Extreme Variations of pCO2 and pH in a Macrophyte Meadow of the Baltic Sea in Summer: Evidence of the Effect of Photosynthesis and Local Upwelling

The impact of ocean acidification on benthic habitats is a major preoccupation of the scientific community. However, the natural variability of pCO₂ and pH in those habitats remains understudied, especially in temperate areas. In this study we investigated temporal variations of the carbonate system in nearshore macrophyte meadows of the western Baltic Sea. These are key benthic ecosystems, providing spawning and nursery areas as well as food to numerous commercially important species. In situ pCO₂, pH (total scale), salinity and PAR irradiance were measured with a continuous recording sensor package dropped in a shallow macrophyte meadow (Eckernförde bay, western Baltic Sea) during three different weeks in July (pCO₂ and PAR only), August and September 2011.The mean (± SD) pCO₂ in July was 383±117 µatm. The mean (± SD) pCO₂ and pH_tot in August were 239±20 µatm and 8.22±0.1, respectively. The mean (± SD) pCO₂ and pH_tot in September were 1082±711 µatm and 7.83±0.40, respectively. Daily variations of pCO₂ due to photosynthesis and respiration (difference between daily maximum and minimum) were of the same order of magnitude: 281±88 µatm, 219±89 μatm and 1488±574 µatm in July, August and September respectively. The observed variations of pCO₂ were explained through a statistical model considering wind direction and speed together with PAR irradiance. At a time scale of days to weeks, local upwelling of elevated pCO₂ water masses with offshore winds drives the variation. Within days, primary production is responsible. The results demonstrate the high variability of the carbonate system in nearshore macrophyte meadows depending on meteorology and biological activities. We highlight the need to incorporate these variations in future pCO₂ scenarios and experimental designs for nearshore habitats.     

Temperature and CO2 additively regulate physiology,morphology and genomic responses of larval sea urchins,Strongylocentrotus purpuratus

Ocean warming and ocean acidification, both consequences of anthropogenic production of CO₂, will combine to influence the physiological performance of many species in the marine environment. In this study, we used an integrative approach to forecast the impact of future ocean conditions on larval purple sea urchins (Strongylocentrotus purpuratus) from the northeast Pacific Ocean. In laboratory experiments that simulated ocean warming and ocean acidification, we examined larval development, skeletal growth, metabolism and patterns of gene expression using an orthogonal comparison of two temperature (13°C and 18°C) and pCO₂ (400 and 1100 μatm) conditions. Simultaneous exposure to increased temperature and pCO₂ significantly reduced larval metabolism and triggered a widespread downregulation of histone encoding genes. pCO₂ but not temperature impaired skeletal growth and reduced the expression of a major spicule matrix protein, suggesting that skeletal growth will not be further inhibited by ocean warming. Importantly, shifts in skeletal growth were not associated with developmental delay. Collectively, our results indicate that global change variables will have additive effects that exceed thresholds for optimized physiological performance in this keystone marine species.     

Rapid shifts in picoeukaryote community structure in response to ocean acidification

Rapid shifts in picoeukaryote community structure were observed during a CO₂ perturbation experiment in which we followed the development of phytoplankton blooms in nutrient-amended mesocosms under the present day or predicted future atmospheric pCO₂ (750 μatm, seawater pH 7.8). Analysis of rbcL clone libraries (encoding the large subunit of RubisCO) and specific quantitative PCR assays showed that two prasinophytes closely related to Micromonas pusilla and Bathycoccus prasinos were present, but responded very differently to high CO₂/acidification. We found that the abundance of Micromonas-like phylotypes was significantly higher (>20-fold) under elevated CO₂/low pH, whereas the Bathycoccus-like phylotypes were more evenly distributed between treatments and dominated the prasinophyte community under ambient conditions.     

Growth Attenuation with Developmental Schedule Progression in Embryos and Early Larvae of Sterechinus neumayeri Raised under Elevated CO2

