191–198

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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e

Impacts of elevated CO2 on organic carbon dynamics in nutrient depleted Okhotsk Sea surface waters Takeshi Yoshimura a,⁎, Jun Nishioka b, Koji Suzuki c, Hiroshi Hattori d, Hiroshi Kiyosawa e, Yutaka W. Watanabe c a

Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko, Chiba 270-1194, Japan Institute of Low Temperature Science, Hokkaido University, North 19 West 8, Kita-ku, Sapporo, Hokkaido 060-0819, Japan Faculty of Environmental Earth Science, Hokkaido University, North 10 West 5, Kita-ku, Sapporo, Hokkaido 060-0810, Japan d Department of Marine Biology and Sciences, Tokai University, 5-1-1-1 Minamisawa, Minami-ku, Sapporo, Hokkaido 005-8601, Japan e Marine Biological Research Institute of Japan, 4-3-16 Yutaka-cho, Shinagawa, Tokyo 142-0042, Japan b c

a r t i c l e

i n f o

Article history: Received 13 April 2010 Received in revised form 30 August 2010 Accepted 1 September 2010 Keywords: Carbon dioxide Ocean acidification Organic carbon Phytoplankton Sea of Okhotsk

a b s t r a c t Increasing CO2 in seawater (i.e. ocean acidification) may have various and potentially adverse effects on phytoplankton dynamics and hence the organic carbon dynamics. We conducted a CO2 manipulation experiment in the Sea of Okhotsk in summer 2006 to investigate the response of the organic carbon dynamics. During the 14-day incubation of nutrient depleted and 200 μatm in situ pCO2 surface water with a natural plankton assemblage under 150, 280, 480, and 590 μatm pCO2, the amount of net dissolved organic carbon accumulation was significantly lower at N480 μatm pCO2 than at 150 μatm pCO2, while differences in net particulate organic carbon accumulation between the treatments were small and did not show a clear relationship with the pCO2. This is the first report to show a decreased net organic carbon production of natural plankton community under elevated pCO2. Phytoplankton pigment analysis suggests that the relative contribution of fucoxanthin-containing phytoplankton such as diatoms to the phytoplankton biomass was lower at N 280 μatm pCO2 than at 150 μatm pCO2. Different pCO2 conditions may alter the organic carbon dynamics through changes in plankton processes. We conclude that the continuing increase in atmospheric CO2 in a time scale from the last half century to the end of this century has potential to affect the carbon cycle in nutrient depleted subpolar surface waters. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Increasing anthropogenic CO2 in the atmosphere through mainly the burning of fossil fuels is penetrating rapidly into the surface ocean (Sabine et al., 2004), and lowering the pH in surface seawater as a result of the acidifying effects of CO2 in water (Caldeira and Wickett, 2003). Surface ocean pH has already decreased by 0.1 U since the industrial revolution (Bindoff et al., 2007), and has been projected to decrease an additional 0.14–0.35 U within this century (Meehl et al., 2007). Although ocean acidification will potentially have significant direct impacts on a wide range of marine organisms (Raven et al., 2005; Kleypas et al., 2006), the responses of individual organisms, populations, communities, and ecosystems are largely unknown (Vézina and Hoegh-Guldberg, 2008; Doney et al., 2009). Considerable research effort has mainly been focused on predicting the impact of ocean acidification on calcifying marine organisms (Shirayama and Thornton, 2005; Gazeau et al., 2007; Hoegh-Guldberg et al., 2007;

⁎ Corresponding author. Tel.: + 81 4 7182 1181; fax: + 81 4 7183 2966. E-mail address: [email protected] (T. Yoshimura). 0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2010.09.001

Kurihara, 2008). For phytoplankton, the impact of CO2 increase on coccolithophores has been studied, and their diverse response to ocean acidification was demonstrated (Riebesell et al., 2000; Langer et al., 2006, 2009; Iglesias-Rodriguez et al., 2008). However, studies on planktonic primary producers including non-calcifying phytoplankters in relation to ocean acidification are still limited, despite the established importance of these organisms in marine carbon cycles (Riebesell, 2004; Rost et al., 2008). Changes in planktonic processes may have an impact on the oceanic CO2 uptake process, which moderate the increase in atmospheric CO2 concentration. Therefore, manipulative experiments are required for assessing the impacts of acidification on organic carbon dynamics (Orr et al., 2009). The results should be integrated into a biogeochemical modeling approach, as shown for impacts of future climate change on phytoplankton dynamics using an ecosystem model by Boyd and Doney (2002). Several previous studies using natural phytoplankton assemblages have focused on the impact of CO2 increase on primary production. For example, Tortell et al. (2002) and Kim et al. (2006) showed that a constant primary production with accelerated growth of the diatom occurred under elevated CO2. Hare et al. (2007) also reported constant primary production under elevated CO2 in the Bering Sea although a

