External Carbonic Anhydrase Activity Research Articles

Inorganic carbon utilization: A target of silver nanoparticle toxicity on a submerged macrophyte

Submerged macrophytes play an important role in the global carbon cycle through diversified pathways of inorganic carbon (Ci) utilization distinct from terrestrial plants. However, the effects of silver nanoparticles (AgNPs), an emerging contaminant, were unknown on the Ci utilization of submerged macrophytes. In Ottelia alismoides, the only known submerged macrophyte with three pathways of Ci utilization, before absorption, AgNPs inhibited the external carbonic anhydrase activity thus reducing the capacity of the plant to use HCO3−. After entering the plant, AgNPs mainly aggregated at the cell wall and in the chloroplast. The internalized AgNPs inhibited ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) activity blocking CO2 fixation and disturbed C4 and crassulacean acid metabolism (CAM) by inhibiting phosphoenolpyruvate carboxylase (PEPC), pyruvate phosphate dikinase (PPDK), and NAD-dependent malic enzyme (NAD-ME) activities to alter intracellular malate biosynthesis and decarboxylation. Overall, our findings indicate that the Ci utilization of the submerged macrophyte is a target of AgNPs toxicity that might affect the carbon cycle in aquatic systems.

Cell size influences inorganic carbon acquisition in artificially selected phytoplankton.

Cell size influences the rate at which phytoplankton assimilate dissolved inorganic carbon (DIC), but it is unclear whether volume-specific carbon uptake should be greater in smaller or larger cells. On the one hand, Fick's Law predicts smaller cells to have a superior diffusive CO2 supply. On the other, larger cells may have greater scope to invest metabolic energy to upregulate active transport per unit area through CO2 -concentrating mechanisms (CCMs). Previous studies have focused on among-species comparisons, which complicates disentangling the role of cell size from other covarying traits. In this study, we investigated the DIC assimilation of the green alga Dunaliella tertiolecta after using artificial selection to evolve a 9.3-fold difference in cell volume. We compared CO2 affinity, external carbonic anhydrase (CAext ), isotopic signatures (δ13 C) and growth among size-selected lineages. Evolving cells to larger sizes led to an upregulation of CCMs that improved the DIC uptake of this species, with higher CO2 affinity, higher CAext and higher δ13 C. Larger cells also achieved faster growth and higher maximum biovolume densities. We showed that evolutionary shifts in cell size can alter the efficiency of DIC uptake systems to influence the fitness of a phytoplankton species.

Resilience of the temperate coral Oculina arbuscula to ocean acidification extends to the physiological level

Both juvenile and adult life stages of the temperate scleractinian coral Oculina arbuscula are resilient to the effects of moderate ocean acidification (OA) in contrast to many tropical corals in which growth and calcification rates are suppressed. Here, potential mechanisms of resilience to OA related to photosynthetic physiology and inorganic carbon processing were studied in adult O. arbuscula colonies. After exposing colonies to ambient and elevated carbon dioxide (CO2) treatments for 7 weeks, photosynthetic performance was characterized using photosynthesis versus irradiance experiments, chlorophyll fluorescence kinetics, and algal pigment content. Inorganic carbon-processing capabilities were assessed by measurement of internal and external carbonic anhydrase activity of the coral host, internal carbonic anhydrase activity of symbiotic algae, and the reliance of photosynthesis on external carbonic anhydrase. Photosynthetic physiology was unaffected by OA ruling out the possibility that resilience was mediated by increased photosynthetic energy supply. Carbonic anhydrase activities were maintained at elevated CO2 suggesting no major rearrangements of the inorganic carbon-processing machinery, but this could be a sign of resilience since tropical corals often down-regulate carbonic anhydrases at high CO2. The general lack of effect of ocean acidification on these physiological traits suggests other characteristics, such as maintenance of calcifying fluid pH and ability to acquire energy from heterotrophy, may be more important for the resilience of O. arbuscula to OA.

