The influence of abrupt increases in seawater pCO2 on plankton productivity in the subtropical North Pacific Ocean

Donn A. Viviani; Daniela Böttjer; Ricardo M. Letelier; Matthew J. Church

doi:10.1371/journal.pone.0193405

Abstract

We conducted a series of experiments to examine short-term (2–5 days) effects of abrupt increases in the partial pressure of carbon dioxide (pCO₂) in seawater on rates of primary and bacterial production at Station ALOHA (22°45’ N, 158° W) in the North Pacific Subtropical Gyre (NPSG). The majority of experiments (8 of 10 total) displayed no response in rates of primary production (measured by ¹⁴C-bicarbonate assimilation; ¹⁴C-PP) under elevated pCO₂ (~1100 μatm) compared to ambient pCO₂ (~387 μatm). In 2 of 10 experiments, rates of ¹⁴C-PP decreased significantly (~43%) under elevated pCO₂ treatments relative to controls. Similarly, no significant differences between treatments were observed in 6 of 7 experiments where bacterial production was measured via incorporation of ³H-leucine (³H-Leu), while in 1 experiment, rates of ³H-Leu incorporation measured in the dark (³H-Leu_Dark) increased more than 2-fold under high pCO₂ conditions. We also examined photoperiod-length, depth-dependent (0–125 m) responses in rates of ¹⁴C-PP and ³H-Leu incorporation to abrupt pCO₂ increases (to ~750 μatm). In the majority of these depth-resolved experiments (4 of 5 total), rates of ¹⁴C-PP demonstrated no consistent response to elevated pCO₂. In 2 of 5 depth-resolved experiments, rates of ³H-Leu_Dark incorporation were lower (10% to 15%) under elevated pCO₂ compared to controls. Our results revealed that rates of ¹⁴C-PP and bacterial production in this persistently oligotrophic habitat generally demonstrated no or weak responses to abrupt changes in pCO₂. We postulate that any effects caused by changes in pCO₂ may be masked or outweighed by the role that nutrient availability and temperature play in controlling metabolism in this ecosystem.

Citation: Viviani DA, Böttjer D, Letelier RM, Church MJ (2018) The influence of abrupt increases in seawater pCO₂ on plankton productivity in the subtropical North Pacific Ocean. PLoS ONE 13(4): e0193405. https://doi.org/10.1371/journal.pone.0193405

Editor: João Miguel Dias, Universidade de Aveiro, PORTUGAL

Received: June 22, 2017; Accepted: February 9, 2018; Published: April 25, 2018

Copyright: © 2018 Viviani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The data underlying this study have been uploaded to the Biological and Chemical Oceanography Data Management Office (BCO-DMO) and are accessible using the following link: https://www.bco-dmo.org/dataset/726341.

Funding: Funding for this study was derived from the National Science Foundation (https://www.nsf.gov/), under grants OCE-0850827 (MJC and RML) and 1260164 (MJC). Additional support was provided by the Center for Microbial Oceanography: Research and Education (CMORE; NSF grant EF04-24599) and Hawaii Ocean Time-series (HOT; NSF grant OCE-09-26766). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Human socioeconomic activities, specifically fossil fuel combustion, cement production, and changes in land use, have resulted in progressive increases in atmospheric and oceanic carbon dioxide (CO₂) inventories [1]. The ocean is a globally important net sink for CO₂, and as such, increases in atmospheric CO₂ have raised seawater pCO₂, with concomitant decreases in seawater pH [2–5]. However, studies examining the effects of changes in seawater carbonate chemistry on plankton productivity in open ocean ecosystems are relatively scarce. While an appropriate null hypothesis could be that ocean acidification may lead to no significant changes in microbial contributions to biogeochemical cycling [6], testing such a hypothesis demands rigorous experimental evidence. Previous results and observations suggest that, either as individual species or microbial assemblages, marine microbial physiology may be affected by increases in pCO₂ [7–14]. However, the reported signs and magnitudes of the effects vary [14]. Whether these changes in microbial physiology are large enough to impact ocean biogeochemical cycles remains an important unanswered question.

To date, relatively little is known about the capacity of phytoplankton to adapt or acclimate to changes in the seawater carbonate system, which are likely to have complex influences on ocean biology. Most contemporary lineages of phytoplankton evolved during periods in Earth’s history when atmospheric and oceanic CO₂ inventories were considerably greater than today [15,16]. Indeed, for many algal species ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the enzyme that catalyzes the initial steps of carbon fixation, is less than half saturated at present day pCO₂ [17]. As a consequence, many algae, including cyanobacteria, appear to possess mechanisms for concentrating CO₂ [18]. Experiments examining the effects of elevated pCO₂ on natural phytoplankton communities have yielded enigmatic results; in several studies, rates of production have increased under elevated pCO₂ [9,14,19], although in other cases, no significant changes in rates of production have been observed [20,21]. There is also compelling evidence that the decline in carbonate ion concentrations that accompanies decreases in seawater pH can be detrimental to the growth of calcifying microorganisms [7], although this appears species-specific [22] and may not apply in the tropical oceans [23]. Additionally, decreases in seawater pH could affect other aspects of seawater chemistry, including altering availability of nitrogen substrates (i.e. decreasing ammonia relative to ammonium) and iron, with concomitant impacts on key processes in the marine nitrogen cycle [13,24].

