High CO2 Under Nutrient Fertilization Increases Primary Production and Biomass in Subtropical Phytoplankton Communities: A Mesocosm Approach

Introduction

With a surface area of more than 200 million square kilometers, subtropical oligotrophic waters form the largest ecosystem of the worlds' surface oceans, covering more than 60% of total ocean surface (Longhurst et al., 1995). These extensive areas are typically characterized by a deep and nutrient-poor mixed layer, which is prevented from mixing with deeper nutrient-rich waters by a strong, almost permanent thermocline. Consequently, both phytoplankton biomass and primary production are low during most of the year. Despite its low productivity per surface area (Longhurst et al., 1995), more than 30 million tons of carbon dioxide (CO₂) are photosynthetically fixed into organic compounds every day, contributing nearly one third of total oceanic primary production (Field et al., 1998; Behrenfeld et al., 2001, 2006), and thus playing a key role in the global carbon cycle (Falkowski, 1994; Falkowski et al., 2000).

Climate change is inducing physical and chemical changes in the marine environment, with profound consequences for ocean productivity (Bopp et al., 2001; Gruber, 2011; Doney et al., 2012; IPCC, 2014). The anthropogenic release of CO₂ through human activity since the beginning of the industrial revolution is leading to an increase of the partial pressure of this greenhouse gas in the ocean, and consequently both pH and calcium carbonate saturation states (Ω) are declining rapidly, a process termed “ocean acidification” (Caldeira and Wickett, 2003; Sarmiento et al., 2004). Furthermore, as a consequence of increasing atmospheric greenhouse gas concentrations, the surface ocean is warming at a higher rate than the deep ocean, which may lead to a strengthening of the water column stratification (IPCC, 2014 and references therein).

Whereas increasing oceanic CO₂ levels are hypothesized to boost ocean productivity by relieving CO₂ limitation of the Ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO) enzyme (Beardal and Raven, 2004; Reinfelder, 2011; Mackey et al., 2015), a more stratified ocean would lead to a decrease in nutrient supply to the euphotic layer and therefore a potential reduction of autotrophic productivity (Bopp et al., 2001; Steinacher et al., 2010). Nevertheless, it has been predicted that the heterogeneous warming of oceans and continents, may enhance upwelling-favorable winds in Eastern Boundary Current Systems (Bakun, 1990; Sydeman et al., 2014; García-Reyes et al., 2015). Stronger cross-shore wind gradients would lead to an intensification of the eddy kinetic energy fields across eastern boundary regions of the subtropical Gyres, favoring the upward pumping of nutrients driven by upwelling processes. Phytoplankton thriving in a future warmer and acidified oligotrophic subtropical ocean could therefore be patchily fertilized by increased mesoscale and submesoscale processes inducing nutrient pumping into the ocean surface.

Investigations of CO₂-related effects on marine productivity have experienced a remarkable surge over the last years (Riebesell and Gattuso, 2015). Theoretical studies based on chlorophyll-dependent models predict a decrease in phytoplankton chlorophyll concentration, and consequently in primary production, in a warmer, acidified and more stratified ocean (Behrenfeld et al., 2006; Marinov et al., 2010; Steinacher et al., 2010). On the other hand, experimental studies have reported contrasting results about the potential effects of increasing CO₂ on marine productivity in natural assemblages (Table 1). Whereas, some authors reported increased photosynthetic rates with increasing CO₂ (Hein and Sand-Jensen, 1997; Riebesell et al., 2007; Bellerby et al., 2008; Tortell et al., 2008; Egge et al., 2009; Engel et al., 2013; Eberlein et al., 2017), others have not observed significant relationships between marine productivity and ocean acidification (Tortell et al., 2002; Delille et al., 2005; Hare et al., 2007; Feng et al., 2009; Tanaka et al., 2013; Maugendre et al., 2017). Most of the experiments, however, have been carried out in nutrient-rich systems, with only a few performed in low-nutrient regions (Yoshimura et al., 2009; Maugendre et al., 2017). Thus, there is a severe lack of information on how ocean acidification could affect primary production in subtropical oligotrophic regions.

TABLE 1

Table 1. Published studies on the effect of ocean acidification on primary production in plankton communities.

This study investigates how an acidified and patchily fertilized subtropical ocean impacts marine autotrophic productivity. For this, we carried out a mesocosm experiment off the coast of Gran Canaria (Canary Islands) during the autumn of 2014. We studied the response of size-fractionated primary production, chlorophyll a and biomass of the phytoplankton community to enhanced CO₂ levels and nutrient fertilization to investigate which size-fraction, if any, responds more readily to these short-term perturbations.