Physiological differences in photosynthetic inorganic carbon utilization between gametophytes and sporophytes of the economically important red algae Pyropia haitanensis

Despite the lifecycle of the red seaweed Pyropia/Porphyra being extensively studied, photosynthesis and the CO2 concentrating mechanisms (CCMs) have comparatively not been well studied between the gametophyte phase (leafy thallus) and the sporophyte phase (filamentous conchocelis). This study found that the net photosynthetic rates of the thalli were about twice that of the conchocelis of Py. haitanensis, and the maximal quantum yield of photosystem II (Fv/Fm) and thallus respiration were significantly higher and lower than the conchocelis, respectively. Additionally, two phases did not show significant differences in phycocyanin (PC) content, but significantly higher contents of phycoerythrin (PE), allophycocyanin (APC), and chlorophyll α (Chl α) were observed in the thalli compared with the conchocelis. The external carbonic anhydrase (eCA) activity of the thalli was also higher than that of the conchocelis, but no differences in internal CA (iCA) activity were detected between the two phases. As for carbon acquisition for photosynthesis, the major carbon source of the thalli was seawater HCO3−, which was absorbed via eCA-catalyzed conversion to CO2. Conversely, the conchocelis used HCO3− transporters to directly absorb seawater HCO3−, which contributed equally to photosynthesis compared with the eCA- or iCA-mediated pathway. The discovery of different CCMs between the thallus and conchocelis phases could further our understanding of lifecycle regulation in Pyropia. In addition, a decrease in pH from 9 to 6 significantly increased the photosynthetic rates of both the thalli and conchocelis by about 100% and 600%, respectively. Thus, both phases have potential for carbon capture from flue gases and carbon sequestration.

Insights into carbon acquisition and photosynthesis in Karenia brevis under a range of CO2 concentrations

Karenia brevis is a marine dinoflagellate commonly found in the Gulf of Mexico and important both ecologically and economically due to its production of the neurotoxin brevetoxin, which can cause respiratory illness in humans and widespread death of marine animals. K. brevis strains have previously shown to be sensitive to changes in CO2, both in terms of growth as well as toxin production. Our study aimed to understand this sensitivity by measuring underlying mechanisms, such as photosynthesis, carbon acquisition, and photophysiology. K. brevis (CCFWC-126) did not show a significant response in growth, cellular composition of carbon and nitrogen, nor in photosynthetic rates between pCO2 concentrations of 150, 400, or 780 µatm. However, a strong response in its acquisition of inorganic carbon was found. Half saturation values for CO2 increased from 1.5 to 3.3 µM, inorganic carbon preference switched from HCO3− to CO2 (14–56% CO2 usage), and external carbonic anhydrase activity was downregulated by 23% when comparing low and high pCO2. We conclude that K. brevis must employ an efficient and regulated CO2 concentrating mechanism (CCM) to maintain constant carbon fixation and growth across pCO2 levels. No statistically significant correlation between CO2 and brevetoxin content was found, yet a positive trend with enhanced pCO2 was detected. This study is the first explaining how this socioeconomically important species is able to efficiently supply inorganic carbon for photosynthesis, which can potentially prolong bloom situations. This study also highlights that elevated CO2 concentrations, as projected for a future ocean, can affect underlying physiological processes of K. brevis, some of which could lead to increases in cellular brevetoxin production and therefore increased impacts on coastal ecosystems and economies.

Increased CO2 and iron availability effects on carbon assimilation and calcification on the formation of Emiliania huxleyi blooms in a coastal phytoplankton community

In the present work, we exposed a natural phytoplankton community to either present (390-μatm, LC) or future CO2 levels predicted for year-2100 (900-μatm, HC) combined with ambient (4.5 nmol L−1, −DFB) or high (12 nmol L−1, +DFB) dissolved iron (dFe) levels, during 25 days by using mesocosms. We report on changes in carbon assimilation processes (acquisition, fixation, and calcification) of the phytoplankton community due to increased dissolved CO2 and dFe and to the interaction of both factors. The isotopic disequilibrium assay results showed that inorganic carbon (Ci) acquisition by the community was unaffected by CO2 and Fe availability. The main Ci source for photosynthesis was HCO3− and external carbonic anhydrase activity was only detected at the beginning of the experiment, suggesting a relevant role for HCO3− transporters in the phytoplankton community developed in all treatments. However, there was a significant effect of both factors on particulate organic carbon (POC) content, particulate calcium production and carbon fixation rates. Increased dFe at LC conditions led to the highest values of carbon fixation and POC of all treatments, promoting a massive Emiliania huxleyi bloom. This response was not observed in the HC treatments. The latter indicates a negative impact of increased CO2 on the formation of E. huxleyi blooms, in agreement with the observed significant reduction in calcium production under HC. Our results suggest that ocean acidification can decrease primary production under iron-replete conditions in E. huxleyi blooming areas, affecting the biological carbon pump in coastal ecosystems.