Numerous laboratory-based pCO₂ manipulation studies have examined the response of specific organisms to changes in seawater carbonate chemistry, where growth conditions are controlled and organisms are generally examined in isolation [12,25]. Other studies have examined natural planktonic communities, including work in the oligotrophic open ocean [8,9,20,21]. Recent mesocosm experiments conducted in nearshore waters found that elevated pCO₂ shifted the partitioning of carbon fixed via photosynthesis from the particulate to the dissolved phase [26] and increased bacterial growth [10]. These intriguing results highlight the need for experiments examining the effects of increased pCO₂ on plankton growth in the open ocean, where a major fraction of global productivity occurs [27] with a significant fraction of the production being partitioned into the dissolved phase which supports the microbial food web [28]. Increasing seawater pCO₂ could impact heterotrophic bacterial growth, through both direct changes to metabolic rates, for example alteration of enzymatic activities [29–31], or indirectly through changes in organic matter production or substrate lability [10,32]. Even small changes in rates of bacterial consumption of organic matter could have a large impact on carbon and nutrient cycling.

In this study, we conducted abrupt perturbations to the seawater carbonate system in the waters of the oligotrophic North Pacific Subtropical Gyre (NPSG) to artificially alter seawater pCO₂ to conditions projected for the surface ocean within the next 50 to 100 years [1]. During these experiments, we examined how such abrupt changes in pCO₂ influenced rates of primary and bacterial production in the near-surface ocean during a series of incubation experiments (2–5 days in duration). We also conducted a series of short-term (~12 hours; photoperiod) depth-resolved experiments to evaluate possible influences of increased pCO₂ on microbial production throughout the euphotic zone (0–125 m). Experiments were conducted in the open ocean of the NPSG, one of the largest biomes on the planet, and hence in an ecosystem that plays a major role in the global cycles of bioelements.

Materials and methods

Experimental design

All seawater carbonate system manipulation experiments were performed on Hawaii Ocean Time-series (HOT) program cruises to Station ALOHA (22° 45’ N 158° W), the field site of the HOT program (between June 2010 and September 2012) or during two process cruises conducted in the vicinity of Station ALOHA (August 2010 and March 2011; Fig 1). Two types of carbonate system manipulation experiments were performed. The first kind of experiments (hereafter “bubbling”) were performed as described in Böttjer et al. [21]. Briefly, near-surface (5–25 m) ocean seawater was collected near midnight using polyvinyl chloride sampling bottles attached to a conductivity-temperature-density (CTD) rosette, and subsampled from CTD rosette bottles under minimal light into acid-washed 20 L polycarbonate carboys fitted with sterile caps with ports for introducing and venting gases. After filling, carboys were placed into shaded (~50% surface irradiance) surface seawater-cooled incubators. Targeted pCO₂ levels were attained by bubbling control carboys with air (~387 μatm pCO₂) and treatment carboys with a mixture of air and CO₂ (targeting ~750 or ~1100 μatm pCO₂) for 6–8 hours (< 3 L min^-1). Mixing and delivery of air or mixed air and CO₂ was regulated by use of mass flow controllers. During the initial 6–8 hours of bubbling, subsamples were collected regularly for measurements of seawater pH (see below for methods), and together with measurements of total alkalinity (TA) were used to estimate seawater pCO₂ using the ‘seacarb’ package [33] in the R statistical environment, with default settings for the carbonate dissociation constants [34–37]. Once the target pCO₂ was reached, the rate of bubbling was reduced (≤ 1.5 L min^-1) for the duration of the experiment. Subsequent sampling was conducted before dawn at each time point, with the initial sample taken post equilibration considered the beginning of the experiment. Sampling was performed by applying positive pressure to the carboys and subsampling for measurements of TA, dissolved inorganic carbon (DIC), chlorophyll a, ¹⁴C-based primary productivity (¹⁴C-PP), and rates of ³H-leucine (³H-Leu) incorporation (as a proxy for bacterial production).

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Fig 1. Chlorophyll concentrations and station locations.

Satellite ocean color image depicting distributions and concentrations of near-surface ocean chlorophyll a (μg/ L^-1) in the proximity of the Hawaiian Islands on August 21, 2010 (panel A). This image was derived from MODIS Aqua data using the color index (CI) algorithm [38] with no flags applied. The locations of the three stations occupied in August 2010 are also indicated; Station ALOHA is depicted as a circle, while stations S1 and S2 are depicted by triangles. Depth profiles of chlorophyll a during the depth-dependent experiments are also shown (panel B).

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Additional CO₂ perturbation experiments were conducted at either ambient (~387 μatm) or elevated (~750 μatm) seawater pCO₂ to evaluate depth-dependent responses in ¹⁴C-PP and ³H-Leu incorporation to perturbation of the seawater carbonate system. For these experiments, samples were incubated in situ to simulate the vertical gradients in light and temperature representative of the depths from which samples were originally collected and rates of ¹⁴C-PP and ³H-Leu incorporation were measured. Seawater was collected before dawn from six euphotic zone depths (5, 25, 45, 75, 100, and 125 m) and subsampled from the CTD rosette bottles under minimal light into acid-washed 20 L polycarbonate carboys. These carboys were left untreated (controls) or amended with trace metal grade hydrochloric acid (43 mL of 0.1 N HCl) to increase pCO₂ to ~750 μatm (elevated pCO₂ treatments) while minimizing potential changes to TA through additions of sodium bicarbonate (4 mmol). Once the carboys had been amended, seawater from each depth was subsampled into triplicate acid-cleaned 500 mL polycarbonate bottles and acid-cleaned 40 mL polycarbonate centrifuge tubes for subsequent measurements of ¹⁴C-PP and ³H-Leu incorporation, respectively. Following addition of radioactive substrates (see below), these bottles and tubes were affixed to a free-drifting array and incubated in situ at the depths of sample collection for the duration of the photoperiod (dawn to dusk).