Methods

Set-up and Sampling

The experiment was carried out in Gando Bay (27°55′ 41′′ N, 15° 21′ 55′′ W), Gran Canaria (Canary Island), as part of the BIOACID (Biological Impacts of Ocean ACIDification) project. Nine KOSMOS (Kiel Off-Shore Mesocosms for future Ocean Simulations; Riebesell et al., 2013) were deployed and enclosed ~35 m³ of low-nutrient low-chlorophyll water off the east coast of Gran Canaria. In order to achieve a pCO₂ gradient from ~400 μatm to partial pressures corresponding to the year 2150 (~1,000 μatm) according to the RCP8.5 scenario (IPCC, 2014), seven mesocosms were gradually enriched at the start of the experiment over a period of 7 days (from t₀ to t₆), via the addition of different amounts of filtered CO₂-saturated seawater. Unfortunately, one of the high-CO₂ mesocosms (M6) was damaged on t₂₆, and hence was not considered in the data analyses. Two more CO₂ addition were carried out on t₂₄ and t₃₈ to counteract the loss of CO₂ due to outgassing and biological uptake. The average pCO₂ concentrations along the whole experiment for each of the remaining six mesocosms were: M5 (448 μatm), M3 (563 μatm), M7 (668 μatm), M4 (716 μatm), M2 (887 μatm), and M8 (1025 μatm). Two other mesocosms -M1 (369 μatm) and M9 (352 μatm)- remained untreated, serving as controls (ambient pCO₂) (Figure 1). The volumes of CO₂-saturated seawater added to achieve the pCO₂ gradient varied from 77 L in M5 to 382 L in M8. After 24 days, between 7.5 and 9.0 m³ of nutrient-rich deep water was added to each mesocosm with the purpose of simulating a natural fertilization event (Figure 2). During the 56 day experimental period, integrated water samples (0–13 m) were collected by means of depth-integrated water samplers (IWS, HYDRO-BIOS, Kiel), on days −1, 1, 5, 9, 13, 17, 21, 25, 27, 29, 31, 33, 35, 37, 41, 45, 50, and 55, from each of the nine mesocosms, plus an extra sample from ambient waters outside the mesocosms (referred to as Atlantic; A). Once on land, subsamples were taken for primary production, chlorophyll a, phytoplankton abundances, as well as for dissolved inorganic carbon and inorganic nutrients. More detailed information concerning the experimental set-up (CO₂ manipulation, deep water addition, sampling, etc.) is provided by Taucher et al. (2017).

FIGURE 1

Figure 1. Temporal development of pCO₂ (μatm) over the course of the experiment in the mesocosms (MX) and the surrounding Atlantic waters (A). Vertical lines separate the three phases of the experiment. Values in parentheses indicate average pCO₂ concentrations for each mesocosm along the whole experiment.

FIGURE 2

Figure 2. Temporal development of (A) nitrate + nitrite (NO_x⁻) (μmol·L⁻¹), (B) phosphate (μmol·L⁻¹), (C) silicate (μmol·L⁻¹) and (D) ammonium (μmol·L⁻¹) over the course of the experiment. Vertical lines separate the three phases of the experiment.

pCO₂ and Inorganic Nutrients

Partial pressure of CO₂ (pCO₂) was derived from total alkalinity (TA) and dissolved inorganic carbon (DIC) data, following Pierrot et al. (2006) and Lueker et al. (2000). TA was measured by means of a Metrohm 862 Compact Titrosampler and a 907 Titrando unit. DIC was determined by infrared absorption using a LI-COR LI-700 on an ARICA system (MIRANDA, Kiel). Inorganic nutrients (${\text{NO}}_3^{-}$, ${\text{NO}}_2^{-}$, ${\text{PO}}_4^{3-}$, and Si(OH)₄) were determined by colorimetric methods following Murphy and Riley (1962) and Hansen and Grasshoff (1983). Ammonium (${\text{NH}}_4^{+}$) was fluorometrically analyzed following Holmes et al. (1999). A SEAL Analytical QuAAtro AutoAnalyzer connected to a JASCO Model FP-2020 Intelligent Fluorescence Detector and a SEAL Analytical XY2 autosampler was used to measure ${\text{NO}}_3^{-}$, ${\text{NO}}_2^{-}$, ${\text{PO}}_4^{3-}$, Si(OH)₄, and ${\text{NH}}_4^{+}$ For details on the methodology of these measurements see Taucher et al. (2017).

Chlorophyll a

For chlorophyll a analysis, 500 ml of sea water were sampled and filtered sequentially through 20, 2, and 0.2 μm pore-size Whatman polycarbonate filters under low vacuum pressure. Filters were kept frozen at −20°C until analysis. Before chlorophyll determination, pigments were extracted using 10 ml of 90% acetone at 4°C in the dark for 24 h. Extracts were measured fluorometrically, before and after acidification, by means of a Turner Designs bench fluorometer 10-AU, previously calibrated with pure chlorophyll a (Sigma Chemical), following Holm-Hansen et al. (1965). The collected material on the 20, 2, and 0.2 μm filters was used to measure the corresponding microplankton (Chl_Micro), nanoplankton (Chl_Nano) and picoplankton (Chl_Pico) chlorophyll concentrations, respectively. Total chlorophyll (Chl_Tot) was derived from the sum of the three size fractions.