Inorganic carbon is scarce for symbionts in scleractinian corals

AbstractOcean acidification and changing sea surface temperatures stand to affect the interactions of corals and their Symbiodinium symbionts with regard to the inorganic carbon used for photosynthesis. However, there have been few investigations on the availability of dissolved inorganic carbon (DIC) for algal symbionts in hospite. This study compared the DIC‐associated photosynthetic kinetic parameters of three Caribbean corals and their freshly isolated symbionts, as well as components of the DIC concentrating systems of both corals and symbionts. Species level differences were found in the extent of inorganic carbon saturation among the coral taxa studied. Only Orbicella faveolata was photosynthesizing at maximum rates under current seawater conditions, while Porites astreoides and Siderastrea radians were at or below half DIC saturation. O. faveolata also had significantly more external carbonic anhydrase activity, indicating that this species could produce more CO2 at the coral surface than P. astreoides or S. radians. In contrast and despite differences in Symbiodinium type, the symbionts of all the corals had a similar, very low DIC half saturation constant for photosynthesis and high levels of internal carbonic anhydrase activity, showing that they live in a carbon scarce environment and invest a great deal of energy in concentrating carbon at the site of photosynthesis. Considering the diffusional dynamics of the system and the relationship of host to symbiont kinetic parameters, the most likely cause of this scarcity is host regulation of DIC delivery to the symbionts.

Effects of future climate conditions on photosynthesis and biochemical component of Ulva pertusa (Chlorophyta)

Ulva pertusa, a common bloom-forming green alga, was used as a model system to examine the effects of elevated carbon dioxide (CO2) and temperature on growth and photosynthetic performance. To do this, U. pertusa was grown under four temperature and CO2 conditions; ambient CO2 (400 μatm) and temperature (16°C) (i.e., present), elevated temperature only (19°C) (ET; i.e., warming), elevated CO2 only (1,000 μatm) (EC; i.e., acidification), and elevated tempera ture and CO2 (ET and EC; i.e., greenhouse), and its steady state photosynthetic performance evaluated. Maximum gross photosynthetic rates (GPmax) were highest under EC conditions and lowest under ET conditions. Further, ET conditions resulted in decreased rate of dark respiration (Rd), but growth of U. pertusa was higher under ET conditions than under ambient temperature conditions. In order to evaluate external carbonic anhydrase (eCA) activity, photosynthesis was measured at 70 μmol photons m -2 s -1 in the presence or absence of the eCA inhibitor acetazolamide (AZ), which inhibited photosynthetic rates in all treatments, indicating eCA activity. However, while AZ reduced U. pertusa photosynthesis in all treatments, this reduction was lower under ambient CO2 conditions (both present and warming) compared to EC conditions (both acidification and greenhouse). Moreover, Chlorophyll a and glucose contents in U. pertusa tissues declined under ET conditions (both warming and greenhouse) in conjunction with reduced GPmax and Rd. Overall, our results indicate that the interaction of EC and ET would offset each other’s impacts on photosynthesis and biochemical composition as related to carbon balance of U. pertusa.

Effects of simultaneous increase in temperature and ocean acidification on biochemical composition and photosynthetic performance of common macroalgae from Kongsfjorden (Svalbard)

