eCA Activity Research Articles

The Role of Extracellular Carbonic Anhydrase in Biogeochemical Cycling: Recent Advances and Climate Change Responses.

Climate change has been predicted to influence the marine phytoplankton community and its carbon acquisition strategy. Extracellular carbonic anhydrase (eCA) is a zinc metalloenzyme that catalyses the relatively slow interconversion between HCO3− and CO2. Early results indicated that sub-nanomolar levels of eCA at the sea surface were sufficient to enhance the oceanic uptake rate of CO2 on a global scale by 15%, an addition of 0.37 Pg C year−1. Despite its central role in the marine carbon cycle, only in recent years have new analytical techniques allowed the first quantifications of eCA and its activity in the oceans. This opens up new research areas in the field of marine biogeochemistry and climate change. Light and suitable pH conditions, as well as growth stage, are crucial factors in eCA expression. Previous studies showed that phytoplankton eCA activity and concentrations are affected by environmental stressors such as ocean acidification and UV radiation as well as changing light conditions. For this reason, eCA is suggested as a biochemical indicator in biomonitoring programmes and could be used for future response prediction studies in changing oceans. This review aims to identify the current knowledge and gaps where new research efforts should be focused to better determine the potential feedback of phytoplankton via eCA in the marine carbon cycle in changing oceans.

Use of the particle tracking method for modelling the transport and deceasing of Escherichia coli in the sea

We discuss modeling of the transport and decease of bacteria Escherichia coli in marine environments. We calculated the spatially variable decease coefficient of E. coli from the extrapolated measurement data on environmental parameters in accordance with Mancini's equation for bacteria stemming from the Rižana River and entering into the computational domain – the eastern part of the Koper Bay. Using pre-computed circulation data and the modified particle-tracking model Nafta3D we performed two-day simulations of an instantaneous and a continuous release from a single source. We analyzed locations of two particle types: free E. coli (ECF) and E. coli bound to suspended solids (ECA). We then analyzed the ECA and ECF activity decrease with a newly developed decease model, applying a first-order kinetics equation. In the given conditions a vast majority of ECA remained in the second basin of the Port of Koper and sank to the bottom layers. The decease time of 90% of the ECA (T90) was about 48 hours. The ECF remained closer to the surface; they moved more quickly and for the most part abandoned the second basin. The determined T90 was 6 and 10 hours in instantaneous and continuous release, respectively.

Role of Extracellular Carbonic Anhydrase in Dissolved Inorganic Carbon Uptake in Alkaliphilic Phototrophic Biofilm.

Alkaline Soda Lakes are extremely productive ecosystems, due to their high dissolved inorganic carbon (DIC) concentrations. Here, we studied the dynamics of the carbonate system, in particular, the role of extracellular carbonic anhydrase (eCA) of an alkaliphilic phototrophic biofilm composed of bacteria enriched from soda lake benthic mats. By using measurements with microsensors and membrane inlet mass spectrometry, combined with mathematical modeling, we show how eCA controls DIC uptake. In our experiments, the activity of eCA varied four-fold, and was controlled by the bicarbonate concentration during growth: a higher bicarbonate concentration led to lower eCA activity. Inhibition of eCA decreased both the net and the gross photosynthetic productivities of the investigated biofilms. After eCA inhibition, the efflux of carbon dioxide (CO2) from the biofilms increased two- to four-fold. This could be explained by the conversion of CO2, leaking from cyanobacterial cells, by eCA, to bicarbonate. Bicarbonate is then taken up again by the cyanobacteria. In suspensions, eCA reduced the CO2 leakage to the bulk medium from 90 to 50%. In biofilms cultivated at low bicarbonate concentration (~0.13 mM), the oxygen production was reduced by a similar ratio upon eCA inhibition. The role of eCA in intact biofilms was much less significant compared to biomass suspensions, as CO2 loss to the medium is reduced due to mass transfer resistance.

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.