Phytoplankton Abundance and Biomass

Prochlorococcus and Synechococcus type cyanobacteria and small photosynthetic eukaryotic cells (picoeukaryotes) were enumerated with a FACScalibur (Becton and Dickinson) flow cytometer. Picoeukaryotes, Prochlorococcus and Synechococcus samples (about 1 mL) were analyzed in fresh material 30–60 min after subsampling from the carboys. Prochlorococcus were recurrently observed in Atlantic waters, but vanished inside all the mesocosms after 2 days. Thus, we did not use their abundances to compute biomass. Phytoplankton groups were identified by their signatures in a plot of side scatter (SSC) vs. red (FL3) and orange (FL2) fluorescence. Samples were run at 60 μL min⁻¹. A suspension of yellow–green 1 μm latex beads (~10⁵ beads mL⁻¹) was added as an internal standard (Polysciences, Inc.). Pigmented nanoeukaryotes (2–20 μm) were counted on fresh samples with a Cytobuoy cytometer (Dubelaar and Gerritzen, 2000), provided with flow-image. Samples (about 3 ml) were analyzed in vivo for 7 min at a flow rate of 300 μL min⁻¹. Microphytoplankton (mostly diatoms and dinoflagellates) were fixed with alkaline Lugol's iodine (1% final concentration), sedimented in Utermöhl chambers and counted by means of an inverted microscope (Utermöhl, 1931).

Biomass of Synechococcus and picoeukaryotes were estimated by multiplying their abundances by the average cell carbon content obtained for each group, using the conversion factors obtained by MF Montero (unpublished) from samples collected in coastal waters of Gran Canaria: 120 fgC/cell (Synechococcus), 420 fgC/cell (picoeukaryotes). Nanoeukaryotes' abundances were converted into biomass using an estimated average biovolume of 20 μm⁻³ for organisms between 2 and 6 μm and a biovolume of 125 μm⁻³ for organisms between 6 and 11 μm, applying the conversion factor of 220 fgC μm⁻³ proposed by Borsheim and Bratbak (1987). Biovolumes and conversion factors used for diatoms and dinoflagellates were calculated following Menden-Deuer and Lessard (2000). Picoplankton biomass (B_Pico) was calculated as the sum of the biomasses of Synechococcus and picoeukaryotes, nanoplankton biomass (B_Nano) as the sum of all nanoeukaryotes' biomass, and microplankton biomass (B_Micro) as the sum of the diatoms and dinoflagellates biomass. Total biomass (B_Tot) refers to the sum of all size fractions.

¹⁴C-Based Primary Production

Primary production was measured using the ¹⁴C method. Four culture flasks per mesocosm, and per ambient Atlantic seawater sample, were filled with 70 ml of water, and inoculated with 15 μCi of ¹⁴C-labeled sodium bicarbonate solution (NaH¹⁴CO₃; Perkin Elmer). Three of them were in vitro incubated for 12 h in a temperature-controlled chamber reproducing in situ daily average light and temperature, while the remaining flask was incubated at the same temperature under complete darkness to measure the dark carbon uptake. Sixty milliliter of the samples were filtered with a vacuum pump sequentially through 20, 2, and 0.2 μm pore-size Whatman polycarbonate filters, to allow calculation of the particulate organic carbon fixed by microplankton (PP_Micro), nanoplankton (PP_Nano) and picoplankton (PP_Pico), respectively. The total particulate organic carbon production (PP_POC) was derived from the sum of the three size fractions. Filters were then placed in 4 ml scintillation vials and exposed to concentrated HCl fumes overnight to remove ¹⁴C-labeled inorganic carbon. To estimate the amount of carbon fixation into the dissolved organic carbon fraction (PP_DOC), 5 ml of water sample was gently filtered onto 0.2 μm Whatman polycarbonate filter under low vacuum pressure. The filtrate was transferred to a 20 ml scintillation vial. Liquid samples were acidified with 100 μl of 50% HCl and placed in an orbital oscillator for 24 h. Finally, scintillation cocktail (Ultima Gold XR) was added to every sample, thoroughly mixed, and stored in darkness for another 24 h, prior to measuring radioactivity in a scintillation counter Beckman LS-6500. Primary production (μg C·L⁻¹·h⁻¹) was calculated according to:

$\begin{array}{l}\text{PP}=\left[\frac{{\text{V}}_{\text{S}}}{{\text{V}}_{\text{F}}}\right]·\frac{\text{DIC}·(\text{DP}{\text{M}}_{\text{S}}-\text{DP}{\text{M}}_{\text{D}})}{{\text{DPM}}_A·{\text{t}}_{\text{i}}}\end{array}$

where V_S is the volume of the sample (ml); V_F the filtered volume (ml); DIC the dissolved inorganic carbon of the sample (μg C·L⁻¹); DPM_S the disintegration per minute of the samples; DPM_D the disintegration per minute of the dark-incubated samples; DPM_A total initial addition of ¹⁴C and t_i the incubation time (h).