Increased temperature and ocean acidification are two main factors threatening Arctic coastal ecosystems. To explore the consequences of increased average temperature and ocean acidification in Kongsfjorden, the performance of six common species of macroalgae, the chlorophyte Monostroma arcticum, the rhodophytes Phycodrys rubens, and Ptilota plumosa, and the phaeophytes Alaria esculenta, Desmarestia aculeata and Saccorhiza dermatodea, was tested after 9 days of culture at 4 and 10 °C in combination with current (ca. 390 ppmv) and future (1000 ppmv) levels of atmospheric CO2 under solar radiation in summer. Temperature affected mainly the photosynthetic performance as measured by PAM fluorescence, particularly the initial slope (α) of ETR curves, the light saturation parameter (E k ), and F v /F m values, as well as the protein content, especially in the phaeophytes. On the other hand, CO2 affected mainly the internal accumulation of carbohydrates and lipids. The C:N balance was largely unaffected. External carbonic anhydrase activity was not inhibited at high CO2, and nitrate reductase activity remained unaffected. The resulting growth rate was not altered by treatments in three out of the six species studied. On the other hand, P. rubens showed a positive effect of increasing temperature, D. aculeata was negatively influenced by CO2, and S. dermatodea was positively affected by CO2. Significant interactions between CO2 and temperature were found in 20 % of the analyses. Whether additive or synergistic, the co-occurrence of a higher temperature with other stressors such as elevated CO2 increases the probability of community changes by modifying the performance of these species.

Effects of ocean acidification on the photosynthetic performance, carbonic anhydrase activity and growth of the giant kelp Macrocystis pyrifera.

Under ocean acidification (OA), the 200 % increase in CO2(aq) and the reduction of pH by 0.3-0.4 units are predicted to affect the carbon physiology and growth of macroalgae. Here we examined how the physiology of the giant kelp Macrocystis pyrifera is affected by elevated pCO2/low pH. Growth and photosynthetic rates, external and internal carbonic anhydrase (CA) activity, HCO3 (-) versus CO2 use were determined over a 7-day incubation at ambient pCO2 400 µatm/pH 8.00 and a future OA treatment of pCO2 1200 µatm/pH 7.59. Neither the photosynthetic nor growth rates were changed by elevated CO2 supply in the OA treatment. These results were explained by the greater use of HCO3 (-) compared to CO2 as an inorganic carbon (Ci) source to support photosynthesis. Macrocystis is a mixed HCO3 (-) and CO2 user that exhibits two effective mechanisms for HCO3 (-) utilization; as predicted for species that possess carbon-concentrating mechanisms (CCMs), photosynthesis was not substantially affected by elevated pCO2. The internal CA activity was also unaffected by OA, and it remained high and active throughout the experiment; this suggests that HCO3 (-) uptake via an anion exchange protein was not affected by OA. Our results suggest that photosynthetic Ci uptake and growth of Macrocystis will not be affected by elevated pCO2/low pH predicted for the future, but the combined effects with other environmental factors like temperature and nutrient availability could change the physiological response of Macrocystis to OA. Therefore, further studies will be important to elucidate how this species might respond to the global environmental change predicted for the ocean.

Ocean acidification modulates the response of two Arctic kelps to ultraviolet radiation

The combined effects of ocean acidification and ultraviolet radiation (UVR) have been studied in the kelps Alaria esculenta and Saccharina latissima from Kongsfjorden (Svalbard), two major components of the Arctic macroalgal community, in order to assess their potential to thrive in a changing environment. Overall results revealed synergistic effects, however with a different amplitude in the respective species. Changes in growth, internal N, C:N ratio, pigments, optimum quantum yield (Fv/Fm) and electron transport rates (ETR) following CO2 enrichment and/or UVR were generally more pronounced in S. latissima than in A. esculenta. The highest growth rates were recorded under simultaneous CO2 enrichment and UVR in both species. UVR-mediated changes in pigment content were partially prevented under elevated CO2 in both species. Similarly, UVR led to increased photosynthetic efficiency (α) and ETR only if CO2 was not elevated in A. esculenta and even under high CO2 in S. latissima. Increased CO2 did not inhibit external carbonic anhydrase (eCA) activity in the short-term but in the mid-term, indicating a control through acclimation of photosynthesis rather than a direct inhibition of eCA by CO2. The higher benefit of simultaneous CO2 enrichment and UVR for S. latissima respect to A. esculenta seems to involve higher C and N assimilation efficiency, as well as higher ETR, despite a more sensitive Fv/Fm. The differential responses shown by these two species indicate that ongoing ocean acidification and UVR could potentially change the dominance at lower depths (4–6m), which will eventually drive changes at the community level in the Arctic coastal ecosystem. These results support an existing consideration of S. latissima as a winner species in the global change scenario.