The Southern Ocean, a region that will be an ocean acidification hotspot in the near future, is home to a uniquely adapted fauna that includes a diversity of lightly-calcified invertebrates. We exposed the larvae of the echinoid Sterechinus neumayeri to environmental levels of CO₂ in McMurdo Sound (control: 410 µatm, Ω = 1.35) and mildly elevated pCO₂ levels, both near the level of the aragonite saturation horizon (510 µatm pCO₂, Ω = 1.12), and to under-saturating conditions (730 µatm, Ω = 0.82). Early embryological development was normal under these conditions with the exception of the hatching process, which was slightly delayed. Appearance of the initial calcium carbonate (CaCO₃) spicule nuclei among the primary mesenchyme cells of the gastrulae was synchronous between control and elevated pCO₂ treatments. However, by prism (7 days after the initial appearance of the spicule nucleus), elongating arm rod spicules were already significantly shorter in the highest CO₂ treatment. Unfed larvae in the 730 µatm pCO₂ treatment remained significantly smaller than unfed control larvae at days 15–30, and larvae in the 510 µatm treatment were significantly smaller at day 20. At day 30, the arm lengths were more differentiated between 730 µatm and control CO₂ treatments than were body lengths as components of total length. Arm length is the most plastic morphological aspect of the echinopluteus, and appears to exhibit the greatest response to high pCO₂/low pH/low carbonate, even in the absence of food. Thus, while the effects of elevated pCO₂ representative of near future climate scenarios are proportionally minor on these early developmental stages, the longer term effects on these long-lived invertebrates is still unknown.     

Microcystin Production by Microcystis aeruginosa in a Phosphorus-Limited Chemostat

The production of microcystins (MC) from Microcystis aeruginosa UTEX 2388 was investigated in a P-limited continuous culture. MC (MC-LR, MC-RR, and MC-YR) from lyophilized M. aeruginosa were extracted with 5% acetic acid, purified by a Sep-Pak C₁₈ cartridge, and then analyzed by high-performance liquid chromatography with a UV detector and Nucleosil C₁₈ reverse-phase column. The specific growth rate (μ) of M. aeruginosa was within the range of 0.1 to 0.8/day and was a function of the cellular P content under a P limitation. The N/P atomic ratio of steady-state cells in a P-limited medium varied from 24 to 15 with an increasing μ. The MC-LR and MC-RR contents on a dry weight basis were highest at μ of 0.1/day at 339 and 774 μg g⁻¹, respectively, while MC-YR was not detected. The MC content of M. aeruginosa was higher at a lower μ, whereas the MC-producing rate was linearly proportional to μ. The C fixation rate at an ambient irradiance (160 microeinsteins m⁻² s⁻¹) increased with μ. The ratios of the MC-producing rate to the C fixation rate were higher at a lower μ. Accordingly, the growth of M. aeruginosa was reduced under a P limitation due to a low C fixation rate, whereas the MC content was higher. Consequently, increases in the MC content per dry weight along with the production of the more toxic form, MC-LR, were observed under more P-limited conditions.     

Effect of Altered pO(2) in the Aerial Part of Soybean on Symbiotic N(2) Fixation

Dry matter accumulation, nitrogen content and N₂ fixation rates of soybean (Glycine max [L.] Merr. cv. Wye) plants grown in chambers in which the aerial portion was exposed to a pO₂ of 5, 10, 21, or 30% and a pCO₂ of 300 μl CO₂/l or a pO₂ of 21% and a pCO₂ of 1200 μl CO₂/l during the complete growth cycle were measured. Total N₂[C₂H₂] fixed was increased by CO₂/O₂ ratios greater than those in air and was decreased by ratios smaller than those in air; the effects on N₂ fixation of decreased pO₂ or elevated pCO₂ were quantitatively similar during the period of vegetative growth. Decreased pO₂ produced a smaller increase then elevated pCO₂ during the reproductive period, presumably because of the decreased sink activity of the arrested reproductive growth under subambient pO₂. At a pO₂ of 5% and a pCO₂ of 300 μl CO₂/l total N₂ fixed was increased 125% and per cent nitrogen content in the vegetative parts was increased relative to air while that in the seed was decreased. Dry matter production was increased and reproductive growth was arrested as previously reported for plants receiving only fertilizer nitrogen. At a pO₂ of 30% and a pCO₂ of 300 μl CO₂/l total N₂ fixed was decreased 50% and per cent nitrogen content in the vegetative part was increased relative to air while that in the reproductive structures was unaffected. Dry matter production was similarly decreased in both vegetative and reproductive structures. These effects of altered pO₂ in the aerial part on N₂ fixation are consistent with the hypothesis that the amount of photosynthate available to the nodule may be the most significant primary factor limiting N₂ fixation while sink activity of the reproductive structures may be a secondary factor.     