The percentage of extracellular carbon release (PER) was calculated as:

$\begin{array}{l}PER(\%)=\frac{P{P}_{DOC}}{P{P}_{TOT}}x100\end{array}$

being PP_TOT the sum of PP_DOC and PP_POC

Statistical Analysis

To investigate the potential effects of ocean acidification on autotrophic productivity throughout the three phases of the experiment, model II (Reduced Major Axis) linear regressions (Sokal and Rohlf, 2013) between primary production, chlorophyll and biomass and pCO₂ concentrations were performed for each phase using Matlab (The MathWorks, Inc, Natick, Massachusetts, United States). For that purpose, all datasets were averaged per mesocosm and phase. The confidence level for all analysis was set at 95% (p < 0.05).

Results

Temporal Development of Size-Fractionated Chlorophyll a and Autotrophic Biomass

Nutrient concentrations (Figure 2), together with the development of chlorophyll and biomass (Figures 3, 4), allowed the differentiation of three well-defined phases over the experimental period: The pre-bloom phase (I), from t₁ to t₂₃; the bloom phase (II), from t₂₅ to t₃₅; and the post-bloom phase (III), from t₃₇ until the end of the experiment (t₅₅).

FIGURE 3

Figure 3. Temporal development of chlorophyll a (μg · L⁻¹) in different size fractions: (A) total (Chl_Tot), (B) microplankton (Chl_Micro), (C) nanoplankton (Chl_Nano) and (D) picoplankton (Chl_Pico) over the course of the experiment. Vertical lines defined the three phases of the experiment.

FIGURE 4

Figure 4. Temporal development of biomass (μg C·L⁻¹) in different size fractions: (A) total (B_Tot), (B) microplankton (B_Micro), (C) nanoplankton (B_Nano) and (D) picoplankton (B_Pico) over the course of the experiment. Vertical lines defined the three phases of the experiment.

During phase I, the waters inside the mesocosms were characterized by relatively low nutrient concentrations (similar to ambient Atlantic waters), low Chl_Tot, and low B_Tot (Figures 2– 4). Two to three days after enclosing the water inside the mesocosm bags (t₁-t₂), the concentration of all inorganic nutrients slightly increased both inside the mesocosms and in Atlantic waters. This suggests the influence of external inputs; perhaps caused by dust deposition on the surface waters (since during those days there was aerosols deposition with dust originating from NW Africa). Nitrate, phosphate and silicate displayed relative maxima around t₅, dropping after t₁₀ and reaching minimum values at t₂₃. Ammonia peaked around t₁₀₋₁₅ and dropped to minimum values around t₂₀. The largest decrease in the meosocosm nutrient concentrations compared to the Atlantic waters was in the silicates, as a result of its consumption by diatoms (Taucher et al., 2017).

Total chlorophyll and biomass increased from t₀ to t₂₃, following a general inverse trend with nutrient concentrations (Figure 3). However, there were contrasting patterns between chlorophyll and biomass in the different size-fractions. There were no significant differences in chlorophyll concentrations between the mesocosms and Atlantic waters in any of the size fractions (Figure 3). In contrast, there were significant differences in biomass in the largest size fractions between mesocosms and Atlantic waters, suggesting a dominance of large mixotrophic organisms inside the mesocosms (Figure 4). Figure 5 illustrates the relative contributions of the different size fractions (averaged from all the mesocosms) to chlorophyll, biomass and primary production over the course of the experiment. During phase I, picophytoplankton (Pico) contributed >40% to Chl_Tot, but < 40% to B_Tot, with no significant differences with ambient waters. Nanophytoplankton (Nano) contributed 30–35% to Chl_Tot, but dominated in biomass (about 50%), although differences between mesocosms and Atlantic waters were not significant. Diatoms and dinoflagellates (Micro) contributed about 25% to Chl_Tot but only 10–20% to B_Tot, with clear differences with respect to Atlantic waters, particularly after t₁₀.

FIGURE 5

Figure 5. Contribution (%) of each size fraction to (A) total particulate primary production (PP), (B) total biomass, and (C) total chlorophyll (Chl) in phase I (t₁-t₂₄), phase II (t₂₅-t₃₅) and phase III (t₃₆-t₅₅). Micro, microplankton; Nano, nanoplankton; Pico, picoplankton.