A new model for the calcification of the green macro-alga Halimeda opuntia (Lamouroux)

Halimeda opuntia is a cosmopolitan marine calcifying green alga in shallow tropical marine environments. Besides Halimeda’s contribution to a diverse habitat, the alga is an important sediment producer. Fallen calcareous segments of Halimeda spp. are a major component of carbonate sediments in many tropical settings and play an important role in reef framework development and carbonate platform buildup. Consequently the calcification of H. opuntia accounts for large portions of the carbonate budget in tropical shallow marine ecosystems. Earlier studies investigating the calcification processes of Halimeda spp. have tended to focus on the microstructure or the physiology of the alga, thus overlooking the interaction of physiological and abiotic processes behind the formation of the skeleton. By analyzing microstructural skeletal features of Halimeda segments with the aid of scanning electron microscopy and relating their occurrence to known physiological processes, we have been able to identify the initiation of calcification within an organic matrix and demonstrate that biologically induced cementation is an important process in calcification. For the first time, we propose a model for the calcification of Halimeda spp. that considers both the alga’s physiology and the carbon chemistry of the seawater with respect to the development of different skeletal features. The presence of an organic matrix and earlier detected external carbonic anhydrase activity suggest that Halimeda spp. exhibit biotic precipitation of calcium carbonate, as many other species of marine organisms do. On the other hand, it is the formation of micro-anhedral carbonate through the alga’s metabolism that leads to a cementation of living segments. Precisely, this process allows H. opuntia to contribute substantial amounts of carbonate sediments to tropical shallow seas.

CO2 acquisition in Chlamydomonas acidophila is influenced mainly by CO2, not phosphorus, availability.

The extremophilic green microalga Chlamydomonas acidophila grows in very acidic waters (pH 2.3-3.4), where CO2 is the sole inorganic carbon source. Previous work has revealed that the species can accumulate inorganic carbon (Ci) and exhibits high affinity CO2 utilization under low-CO2 (air-equilibrium) conditions, similar to organisms with an active CO2 concentrating mechanism (CCM), whereas both processes are down-regulated under high CO2 (4.5% CO2) conditions. Responses of this species to phosphorus (Pi)-limited conditions suggested a contrasting regulation of the CCM characteristics. Therefore, we measured external carbonic anhydrase (CAext) activities and protein expression (CAH1), the internal pH, Ci accumulation, and CO2-utilization in cells adapted to high or low CO2 under Pi-replete and Pi-limited conditions. Results reveal that C. acidophila expressed CAext activity and expressed a protein cross-reacting with CAH1 (the CAext from Chlamydomonas reinhardtii). Although the function of this CA remains unclear, CAext activity and high affinity CO2 utilization were the highest under low CO2 conditions. C. acidophila accumulated Ci and expressed the CAH1 protein under all conditions tested, and C. reinhardtii also contained substantial amounts of CAH1 protein under Pi-limitation. In conclusion, Ci utilization is optimized in C. acidophila under ecologically relevant conditions, which may enable optimal survival in its extreme Ci- and Pi-limited habitat. The exact physiological and biochemical acclimation remains to be further studied.

Nutrient availability affects the response of the calcifying chlorophyte Halimeda opuntia (L.) J.V. Lamouroux to low pH

Atmospheric carbon dioxide emissions cause a decrease in the pH and aragonite saturation state of surface ocean water. As a result, calcifying organisms are expected to suffer under future ocean conditions, but their physiological responses may depend on their nutrient status. Because many coral reefs experience high inorganic nutrient loads or seasonal changes in nutrient availability, reef organisms in localized areas will have to cope with elevated carbon dioxide and changes in inorganic nutrients. Halimeda opuntia is a dominant calcifying primary producer on coral reefs that contributes to coral reef accretion. Therefore, we investigated the carbon and nutrient balance of H. opuntia exposed to elevated carbon dioxide and inorganic nutrients. We measured tissue nitrogen, phosphorus and carbon content as well as the activity of enzymes involved in inorganic carbon uptake and nitrogen assimilation (external carbonic anhydrase and nitrate reductase, respectively). Inorganic carbon content was lower in algae exposed to high CO₂, but calcification rates were not significantly affected by CO₂ or inorganic nutrients. Organic carbon was positively correlated to external carbonic anhydrase activity, while inorganic carbon showed the opposite correlation. Carbon dioxide had a significant effect on tissue nitrogen and organic carbon content, while inorganic nutrients affected tissue phosphorus and N:P ratios. Nitrate reductase activity was highest in algae grown under elevated CO₂ and inorganic nutrient conditions and lowest when phosphate was limiting. In general, we found that enzymatic responses were strongly influenced by nutrient availability, indicating its important role in dictating the local responses of the calcifying primary producer H. opuntia to ocean acidification.