Measurements of TA, DIC, and pH

Seawater samples for DIC and TA were collected from each carboy at every time point to evaluate the stability of the carbonate system during bubbling. Samples for determination of carbon system components (TA, DIC, and pH) were collected and analyzed following HOT program protocols [5,39]. DIC and TA samples were collected from carboys into precombusted 300 mL borosilicate bottles. Care was taken to avoid introducing bubbles into the sample during filling, and bottles were allowed to overflow three times during filling. Once filled, each sample was immediately fixed with 100 μL of a saturated solution of mercuric chloride; bottles were capped with a grease seal, and stored in the dark for later analysis. DIC concentrations were determined coulometrically using a Versatile INstrument for the Determination of Total inorganic carbon and Titration Alkalinity 3S (VINDTA) system [40]. TA was determined using an automated, closed-cell potentiometric titration. The precision and accuracy of these measurements were validated by comparison to a certified seawater CO₂ reference sample [40], with accuracies of approximately ±3 μmol L^-1 for TA and ±1 μmol L^-1 for DIC. Seawater pH (measured at 25°C) was analyzed using spectrophotometric detection of m-cresol purple with a precision of 0.001 [5,41].

Measurements of ¹⁴C-PP

Rates of ¹⁴C-PP were measured at each sampling time point during the bubbling experiments. At each pre-dawn sampling, seawater was subsampled from the carboys into acid cleaned 500 mL polycarbonate bottles, and each bottle was amended with ~1.85 MBq ¹⁴C-bicarbonate. The total radioactivity added to each sample bottle was determined post-incubation by subsampling 250 μL aliquots of seawater into scintillation vials containing 500 μL of β-phenylethylamine. Bottles were placed in shaded (~50% irradiance) surface seawater-cooled incubators for the duration of the photoperiod. After sunset, 100 mL from each sample bottle was filtered at low vacuum (<50 mm Hg) onto 25 mm diameter, 0.2 μm porosity polycarbonate membrane filters. The filters were then stored frozen in 20 mL scintillation vials until analysis at the shore-based laboratory. At the shore-based laboratory, filters were acidified by the addition of 1 mL of 2 N hydrochloric acid, and allowed to passively vent for at least 24 hours in a fume hood to remove all inorganic ¹⁴C, followed by addition of 10 mL Ultima Gold LLT liquid scintillation cocktail. The resulting radioactivity was determined on a Perkin Elmer 2600 liquid scintillation counter.

Measurements of ¹⁴C-PP from the depth-dependent experiments were conducted similarly, except that the samples were incubated in situ at the respective depths of collection on a free-drifting array for the duration of the photoperiod, and the incubations were terminated via sequential size fractionated filtration onto 10 μm, 2 μm, and 0.2 μm pore sized polycarbonate filters (25 mm diameter). Filters were treated as previously described for subsequent determination of ¹⁴C activity.

³H-leucine incorporation measurements

We measured ³H-Leu incorporation into plankton protein as a proxy measurement for bacterial production [42, 43]. Rates of ³H-Leu incorporation were measured following incubations conducted in both the light (³H-Leu_Light) and in the dark (through use of black cloth bags; ³H-Leu_Dark [44]). From the bubbling experiments, 125 mL polyethylene amber bottles were subsampled from each carboy in the pre-dawn hours; 6 aliquots of 1.5 mL were then subsampled from each bottle into 2 mL microcentrifuge tubes (Axygen) containing 20 nmol L^{-1 3}H-leucine (final concentration). In addition, 1.5 mL of seawater was subsampled into a 2 mL microcentrifuge tube containing 20 nmol L^{-1 3}H-leucine (final concentration) and 100 μL of 100% (w/v) trichloroacetic acid (TCA); these samples served as time zero “blanks”. Samples were incubated for 2 to 12 hours in the same surface seawater-cooled incubator described previously. To terminate incubations, 100 μL of 100% TCA was added to each microcentrifuge tube and tubes were frozen (-20°C) for later processing.

For those experiments incubated in situ on the free-drifting array, water was subsampled from each of the control and treatment carboys into 40 mL polycarbonate centrifuge tubes and each tube was inoculated with ³H-leucine to a final concentration of 20 nmol L^-1. Time zero blanks were immediately subsampled from each tube; for these samples, 1.5 mL of seawater was aliquoted into 2 mL microcentrifuge tubes containing 100 μL of 100% TCA. The 40 mL tubes were then affixed to the same free drifting array utilized for the ¹⁴C-bicarbonate assimilation measurements and samples were incubated under ambient light and in the dark (by placing the tubes in a darkened cloth bag). The array was then deployed for the duration of the photoperiod. After sunset, the array was recovered, and triplicate 1.5 mL subsamples were removed from each of the polycarbonate tubes and aliquoted into 2 mL microcentrifuge tubes containing 100 μL of 100% TCA. The microcentrifuge tubes were frozen (-20°C) for later processing, following the procedures described in Smith and Azam [45].