Interactive Effects of Ocean Acidification and Nitrogen-Limitation on the Diatom Phaeodactylum tricornutum

Climate change is expected to bring about alterations in the marine physical and chemical environment that will induce changes in the concentration of dissolved CO₂ and in nutrient availability. These in turn are expected to affect the physiological performance of phytoplankton. In order to learn how phytoplankton respond to the predicted scenario of increased CO₂ and decreased nitrogen in the surface mixed layer, we investigated the diatom Phaeodactylum tricornutum as a model organism. The cells were cultured in both low CO₂ (390 μatm) and high CO₂ (1000 μatm) conditions at limiting (10 μmol L⁻¹) or enriched (110 μmol L⁻¹) nitrate concentrations. Our study shows that nitrogen limitation resulted in significant decreases in cell size, pigmentation, growth rate and effective quantum yield of Phaeodactylum tricornutum, but these parameters were not affected by enhanced dissolved CO₂ and lowered pH. However, increased CO₂ concentration induced higher rETR_max and higher dark respiration rates and decreased the CO₂ or dissolved inorganic carbon (DIC) affinity for electron transfer (shown by higher values for K_{1/2 DIC} or K_{1/2 CO2}). Furthermore, the elemental stoichiometry (carbon to nitrogen ratio) was raised under high CO₂ conditions in both nitrogen limited and nitrogen replete conditions, with the ratio in the high CO₂ and low nitrate grown cells being higher by 45% compared to that in the low CO₂ and nitrate replete grown ones. Our results suggest that while nitrogen limitation had a greater effect than ocean acidification, the combined effects of both factors could act synergistically to affect marine diatoms and related biogeochemical cycles in future oceans.     

Combined Effects of CO2 and Light on the N2-Fixing Cyanobacterium Trichodesmium IMS101: A Mechanistic View