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decrease in diatoms in phytoplankton community was observed. On the other hand, enhanced primary production under elevated CO2 was demonstrated in Riebesell et al. (2007) without a shift in the community structure and in Tortell et al. (2008) with increased diatom growth relative to other phytoplankton groups. These studies indicate that an increase in CO2 can alter the organic carbon dynamics in the sea in different ways. However, it should be noted that all these results were obtained from phytoplankton blooms induced by macroand/or micronutrient enrichments or a dilution with nutrient rich water. At present, macro- and/or micronutrients are depleted in most surface areas of the oceans. Physiological responses of phytoplankton and hence the organic carbon production can be expected to differ between nutrient depleted and replete conditions. Additionally, in such nutrient depleted waters, regenerated production via the microbial loop may become dominant (Azam et al., 1983), therefore the response of grazing processes should be also considered. Further studies are clearly essential to address the effects of CO2 on the organic carbon dynamics in nutrient depleted waters. We thus conducted a CO2 manipulated bottle incubation experiment using the natural plankton community collected from nutrient depleted surface waters in the Sea of Okhotsk in summer 2006, in the framework of the Plankton Ecosystem Response to CO2 Manipulation Study (PERCOM). In the Sea of Okhotsk, the spring phytoplankton bloom occurs from May through July (Sorokin and Sorokin, 1999; Nakatsuka et al., 2004). The vertical profiles of nutrients reported in Nakatsuka et al. (2004) suggest that more than 20 μmol L− 1 of nitrate (NO− 3 ) in the surface was consumed during the phytoplankton bloom. The phytoplankton bloom depletes the NO− 3 and silicic acid (Si(OH)4) in the surface, and a low nutrient environment occurs in the summer season (Nakatsuka et al., 2004). Andreev and Pavlova (2010) reported seawater pCO2 (adjusted to 0 °C) of 130–230 μatm in the Si(OH)4 depleted water. We thus sampled an approximate steady state nutrient-recycling food web with low pCO2. Care was taken to ensure that no nutrient enrichment of the incubation bottles occurred and we measured changes over time in the organic carbon accumulation and phytoplankton community structure, and discuss the effects of CO2 on these parameters. 2. Materials and methods 2.1. Sampling and experimental setup The study was carried out aboard the R/V Professor Khromov in summer of 2006. Surface seawater sampling for a CO2 manipulation experiment was conducted at a station (49.5°N, 148.25°E; 1050 m bottom depth) in the Sea of Okhotsk on 25 August 2006 (Fig. 1). The water was collected from 10 m depth with acid-cleaned Niskin-X bottles on a Kevlar wire. A total of 160 L of seawater was poured into four 50 L polypropylene carboys (40 L seawater for each of the given pCO2 treatment) through silicon tubing with a 243 μm mesh Teflon net to remove any large plankton. Subsamples were taken from each carboy and poured into three acid-cleaned 9 L polycarbonate bottles (12 bottles in total) for incubation. Similarly, initial samples also were collected from each carboy. All of the sampling was carried out using trace metal clean techniques to avoid any effects of trace elements other than CO2 on phytoplankton growth. Incubation was conducted on deck in a temperature-controlled water-circulating tank for 14 days under an in situ temperature of 13.5 °C and 50% surface irradiance. To control CO2 concentrations in the incubation bottles, air mixtures containing 180, 380, 750, and 1000 ppm CO2, purchased from a commercial gas supply company (Nissan-Tanaka Co., Japan), were bubbled into triplicate incubation bottles. The flow rate of the gas was set at 100 mL min− 1 for the first 24 h, and thereafter maintained at 50 mL min− 1. Subsamples were collected from each incubation bottle on days 1, 3, 5, 9, and 14 at 10:00 A.M. during the course of the experiment. Samples for nutrients and

60˚N

Sea of Okhotsk 55˚N

Study Site (1050 m bottom depth) 50˚N

45˚N

km

40˚N 0

130˚E

Japan Pacific Ocean

200 400

140˚E

150˚E

160˚E

170˚E

Fig. 1. Location of a sampling station (49.5°N, 148.25°E) for the CO2 manipulation experiment in the Sea of Okhotsk. Water was sampled for incubation from 10 m depth.