A modified pH drift assay for inorganic carbon accumulation and external carbonic anhydrase activity in microalgae

Threat of global warming due to carbon dioxide (CO2) emissions has stimulated research into carbon sequestration and emissions reduction technologies. Alkaline scrubbing allows CO2 to be captured as bicarbonate, which can be photochemically fixed by microalgae. The carbon concentrating mechanism (CCM), of which external carbonic anhydrase is a key component, allows organisms to utilise this bicarbonate. In order to select a suitable strain for this application, a screening tool is required. The current method for determining carbonic anhydrase activity, the Wilbur and Anderson assay, was found to be unsuitable as a screening tool as the associated error was unacceptably large and tests on whole cells were inconclusive. This paper presents the development of a new, whole cell assay to measure inorganic carbon uptake and external carbonic anhydrase activity, based on classical pH drift experiments. Spirulina platensis was successfully used to develop a correlation between the specific carbon uptake (C) and the specific pH change (dpH). The relationship is described by the following: C[mmol C (g dry algae)−1 h−1] = 0.064 × (dpH). Inhibitor and salt dissociation tests validated the activity and presence of external carbonic anhydrase and allowed correlation between the Wilbur and Anderson assay and the new whole cell assay. Screening tests were conducted on S. platensis, Scenedesmus sp., Chlorella vulgaris and Dunaliella salina that were found to have carbon uptake rates of 5.76, 5.86, 3.86 and 2.15 mmol C (g dry algae)−1 h−1, respectively. These results corresponded to the species' known bicarbonate utilisation abilities and validated the use of the assay as a screening tool.

Contrasting modes of inorganic carbon acquisition amongst Symbiodinium (Dinophyceae) phylotypes

Growing concerns over ocean acidification have highlighted the need to critically understand inorganic carbon acquisition and utilization in marine microalgae. Here, we contrast these characteristics for the first time between two genetically distinct dinoflagellate species of the genus Symbiodinium (phylotypes A13 and A20) that live in symbiosis with reef-forming corals. Both phylotypes were grown in continuous cultures under identical environmental conditions. Rubisco was measured using quantitative Western blots, and radioisotopic (14) C uptake was used to characterize light- and total carbon dioxide (TCO2 )-dependent carbon fixation, as well as inorganic carbon species preference and external carbonic anhydrase activity. A13 and A20 exhibited similar rates of carbon fixation despite cellular concentrations of Rubisco being approximately four-fold greater in A13. The uptake of CO2 over HCO3 - was found to support the majority of carbon fixation in both phylotypes. However, A20 was also able to indirectly utilize HCO3 - by first converting it to CO2 via external carbonic anhydrase. These results show that adaptive differences in inorganic carbon acquisition have evolved within the Symbiodinium genus, which thus carries fundamental implications as to how this functionally key genus will respond to ocean acidification, but could also represent a key trait factor that influences their productivity when in hospite of their coral hosts.

Zinc Affects Differently Growth, Photosynthesis, Antioxidant Enzyme Activities and Phytochelatin Synthase Expression of Four Marine Diatoms

Zinc-supplementation (20 μM) effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase), and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa). Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses.