The marine diazotrophic cyanobacterium Trichodesmium responds to elevated atmospheric CO₂ partial pressure (pCO₂) with higher N₂ fixation and growth rates. To unveil the underlying mechanisms, we examined the combined influence of pCO₂ (150 and 900 μatm) and light (50 and 200 μmol photons m⁻² s⁻¹) on Trichodesmium IMS101. We expand on a complementary study that demonstrated that while elevated pCO₂ enhanced N₂ fixation and growth, oxygen evolution and carbon fixation increased mainly as a response to high light. Here, we investigated changes in the photosynthetic fluorescence parameters of photosystem II, in ratios of the photosynthetic units (photosystem I:photosystem II), and in the pool sizes of key proteins involved in the fixation of carbon and nitrogen as well as their subsequent assimilation. We show that the combined elevation in pCO₂ and light controlled the operation of the CO₂-concentrating mechanism and enhanced protein activity without increasing their pool size. Moreover, elevated pCO₂ and high light decreased the amounts of several key proteins (NifH, PsbA, and PsaC), while amounts of AtpB and RbcL did not significantly change. Reduced investment in protein biosynthesis, without notably changing photosynthetic fluxes, could free up energy that can be reallocated to increase N₂ fixation and growth at elevated pCO₂ and light. We suggest that changes in the redox state of the photosynthetic electron transport chain and posttranslational regulation of key proteins mediate the high flexibility in resources and energy allocation in Trichodesmium. This strategy should enable Trichodesmium to flourish in future surface oceans characterized by elevated pCO₂, higher temperatures, and high light.The marine filamentous N₂-fixing (diazotrophic) cyanobacteria Trichodesmium spp. bloom extensively in the oligotrophic subtropical and tropical oceans (Carpenter and Capone, 2008). Trichodesmium contributes 25% to 50% of the estimated rates of N₂ fixation in these areas, where the new nitrogen inputs stimulate carbon and nitrogen cycling (Capone and Subramaniam, 2005; Mahaffey et al., 2005). The increases in atmospheric CO₂ partial pressure (pCO₂) and the subsequent impacts on ocean acidification are predicted to influence diazotrophs and specifically Trichodesmium.The reported sensitivity of Trichodesmium to changes in pCO₂ prompted further investigation into the cellular responses and underlying mechanisms, specifically when combined with other environmental parameters such as temperature, nutrient availability, and light. Elevated pCO₂ significantly increased growth and N₂ fixation rates of Trichodesmium cultures (Barcelos é Ramos et al., 2007; Hutchins et al., 2007; Levitan et al., 2007, 2010). The physiological response was also characterized by changes in inorganic carbon acquisition, limited flexibility of carbon-nitrogen ratios, and conservation of photosynthetic activities with increased pCO₂. These manifestations suggested that ATP and reductants [ferredoxin, NAD(P)H] are reallocated in the cells (Levitan et al., 2007, 2010; Kranz et al., 2009, 2010).In Trichodesmium, as in all cyanobacteria, the metabolic pathways of respiration and photosynthesis share several cellular complexes/proteins such as the plastoquinone (PQ) pool, succinate dehydrogenase, and ferredoxin (Fig. 1; Kana, 1993; Bergman et al., 1997; Lin et al., 1998). Energetic currencies [reduced ferredoxin, ATP, NAD(P)H] are also shared and can be allocated and utilized according to cellular requirements. N₂ fixation by nitrogenase and the subsequent assimilation of NH₄⁺ by Gln synthetase requires carbon skeletons from the tricarboxylic acid reactions. Moreover, linear and pseudocyclic photosynthesis can also generate additional ATP and reductants essential for N₂ fixation (Fig. 1; Berman-Frank et al., 2001).Open in a separate windowFigure 1.Schematic representation of major cellular complexes involved in energy flow [electron, ATP, NAD(P)H, carbon skeletons] in Trichodesmium IMS101. Dashed arrows represent movement direction of electrons, and solid arrows represent directions of protons, ATP, and NAD(P)H. Measured protein subunits are represented by gray diamonds. See Kranz et al. (2010) for measurements of O₂ evolution, inorganic carbon fixation, and fluxes of N₂ fixation.To understand the regulation of these metabolic pathways in Trichodesmium under varying pCO₂ levels and light intensities, we designed an experiment to characterize changes in the fluxes of carbon, nitrogen, and oxygen (O₂), related protein pool sizes, and variable fluorescence parameters of PSII. Elevated atmospheric pCO₂ combined with enhanced sea surface temperatures are forecast to stabilize thermal stratification, resulting in a shallower, more acidified, upper mixed layer characterized by higher mean light intensities (Doney, 2006). Thus, Trichodesmium IMS101 cultures were acclimated to past and future pCO₂ levels (150 and 900 μatm) at low and high light (50 and 200 μmol photons m⁻² s⁻¹).In the first part of this combined report (Kranz et al., 2010), we examined the physiological responses to the different acclimation conditions. The combination of elevated pCO₂ and light enhanced the production of particulate organic carbon and nitrogen (270% and 390% increase, respectively) as well as growth rates (180% increase; percentages are calculated from Kranz et al., 2010). Generally, the pCO₂-dependent stimulation was higher in cultures acclimated to low light. The pCO₂ effect was also reflected in other measured physiological parameters, particularly the diel patterns of N₂ fixation and the integrated N₂ fixation rates during the day, which increased approximately 30-fold between the low-pCO₂/low-light and the high-pCO₂/high-light acclimations (Kranz et al., 2010). While at high light, elevated pCO₂ extended the period of high N₂ fixation, which lasted from 5 h after the onset of light throughout the end of the photoperiod, the high-pCO₂ contribution to the integrated N₂ fixation was more significant at low light (Kranz et al., 2010). Light, but not pCO₂, influenced gross photosynthesis as measured by PSII O₂ evolution, which increased by approximately 250% in high-light-acclimated cultures. To supply the Calvin cycle with sufficient CO₂, Trichodesmium possesses a CO₂-concentrating mechanism mainly based on HCO₃⁻ uptake (Kranz et al., 2009, 2010). When Trichodesmium was acclimated to elevated pCO₂ (900 μatm), a decline in the cellular affinity to dissolved inorganic carbon was observed (Kranz et al., 2009), while the specific uptake of CO₂ showed a 9-fold increase between the low-pCO₂/low-light and the high-pCO₂/high-light acclimations (Kranz et al., 2010).Proteins are fundamental cellular components that influence the underlying mechanisms subsequently reflected in the cells’ physiology. In this study, we extend the experimental results presented by Kranz et al. (2010) by examining the influence of pCO₂ at different light regimes on the photosynthetic fluorescence parameters of PSII and on the pool sizes of key proteins involved in carbon and nitrogen fixation and their subsequent assimilation processes.     

Steady-State Growth and Chemical Composition of the Marine Chlorophyte Dunaliella tertiolecta in Nitrogen-Limited Continuous Cultures

The marine chlorophyte Dunaliella tertiolecta was grown in continuous cultures under NH₄⁺-N, NO₂⁻-N, NO₃⁻-N, and urea-N limitations. The effect of the nitrogen cell quota (Q_n) on the steady-state growth rate (μ) was the same regardless of the N source. The relationship between μ and Q_n was well described by the Droop equation, but only up to the true maximum growth rate ^μ (= cell washout rate). The ratio between the minimum cell quota (k_Q) and the maximum cell quota (Q_m) was 0.19. Hence, there is no substitute for determining ^μ experimentally. That there was no difference in growth response to different N sources suggests that no internal pooling of inorganic nitrogen occurred. Both the carbon (Q_c) and phosphorus (Q_p) cell quotas under N limitation increased with increasing μ in a threshold fashion: virtually no change in either cell quota up to ~0.8 ^μ, followed by a rapid and large increase up to ^μ. In addition, in the region of low μ, there was an increase in Q_p with a decreasing medium N/P ratio of between 15 and 5 (by atoms). The results generally indicate the physiological limits in cellular constituency under N limitation. The usefulness of this information, however, in describing the response of natural populations of marine phytoplankton to transient nutrient exposures on the temporal and spatial microscales that most likely exist is of limited value.     