External carbonic anhydrase in three Caribbean corals: quantification of activity and role in CO2 uptake

Scleractinian corals have complicated inorganic carbon (C i) transport pathways to support both photosynthesis, by their symbiotic dinoflagellates, and calcification. The first step in C i acquisition, uptake into the coral, is critical as the diffusive boundary layer limits the supply of CO2 to the surface and HCO3 − uptake is energy intensive. An external carbonic anhydrase (eCA) on the oral surface of corals is thought to facilitate CO2 uptake by converting HCO3 − into CO2, helping to overcome the limitation imposed by the boundary layer. However, this enzyme has not yet been identified or detected in corals, nor has its activity been quantified. We have developed a method to quantify eCA activity using a reaction–diffusion model to analyze data on 18O removal from labeled C i. Applying this technique to three species of Caribbean corals (Orbicella faveolata, Porites astreoides, and Siderastrea radians) showed that all species have eCA and that the potential rates of CO2 generation by eCA greatly exceed photosynthetic rates. This demonstrates that eCA activity is sufficient to support its hypothesized role in CO2 supply. Inhibition of eCA severely reduces net photosynthesis in all species (on average by 46 ± 27 %), implying that CO2 generated by eCA is a major carbon source for photosynthesis. Because of the high permeability of membranes to CO2, CO2 uptake is likely driven by a concentration gradient across the cytoplasmic membrane. The ubiquity of eCA in corals from diverse genera and environments suggests that it is fundamental for photosynthetic CO2 supply.

Size scaling of extracellular carbonic anhydrase activity in centric marine diatoms.

Many microalgae have a surface-associated extracellular carbonic anhydrase (eCA) that converts HCO3 (-) to CO2 for uptake and subsequent photosynthetic fixation. We investigated eCA activity and assessed its importance for photosynthetic CO2 supply in six centric diatom species spanning nearly the full range of cell sizes for centric diatoms (equivalent spherical radius 3-67 μm). Since larger cells are more susceptible to diffusion limitation, we hypothesized that eCA activity would increase with cell size as would its importance for CO2 supply. eCA activity did increase with cell size, increasing with cell radius by a size-scaling exponent of 2.6 ± 0.3. The rapid increase in eCA activity with cell radius keeps the absolute CO2 concentration difference between bulk seawater and the cell surface very low (<~0.2 μM) allowing high rates of CO2 uptake even for large diatoms. Although inhibiting eCA did reduce photosynthesis in the diatoms, there was no overall relationship between the extent of inhibition of photosynthesis and cell size. The only indication that eCA may be more important for larger diatoms was that photosynthesis in the smallest diatoms (<4 μm radius) was only affected by eCA inhibition when CO2 concentrations were very low, while photosynthesis in some larger diatoms was affected even at typical seawater CO2 concentrations. eCA is ubiquitous in centric marine diatoms, in contrast to other taxa where its presence is irregularly distributed among different species, and plays an important role in supplying CO2 for photosynthesis across the size spectrum.

Low temperature reduces the energetic requirement for the CO2 concentrating mechanism in diatoms.

The goal of this study is to investigate the CO2 concentrating mechanism (CCM) of the dominant phytoplankton species during the growing season at Palmer station in the Western Antarctic Peninsula. Key CCM parameters (cellular half-saturation constants for CO2 fixation, carbonic anhydrase activity, CO2 /HCO3 (-) uptake, δ(13) Corg ) in natural phytoplankton assemblages were determined. Those results, together with additional measurements on CO2 membrane permeability from Fragilariopsis cylindrus laboratory cultures, were used to develop a numerical model of the CCM of cold water diatoms. The field data demonstrate that the dominant species throughout the season possess an effective CCM, which achieves near saturation of CO2 for fixation. The model provides a means to examine the role of eCA activity and HCO3 (-) /CO2 uptake in the functioning of the CCM. According to the model, the increase in δ(13) Corg during the bloom results chiefly from decreasing ambient CO2 concentration (which reduces the gross diffusive flux across the membrane) rather than a shift in inorganic carbon uptake from CO2 to HCO3 (-) . The CCM of diatoms in the Western Antarctic Peninsula functions with a relatively small expenditure of energy, resulting chiefly from the low half-saturation constant for Rubisco at cold temperatures.