Changes in carbon uptake mechanisms in two green marine algae by reduced seawater pH

Acidification of the oceans, as a consequence of anthropogenic CO2 emissions has reduced the pH at the surface of oceans by 0.1 unit compared to pre-industrial values. The reduction of seawater pH changes the relative proportion of the inorganic carbon (Ci) species which could potentially affect the modes of Ci assimilation by marine microalgae. In this study the effects of changes in external pH on the modes of Ci uptake over the pH range 5.0 to 7.5 were determined mass spectrometrically in Stichococcus minor Naegeli and S. cylindricus Butcher et Umbauk. Both species were found to tolerate a broad range of pH from pH 5.0 to 9.5 but the optimum for growth of both species was 8.2. Both species were also found to grow over a range of salinities and are best described as brackish water species rather than marine species, since they grow over wide ranges of salinity and pH. Neither species expresses external carbonic anhydrase (CA) activity. In both species, cells grown at pH 5.0, where the bulk of dissolved inorganic carbon (DIC) is in the form of CO2, active HCO3− and CO2 uptake were absent and cells appear to take up CO2 by diffusion. However, active HCO3− uptake was present in cells of both species grown at pH 6.0, 7.0 and 7.5 but active CO2 uptake was not detectable. Cells of both species, when grown at pH 8.2, display both active CO2 uptake and active HCO3− uptake.

Inorganic carbon acquisition in two green marine Stichococcus species

The mechanism of inorganic carbon (C(i)) uptake was examined in the marine green microalgae Stichococcus cylindricus and Stichococcus minor. External carbonic anhydrase (CA) activity was not detected in either species, by potentiometric assay or by mass spectrometry. Photosynthetic characteristics of C(i) uptake indicate that both species have high apparent affinity for CO(2) with a low K(1/2) (CO(2)) of about 10 µm. The O(2) evolution rates in light exceeded the spontaneous CO(2) formation rate by 2.5-fold in both species, which thus have active bicarbonate uptake. Mass spectrometric monitoring of CO(2) and O(2) fluxes showed that rates of O(2) evolution exceeded those of CO(2) depletion by about three- and twofold in S. minor and S. cylindricus, respectively, and also showed, in cells photosynthesizing at pH 8.2, a rapid depletion of CO(2) upon illumination to a CO(2) compensation concentration of 15.42 and 12.03 µm in S. minor and S. cylindricus, respectively. Both species also exhibit active CO(2) uptake: addition of bovine CA at CO(2) compensation concentration caused a rapid rise in CO(2) as the CO(2) -HCO(3) (-) equilibrium was restored. Accumulation of unfixed C(i) by cells at pH 8.2 was calculated to be 84.33 mm in S. cylindricus, and 30.37 mm in S. minor to give internal accumulations of 23- and 8-fold, respectively, compared to the external C(i) concentration.

Different mechanisms of inorganic carbon acquisition in red macroalgae (Rhodophyta) revealed by the use of TRIS buffer

The effects on photosynthesis of acetazolamide (AZ, an inhibitor of the external carbonic anhydrase) and TRIS buffer at pH 8.7 were assessed in 24 species of red macroalgae. Only Palmaria palmata was unaffected by both substances. The rest of species were classified into three groups according to their sensitivity to TRIS and AZ. Photosynthesis of fourteen species was significantly inhibited by both TRIS and AZ. Inhibition by TRIS varied from almost 100% to 25% while AZ produced similar effects. Inhibition by TRIS was completely reverted by increasing the dissolved inorganic carbon concentration (DIC). This species group had half-saturation constants for photosynthesis ( K m(DIC)) ranging from 0.5 to 1.1 mM of DIC. TRIS produced a significant increase of K m(DIC). Altogether, these results indicate that the algae sensitive to TRIS are capable of using HCO 3 − efficiently at pH 8.7. Furthermore, the buffering capacity of TRIS was responsible for its inhibitory effect on photosynthesis suggesting that HCO 3 − use was facilitated by excretion of protons outside the plasma membrane, which creates regions of low pH resulting in a higher-than-ambient CO 2 concentration. In contrast, photosynthesis by two Porphyra species analysed was slightly stimulated by TRIS and completely inhibited by AZ, suggesting that the mechanism was different. In a third group of seaweeds, photosynthesis was insensitive to TRIS but it was significantly inhibited by AZ. These species had relatively high values of K m(DIC) indicating that they relied on purely diffusive entry of CO 2 generated by external carbonic anhydrase activity. Consequently, the results demonstrate that external carbonic anhydrase is widespread among red macroalgae since only P. palmata was insensitive to AZ. The functional significance of this enzyme was quite variable among the tested species.