Rising CO2 Interacts with Growth Light and Growth Rate to Alter Photosystem II Photoinactivation of the Coastal Diatom Thalassiosira pseudonana

We studied the interactive effects of pCO₂ and growth light on the coastal marine diatom Thalassiosira pseudonana CCMP 1335 growing under ambient and expected end-of-the-century pCO₂ (750 ppmv), and a range of growth light from 30 to 380 µmol photons·m⁻²·s⁻¹. Elevated pCO₂ significantly stimulated the growth of T. pseudonana under sub-saturating growth light, but not under saturating to super-saturating growth light. Under ambient pCO₂ susceptibility to photoinactivation of photosystem II (σ_i) increased with increasing growth rate, but cells growing under elevated pCO₂ showed no dependence between growth rate and σ_i, so under high growth light cells under elevated pCO₂ were less susceptible to photoinactivation of photosystem II, and thus incurred a lower running cost to maintain photosystem II function. Growth light altered the contents of RbcL (RUBISCO) and PsaC (PSI) protein subunits, and the ratios among the subunits, but there were only limited effects on these and other protein pools between cells grown under ambient and elevated pCO₂.     

Ocean acidification and temperature increase impact mussel shell shape and thickness: problematic for protection?

Ocean acidification threatens organisms that produce calcium carbonate shells by potentially generating an under‐saturated carbonate environment. Resultant reduced calcification and growth, and subsequent dissolution of exoskeletons, would raise concerns over the ability of the shell to provide protection for the marine organism under ocean acidification and increased temperatures. We examined the impact of combined ocean acidification and temperature increase on shell formation of the economically important edible mussel Mytilus edulis. Shell growth and thickness along with a shell thickness index and shape analysis were determined. The ability of M. edulis to produce a functional protective shell after 9 months of experimental culture under ocean acidification and increasing temperatures (380, 550, 750, 1000 μatm pCO ₂, and 750, 1000 μatm pCO ₂ + 2°C) was assessed. Mussel shells grown under ocean acidification conditions displayed significant reductions in shell aragonite thickness, shell thickness index, and changes to shell shape (750, 1000 μatm pCO ₂) compared to those shells grown under ambient conditions (380 μatm pCO ₂). Ocean acidification resulted in rounder, flatter mussel shells with thinner aragonite layers likely to be more vulnerable to fracture under changing environments and predation. The changes in shape presented here could present a compensatory mechanism to enhance protection against predators and changing environments under ocean acidification when mussels are unable to grow thicker shells. Here, we present the first assessment of mussel shell shape to determine implications for functional protection under ocean acidification.     

Differential Responses of Calcifying and Non-Calcifying Epibionts of a Brown Macroalga to Present-Day and Future Upwelling pCO2

Seaweeds are key species of the Baltic Sea benthic ecosystems. They are the substratum of numerous fouling epibionts like bryozoans and tubeworms. Several of these epibionts bear calcified structures and could be impacted by the high pCO₂ events of the late summer upwellings in the Baltic nearshores. Those events are expected to increase in strength and duration with global change and ocean acidification. If calcifying epibionts are impacted by transient acidification as driven by upwelling events, their increasing prevalence could cause a shift of the fouling communities toward fleshy species. The aim of the present study was to test the sensitivity of selected seaweed macrofoulers to transient elevation of pCO₂ in their natural microenvironment, i.e. the boundary layer covering the thallus surface of brown seaweeds. Fragments of the macroalga Fucus serratus bearing an epibiotic community composed of the calcifiers Spirorbis spirorbis (Annelida) and Electra pilosa (Bryozoa) and the non-calcifier Alcyonidium hirsutum (Bryozoa) were maintained for 30 days under three pCO₂ conditions: natural 460±59 µatm, present-day upwelling1193±166 µatm and future upwelling 3150±446 µatm. Only the highest pCO₂ caused a significant reduction of growth rates and settlement of S. spirorbis individuals. Additionally, S. spirorbis settled juveniles exhibited enhanced calcification of 40% during daylight hours compared to dark hours, possibly reflecting a day-night alternation of an acidification-modulating effect by algal photosynthesis as opposed to an acidification-enhancing effect of algal respiration. E. pilosa colonies showed significantly increased growth rates at intermediate pCO₂ (1193 µatm) but no response to higher pCO₂. No effect of acidification on A. hirsutum colonies growth rates was observed. The results suggest a remarkable resistance of the algal macro-epibionts to levels of acidification occurring at present day upwellings in the Baltic. Only extreme future upwelling conditions impacted the tubeworm S. spirorbis, but not the bryozoans.     

Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The K_m and V_max values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC₅₀ values were 16.00 μM and 9.83 μM, and K_i values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.     

One‐year experiment on the physiological response of the Mediterranean crustose coralline alga,Lithophyllum cabiochae,to elevated pCO2 and temperature

The response of respiration, photosynthesis, and calcification to elevated pCO₂ and temperature was investigated in isolation and in combination in the Mediterranean crustose coralline alga Lithophyllum cabiochae. Algae were maintained in aquaria during 1 year at near‐ambient conditions of irradiance, at ambient or elevated temperature (+3°C), and at ambient (ca. 400 μatm) or elevated pCO₂ (ca. 700 μatm). Respiration, photosynthesis, and net calcification showed a strong seasonal pattern following the seasonal variations of temperature and irradiance, with higher rates in summer than in winter. Respiration was unaffected by pCO₂ but showed a general trend of increase at elevated temperature at all seasons, except in summer under elevated pCO₂. Conversely, photosynthesis was strongly affected by pCO₂ with a decline under elevated pCO₂ in summer, autumn, and winter. In particular, photosynthetic efficiency was reduced under elevated pCO₂. Net calcification showed different responses depending on the season. In summer, net calcification increased with rising temperature under ambient pCO₂ but decreased with rising temperature under elevated pCO₂. Surprisingly, the highest rates in summer were found under elevated pCO₂ and ambient temperature. In autumn, winter, and spring, net calcification exhibited a positive or no response at elevated temperature but was unaffected by pCO₂. The rate of calcification of L. cabiochae was thus maintained or even enhanced under increased pCO₂. However, there is likely a trade‐off with other physiological processes. For example, photosynthesis declines in response to increased pCO₂ under ambient irradiance. The present study reports only on the physiological response of healthy specimens to ocean warming and acidification, however, these environmental changes may affect the vulnerability of coralline algae to other stresses such as pathogens and necroses that can cause major dissolution, which would have critical consequence for the sustainability of coralligenous habitats and the budgets of carbon and calcium carbonate in coastal Mediterranean ecosystems.     

Combined Effects of CO2 and Light on the N2-Fixing Cyanobacterium Trichodesmium IMS101: Physiological Responses