Contextual biogeochemical data, statistics, and data analysis

Seawater samples for contextual biogeochemical analyses were collected and analyzed according to HOT program protocols (http://hahana.soest.hawaii.edu/hot/methods/results.html). Measurements of fluorometric chlorophyll a concentrations were performed as in Letelier et al. [46]. Samples for analysis of nutrient concentrations were subsampled from the CTD rosette bottles into acid washed polyethylene bottles (125 or 500 mL) and stored upright at -20°C. Combined concentrations of nitrate and nitrite (N+N) were analyzed using the high sensitivity chemiluminescent technique [47,48], while concentrations of soluble reactive phosphorus (SRP) were determined via the magnesium-induced co-precipitation (MAGIC) method [49].

Statistical analyses were performed using Matlab (Mathworks). Data that were not normally distributed were log₁₀ transformed prior to subsequent analyses. Statistical differences between rates of ¹⁴C-PP and ³H-Leu incorporation at different pCO₂ levels during our bubbling experiments were determined by two-way analysis of variance (ANOVA), where pCO₂ and time were the factors of variation. For the depth-resolved experiments, we assessed significance for individual depths and for the depth-integrated rates based on the mean and standard deviation of the rates of ¹⁴C-PP and ³H-Leu incorporation in the treatments and controls using Student’s t-tests.

Results

We conducted a total of 10 shipboard pCO₂ manipulation experiments where seawater pCO₂ was altered by bubbling with CO₂-air gas mixtures. We measured rates of ¹⁴C-PP in all 10 of these bubbling experiments, and rates of ³H-Leu incorporation were measured in 8 of these experiments. An additional 5 experiments were conducted to evaluate depth-dependent responses in rates of ¹⁴C-PP and ³H-Leu incorporation to elevated pCO₂. Experiments were conducted in all four seasons, spanning the range of conditions typically observed at Station ALOHA (Table 1). With two exceptions, all experiments were conducted with seawater collected at Station ALOHA; two of the depth-resolved experiments (August 26, 2010 and August 28, 2010) were conducted at sampling sites to the northwest of Station ALOHA (termed S1: 24° 45’ N 160° 45’ W and S2: 25° 35’ N 160° 32’ W) where concentrations of chlorophyll a in near-surface waters were elevated relative to Station ALOHA (Table 1; Fig 1).

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Table 1. Starting conditions for experiments.

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For all bubbling experiments, initial concentrations of N+N and SRP were consistently below 10 nmol L^-1 and 150 nmol L^-1, respectively, consistent with HOT program measurements of these nutrients (Table 1; Fig 2). Rates of particulate ¹⁴C-PP at the beginning of the experiments ranged between 0.17 to 0.93 μmol C L^-1 d^-1, with the higher rates measured at those stations to the northwest of ALOHA where chlorophyll a concentrations were elevated (Table 1). Seawater pCO₂ in the near-surface waters at the time the experiments were conducted ranged between 351 μatm to 419 μatm, consistent with HOT program observations at Station ALOHA [5,21], while sea surface temperatures ranged between ~24 and 26°C (Table 1).

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Fig 2. Initial experimental conditions.

Mixed layer seawater pCO₂ measured during the period of this study (2010–2012) at Station ALOHA (panel A; triangles). Also shown are temperature at Station ALOHA (panel B; diamonds), concentrations of nitrate + nitrite (N+N; panel C; squares) and chlorophyll a (panel D; circles) in near-surface waters (5 m). Grey stars are used to indicate those cruises when bubbling experiments were conducted.

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Concentrations of chlorophyll a and rates of ¹⁴C-PP and ³H-Leu incorporation under elevated pCO₂

In 8 out of 10 bubbling experiments rates of ¹⁴C-PP in the elevated pCO₂ treatments were not significantly different than rates measured in the controls (two-way ANOVA; p>0.05; Table 2; Fig 3). In the remaining 2 bubbling experiments (April 2011 and September 2012) rates of ¹⁴C-PP in the controls were significantly greater than rates measured in the enhanced pCO₂ treatments (two-way ANOVA, p<0.05; Table 2); notably, concentrations of N+N and SRP in both of these experiments were elevated relative to other experiments (Table 1). There were no significant interactions between pCO₂ and time for any of the experiments (two-way ANOVA; p>0.05). The median value of the percent differences between treatments and controls ([treatments—controls] / controls) across all time points was -6% (mean 3%, standard deviation 51%; Table 2), with the treatments differing from the controls during 20% of the sampling occasions.

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Fig 3. Results of pCO₂ bubbling experiments.

Measured rates of ¹⁴C-PP (panel A) and ³H-Leu_Dark incorporation (panel C) from all experiments (n = 8 and n = 7, respectively) where no significant differences (NSD) were observed between controls (white circles) and elevated pCO₂ treatments at 1100 μatm (grey squares) and 750 μatm (grey triangles) are depicted. Also shown are rates of ¹⁴C-PP (panel B) and ³H-Leu_Dark incorporation (panel D) from experiments where significant differences (two-way ANOVA; p<0.05) were observed between controls (open symbols) and elevated pCO₂ (black symbols) treatments. Dates of experiments showing significant differences are given in the legend.