Following the nutrient fertilization at t₂₄, Chl_Tot and B_Tot increased exponentially reaching maximum average values (3.4 ± 0.5 and 244 ± 122 μg C·L⁻¹, respectively) at t₂₉ (Figures 3, 4). Consequently, nutrient concentrations were rapidly utilized inside the mesocosms, dropping to levels similar to the Atlantic waters when the phytoplankton bloom was at its peak (Figure 2). There was an exception with silicates, whose values dropped even below the Atlantic values after the bloom, as result of the large consumption by diatoms during phases II and III (Taucher et al., 2017). M8 and M2, the two mesocosms with highest CO₂ concentrations, were markedly elevated in biomass, both in the Micro and Nano fractions, compared to the other mesocosms (Figure 3). With the depletion of inorganic nutrients, the levels of chlorophyll as well as biomass decreased in the Pico and Nano fractions, with a change in the slopes at t₃₆, when nutrients were almost exhausted. Biomass of Micro decreased more smoothly, maintaining high values in the high CO₂ treatments during the start of phase III. chlorophyll and biomass in phase II were dominated by Micro, contributing 40–60%, with a slightly higher dominance in biomass than in chlorophyll. In contrast, Pico contributed on average only 10–35% to chlorophyll and biomass inside the mesocosms, with values significantly lower than in the Atlantic waters.

During phase III, nutrient concentrations remained low (Figure 2), with nitrate and phosphate occurring at similar levels compared to Atlantic waters, but silicate was almost depleted (due to the consumption by diatoms) to levels < 0.08 μM, significantly below those of Atlantic waters. Only ammonium showed higher concentrations in some of the mesocosms, compared to Atlantic waters and phases I and II, probably due to higher excretion rates of grazers during this phase. Although Chl_Tot and B_Tot decreased with respect to phase II, they were still markedly higher than in Atlantic waters. The contribution of the larger size fractions to chlorophyll and biomass were always higher in the mesocosms than in Atlantic waters, but with apparent differences between chlorophyll and biomass (Figure 5). Pico contributed in general to near 45% of Chl_Tot and 35% to B_Tot in Atlantic waters compared to < 25% of Chl_Tot and < 10% of B_Tot in the mesocosms. Nano and Micro contributed almost evenly to Chl_Tot in the mesocosms, but not to biomass.

Dynamics of Size-Fractionated ¹⁴C-Based Primary Production

Total primary production in the particulate fraction (PP_POC) displayed average rates in the mesocosms (0.93 ± 0.48 μg C·L⁻¹·h⁻¹) that were almost double than in Atlantic waters (0.56 ± 0.24 μg C·L⁻¹·h⁻¹) at the start of the experiment. Like chlorophyll and biomass, the rates increased 4 to 5-fold peaking at t₁₀₋₁₂ in all size fractions (although more for the Pico), to decrease again to initial rates, just before nutrient fertilization (Figure 6). The greatest differences between Atlantic waters and mesocosms were observed in Pico. This fraction contributed >50% to PP_POC in phase I, compared to ~30% in Atlantic waters.

FIGURE 6

Figure 6. Temporal development of primary production (μg C·L⁻¹·h⁻¹) in (A) the total particulate faction (Total), and in the (B) microplankton (Micro), (C) nanoplankton (Nano) and (D) picoplankton (Pico) size fractions, over the course of the experiment. Vertical lines defined the three phases of the experiment. PP is represented in log scale to better illustrate the differences among mesocosms.

After nutrient fertilization (phase II), PP_POC increased more than 15 fold in all mesocosms, peaking at t₂₉ with the highest rates in M2, reaching 35 μg C·L⁻¹·h⁻¹, compared to values < 2 μg C·L⁻¹·h⁻¹ in Atlantic waters (Figure 6). Due to nutrient depletion, total PP_POC rates declined to < 4 μg C·L⁻¹·h⁻¹ at t₃₅ (end of phase II). Micro contributed almost a 75% to total PP_POC, even more than to chlorophyll and biomass (Figure 5), while the contribution of Pico was about 10%.

During phase III, PP_POC rates declined more smoothly than in phase II, following nutrient depletion, with higher rates in the mesocosms with higher CO₂ treatments (see following section). Like in phase II, the major contribution to total PP_POC was due to Micro (60–70%), while Pico and Nano contributed about 15–20% each.