Recent studies on the diazotrophic cyanobacterium Trichodesmium erythraeum (IMS101) showed that increasing CO₂ partial pressure (pCO₂) enhances N₂ fixation and growth. Significant uncertainties remain as to the degree of the sensitivity to pCO₂, its modification by other environmental factors, and underlying processes causing these responses. To address these questions, we examined the responses of Trichodesmium IMS101 grown under a matrix of low and high levels of pCO₂ (150 and 900 μatm) and irradiance (50 and 200 μmol photons m⁻² s⁻¹). Growth rates as well as cellular carbon and nitrogen contents increased with increasing pCO₂ and light levels in the cultures. The pCO₂-dependent stimulation in organic carbon and nitrogen production was highest under low light. High pCO₂ stimulated rates of N₂ fixation and prolonged the duration, while high light affected maximum rates only. Gross photosynthesis increased with light but did not change with pCO₂. HCO₃⁻ was identified as the predominant carbon source taken up in all treatments. Inorganic carbon uptake increased with light, but only gross CO₂ uptake was enhanced under high pCO₂. A comparison between carbon fluxes in vivo and those derived from ¹³C fractionation indicates high internal carbon cycling, especially in the low-pCO₂ treatment under high light. Light-dependent oxygen uptake was only detected under low pCO₂ combined with high light or when low-light-acclimated cells were exposed to high light, indicating that the Mehler reaction functions also as a photoprotective mechanism in Trichodesmium. Our data confirm the pronounced pCO₂ effect on N₂ fixation and growth in Trichodesmium and further show a strong modulation of these effects by light intensity. We attribute these responses to changes in the allocation of photosynthetic energy between carbon acquisition and the assimilation of carbon and nitrogen under elevated pCO₂. These findings are supported by a complementary study looking at photosynthetic fluorescence parameters of photosystem II, photosynthetic unit stoichiometry (photosystem I:photosystem II), and pool sizes of key proteins in carbon and nitrogen acquisition.Human-induced climate change will significantly alter the marine environment within the next century and beyond. Future scenarios predict an increase from currently approximately 380 to about 750 to 1,000 μatm CO₂ partial pressure (pCO₂) in the atmosphere until the end of this century (Raven et al., 2005; Raupach et al., 2007). As the ocean takes up this anthropogenic CO₂, dissolved inorganic carbon (DIC) in the surface ocean increases while the pH decreases (Wolf-Gladrow et al., 1999). Rising global temperatures will increase surface ocean stratification, which may affect the light regime in the upper mixed layer as well as nutrient input from deeper waters (Doney, 2006). Uncertainties remain regarding both the magnitude of the physicochemical changes and the biological responses of organisms, including species and populations of the oceanic primary producers at the basis of the food webs.In view of potential ecological implications and feedbacks on climate, several studies have examined pCO₂ sensitivity in phytoplankton key species (Burkhardt and Riebesell, 1997; Riebesell et al., 2000; Rost et al., 2003; Tortell et al., 2008). Pronounced responses to elevated pCO₂ were observed in N₂-fixing cyanobacteria (Barcelos é Ramos et al., 2007; Hutchins et al., 2007; Levitan et al., 2007; Fu et al., 2008; Kranz et al., 2009), which play a vital role in marine ecosystems by providing a new source of biologically available nitrogen species to otherwise nitrogen-limited regions. Recent studies focused on the impact of different environmental factors on the filamentous Trichodesmium species, which is known for high abundance and the formation of massive blooms in tropical and subtropical areas (Capone et al., 2005; Mahaffey et al., 2005). Higher pCO₂ levels stimulated growth rates, biomass production, and N₂ fixation (Hutchins et al., 2007; Levitan et al., 2007; Kranz et al., 2009) and affected inorganic carbon acquisition of the cells (Kranz et al., 2009). While elevated sea surface temperatures are predicted to shift the spatial distribution of Trichodesmium species toward higher latitudes (Breitbarth et al., 2007), the combined effects of pCO₂ and temperature may favor this species and extend its niche even farther (Hutchins et al., 2007; Levitan et al., 2010a). An increase in the average light intensity, caused by the predicted shoaling of the upper mixed layer, may further stimulate photosynthesis and thus growth and N₂ fixation of Trichodesmium (Breitbarth et al., 2008). To our knowledge, the combined effects of light and pCO₂ have not been studied yet, although these environmental factors are likely to influence photosynthesis and other key processes in Trichodesmium.To understand the responses of an organism to changes in environmental conditions, metabolic processes must be studied. In Trichodesmium, photosynthetically generated energy (ATP and NADPH) is primarily used for the fixation of CO₂ in the Calvin-Benson cycle. A large proportion of this energy, however, is also required for the process of N₂ fixation via nitrogenase and for the operation of a CO₂-concentrating mechanism (CCM). The latter involves active uptake of inorganic carbon, which functions to increase the rate of carboxylation reaction mediated by Rubisco. This ancient and highly conserved enzyme is characterized by low affinities for its substrate CO₂ and a susceptibility to a competing reaction with oxygen (O₂) as substrate (Badger et al., 1998); the latter initiates photorespiration. As cyanobacterial Rubisco possesses one of the lowest CO₂ affinities among phytoplankton (Badger et al., 1998), a considerable amount of resources have to be invested to achieve sufficient rates of carbon fixation and to avoid photorespiration. A first step toward a mechanistic understanding of responses in Trichodesmium has been taken by Levitan et al. (2007), focusing on pCO₂ dependency of nitrogenase activity and photosynthesis. Subsequently, Kranz et al. (2009) described variations in CCM efficiency with pCO₂ and suggested that the observed plasticity in CCM regulation allowed energy reallocation under high pCO₂, which may explain the observed pCO₂-dependent changes in nitrogenase activity, growth, and elemental composition (Barcelos é Ramos et al., 2007; Hutchins et al., 2007; Levitan et al., 2007).In this study, we measured growth responses as well as metabolic key processes in Trichodesmium erythraeum (IMS101) under environmental conditions that likely alter the energy budget and/or energy allocation of the cell. Cultures were acclimated to a matrix of low and high pCO₂ (150 and 900 μatm) at two different light intensities (50 and 200 μmol photons m⁻² s⁻¹). For each of the four treatments, changes in growth rates, elemental ratios, and the accumulation of particulate carbon and nitrogen were measured. Metabolic processes (gross photosynthesis, CCM activity, and O₂ uptake) were obtained by means of membrane-inlet mass spectrometry (MIMS), while N₂ fixation was detected by gas chromatography. As these processes may vary over the diurnal cycle in Trichodesmium (Berman-Frank et al., 2001; Kranz et al., 2009), measurements were performed in the morning and around midday. The results on metabolic processes were accompanied by measurements of the fluorescence of PSII, ratios of the photosynthetic units (PSI:PSII), and pool sizes of key proteins involved in carbon and nitrogen fixation as well as assimilation (Levitan et al., 2010b).     

