Map of stations.

<p>Location of the stations from where water was collected for experiments I, II and III.</p

Flow cytometric analysis.

<p>Cytograms of bacterial samples at the start of experiments in controls and end of experiments in krill treatments, giving a relative estimate of the distribution of cell groups in each sample. The red lines at the controls broadly separate two groups of bacterial assemblages: (i) HNA: cells with high green fluorescence and large size (side scatter) and (ii) LNA: cells with low green fluorescence and small size (side scatter). Notice that experiments II and III present HNA cells at T0 with higher fluorescence and side scatter than in experiment I (see text for explanation).</p

Statistical ANOVAs analyses.

<p>Results of two-way repeated measures ANOVAs to determine the effects of treatment, time evolution, and the interaction between the two factors on bacterial production. The analyses indicate that there are significant differences (p<0.0001) between treatments, time evolution, and their interaction in the three experiments (see text for details). DF = Degree of freedom; F - ratio = Variance ratio.</p

Post hoc Bonferroni’s and Tukey’s mean comparisons tests.

<p>Pairwise numbers and letters between brackets indicate that the means of those values were significantly different at p<0.01. Values for “Treatment” are: A = ammonium, C = control, K = krill. Values for “Time” are 0 = T0, 1 = T1,… 5 = T5 (see text for details).</p

Bacterial metabolism in experiments.

<p>Integrated bacterial production from changes in biomass (BP) and respiration (BR) in the two treatments (+ammonium; +krill excretion products) and controls, along the three experiments. BCD: Bacterial carbon demand (BP+BR). BGE: Bacterial growth efficiency [BP/(BP+BR)]. NGR: Net specific growth rate [ln (BB_T5/BB_T0)/T5, being BB bacterial biomass and T time in days].</p

Time series of oxygen and ammonium concentration.

<p>Time evolution of DO and NH₄⁺ in experimental units receiving krill excretion products, ammonium inputs and controls for the three experiments conducted.</p

Comparative analysis of biological variables <i>in situ</i> and at the onset of experiments.

<p>Upper panel: Physical and biological parameters at 5 m depth, from the three sampling sites (<i>in situ</i>) where water was collected for experiments. Mean and standard deviation values (of 2–3 replicates, in parenthesis) of ammonium (NH₄⁺), dissolved organic carbon (DOC), iron (Fe), chlorophyll a (Chl a), bacterial abundance (BA) and bacterial production from Leucine uptake (BP-Leu). Lower panel: Mean and standard deviation values (of 2–3 replicates, in parenthesis) of NH₄⁺, DOC, BA and BP at the onset (T0) of the three experiments in the two treatments (ammonium, A; krill excretion products, K) and controls (C). Fe = Iron concentrations in the pre-filtered water used for experiments, before adding any treatment.</p

DataSheet1.XLSX

<p>In the surface ocean, microorganisms are both a source of extracellular H₂O₂ and, via the production of H₂O₂ destroying enzymes, also one of the main H₂O₂ sinks. Within microbial communities, H₂O₂ sources and sinks may be unevenly distributed and thus microbial community structure could influence ambient extracellular H₂O₂ concentrations. Yet the biogeochemical cycling of H₂O₂ and other reactive oxygen species (ROS) is rarely investigated at the community level. Here, we present a time series of H₂O₂ concentrations during a 28-day mesocosm experiment where a pCO₂ gradient (400–1,450 μatm) was applied to subtropical North Atlantic waters. Pronounced changes in H₂O₂ concentration were observed over the duration of the experiment. Initially H₂O₂ concentrations in all mesocosms were strongly correlated with surface H₂O₂ concentrations in ambient seawaters outside the mesocosms which ranged from 20 to 92 nM over the experiment duration (Spearman Rank Coefficients 0.79–0.93, p-values < 0.001–0.015). After approximately 9 days of incubation however, H₂O₂ concentrations had increased across all mesocosms, later reaching >300 nM in some mesocosms (2–6 fold higher than ambient seawaters). The correlation with ambient H₂O₂ was then no longer significant (p > 0.05) in all treatments. Furthermore, changes in H₂O₂ could not be correlated with inter-day changes in integrated irradiance. Yet H₂O₂ concentrations in most mesocosms were inversely correlated with bacterial abundance (negative Spearman Rank Coefficients ranging 0.59–0.94, p-values < 0.001–0.03). Our results therefore suggest that ambient H₂O₂ concentration can be influenced by microbial community structure with shifts toward high bacterial abundance correlated with low extracellular H₂O₂ concentrations. We also infer that the nature of mesocosm experiment design, i.e., the enclosure of water within open containers at the ocean surface, can strongly influence extracellular H₂O₂ concentrations. This has potential chemical and biological implications during incubation experiments due to the role of H₂O₂ as both a stressor to microbial functioning and a reactive component involved in the cycling of numerous chemical species including, for example, trace metals and haloalkanes.</p

Image1.TIF

<p>In the surface ocean, microorganisms are both a source of extracellular H₂O₂ and, via the production of H₂O₂ destroying enzymes, also one of the main H₂O₂ sinks. Within microbial communities, H₂O₂ sources and sinks may be unevenly distributed and thus microbial community structure could influence ambient extracellular H₂O₂ concentrations. Yet the biogeochemical cycling of H₂O₂ and other reactive oxygen species (ROS) is rarely investigated at the community level. Here, we present a time series of H₂O₂ concentrations during a 28-day mesocosm experiment where a pCO₂ gradient (400–1,450 μatm) was applied to subtropical North Atlantic waters. Pronounced changes in H₂O₂ concentration were observed over the duration of the experiment. Initially H₂O₂ concentrations in all mesocosms were strongly correlated with surface H₂O₂ concentrations in ambient seawaters outside the mesocosms which ranged from 20 to 92 nM over the experiment duration (Spearman Rank Coefficients 0.79–0.93, p-values < 0.001–0.015). After approximately 9 days of incubation however, H₂O₂ concentrations had increased across all mesocosms, later reaching >300 nM in some mesocosms (2–6 fold higher than ambient seawaters). The correlation with ambient H₂O₂ was then no longer significant (p > 0.05) in all treatments. Furthermore, changes in H₂O₂ could not be correlated with inter-day changes in integrated irradiance. Yet H₂O₂ concentrations in most mesocosms were inversely correlated with bacterial abundance (negative Spearman Rank Coefficients ranging 0.59–0.94, p-values < 0.001–0.03). Our results therefore suggest that ambient H₂O₂ concentration can be influenced by microbial community structure with shifts toward high bacterial abundance correlated with low extracellular H₂O₂ concentrations. We also infer that the nature of mesocosm experiment design, i.e., the enclosure of water within open containers at the ocean surface, can strongly influence extracellular H₂O₂ concentrations. This has potential chemical and biological implications during incubation experiments due to the role of H₂O₂ as both a stressor to microbial functioning and a reactive component involved in the cycling of numerous chemical species including, for example, trace metals and haloalkanes.</p

Distribution of prokaryotic abundace (cells ml^-1 in log units) and the percentage of high-nucleic acid content cells (% HNA) in samples collected during the eruptive phase (left pannels) and the post-eruptive phase (right pannels).

<p>The samples are grouped in different categories by depth (SF: subsurface, 0–70 m; OD: oxygen depleted waters, 70–200 m; DE: deep waters, 200–1900) and location (Control: stations in the control zone, Affected: stations in all affected areas, Volcano: affected stations in the vicinity of the volcano; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118136#pone.0118136.s001" target="_blank">S1 Table</a>).</p