The average rate of primary production contributing to the dissolved organic carbon fraction (PP_DOC; Figure 7A) inside the mesocosms varied strongly during the course of the experiment, between 34.7 μg C·L⁻¹·h⁻¹ in mesocosm M2 during the bloom phase and 0.23 μg C·L⁻¹·h⁻¹ in mesocosm M7 during phase I. Its temporal development matched PP_POC dynamics, increasing with nutrient addition in t₂₄. However, the percentage of extracellular organic carbon release (PER; Figure 7B) decreased from the first week of the experiment (average 26.7 ± 8.6 %), to t₂₉, when primary production reached their maximum values and PER its average minimum value (7.1 ± 3.7 %). In Atlantic waters, PP_DOC was similar (phase I) or lower (phases II and III) than in the experiments, but with higher and less stable PER, ranging from < 10 to 35% (Figure 7B).

FIGURE 7

Figure 7. Temporal development of (A) rates of dissolved primary production (PP_DOC) (μg C·L⁻¹·h⁻¹) and (B) the percentage of extracellular release (PER) (%) [(PP_DOC/PP_TOC) · 100] over the course of the experiment. Vertical lines separate the three phases of the experiment.

CO₂ Effects on Size-Fractionated Chlorophyll, Biomass and Primary Production

A total of 39 linear regressions were conducted to test potential CO₂ effects on total community and size-fractionated primary production, biomass and chlorophyll, and almost two thirds (28) showed statistically significant (p < 0.05) relationships (Tables 2 – 4, respectively). Total particulate primary production (PP_POC) remained unaffected during phase I, in contrast to B_Tot, B_Nano, Chl_Nano and Chl_Tot, which exhibited negative responses to increasing pCO₂. Negative responses (6) were only observed in the oligotrophic phase (phase I). Dissolved carbon production rate (PP_DOC) also presented a strong relationship with pCO₂, but a positive one. No statistically significant correlations with pCO₂ concentrations were found with the remaining parameters in phase I.

TABLE 2

Table 2. Linear regressions statistics of the relationship between average primary production in the total particulate (PP_POC) and different size fractions (PP_Micro, PP_Nano, PP_Pico), as well as in the total dissolved fraction (PP_DOC), vs. pCO₂ levels for the three experimental phases.

After nutrient fertilization in t₂₄, the majority of the CO₂ correlations (23 out of 26) were statistically significant and mostly positive (Tables 2 – 4). Fractionated primary production, biomass and chlorophyll, as well as PP_POC, B_Tot and Chl_Tot were positively related to increasing pCO₂ (except PP_Nano) in phase II. PP_DOC was statistically correlated with pCO₂ in phase III but not in phase II. We also observed strong significant and positive correlations between pCO₂ and both total and size-fractionated primary production, biomass and chlorophyll during phase III (except in the Pico fraction).

Discussion

CO₂ and Nutrient Impacts on Phytoplankton Biomass and Productivity

The eight mesocosms during phase I displayed similar behaviors to the Atlantic ambient waters, showing a phytoplankton community dominated in terms of biomass by small picophytoplankton and nanophytoplankton (Figure 5), characteristic of the oligotrophic subtropical northeast Atlantic (Zubkov et al., 2000a,b; Arístegui and Montero, 2005; Figueiras et al., 2016), although with somewhat higher primary production and biomass.

Due to the low nutrient concentrations, PP_POC was low and relatively stable during phase I, showing no significant relationship with pCO₂. Although, theoretically, carbon fixation should be enhanced by high pCO₂ levels (Giordano et al., 2005; Reinfelder, 2011), the absence of a response of primary production to increasing CO₂ in oligotrophic waters dominated by small phytoplankton has been reported by other authors (Yoshimura et al., 2009; Maugendre et al., 2017). Surprisingly, both chlorophyll and biomass decreased with increasing pCO₂ during phase I (Tables 3, 4), even though primary production remained rather stable. Engel et al. (2013) observed the same behavior in a similar mesocosms study in Arctic waters. They hypothesized that either the enhancement of particle aggregation and settling as a consequence of increasing Transparent Exopolymer Particles (TEP), the increase in remineralization of phytoplankton cells, the nutrient competition between auto- and heterotrophic organisms, or a combination of the preceding processes, could explain the mismatch between PP_POC and its accumulation as biomass. Due to the lack of response of TEPs to increasing pCO₂ in our experiment (data not shown), the first hypothesis would not apply in our case. A more likely explanation would be that a fraction of primary production is channeled through the dissolved fraction increasing the DOC pool inside the mesocosms. This hypothesis agrees with both the observed higher values of PP_DOC in the high-CO₂ mesocosms, and the increase in DOC from t₃ to t₂₃ reported by Zark et al. (2017) in this same study.

TABLE 3

Table 3. Linear regressions statistics of the relationship between average total community biomass (B_Tot) and the biomass of the different size fractions (B_Micro, B_Nano, B_Pico) vs. pCO₂ levels for the three experimental phases.

TABLE 4

Table 4. Linear regressions statistics of the relationship between average total community chlorophyll a (Chl_Tot) and the chlorophyll of the different size fractions (Chl_Micro, Chl_Nano, Chl_Pico) vs. pCO₂ levels for the three experimental phases.