chlorophyll-a (chl-a) were collected every sampling day and samples for other parameters were collected on selected sampling days. 2.2. Sample treatments and analyses Subsamples for total dissolved inorganic carbon (DIC) and total alkalinity (TA) were collected in 120 mL glass vials and poisoned with HgCl2. Concentrations in DIC and TA were measured by coulometric titration (Johnson et al., 1985) and single point titration (Culberson et al., 1970), respectively. CO2 and pH were calculated using the K1 and K2 carbonic acid dissociation constants from Mehrbach et al. (1973) with values refitted from Dickson and Millero (1987) on the pH total hydrogen scale at the in situ temperature of 13.5 °C and salinity of 32.52. The precisions of both DIC and TA were less than 0.1% from duplicate determinations. In addition, certified reference material distributed by Prof. A. G. Dickson (Scripps Institution of Oceanography) was used. Samples for particulate organic carbon (POC) and particulate nitrogen (PN) were collected on pre-combusted (450 °C, 4 h) 25 mm GF/F filters (Whatman) under a gentle vacuum at b0.01 MPa and the filter samples were stored at −20 °C until analysis on land. Before analysis, the filter samples were freeze-dried, and then exposed to HCl fumes overnight in a desiccator to remove inorganic carbon from the samples. Carbon and nitrogen contents on the filter were measured with a CN elemental analyzer (NA-1500, Fisons). Subsamples for dissolved organic carbon (DOC) were filtered through pre-combusted (450 °C, 4 h) 47 mm GF/F filters and collected in pre-combusted (550 °C, 4 h) glass ampoules, which were stored at −20 °C until analysis on land. Dissolved organic carbon was measured by a hightemperature combustion method (TOC-V, Shimadzu). We measured at least one consensus reference material for DOC (CRM; #05-04), distributed by Prof. D. A. Hansell's laboratory (University of Miami), with the samples during every analysis run. Our measurements of 44.5 ± 0.7 μmol DOC L− 1 (mean ± SD, n = 9) for the CRMs were within the consensus values of 44–45 μmol L− 1. Subsamples for nutrients were collected in 10 mL plastic tubes and stored at −20 °C until analysis on land. Nitrate plus nitrite (NO− 3 + 3− NO− 2 ), phosphate (PO4 ), and Si(OH)4 concentrations were determined with a segmented continuous flow analyzer (QuAAtro, Bran + Luebbe). Ammonium (NH+ 4 ) was analyzed fluorometrically (Holmes et al., 1999).

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Size-fractionated chl-a samples were collected sequentially on 10 μm pore size 47 mm polycarbonate filter without vacuum and then 25 mm GF/F filter under the gentle vacuum (b0.01 MPa), and the filters extracted immediately with N,N-dimethylformamide (DMF) at − 20 °C in the dark over 24 h (Suzuki and Ishimaru, 1990). Chlorophyll-a concentrations were measured onboard with a fluorometer (Model 10-AU, Turner Designs) by the non-acidification method of Welschmeyer (1994). For phytoplankton pigment analysis, subsamples from each of the triplicate incubation bottles were pooled together and filtered onto GF/F filters, which were stored at − 85 °C until analysis on land. The pigments extracted in DMF were measured with a high-performance liquid chromatography (CLASS-VP system, Shimadzu) according to the method detailed in Suzuki et al. (2005). For enumeration of ultraphytoplankton and heterotrophic bacteria, subsamples were preserved with paraformaldehyde (PFA; 0.2% in final concentration) and frozen at −85 °C until analysis on land. The samples were analyzed with a flow cytometry (XL ADC system, Beckman Coulter) as outlined in Suzuki et al. (2005). Microscopic examination was conducted on formaldehyde- (1% in final concentration) and glutaraldehyde-preserved (1% in final concentration) samples. Cells of coccolithophores were examined on PFA-preserved (0.2% in final concentration) samples using a scanning electron microscope (SEM; JMS-840A, JEOL) according to the protocol detailed in Hattori et al. (2004). 3. Results and discussion We could create significant gradients in pCO2 and pH among the four treatments by bubbling the four different air CO2 mixtures into the incubation seawater medium (Fig. 2). Mean seawater pCO2 and pH in the four different CO2 treatments during the experiment were 145–148, 275–276, 479–489, and 583–597 μatm and 8.40, 8.18, 7.97, and 7.89, respectively (Table 1). The CO2 systems were manipulated significantly by comparison with the initial value of pCO2 of 195– 200 μatm and pH of 8.30. The low initial pCO2 condition is reasonable because this is post spring bloom water; if we assume NO− 3 consumption of 20 μmol L− 1, as mentioned in Section 1, and the Redfield ratio (mol mol− 1) of C/N = 6.6 (Redfield et al., 1963) during the spring phytoplankton bloom at the study site, this is equivalent to ca. 200 μatm pCO2 drawdown in the seawater which was equilibrated with an atmospheric CO2 concentration of 380 ppm. The initial pCO2 value is also consistent with the values presented in Andreev and Pavlova (2010). It should be noted that the seawater pCO2 did not fully equilibrate with the bubbling air CO2 concentrations, which was reported by the gas manufacturer. This may be attributable to insufficient flow rates of the gas bubbling or deviation of the actual CO2 concentrations from the reported values. Our results clearly show that direct measurements of at least two parameters in a seawater CO2 700 600

8.4

pCO2 (µatm)

pCO2 500

8.3

400

8.2

300

8.1

200

8.0

100

7.9

0

Initial

150

280

480

590

pH (total hydrogen scale)

8.5 pH

7.8

pCO2 treatment (µatm) Fig. 2. Seawater pCO2 and pH (mean with range) at the initial and during the course of the incubation experiment in the four different pCO2 treatments.

