Response of two marine bacterial isolates to high CO2 concentration

Experimental results related to the effects of ocean acidification on planktonic marine microbes are still rather inconsistent and occasionally contradictory. Moreover, laboratory or field experiments that address the effects of changes in CO₂ concentrations on heterotrophic microbes are very scarce, despite the major role of these organisms in the marine carbon cycle. We tested the direct effect of an elevated CO₂ concentration (1000 ppmv) on the biomass and metabolic rates (leucine incorporation, CO₂ fixation and respiration) of 2 isolates belonging to 2 relevant marine bacterial families, Rhodobacteraceae (strain MED165) and Flavobacteriaceae (strain MED217). Our results demonstrate that, contrary to some expectations, high pCO₂ did not negatively affect bacterial growth but increased growth efficiency in the case of MED217. The elevated partial pressure of CO₂ ( pCO₂) caused, in both cases, higher rates of CO₂ fixation in the dissolved fraction and, in the case of MED217, lower respiration rates. Both responses would tend to increase the pH of seawater acting as a negative feedback between elevated atmospheric CO₂ concentrations and ocean acidification.

The response of Thalassiosira pseudonana to long-term exposure to increased CO2 and decreased pH

The effect of ocean acidification conditions has been investigated in cultures of the diatom Thalassiosira pseudonana CCMP1335. Expected end-of-the-century pCO2 (aq) concentrations of 760 µatm (equivalent to pH 7.8) were compared with present-day condition (380 µatm CO2, pH 8.1). Batch culture pH changed rapidly because of CO2 (aq) assimilation and pH targets of 7.8 and 8.1 could not be sustained. Long-term (~100 generation) pH-auxostat, continuous cultures could be maintained at target pH when cell density was kept low (<2×105 cells mL−1). After 3 months continuous culture, the C:N ratio was slightly decreased under high CO2 conditions and red fluorescence per cell was slightly increased. However, no change was detected in photosynthetic efficiency (Fv/Fm) or functional cross section of PS II (σPSII). Elevated pCO2 has been predicted to be beneficial to diatoms due to reduced cost of carbon concentration mechanisms. There was reduced transcription of one putative δ-carbonic anhydrase (CA-4) after 3 months growth at increased CO2 but 3 other δ-CAs and the small subunit of RUBISCO showed no change. There was no evidence of adaptation or clade selection of T. pseudonana after ~100 generations at elevated CO2. On the basis of this long-term culture, pH change of this magnitude in the future ocean may have little effect on T. pseudonana in the absence of genetic adaption.

Temperate and cold water sea urchin species in an acidifying world: coping with change?

Anthropogenic carbon dioxide (CO2) emissions are increasing the atmospheric CO2 concentration and the oceans are absorbing around 1/3 them. The CO2 hydrolysis increases the H+ concentration, decreasing the pH, while the proportions of the HCO3- and CO32- ions are also affected. This process already led to a decrease of 0.1 pH units in surface seawater. According to “business-as-usual” models, provided by the Intergovernmental Panel on Climate Change (IPCC), the pH is expected to decrease 0.3-0.5 units by 2100 and 0.7-0.8 by 2300. As a result the surface ocean carbonates chemistry will also change: with increasing pCO2, dissolved inorganic carbon will increase and the equilibrium of the carbonate system will shift to higher CO2 and HCO3– levels, while CO32– concentration will decrease. Surface seawaters will progressively become less saturated towards calcite and aragonite saturation state and some particular polar and cold water regions could even become completely undersaturated within the next 50 years.

Responses of marine organisms to environmental hypercapnia, i.e. to an excess of CO2 in the aquatic environment, can be extremely variable and the degree of sensitivity varies between species and life stages. Sea urchins are key stone species in many marine ecosystems. They are considered to be particularly vulnerable to ocean acidification effects not only due to the nature of their skeleton (magnesium calcite) whose solubility is similar or higher than that of aragonite, but also because they lack an efficient ion regulatory machinery, being therefore considered poor acid-base regulators. Populations from polar regions are expected to be at an even higher risk since the carbonate chemical changes in surface ocean waters are happening there at a faster rate.

The goal of this work was to study the effects of low seawater pH exposure of different life stages of sea urchins, in order to better understand how species from different environments and/or geographic origins would respond and if there would be scope for possible adaptation and/or acclimatization.

CO2 induced acidification impacts sea urchin larval development II: gene expression patterns in pluteus larvae

Extensive use of fossil fuels is leading to increasing CO2 concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO2. As a result, the oceans’ surface pH is expected to decrease by ca. 0.4 units by the year 2100, a major change with potentially negative consequences for some marine species. Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa. In order to investigate sensitivity of pre-feeding (2 days post-fertilization) and feeding (4 and 7 days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO2 41 Pa e.g. 399 μatm) and CO2 acidified seawater with pH of 7.7 (pCO2 134 Pa e.g. 1318 μatm) and investigated growth, calcification and survival. At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR.