Nutrient fertilization triggered autotrophic phytoplankton community growth, with higher maximum biomass build-up in the high-CO₂ mesocosms. The bloom that occurred during phase II, coincided with an increase in primary production and biomass inside all mesocosms. As a consequence, major nutrients were depleted to values similar (such as ${\text{NO}}_3^{-}$ + NO₂ and ${\text{PO}}_4^{3-}$) or lower (Si(OH)₄) to phase I. Linear regressions between PP_POC, B_Tot and Chl_Tot vs pCO₂ revealed significantly positive relationships in phase II, although the strongest relationships of PP_POC, B_Tot and Chl_Tot vs pCO₂ occurred in phase III. A positive effect of increasing pCO₂ in seawater on primary production has been reported by other authors, from single species' experiments (Beardal and Raven, 2004; Fu et al., 2007; Sobrino et al., 2008) to whole community level experiments (Egge et al., 2009; Engel et al., 2013). The positive effect of enhanced pCO₂ on primary production has been attributed to the relieve of CO₂ limitation of RubisCO (Giordano et al., 2005; Riebesell et al., 2007; Reinfelder, 2011; Mackey et al., 2015). The increase in the availability of CO₂ should thus produce an increase in photosynthetic rates, by relieving carbon limitation, or indirectly by lowering the energy required to concentrate CO₂ against a smaller concentration gradient.

In summary, our results show that all the positive significant correlations between plankton productivity and biomass and pCO₂ occur after nutrients addition, suggesting a synergistic effect of nutrients and CO₂. Indeed, as far as we know, the only ocean acidification mesocosm study carried out to test primary production responses in oligotrophic regions, report non-statistically significant effects of increasing pCO₂ on primary production under nutrient deplete conditions (Maugendre et al., 2017).

Community Structure Response to Elevated CO₂ and Nutrient Fertilization

During phase I, Pico contributed > 40% to Chl and > 50% to primary production, although Nano was the dominant size fraction in terms of biomass. A marked change in the phytoplankton community occurred after the fertilization through deep-water addition at the beginning of phase II and during phase III, where the community shifted to larger microphytoplankton (mostly diatoms; Taucher et al., 2017) with > 57% dominance in biomass and > 60% in primary production (Figure 5). Changes from small to large size fractions of phytoplankton have been described in the Canary Islands region, associated with transitional changes from oligotrophic to eutrophic conditions across upwelling filaments, eddies and fronts (Basterretxea and Arístegui, 2000; Arístegui et al., 2004).

Our results show that phytoplankton size-groups generally remained unresponsive during phase I to the increases in pCO₂, whereas all of them benefitted from the nutrient addition. This disagrees with a study by Egge et al. (2009), who reported non-significant changes in primary production among size fractions in a mesocosm experiment in Bergen (Norway), although differences in chlorophyll and biomass at group level have been observed during other mesocosm experiments (Brussaard et al., 2013; Schulz et al., 2017). For this present study, Taucher et al. (2017), using a non-metric multidimensional scaling (NMDS) analysis, described a significant effect of CO₂ on the whole planktonic community (including protozoa and mesozooplankton). The effect is apparent even in phase I, but became more pronounced after nutrient fertilization. They reported that the response of the community structure to CO₂ treatments emerges not from one or two dominant species but from overall shifts across the entire plankton community. At the phytoplankton level, we find in most cases a significant effect of CO₂ on primary production, biomass and chlorophyll a in all the size fractions during phases II and III, pointing to a size independent stimulation of nutrients and CO₂ over all the phytoplankton groups, which particularly benefited the increase of diatoms in absolute terms (Taucher et al., 2017). However, a closer look to the linear correlations reveals significant differences among regression slopes, indicating differences in size fractions responses. Indeed, the relative increase of primary production after nutrient additions display a clear size-related pattern, where small PP_Pico was enhanced about 250%, PP_Nano up to 150% and PP_Micro about 100% in the two high-CO₂ mesocosms compared to the two low-CO₂ mesocosms, Surprisingly, the highest relative change in biomass was observed in B_Micro followed by B_Nano and B_Pico. This could be partly explained by a more intense grazing pressure on the smallest size fractions compared to the largest ones.

A number of experimental and modeling studies have suggested the existence of potential winners and losers in a future acidified ocean (Dutkiewicz et al., 2013; Kroeker et al., 2013; Bach et al., 2017). In our mesocosm experiment, the shift from a cyanobacteria-dominated community to a large diatom-dominated community in the high CO₂ scenarios after nutrient fertilization, as well as the distinct size-related relative change observed among size-fractions, seems to support this hypothesis. Nevertheless, the fact that all phytoplankton size classes increased after nutrient addition in high CO₂ treatments, suggests that the entire phytoplankton community in subtropical regions would benefit in an acidified and patchily fertilized ocean, although larger cells could outcompete the smaller ones under high nutrient inputs.