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Table 1 The measured dissolve inorganic carbon (DIC) and total alkalinity (TA), and the calculated pCO2 and pH (total hydrogen scale) under in situ temperature of 13.5 °C and salinity of 32.52. pCO2 treatment (μatm) Initial (mean± 1 standard deviation, n = 4) Day 9

Day 14

150 280 480 590 150 280 480 590

DIC (μmol kg− 1)

TA (μmol kg− 1)

pCO2 (μatm)

pHtotal

1920.10 ± 2.19

2241.84 ± 0.55

196.63 ± 2.22

8.30 ± 0.00

1859.49 1987.86 2079.63 2107.63 1857.34 1989.47 2084.21 2113.96

2242.36 2243.39 2239.46 2237.79 2244.42 2244.42 2241.12 2241.12

147.57 275.24 479.01 583.29 145.07 276.33 488.85 597.05

8.40 8.18 7.97 7.90 8.40 8.18 7.97 7.89

system are necessary to confirm the actual CO2 condition in the experimental system. Hereafter we will refer to the seawater pCO2 as 150, 280, 480, and 590 μatm for the four different CO2 treatments. The highest seawater pCO2 (590 μatm) in the present study corresponds to the projected atmospheric CO2 level at 2060–2070 in the A1B and A2 scenarios or at 2090–2100 in the B2 scenario of the SRES CO2 emission scenarios (Prentice et al., 2001). However, the deviation of the surface seawater pCO2 from the atmospheric CO2 concentration (200 μatm in seawater vs. 380 ppm in atmosphere during summer at present in the study area) should be considered, when future CO2 conditions will be discussed. If we assume a 180 μatm lower surface water pCO2 than the atmospheric CO2 concentration, each manipulated pCO2 condition was equivalent to the atmospheric CO2 concentrations of 330, 460, 660, and 770 ppm, respectively. These concentrations correspond to the atmospheric CO2 levels in the 1970s, projected 2030s, 2070s, and 2090s in the A2 scenario, respectively. In addition, note that the in situ pCO2 condition in the examined system (200 μatm) corresponded to the intermediate between the two lower pCO2 treatments (150 and 280 μatm) in the present experiment. The initial condition of our incubation water represented depleted macronutrients and low phytoplankton biomass in terms of chl-a concentration (Table 2). Nitrogenous nutrients were completely depleted, and PO3− and Si(OH)4 concentrations were 0.25 and 4 1.06 μmol L− 1, respectively, before incubation. Initial chl-a biomass was comprised of 0.30 μg L− 1 of small-sized (b10 μm) algal cells and 0.01 μg L− 1 of large-sized (N10 μm) ones. These conditions indicate that we sampled a low nutrient and low biomass phytoplankton community which was formed after a massive spring phytoplankton bloom, as reported to occur around June (Sorokin and Sorokin, 1999; Nakatsuka et al., 2004). In our initial samples, phytoplankton cells larger than 5 μm in size were rarely observed using light microscopy. Diatoms were scarcely observed in that size range. Some dinoflagellates such as, Prorocentrum, Gonyaulax, and Ceratium were identified, but their abundance was low. The ultraplankton community (ca. b5 μm in size), revealed by flow cytometry, was comprised of Synechococcus, eukaryotic ultraphytoplankton, and heterotrophic bacteria. The initial abundance of the cells was 9.0 × 103, 1.8 × 104, and 3.8 × 105 cells mL− 1, respectively.

Table 2 Initial and final (day 14) conditions of nutrients and chlorophyll-a (chl-a) concentrations (mean ± 1 standard deviation) pooled for all treatments. NH+ 4 (μmol L− 1)

PO3− NO− Si(OH)4 Small3 + 4 NO− (μmol L− 1) (μmol L− 1) sized chl-a 2 −1 (μg L−1) (μmol L )

Initial 0.09 ± 0.02 0.05±0.02 Final 0.10 ± 0.06 0.02±0.01

0.25±0.01 0.22±0.01

1.06±0.05 0.68±0.12

Largesized chl-a (μg L−1)

0.30±0.01 0.01±0.00 0.21±0.07 0.01±0.00

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During the 14-day incubation experiment under nutrient depleted conditions, chl-a concentrations fluctuated between 0.1 and 0.4 μg L− 1, but the variation pattern was similar in all treatments (Fig. 3a and b). Chlorophyll-a was mainly derived from small-sized cells throughout the experiment. If we set the endpoint as the end of the experiment of day 14, net chl-a production (the end minus the initial value) ranged from −0.04 to −0.16 μg L− 1 between the treatments (Fig. 4a). No

significant difference in the net chl-a production was observed bet− ween the treatments. Changes in the concentrations of NH+ 4 , NO3 + − 3− NO2 , and PO4 were small (Table 2). On the other hand, Si(OH)4 was consumed to 53% of the initial concentration (Fig. 3e and Table 2); the consumption in 150 μatm was significantly larger than the other treatments (Fig. 4b; Tukey's test, p b 0.05). The significant and not significant differences in the Si(OH)4 consumption and net chl-a