After one week of development, we observed a significant difference in growth. Maximum differences in size were detected at day 4 (ca. 10 % reduction in body length). A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (Two way ANOSIM: Global R = 1) while acidification effects were less pronounced (Global R = 0.518). Significant differences in gene expression patterns (ANOSIM R = 0.938, SIMPER: 4.3% difference) were also detected at day 4 leading to the hypothesis that differences between CO2 treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO2 effect. We found an up regulation of metabolic genes (between 10 to 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes (between 23 and 36% in msp130, SM30B, SM50 at day 4). Ion regulation was mainly impacted by up regulation of Na+/K+-ATPase at day 4 (15%) and down regulation of NHE3 at day 4 (45%). We conclude that in studies in which a stressor induces an alteration in the speed of development, it is crucial to employ experimental designs with a high time resolution in order to correct for developmental artifacts. This helps prevent misinterpretation of stressor effects on organism physiology.

CO2 induced seawater acidification impacts sea urchin larval development I: elevated metabolic rates decrease scope for growth and induce developmental delay

Anthropogenic CO2 emissions are acidifying the world’s oceans. A growing body of evidence is showing that ocean acidification impacts growth and developmental rates of marine invertebrates. Here we test the impact of elevated seawater pCO2 (129 Pa, 1271 μatm) on early development, larval metabolic and feeding rates in a marine model organism, the sea urchin Strongylocentrotus purpuratus. Growth and development was assessed by measuring total body length, body rod length, postoral rod length and posterolateral rod length. Comparing these parameters between treatments suggests that larvae suffer from a developmental delay (by ca. 8%) rather than from the previously postulated reductions in size at comparable developmental stages. Further, we found maximum increases in respiration rates of + 100 % under elevated pCO2, while body length corrected feeding rates did not differ between larvae from both treatments. Calculating scope for growth illustrates that larvae raised under high pCO2 spent an average of 39 to 45% of the available energy for somatic growth, while control larvae could allocate between 78 and 80% of the available energy into growth processes. Our results highlight the importance of defining a standard frame of reference when comparing a given parameter between treatments, as observed differences can be easily due to comparison of different larval ages with their specific set of biological characters.

Moderate seawater acidification does not elicit long-term metabolic depression in the blue mussel Mytilus edulis

Marine organisms are exposed to increasingly acidic oceans, as a result of equilibration of surface ocean water with rising atmospheric CO2 concentrations. In this study, we examined the physiological response of Mytilus edulis from the Baltic Sea, grown for 2 months at 4 seawater pCO2 levels (39, 113, 243 and 405 Pa/385, 1,120, 2,400 and 4,000 μatm). Shell and somatic growth, calcification, oxygen consumption and excretion rates were measured in order to test the hypothesis whether exposure to elevated seawater pCO2 is causally related to metabolic depression. During the experimental period, mussel shell mass and shell-free dry mass (SFDM) increased at least by a factor of two and three, respectively. However, shell length and shell mass growth decreased linearly with increasing pCO2 by 6–20 and 10–34%, while SFDM growth was not significantly affected by hypercapnia. We observed a parabolic change in routine metabolic rates with increasing pCO2 and the highest rates (+60%) at 243 Pa. excretion rose linearly with increasing pCO2. Decreased O:N ratios at the highest seawater pCO2 indicate enhanced protein metabolism which may contribute to intracellular pH regulation. We suggest that reduced shell growth under severe acidification is not caused by (global) metabolic depression but is potentially due to synergistic effects of increased cellular energy demand and nitrogen loss.

The influence of pCO2 and temperature on gene expression of carbon and nitrogen pathways in Trichodesmium IMS101

Growth, protein amount, and activity levels of metabolic pathways in Trichodesmium are influenced by environmental changes such as elevated pCO2 and temperature. This study examines changes in the expression of essential metabolic genes in Trichodesmium grown under a matrix of pCO2 (400 and 900 µatm) and temperature (25 and 31°C). Using RT-qPCR, we studied 21 genes related to four metabolic functional groups: CO2 concentrating mechanism (bicA1, bicA2, ccmM, ccmK2, ccmK3, ndhF4, ndhD4, ndhL, chpX), energy metabolism (atpB, sod, prx, glcD), nitrogen metabolism (glnA, hetR, nifH), and inorganic carbon fixation and photosynthesis (rbcL, rca, psaB, psaC, psbA). nifH and most photosynthetic genes exhibited relatively high abundance and their expression was influenced by both environmental parameters. A two to three orders of magnitude increase was observed for glnA and hetR only when both pCO2 and temperature were elevated. CO2 concentrating mechanism genes were not affected by pCO2 and temperature and their expression levels were markedly lower than that of the nitrogen metabolism and photosynthetic genes. Many of the CO2 concentrating mechanism genes were co-expressed throughout the day. Our results demonstrate that in Trichodesmium, CO2 concentrating mechanism genes are constitutively expressed. Co-expression of genes from different functional groups were frequently observed during the first half of the photoperiod when oxygenic photosynthesis and N2 fixation take place, pointing at the tight and complex regulation of gene expression in Trichodesmium. Here we provide new data linking environmental changes of pCO2 and temperature to gene expression in Trichodesmium. Although gene expression indicates an active metabolic pathway, there is often an uncoupling between transcription and enzyme activity, such that transcript level cannot usually be directly extrapolated to metabolic activity.