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Table 2. Percent difference between controls and elevated pCO₂ treatments.

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In addition, during most experiments (7 of 10) concentrations of chlorophyll a in the elevated pCO₂ treatments were not significantly different than in the controls (two-way ANOVA; p>0.05; Table 2). In a single experiment (September 2010), chlorophyll a concentrations in the elevated pCO₂ treatments were greater than those in the controls. In contrast, rates of ¹⁴C-PP and concentrations of chlorophyll a were greater in controls relative to the elevated pCO₂ treatments in two of the experiments (April 2011 and September 2012; two-way ANOVA; p<0.05; Table 2; Fig 3). We also normalized our measured rates of ¹⁴C-PP to concentrations of chlorophyll a, and in 9 of 10 experiments there was no significant difference between controls and elevated pCO₂ treatments (two-way ANOVA; p>0.05; S1 Fig). In the experiment conducted in March 2011, chlorophyll a normalized rates of ¹⁴C-PP were greater in elevated pCO₂ treatments than in controls (two-way ANOVA; p<0.05; S1 Fig).

We also examined possible responses in rates of ³H-Leu incorporation during the seawater carbonate system manipulation experiments (Table 2). In total, rates of ³H-Leu_Dark incorporation were determined in 7 of the bubbling experiments, with coincident measurements of rates of ³H-Leu_Light incorporation in 6 of these 7 experiments (Table 2). In the enhanced pCO₂ treatments rates of ³H-Leu_Dark incorporation were similar to those measured in the controls, ranging between 7 and 41 pmol Leu L^-1 h^-1, with measurements at subsequent time points ranging from 4 to 98 pmol Leu L^-1 h^-1 (Fig 3). Rates of ³H-Leu_Light incorporation in the controls ranged between 9 and 61 pmol Leu L^-1 h^-1 at the beginning of the experiments, and between 21 and 84 pmol Leu L^-1 h^-1 at subsequent time points. In the enhanced pCO₂ treatments, rates of ³H-Leu_Light incorporation at the beginning of the experiments ranged from 15 to 65 pmol Leu L^-1 h^-1, and from 17 to 99 pmol Leu L^-1 h^-1 at subsequent time points. In 5 out of the 7 experiments where rates were measured, ³H-Leu_Dark incorporation rates increased significantly over time in both the controls and treatments (two-way ANOVA, p<0.05; Fig 3). Similarly, in 4 of the 6 experiments in which ³H-Leu_Light incorporation was measured rates increased significantly over the duration of the experiment in both the controls and treatments (two-way ANOVA, p<0.05). However, in the majority of experiments there were no significant differences in the enhanced pCO₂ treatments relative to the controls (two-way ANOVA, p>0.05; Table 2). In a single experiment (August 2010) rates of ³H-Leu_Dark incorporation in the pCO₂ treatments (750 μatm) were significantly greater than the controls (two-way ANOVA, p<0.05; Table 2; Fig 3). The median values of the percent differences ([CO₂ treatments—ambient controls]/controls) in rates of ³H-Leu_Dark and ³H-Leu_Light incorporation across all time points were 2% (mean 19%, standard deviation 78%) and 1% (mean 22%, standard deviation 87%) respectively (Table 2). The resulting differences (%) were significantly different from zero in less than 20% of experimental time points (13% and 15% for ³H-Leu_Dark and ³H-Leu_Light incorporation, respectively).

Depth-dependent responses in ¹⁴C-PP and ³H-Leu incorporation to elevated pCO₂

In addition to conducting pCO₂ perturbation experiments where near-surface ocean water was bubbled continuously for up to 5 days, we also conducted 5 experiments where we examined short-term, daytime (dawn to dusk), depth-dependent responses in rates of ¹⁴C-PP and ³H-Leu incorporation to perturbations in seawater pCO₂. For these experiments, seawater pCO₂ at 6 discrete depths in the upper ocean was perturbed through the addition of acid (and bicarbonate to maintain constant alkalinity) and incubated in situ on a free-drifting array. For samples in the upper euphotic zone (<45 m), the pCO₂ derived from measurements of DIC and TA was within ~20% of the target pCO₂ (750 μatm), while in the lower euphotic zone (>75 m) the derived pCO₂ values were uniformly greater (by 2–52%) than the target pCO₂ (S2 Fig). This was likely due to a combination of depth-dependent natural increases in pCO₂ and the greater variability of the seawater carbonate system in the lower euphotic zone compared to surface waters [50].

Rates of ¹⁴C-PP were measured from size fractionated water samples (>10 μm, 2–10 μm, and 0.2–2 μm) from all six depths from both the controls and pCO₂-perturbed treatments (Table 3; Fig 4). Overall, rates of ¹⁴C-PP in all of the size fractions were greatest at stations S1 and S2, where concentrations of chlorophyll a were also elevated (Fig 1). Rates of ¹⁴C-PP in the >10 μm size fraction at these two stations ranged from 0.4 to 0.5 μmol C L^-1 d^-1, approximately an order of magnitude greater than rates observed at ALOHA (Fig 4). The resulting depth-integrated upper euphotic zone (0–45 m) rates of ¹⁴C-PP in the >10 μm size fraction ranged between 1.0 and 17.7 mmol C m^-2 d^-1, with average rates at S1 and S2 (August 26 and 28, 2010) ~11-fold greater than at ALOHA (Table 3). Similarly, rates measured at S1 and S2 were elevated in the 2–10 μm and 0.2–2 μm size fractions, with depth-integrated (0–45 m) rates at these stations ranging from 3.1 to 6.8 and 8.8 to 13.2 mmol C m^-2 d^-1, respectively (Table 3) compared to 1.6 to 3.2 mmol C m^-2 d^-1 and 3.8 to 6.0 mmol C m^-2 d^-1, respectively at Station ALOHA. In the lower euphotic zone (75–125 m), rates of ¹⁴C-PP in the two larger size fractions at Station ALOHA were 2- to 5- fold lower than in the upper euphotic zone, with rates in the 0.2–2 μm size fraction in the lower euphotic zone as much as 2.5-fold lower than the upper ocean (Table 3).