Fig. 3. Time course of the concentrations (mean ± 1 standard deviation of triplicate bottles) in (a) small-sized (b10 μm) chlorophyll-a, (b) large-sized (N10 μm) chlorophyll-a, (c) particulate organic carbon (POC), (d) dissolved organic carbon (DOC), (e) silicic acid (Si(OH)4), (f) heterotrophic bacteria, (g) Synechococcus, and (h) autotrophic ultraeukaryotes for the four pCO2 treatments.

T. Yoshimura et al. / Journal of Experimental Marine Biology and Ecology 395 (2010) 191–198

0.2

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(a) Si(OH)4 (µmol L-1)

Chl-a (µg L-1)

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b

b

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ab

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-2 150

280

480

590

150

280

b

b

480

590

pCO2 (µatm)

pCO2 (µatm)

Fig. 4. Differences between the initial and end values of the experiment (the end minus the initial value) in (a) total chlorophyll-a, (b) silicic acid (Si(OH)4), (c) particulate organic carbon (POC), and (d) dissolved organic carbon (DOC). Significant differences were observed between the treatments shown with different letters (“a” and “b”) near bars, while “ab” indicates data that was not significantly different from “a” and “b” (Tukey's test, p b 0.05).

production, respectively, between the treatments were similarly observed if we had set the endpoint as day 5 or day 9 (Fig. 3). Significant changes in organic carbon accumulation were observed among the treatments and the effects of pCO2 change were greater in DOC than in POC. During the incubation, POC concentrations fluctuated, but the variation pattern was similar in all treatments (Fig. 3c). Net accumulation of POC during the 14-day incubation ranged from 1.2 ± 1.4 μmol L− 1 in 280 μatm to −1.7 ± 0.5 μmol L− 1 in 590 μatm (Fig. 4c). Net POC accumulation in 280 μatm was significantly higher than that in 480 μatm and 590 μatm (Tukey's test, p b 0.05). Similar dynamics were observed for PN. The ratios of POC to PN (mol mol− 1) gradually increased from 6.7 ± 0.3 at the initial stage to 7.9 ± 0.2 at the end of the experiment without a significant difference between the treatments. On the other hand, DOC concentrations showed slight increases during the experiment, and the increase was pronounced at 150 μatm (Fig. 3d). Net DOC accumulations ranged from 4.9 ± 2.8 μmol L− 1 in 150 μatm to 0.49 ± 0.50 μmol L− 1 in 590 μatm (Fig. 4d). Net DOC accumulation in 150 μatm was significantly higher than that in 480 μatm and 590 μatm (Tukey's test, p b 0.05). Difference in net DOC accumulation of 4.4 μmol L− 1 between 150 μatm and 590 μatm was larger than that of POC of 2.9 μmol L− 1 between 280 μatm and 590 μatm. Significant differences in net POC and DOC accumulation among the treatments were not found until day 9, but were found for day 14 (Fig. 3c and d). The difference in net DOC accumulation between different pCO2 can be explained by changes in production and/or decomposition processes. Phytoplankton exudation, and zooplankton grazing and metabolism are considered to be major processes for DOC production (Nagata, 2000), and bacterial processes are critical for DOC decomposition. These will be discussed later. For the effect of CO2 increase on plankton abundance, we carried out light microscope observations but phytoplankton cells in the N5 μm fraction were rarely observed. No coccolithophores were

detected either at the initial or at the end of the experiment with SEM, so we cannot determine the impact of pCO2 increase on coccolithophores. Cell abundance of heterotrophic bacteria was stable at around 4 × 105 cells mL− 1 throughout the incubation period (Fig. 3f), and no significant difference in the net change in bacterial abundance was observed between the treatments throughout the experiment. Synechococcus and autotrophic ultra-eukaryotes showed decreasing trends from 9 × 103 to around 2 × 103 cells mL− 1 and from 1.8 × 104 to 0.4–1.0 × 104 cells mL− 1, respectively, during the experiment (Fig. 3g and h). No significant differences in the net change in the abundance of Synechococcus and ultra-eukaryotes were observed between the treatments throughout the experiment. Microscopic observations of microzooplankton showed that the tintinnid ciliate Parafavella denticulata (several 100s of individuals L− 1), small copepods Oithona spp. (several 10s of individuals L− 1, respectively), and copepod nauplii (several 10s of individuals L− 1) remained at relatively constant abundance in the bottles throughout the incubation period (Table 3). Unfortunately, statistically significant data were not obtained for determining pCO2 dependent changes in microzooplankton abundance.