Effects of High Dissolved Inorganic and Organic Carbon Availability on the Physiology of the Hard Coral Acropora millepora from the Great Barrier Reef

Coral reefs are facing major global and local threats due to climate change-induced increases in dissolved inorganic carbon (DIC) and because of land-derived increases in organic and inorganic nutrients. Recent research revealed that high availability of labile dissolved organic carbon (DOC) negatively affects scleractinian corals. Studies on the interplay of these factors, however, are lacking, but urgently needed to understand coral reef functioning under present and near future conditions. This experimental study investigated the individual and combined effects of ambient and high DIC (pCO₂ 403 μatm/ pH_Total 8.2 and 996 μatm/pH_Total 7.8) and DOC (added as Glucose 0 and 294 μmol L^-1, background DOC concentration of 83 μmol L^-1) availability on the physiology (net and gross photosynthesis, respiration, dark and light calcification, and growth) of the scleractinian coral Acropora millepora (Ehrenberg, 1834) from the Great Barrier Reef over a 16 day interval. High DIC availability did not affect photosynthesis, respiration and light calcification, but significantly reduced dark calcification and growth by 50 and 23%, respectively. High DOC availability reduced net and gross photosynthesis by 51% and 39%, respectively, but did not affect respiration. DOC addition did not influence calcification, but significantly increased growth by 42%. Combination of high DIC and high DOC availability did not affect photosynthesis, light calcification, respiration or growth, but significantly decreased dark calcification when compared to both controls and DIC treatments. On the ecosystem level, high DIC concentrations may lead to reduced accretion and growth of reefs dominated by Acropora that under elevated DOC concentrations will likely exhibit reduced primary production rates, ultimately leading to loss of hard substrate and reef erosion. It is therefore important to consider the potential impacts of elevated DOC and DIC simultaneously to assess real world scenarios, as multiple rather than single factors influence key physiological processes in coral reefs.     

Responses of Two Scleractinian Corals to Cobalt Pollution and Ocean Acidification

The effects of ocean acidification alone or in combination with warming on coral metabolism have been extensively investigated, whereas none of these studies consider that most coral reefs near shore are already impacted by other natural anthropogenic inputs such as metal pollution. It is likely that projected ocean acidification levels will aggravate coral reef health. We first investigated how ocean acidification interacts with one near shore locally abundant metal on the physiology of two major reef-building corals: Stylophora pistillata and Acropora muricata. Two pH levels (pH_T 8.02; pCO₂ 366 μatm and pH_T 7.75; pCO₂ 1140 μatm) and two cobalt concentrations (natural, 0.03 μg L^-1 and polluted, 0.2 μg L^-1) were tested during five weeks in aquaria. We found that, for both species, cobalt input decreased significantly their growth rates by 28% while it stimulated their photosystem II, with higher values of rETR_max (relative Electron Transport Rate). Elevated pCO₂ levels acted differently on the coral rETR_max values and did not affect their growth rates. No consistent interaction was found between pCO₂ levels and cobalt concentrations. We also measured in situ the effect of higher cobalt concentrations (1.06 ± 0.16 μg L^-1) on A. muricata using benthic chamber experiments. At this elevated concentration, cobalt decreased simultaneously coral growth and photosynthetic rates, indicating that the toxic threshold for this pollutant has been reached for both host cells and zooxanthellae. Our results from both aquaria and in situ experiments, suggest that these coral species are not particularly sensitive to high pCO₂ conditions but they are to ecologically relevant cobalt concentrations. Our study reveals that some reefs may be yet subjected to deleterious pollution levels, and even if no interaction between pCO₂ levels and cobalt concentration has been found, it is likely that coral metabolism will be weakened if they are subjected to additional threats such as temperature increase, other heavy metals, and eutrophication.