Impact of ocean acidification on the metabolism of calcifying planktonic organisms

Atmospheric pCO2 is predicted to double by the end of the century, and this increase is expected to lead to both global warming and ocean acidification, both being enhanced in polar oceans. Because of the freshening and increased carbon uptake in response to sea ice retreat, pH should decrease by more than 50% by 2050 in the Arctic. Marine calcifying organisms are particularly important in high latitudes as a food source for different species and for carbon fluxes, and will likely be directly affected as shells and other structures of calcium carbonates dissolve with lower pH. It is unclear though how those organisms will react, adapt and survive within this carbonate undersaturation scenario. Marine calcifying organisms include strictly planktonic as well as meroplanktonic organisms. Meroplankton spend only part of their life in a planktonic stage and include benthic larvae. In order to understand the effect of decreasing pH on the metabolism of calcifying organisms, perturbation experiments were performed on two meroplanktonic organisms: benthic gastropods and clams larvae, and one strictly planktonic organism: pteropods. During these experiments, oxygen was monitored every 4-8 hours in order to measure the organisms’ respiration at regular sea water pH (8.1) and at the lower pH predicted for the next 100 years (7.7). The increase of respiration at lower pH reflects a change in the organisms’ metabolism probably due to stress. By affecting calcifying organism metabolism, ocean acidification is likely to lead to changes in food web structure, carbon fluxes and benthic communities.

Synergistic effect of ocean acidification and elevated temperature on the physiological ecology of the intertidal crab Porcellana platycheles

Ongoing global environmental changes pose an unprecedented threat to global biodiversity; in particular increasing environmental temperatures and decreasing ocean pH (Ocean Acidification or OA, as a result of increased seawater pCO2−). The extent to which these two drivers will act synergistically, reducing the thermal tolerance window of individual species, and so potentially affect their large-scale distribution, is only beginning to be understood. Here we present a formal test on the potential synergistic effect of elevated temperatures and hypercapnic sea water on the rate of O2 uptake (as a proxy for metabolism), tolerance to heat, and the degree of exoskeleton calcification in the intertidal porcellanid crab Porcellana platycheles. Eighty individuals crabs were haphazardly assigned to one of four treatments, and kept for 40 days at either 15.0 °C (seasonal ambient) or 20.0 °C (+ 5 °C), and at either pH 8.0 (seasonal ambient) or 7.4. In Porcellana platycheles metabolic activity and tolerance to heat were positively affected by increasing temperature, whilst the degree of exoskeleton calcification was negatively affected. No effect of pH was detectable. It is therefore suggested that P. platycheles may not be affected by medium-term exposure to the predicted level of OA, but that acclimation to elevated temperatures may result in improved tolerance of high temperatures despite an increase in metabolic costs and a decrease of calcification. Our results are discussed within a broader ecological and evolutionary context, with particular emphasis on the idea that intertidal species may be to some extent exapted to hypercapnic exposure.

Impacts of hypercapnia and temperature on physiological performance of marine invertebrates from the Baltic Sea

Anthropogenic CO2 emissions cause rising ocean pCO2 and decreased ocean pH. This progressing ocean acidification has been shown to compromise physiological performance of many marine benthic organisms. In this study, Baltic blue mussel Mytilus edulis and common starfish Asterias rubens from Kiel Fjord were exposed to 6 different hypercapnic levels between 0.04 and 0.41 kPa pCO2 for 1–2 weeks, respectively, at 12 °C. The experiments revealed extracellular acidosis in both species with no active accretion of HCO3−. Even at moderate pCO2 levels (e.g. 0.08 kPa) pHe was significant decreased. Although shell dissolution occurred in the highest pCO2 treatment, no HCO3− or Ca2+ accumulation was observed in extracellular fluids. Gradients of pCO2 between body fluids and ambient sea water were stable at lower pCO2 levels and decreased at 0.41 kPa, which might indicate a metabolic depression. Ongoing metabolic rate and filtration rate determinations will resolve this issue. Calcification rates were only minorly impacted in the lower pCO2 treatments and became negative at 0.41 kPa pCO2. Field measurements conducted in Kiel Fjord revealed a large annual variability of surface pH and pCO2. During winter and spring, surface pH averaged 8.1 but mean summer pH values were < 7.8 and the highest measured pCO2 value exceeded 0.2 kPa (ca 2000 μatm), thus even the maximum average pCO2 values modelled for the surface ocean of the year 2300.

Experiments will be repeated at summer temperatures of 18–22 °C in June 2009 to clarify if thermal extremes increase the species' sensitivity towards hypercapnia.