Table 3 Initial and final (day 14) conditions of microzooplankton abundance. Duplicate measurements for initial and single measurements for final samples were performed. ND, not detected. Initial (individuals L− 1) Parafavella denticulata Oithona spp. Copepod nauplii

Final (individuals L− 1) 150 μatm

280 μatm

480 μatm

590 μatm

124–176

310

190

230

ND

14–35 24–55

50 ND

10 20

30 20

30 10

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While apparent changes in chl-a biomass were small under the nutrient depleted conditions, fucoxanthin/chl-a ratios showed variations between the treatments (Fig. 5). The initial fucoxanthin concentration was 0.04 μg L− 1, and at the end of the incubation the concentrations decreased with the increase in pCO2: 0.05 μg L− 1 in 150 μatm, 0.03 μg L− 1 in 280 μatm, and 0.02 μg L− 1 in 480 and 590 μatm. The fucoxanthin/chl-a ratio (g g− 1) increased from 0.12 before incubation to 0.16–0.30 at the end of the experiment with variation among the treatments. The increase in the fucoxanthin/chl-a ratios during the incubation in all treatments may be explained by the increased abundance of diatom populations in the containers (i.e. bottle effect), as reported in Venrick et al. (1977). At the end of the experiment, the fucoxanthin/chl-a ratio (g g− 1) was higher at 150 μatm pCO2 than N280 μatm pCO2 (Fig. 5). Since the pigment samples were pooled from the triplicate bottles in each treatment, the statistical significance for these pigments values could not be evaluated. It is known that fucoxanthin is contained in diatoms, prymnesiophytes, chrysophytes, and raphidophytes (Jeffrey et al., 1997). In this study, 19′-butanoyloxyfucoxanthin and 19′-hexanoyloxyfucoxanthin, which are chrysophyte and cryptophyte markers, respectively (Jeffrey et al., 1997), were very low throughout the experiment. In addition, raphidophytes were not detected using light microscopy. These results suggest a higher relative contribution of diatoms to the phytoplankton biomass in the lower pCO2 if we can assume a constant cellular fucoxanthin/chl-a ratio under different pCO2 conditions. Since constant fucoxanthin/chl-a ratios were confirmed in six diatom isolates under 180–1000 μatm pCO2 conditions (Endo et al., unpublished data), our results support the conclusion that a higher relative contribution of diatoms occur under 150 μatm pCO2. This is consistent with significantly larger Si(OH)4 consumption in 150 μatm treatment (Fig. 4b). Although diatom cells larger than ca. 5 μm in size were rarely observed using light microscopy at the end of the experiment, smaller diatoms, which are difficult to be identified by microscopy, could be rather abundant in the nutrient depleted conditions. It is well known that small cells can acquire nutrients efficiently due to their high surface area to volume ratio (Malone, 1980). Komuro et al. (2005) also demonstrated using SEM the predominance of the small-sized diatom Fragilariopsis pseudonana in the subarctic NW Pacific during summer, when iron is often depleted (Tsuda et al., 2003). From the viewpoint of bottom up processes, this change may be supported by the characteristic of carbon acquisition in diatoms; they have a CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) which is relatively efficient under lower CO2 concentrations (Tortell, 2000), and relatively efficient carbon concentrating mechanisms (CCMs) compared to other algal groups (Rost et al., 2003). From the viewpoint of top-down process, zooplankton grazing can have a significant impact on the composition of phytoplankton (e.g. Suzuki et al., 2002).

Fucox/chl-a ratio

0.3

0.2

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150

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pCO2 (µatm) Fig. 5. Fucoxanthin (fucox) to chl-a ratios at the initial and end of the 14-day incubation experiment. Pooled samples of triplicate bottles were analyzed.