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Fig 4. Depth-resolved measurements of size fractionated ¹⁴C-PP during cruises in August 2010 and March 2011.

Rates of ¹⁴C-PP in the >10 μm size fraction (panels A and D), 2–10 μm size fraction (panels B and E), and 0.2–2 μm size fraction (panels C and F) under ambient (387 μatm; panels A-C) and elevated pCO₂ (750 μatm) conditions (panels D-F).

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Table 3. Depth-integrated rates of ¹⁴C-PP.

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Consistent with results from the bubbling experiments, overall abrupt increases in pCO₂ had little or no effect on rates of ¹⁴C-PP in these depth-resolved experiments. For at least one of the depths examined in 3 of the depth-resolved experiments, rates of ¹⁴C-PP in the controls were greater than the pCO₂-elevated treatments (t-Test; p<0.05; Fig 5). However, in 1 of the 5 experiments (occurring in August 2010) rates of ¹⁴C-PP in the pCO₂-elevated treatments were greater than in the controls for both the >10 μm and 2–10 μm size fractions at a single depth (25 m; t-Test; p<0.005 and p<0.05, respectively; Fig 5). There were no consistent differences in depth-integrated rates of ¹⁴C-PP in the upper euphotic zone between controls and elevated pCO₂ treatments (0–45 m; Table 3; t-Test; p>0.05). In 4 of the 5 depth-resolved experiments there were no consistent differences in rates of ¹⁴C-PP between controls and pCO₂ elevated treatments in the lower euphotic zone. In one of the experiments (August 2010) conducted at Station ALOHA, rates of ¹⁴C-PP in the lower euphotic zone (75–125 m) were significantly greater in the >10 μm and 2–10 μm size fractions in the pCO₂ perturbed treatments relative to the controls (t-Test; p<0.05; Table 3).

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Fig 5. Percent differences in ¹⁴C-PP between controls and elevated pCO₂ treatments.

Shown are percent differences ([treatments (750 μatm) - controls (387 μatm)] / controls) for rates of ¹⁴C-PP from depth-resolved experiments during cruises in August 2010 and March 2011 for >10 μm size class (panel A), 2–10 μm size class (panel B), and the 0.2–2 μm size class (panel C). Dashed line indicates zero. Bold symbols indicate significant differences between controls and treatments.

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Similar to rates of ¹⁴C-PP, rates of ³H-Leu_Dark and ³H-Leu_Light incorporation were greater at stations S1 and S2 than at ALOHA (one-way ANOVA; p<0.0001 for both; Fig 6). The resulting depth-integrated (0–125 m) rates of ³H-Leu_Dark and ³H-Leu_Light incorporation were significantly greater in controls than in elevated pCO₂ treatments in 2 of 5 and 5 of 5 depth-resolved experiments, respectively (t-Test; p<0.05; Table 4). In the upper euphotic zone (0–45 m), rates of ³H-Leu_Dark and ³H-Leu_Light incorporation were significantly lower in the elevated pCO₂ treatments than in the controls in 2 of 5, and 3 of 5 experiments, respectively (t-Test; p<0.05; Table 4). In 4 of the 5 experiments, rates of ³H-Leu_Light incorporation in the lower euphotic zone (75–125 m) were significantly lower in the elevated pCO₂ treatments than the controls (t-Test; p<0.05; Table 4).

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Fig 6. Depth-resolved rates of ³H-Leu incorporation in August 2010 and March 2011.

Rates of ³H-Leu incorporation (pmol Leu L^-1 h^-1) in the dark for both ambient and elevated seawater pCO₂ (~390 and 750 μatm, respectively) are shown (panels A and C, respectively), as are rates in the light for both ambient and elevated pCO₂ (panels B and D, respectively). Also shown are percent differences between treatments ([treatments—controls] / controls) for ³H-Leu_Dark incorporation (panel E) and ³H-Leu_Light incorporation (panel F).

https://doi.org/10.1371/journal.pone.0193405.g006

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Table 4. Depth-integrated rates of ³H-Leu incorporation.

https://doi.org/10.1371/journal.pone.0193405.t004

Discussion

The overarching goals of this study were to examine whether abrupt changes to the ocean carbonate system would impact organic matter productivity and bacterial growth in the NPSG. Our experiments were not designed to investigate adaptations at the gene, species, or community level. To address these objectives, two types of experiments were conducted: 1) Manipulation of the near-surface (5–25 m) seawater carbonate system by gentle bubbling with air or a mixture of air and CO₂ and subsequent daily measurements of ¹⁴C-PP and ³H-Leu incorporation over 2 to 5 day incubation periods; and 2) Perturbation of the seawater carbonate system through the addition of acid (and bicarbonate to keep TA unchanged) at different depths throughout the euphotic zone, examining subsequent depth-dependent responses in rates of ¹⁴C-PP and ³H-Leu incorporation during in situ incubations lasting over the course of a photoperiod (~12 hours).