Biogeochemical Implications

Oligotrophic regions are expanding at an annual rate of 0.8–4.3%, with the North Atlantic subtropical Gyre showing the fastest annual expansion (Sarmiento et al., 2004; Polovina et al., 2008). Approximately 0.8 million km² of productive waters are being replaced annually by warmer stratified oligotrophic waters. Most models predict a decline in primary production as well as in the downward carbon fluxes in these regions by the end of the century (Riebesell et al., 2009; Gruber, 2011; Bopp et al., 2013), due to a reduction of vertical nutrient supply related with the shoaling of the mixed layer depth (Bopp et al., 2001; Marinov et al., 2010; Steinacher et al., 2010). Nevertheless, these models do not take into account the effect of mesoscale and submesoscale processes, such as eddies or fronts, which are known to play a key role in enhancing primary production (e.g. Mcgillicuddy et al., 2007; Sangrà et al., 2009) and export fluxes (Omand et al., 2015) in the ocean. Under an scenario of an acidified and warmer ocean, leading to an intensification of cross-shore wind gradients and eddy kinetic energy across eastern boundary regions (Bakun, 1990; Sydeman et al., 2014; García-Reyes et al., 2015), mesoscale variability would increase mixing and upwelling of deeper nutrient-rich water into the euphotic zone (Renault et al., 2016; Xiu et al., 2018). Our data suggest that a patchy nutrient pumping in a more acidified ocean would increase primary productivity in subtropical warm regions. Furthermore, community structure would shift from small to large cells, like diatoms, potentially leading to a more efficient carbon export to the deep ocean. The concomitant increase in dissolved organic carbon production (PP_DOC) may also contribute to the biological carbon pump, through particle aggregation and the subsequent increase of sinking rates (Engel et al., 2004, 2014; Schartau et al., 2007).

It has been estimated that mesoscale and submesoscale features account for 20–30% of the new primary production in the world's ocean (Mcgillicuddy et al., 2007). If global warming reinforces wind regimes in eastern boundary regions as predicted (Bakun, 1990; Sydeman et al., 2014), increasing the eddy kinetic energy field, new production could increase as well, counteracting the effect of enhanced stratification in subtropical regions. Nevertheless, further research is needed to constrain the synergistic or antagonistic effects of climate drivers on primary production and plankton community structure in subtropical oligotrophic waters, the most extensive ecosystems of the world's ocean.

Conclusions

This is the first mesocosm study addressing the response, in terms of primary production and community structure, of size-fractionated (0.2–2, 2–20, and >20 μm) natural plankton communities in subtropical oligotrophic regions to increasing CO₂ concentrations and nutrient fertilization. Our results reveal a non-significant CO₂-related effect on PP and B under nutrient depleted conditions, with a phytoplankton dominance of small cyanobacteria. After nutrient fertilization, however, the community shifts toward larger phytoplankton, with a diatom-dominated community, showing a significant marked increase in PP, B and chlorophyll under higher CO₂ conditions in all groups. Our data suggest that in a future acidified subtropical ocean, mesoscale and submesoscale features—which are predicted to enhance under global warming in eastern boundary regions—would drive nutrient pumping to the surface ocean favoring the development of diatoms and increasing new production in the global ocean.

Author Contributions

UR and JA: conceived and designed the experiment. All authors performed the experiment and analyzed the data. Wrote the paper: NH-H and JA with inputs from all co-authors.

Funding

The KOSMOS project was funded by the German Federal Ministry of Education and Research (BMBF) in the framework of the coordinated project BIOACID—Biological Impacts of Ocean Acidification, phase 2 (FKZ 03F06550). UR received additional funding from the Leibniz Award 2012 by the German Research Foundation (DFG). The Natural Environment Research Council provided funding for EA and ME as part of the UK Ocean Acidification Programme (NE/H017348/1). JA was supported by a Helmholtz International Fellow Award, 2015 (Helmholtz Association, Germany). NH-H was partially supported by KOSMOS funding during the experimental work, and by a grant of the Agencia Canaria de Investigación, Innovación y Sociedad de la Información (ACIISI) during the writing stage (TESIS2015010036). IB was supported by a FPI fellowship (BES-2016-078407) from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO). NH-H, MM, IB, and JA benefited also from the FLUXES project (CTM2015-69392-C3-1-R) funded by the Spanish government (Plan Nacional I+D).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We would like to thank the KOSMOS and the Plataforma Oceánica de Canarias (PLOCAN) teams assisting with all aspects of the organization and logistical support before, during and after this mesocosms campaign, as well as to Minerva Espino and Acorayda González (IOCAG, ULPGC) for their technical support in the sampling and analyses of phytoplankton and productivity data.

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