Although we cannot determine which of the processes are responsible for the change in the fucoxanthin/chl-a ratio, effects of pCO2 on phytoplankton physiology and/or the top-down control of zooplankton can be considered to cause the variation of the relative diatom contribution to phytoplankton biomass. For understanding the change in net DOC accumulation among the treatments, both production and decomposition processes need to be considered. Dissolved organic carbon production can change with the change in the phytoplankton community structure because the amount of exudation of photosynthetic products as DOC is highly species-specific (Nagata, 2000). Therefore, suggested changes in phytoplankton community structure among the treatments (Fig. 5) can have some impact on DOC production. Further, zooplankton grazing is generally considered as a more important source of DOC (Nagata, 2000), although the present data cannot provide information on whether the grazing process is sensitive to pCO2 change. In addition to these processes, bacterial decomposition can also be responsible for the change in net DOC accumulation. Temporally stable and indistinguishable dynamics in cell abundance of heterotrophic bacteria between the treatments (Fig. 3f) partly support the relatively constant DOC decomposition process, although we have no data on rate processes such as bacterial production. Additionally, bacterial DOC decomposition, which is an enzymatically-driven reaction, may be sensitive to the change in pH, as reported in Yamada and Suzumura (2010). Our present data is not enough to determine the cause of the differences in DOC dynamics under different pCO2 conditions. However the unidentified process for altering the net DOC accumulation could be also responsible for altering the phytoplankton community structure. Although the present results indicate a pCO2 dependent change in net organic carbon accumulation and this is inferred to have resulted from the change in plankton dynamics, care must be taken whether the responses of the plankton community enclosed in the bottles can be extrapolated to the natural ecosystem. Drawbacks of bottle incubation experiments have been described (Venrick et al., 1977; Carpenter and Lively, 1980). The present study deals with an oligotrophic nutrient-recycling system after removing large plankton. A companion study of Liu et al. (2009), carried out at the same site on the same cruise, revealed that the growth rate of the in situ phytoplankton assemblage was estimated to be 0.38 d− 1 using a dilution method, representing the mean doubling time of the phytoplankton cells which was approximately 2 days at the beginning of the experiment. Bottling of such a community potentially changes the interaction between phytoplankton and zooplankton, and perturbs the nutrient regime. In fact, an increase in the POC/PN ratio during the 14-day incubation may imply enhancement of nitrogen deficiency through the decreased nutrient regeneration rate. However, the microzooplankton abundance (Table 3) is comparable or relatively high compared to the open Pacific waters reported in Gómez (2007), therefore significant grazing could be expected to control the small phytoplankton biomass such as Synechococcus (Fig. 3g) and autotrophic ultra-eukaryotes (Fig. 3h) in the bottles. The possible underlying reason for the decreasing trends in the abundance of small phytoplankton is the resulting consequence of trophic cascading effects, as was shown as mesozooplankton mediated control on the lower trophic level biomass in the mesocosm study by Zöllner et al. (2009). No detectable NH+ 4 accumulation during the incubation (Table 2) suggests that the system was not a simple decline of autotrophic activity (i.e. little phytoplankton growth and overwhelming heterotrophic decomposition) but the phytoplankton assemblage seemed to be actively growing. Some oscillations of chl-a biomass during the incubation (Fig. 3a) may suggest the existence of an interaction between phytoplankton growth and microzooplankton grazing in the bottles. We thus conclude that changes in pCO2 have the potential to alter the organic carbon dynamics, probably through changes in plankton processes.

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Further confirmation is required to apply the present results to future projections. In particular, it is difficult to know if the observed change in DOC production and phytoplankton composition between 150 μatm and N480 μatm mimicked the consequences of the pCO2 change between the present and future, because the in situ pCO2 condition was intermediate between the two lower pCO2 treatments in the present experiment. However, in the case that the present results are not applicable to project future change from the present, our results suggest that changes have already occurred within the last half century. Therefore, the potential direction since the 1970s to 2100 of nutrient depleted surface ocean biogeochemistry in the subpolar region in a high CO2 world could be to a lower DOC production and a lower diatom dominance. These considerations lead us to speculate that the biological carbon pump may weaken in post spring bloom period in the Sea of Okhotsk because diatom production is responsible for the carbon transport to the depth even in the nutrient depleted low productive season in the Sea of Okhotsk (Nakatsuka et al., 2004), as reported in other subarctic regions (Honda et al., 1997, 2002; Takahashi et al., 2000). Further, a decrease in DOC production can have significant impacts on biogeochemical cycles of carbon and nutrients through the change in bacterial activity (Thingstad et al., 2008). The trends drawn from the present study lead to opposite conclusions to various recent studies carried out under nutrient replete conditions. First, in previous studies, the organic carbon production and growth of diatoms accelerated under high CO2 conditions, and it was concluded that ocean acidification may act as a negative feedback on global environmental issues associated with increasing atmospheric CO2 (Riebesell et al., 2007; Tortell et al., 2008). Second, previous studies have demonstrated that DOC production may be enhanced under high CO2; Engel et al. (2004) revealed that the formation of transparent exopolymer particles, which are made of the aggregation of dissolved carbohydrates, was accelerated under high CO2 conditions. A similar process was suggested by Arrigo (2007) for discussing the enhanced biological carbon consumption at elevated pCO2 in Riebesell et al. (2007). These previous results were explained by the potential enhancement of photosynthetic carbon assimilation under high CO2 through lowering the energetic costs associated with carbon acquisition, as found for natural assemblages (Hein and SandJensen, 1997; Tortell et al., 2008) and isolated single strains (Riebesell et al., 1993; Rost et al., 2003). Under such conditions, some parts of the synthesized organic carbon fail to be incorporated into proteins and lipids and can be exuded to outside of the cell as DOC (Nagata, 2000). As suggested in this study, nutrient conditions may alter the consequent response of organic carbon production and diatoms to ocean acidification. We should verify these responses under both nutrient limited and replete conditions.