We detected no consistent changes in rates of either ¹⁴C-PP or ³H-Leu incorporation in response to elevated pCO₂ over the course of our bubbling experiments. This lack of a consistent effect of enhanced pCO₂ on either ¹⁴C-PP or ³H-Leu incorporation suggests that the contemporary microbial assemblages in this region of the NPSG appear relatively resilient to rapid increases in seawater pCO₂. Such observations are in agreement with results from other studies conducted in oligotrophic ocean ecosystems [20,21]. However, several studies have reported small to moderate increases in rates of ¹⁴C-PP [7,51] and bacterial production [31] under elevated pCO₂ in more eutrophic nearshore ecosystems.

Similar to the lack of response to increased pCO₂ observed in the bubbling experiments, rates of production in the depth-resolved experiments also demonstrated no significant or consistent response to increases in seawater pCO₂. Intriguingly, in one of our experiments (conducted in August 2010), rates of ¹⁴C-PP by larger phytoplankton (>2 μm) in the lower euphotic zone demonstrated greater rates of production under elevated pCO₂. These dimly lit waters and larger phytoplankton size classes account for a relatively small fraction of the euphotic zone productivity in the NPSG [52], so the resulting stimulation by pCO₂ resulted in no significant change in the depth-integrated (0–125 m) productivity from this experiment. Although the changes were modest (~20% and ~50%, for 2–10 μm and >10 μm, respectively), the apparent stimulation of productivity by larger phytoplankton may reflect ecological adaptations of phytoplankton in these dimly lit waters. The lower euphotic zone of the NPSG is dynamic with respect to changes in the seawater carbonate system, as a result of the large vertical gradient in DIC concentrations [5] together with the greater influence of mesoscale variability in the lower euphotic zone compared to surface waters [50]. Hence, the observed response to rapid perturbation in pCO₂ could reflect an adaptive response by phytoplankton communities to abrupt changes in the seawater carbonate system in the lower euphotic zone. In addition, this observation could reflect carbon limitation of phytoplankton growing in the lower euphotic zone during summer months. Net production of oxygen (O₂) in the sub-mixed layer waters of the NPSG results in accumulation of dissolved O₂ throughout the spring, with supersaturating concentrations through the summer and early fall [53]. We speculate that the enhanced rates of ¹⁴C-PP during this single experiment may reflect alleviation of CO₂ limitation of the larger phytoplankton growing in waters with elevated O₂: CO₂ ratios, where competitive binding of O₂ by RuBisCO could decrease photosynthetic efficiency and increase photorespiration [17,54].

We also sought to examine the sensitivity of bacterial production to abrupt increases in seawater pCO₂ during our depth-resolved experiments. While we observed no consistent response in rates of ³H-Leu_Light or ³H-Leu_Dark incorporation to the pCO₂ treatments during the bubbling experiments, rates of ³H-Leu incorporation in our depth-resolved experiments were frequently sensitive to changes in pCO₂. In all 5 of the depth-resolved experiments, euphotic zone (0–125 m) rates of ³H-Leu_Light incorporation were always significantly lower in the enhanced pCO₂ treatments than in controls. In contrast, rates of ³H-Leu_Dark incorporation did not vary in a consistent manner, with rates of ³H-Leu_Dark incorporation greater in the controls than the pCO₂ treatments in 2 of 5 experiments. We incubated samples in both the light and dark to evaluate how elevated pCO₂ might alter the known photostimulation of ³H-Leu incorporation previously reported in the euphotic zone of the NPSG [44,55]. Based on flow cytometric sorting of picoplankton populations, Björkman et al. [56] determined that Prochlorococcus incorporation of ³H-Leu in the light was a major factor controlling this photostimulation. Given the large contribution of Prochlorococcus to rates of ¹⁴C-PP (39% ±20%) and ³H-Leu incorporation (20% in the dark and 60% in the light) at Station ALOHA [56,57], our results suggest Prochlorococcus growth may be relatively insensitive to, or perhaps negatively affected by, abrupt increases in pCO₂. These results are consistent with previous findings in culture that suggest that while Synechococcus growth responds to elevated pCO₂, Prochlorococcus growth appears largely insensitive to variations in pCO₂ [11].

Several previous studies have reported increased rates of ³H-Leu_Dark incorporation under elevated pCO₂ [10], suggesting a shift in the partitioning of primary production from the particulate to the dissolved pool [26,58,59], with subsequent increased growth by heterotrophic bacteria on this newly available DOM [31,60]. However, other studies that have specifically measured rates of dissolved organic carbon production under elevated pCO₂ have reported inconsistent responses [61,62]. In our experiments, based on both bubbling and depth-resolved experiments, rates of ³H-Leu_Dark incorporation were most often unchanged under conditions of elevated pCO₂. Similarly, in the near-surface waters (represented by our bubbling experiments) rates of ³H-Leu_Light incorporation were unaffected by increases in pCO₂, but in the deeper regions of the euphotic zone (75–125 m), rates of ³H-Leu_Light incorporation were significantly lower under elevated pCO₂ treatments relative to the controls in 4 out of 5 experiments.