4. Conclusion The present study detected decreased net DOC accumulation at N480 μatm pCO2 compared to 150 μatm pCO2 under nutrient depleted conditions. A decrease in relative contribution of diatoms at higher pCO2 may cause the CO2 dependent change in DOC dynamics. These results contrasted with recent studies, conducted under nutrient replete conditions. The organic carbon and plankton dynamics are fully coupled, so we need a better understanding of the impact of ocean acidification on these critical components of marine biogeochemistry. Previous experimental results have recently been incorporated into a simulation model to assess the impacts of ocean acidification on the biological carbon pump (Oschlies et al., 2008; Hofmann and Schellnhuber, 2009). The present study indicates the possibility that responses of plankton to pCO2 increase are different between nutrient replete and depleted conditions. Further research is required based on in situ CO2 manipulation experiments using a nutrient limited plankton ecosystem as well as the phytoplankton

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blooming processes and eutrophic coastal systems, and results incorporated into global models. Contributors All authors have approved the article for publication. Individual author roles are as follows: Yoshimura — research design and management, field operations, data analysis and interpretation, and manuscript drafting; Nishioka — research design and management, field operations, data analysis, and manuscript drafting assistance; Suzuki — field operations, data analysis and interpretation, and manuscript drafting assistance; Hattori — data analysis and interpretation; Kiyosawa — data analysis and interpretation; and Watanabe — data analysis and interpretation, and manuscript drafting assistance. Disclosure statement The authors declare no actual or potential conflict of interest. Role of the funding source The research presented in this paper was made possible through funding from the Central Research Institute of Electric Power Industry. The sponsor had no participation in the study design, data analysis, interpretation of data, and paper submission. Acknowledgements We wish to thank the participants of Amur-Okhotsk Project and the captain and crew aboard the R/V Professor Khromov for field assistance. We thank K. Sugita and A. Tsuzuku for their help on land in preparing the experiments, A. Murayama for nutrients analysis, N. Saotome for POC analysis, and K. Sugie for discussing the results. We also thank C. Norman for his comments to improve the manuscript. Fig. 1 was reproduced using Online Map Creation (http://www. aquarius.geomar.de/omc/omc/). We acknowledge the editor and two anonymous reviewers for providing valuable comments that improved the manuscript significantly. This work was supported by a grant #060215 from CRIEPI.[ST] References Andreev, A.G., Pavlova, G.Y., 2010. Okhotsk Sea. In: Liu, K.-K., Atkinson, L., Quiñones, R., Talaue-McManus, L. (Eds.), Carbon and Nutrient Fluxes in Continental Margins: a Global Synthesis. Global Change—the IGBP Series. Springer, Berlin. Arrigo, K.R., 2007. Carbon cycle: marine manipulations. Nature 450, 491–492. Azam, F., Fenchel, T., Field, J.G., Gray, J.S., Meyer-Reil, L.A., Thingstad, F., 1983. The ecological role of water-column microbes in the sea. Mar. Ecol. Prog. Ser. 10, 257–263. Bindoff, N.L., Willebrand, J., Artale, V., Cazenave, A., Gregory, J., Gulev, S., Hanawa, K., Le Quéré, C., Levitus, S., Nojiri, Y., Shum, C.K., Talley, L.D., Unnikrishnan, A., 2007. Observations: oceanic climate change and sea level. In: Solomon, S., Qin, D., Manning, M., Chen, Z., Marquis, M., Averyt, K.B., Tignor, M., Miller, H.L. (Eds.), Climate Change 2007: the Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, United Kingdom. Boyd, P.W., Doney, S.C., 2002. Modelling regional responses by marine pelagic ecosystems to global climate change. Geophys. Res. Lett. 29, 1806 doi:10.1029/ 2001GL014130. Caldeira, K., Wickett, M.E., 2003. Anthropogenic carbon and ocean pH. Nature 425, 365. Carpenter, E.J., Lively, J.S., 1980. Review of estimates algal growth using l4C techniques. In: Falkowski, P. (Ed.), Primary Productivity in the Sea. Plenum Press, New York, USA, pp. 161–178. Culberson, C.H., Pytkowicz, R.M., Hawley, J.E., 1970. Seawater alkalinity determination by the pH method. J. Mar. Res. 28, 15–21. Dickson, A.G., Millero, F.J., 1987. A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media. Deep-Sea Res. 40, 107–118. Doney, S.C., Fabry, V.J., Feely, R.A., Kleypas, J.A., 2009. Ocean acidification: the other CO2 problem. Annu. Rev. Mar. Sci. 1, 169–192. Engel, A., Delille, B., Jacquet, S., Riebesell, U., Rochelle-Newall, E., Terbrüggen, A., Zondervan, I., 2004. Transparent exopolymer particles and dissolved organic carbon production by Emiliania huxleyi exposed to different CO2 concentrations: a mesocosm experiment. Aquat. Microb. Ecol. 34, 93–104.

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