In general, we found that abrupt increases in pCO₂ have little or no influence on rates of ¹⁴C-PP and ³H-Leu_Light or ³H-Leu_Dark incorporation at Station ALOHA. On a single occasion, we did observe apparent stimulation of ¹⁴C-PP by larger phytoplankton dwelling in the lower euphotic zone, an observation we hypothesize could reflect seasonally-dependent carbon limitation by phytoplankton growing in these dimly lit waters. However, the majority of our experiments suggest that contemporary microbial growth in the euphotic zone at Station ALOHA is relatively resilient to abrupt increases in pCO₂. Such results are somewhat surprising given the low temporal fluctuations in seawater pCO₂ this habitat experiences; however, we suspect that in this persistently oligotrophic environment, both rates of ¹⁴C-PP and ³H-Leu incorporation are strongly controlled by the availability of growth-limiting substrates, whether in the form of inorganic nutrients or in the form of labile dissolved organic carbon. In particular, decadal-scale (2006–2016) rates of ¹⁴C-PP (at 25 m) at Station ALOHA varied more than threefold (from 0.28 to 0.98 μmol C L^-1 d^-1), approximately equivalent to the largest variation in rates of ¹⁴C-PP we measured between control and treatment rates during our experiments. Consequently, even large perturbations to the carbonate system appear to have only a weak influence on microbial growth in this ecosystem. Additionally, the short division times and large population sizes of open ocean phytoplankton may provide some capacity to adapt to or evolve in response to anthropogenic changes to the ocean carbonate system [63]. In contrast, it is likely that increasing ocean temperatures will exert a relatively stronger influence on microbial metabolism. Temperature effects could manifest directly, for example by changing microbial metabolic rates or growth efficiencies [64,65] or indirectly; through increased vertical stratification with concomitant reduction in nutrient supply and expansion of the oligotrophic gyres [66]. The combination of temperature-driven increases in respiration and decreased nutrient supply to the euphotic zone would likely decrease rates of net community production, with decreases in the amount of organic carbon available for upper trophic levels and export to the deep ocean. Hence, based on our observations together with those from previous reports, responses in planktonic metabolism to elevated pCO₂ appear variable and likely depend on the types of organisms present and the environmental conditions under which they grow [61,67,68]. It remains an open question whether our findings reflect physiological flexibility by the resident microbial community in acclimating to changes in the carbonate system, or whether the growth of these organisms is so tightly regulated by resource availability that any influence due to variations in the carbonate system are obscured by these other controlling factors. This question could be addressed by carrying out similar perturbation experiments that examine whether microbial growth responds to elevated pCO₂ coincident with alterations in the availability of growth-limiting nutrients.

Supporting information

S1 Fig. Rates of chlorophyll normalized ¹⁴C-PP from bubbling experiments.

Chlorophyll normalized rates of ¹⁴C-PP from pCO₂ bubbling experiments during this study where no significant difference was observed between controls (open circles) and elevated pCO₂ (grey squares) treatments (panel A) and for an experiment where a significant difference was found (two-way ANOVA; p<0.05) between controls (open triangle) and elevated pCO₂ (grey triangle) treatments (panel B).

https://doi.org/10.1371/journal.pone.0193405.s001

(TIF)

S2 Fig. Ratio of calculated pCO₂ versus targeted pCO₂ from in situ array experiments.

Solid line depicts the 1:1 ratio.

https://doi.org/10.1371/journal.pone.0193405.s002

(TIF)

Acknowledgments

We thank the scientists and staff of the HOT program for their assistance at sea and in the laboratory, in particular Daniel Sadler for his assistance with carbonate system manipulations and experimental set up. We thank Drs. Sasha Tozzi (Alltech) and Zbigniew Kolber (University of California Santa Cruz) for assistance with incubation experiments, and Dr. Craig Nelson (University of Hawaiʻi at Mānoa) for discussions that improved this manuscript. We would also like to thank Dr. John Dore (Montana State University) for his assistance with the HOT pCO₂ and pH data. We extend our gratitude to the officers and crew of the R/V Kilo Moana and the R/V Kaimikai-o-Kanaloa.

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Figures

Abstract

Introduction

Materials and methods

Experimental design

Measurements of TA, DIC, and pH

Measurements of 14C-PP

3H-leucine incorporation measurements

Contextual biogeochemical data, statistics, and data analysis

Results

Concentrations of chlorophyll a and rates of 14C-PP and 3H-Leu incorporation under elevated pCO2

Depth-dependent responses in 14C-PP and 3H-Leu incorporation to elevated pCO2

Discussion

Supporting information

S1 Fig. Rates of chlorophyll normalized 14C-PP from bubbling experiments.

S2 Fig. Ratio of calculated pCO2 versus targeted pCO2 from in situ array experiments.

Acknowledgments

References

Measurements of ¹⁴C-PP

³H-leucine incorporation measurements

Concentrations of chlorophyll a and rates of ¹⁴C-PP and ³H-Leu incorporation under elevated pCO₂

Depth-dependent responses in ¹⁴C-PP and ³H-Leu incorporation to elevated pCO₂

S1 Fig. Rates of chlorophyll normalized ¹⁴C-PP from bubbling experiments.

S2 Fig. Ratio of calculated pCO₂ versus targeted pCO₂ from in situ array experiments.