Effect of silicate and inorganic carbon availability on the growth and competition of a diatom and two red tide dinoflagellates

:The influence of silicate and inorganic carbon availability on the growth of three common algal species, namely, the diatom Skeletonema costatum (Coscinodiscophyceae) and the dinoflagellates Prorocentrum minimum and Alexandrium tamarense (Dinophyceae), was studied. Competition experiments using these three species were performed under different conditions to determine the dominance of red tide algal species in certain marine environments. Skeletonema costatum outgrew the two dinoflagellates in the silicate-replete medium as a result of its high growth rate. However, the cell density of S. costatum decreased with reduced silicate and changed inorganic carbon availabilities. Skeletonema costatum stopped growing when the inorganic carbon concentration was doubled at a constant pH of 7.7, which may have resulted from the enhanced growth of the two dinoflagellates. Prorocentrum minimum and A. tamarense became dominant at constant pH 7.7 conditions. As a component of the CO2 concentrating mechanism (CCM), extracellular carbonic anhydrase (eCA) showed significantly lower activities in S. costatum under low-silicate and constant pH 7.7 conditions; whereas, the P. minimum eCA activity remained almost unaffected. Different eCA responses to reduced silicate and varied carbon sources suggested that the species differed in inorganic carbon acquisition. The results suggested that patterns of species dominance of bloom-forming microalgae may be related to silicate and inorganic carbon availabilities.

Inorganic C utilization and C isotope fractionation by pelagic and sea ice algal assemblages along the Antarctic continental shelf

Physiological characteristics of inorganic C uptake were examined in Southern Oceanice algae and phytoplankton assemblages. Ice algal and phytoplankton assemblages were largelydominated by diatoms and Phaeocystis antarctica, and showed a high capacity for HCO3−utilization, with direct HCO3− transport accounting for ~60% of total inorganic C uptake. Extracellularcarbonic anhydrase (eCA) was detectable in all samples, but with significantly lower activityin sea ice algae. Neither HCO3− transport nor eCA activity was related to the in situ partial pressureof CO2 (pCO2) or taxonomic composition of samples. The half-saturation constant (KS) for inorganicC ranged from ~100 to 5000 μM, and showed significantly more variability among sea ice algaethan phytoplankton assemblages. For the phytoplankton assemblages, there were significant positivecorrelations between in situ pCO2 and KS (higher C substrate affinity in low pCO2 waters), andalso between KS and maximum C uptake rates (Vmax). In contrast, KS and Vmax in sea-ice algal assemblageswere not correlated to each other, or to any other measured variables. The C isotopecomposition of particulate organic carbon (δ13C-POC) in the phytoplankton assemblages showedmodest variability (range −30 to −24.6‰) and was significantly correlated to the ratio of inferredgrowth rates (derived from Vmax) and in situ CO2 concentrations, but not to any measured C uptakeparameters. δ13C-POC in sea ice algal samples (range −25.7 to −12.9‰) was significantly heavierthan in the phytoplankton assemblages, and not correlated to any other variables. Our results provideevidence for the widespread occurrence of carbon-concentrating mechanisms in SouthernOcean sea ice algae and phytoplankton assemblages.

Quantification of Extracellular Carbonic Anhydrase Activity in Two Marine Diatoms and Investigation of Its Role

Many microalgae induce an extracellular carbonic anhydrase (eCA), associated with the cell surface, at low carbon dioxide (CO2) concentrations. This enzyme is thought to aid inorganic carbon uptake by generating CO2 at the cell surface, but alternative roles have been proposed. We developed a new approach to quantify eCA activity in which a reaction-diffusion model is fit to data on (18)O removal from inorganic carbon. In contrast to previous methods, eCA activity is treated as a surface process, allowing the effects of eCA on cell boundary-layer chemistry to be assessed. Using this approach, we measured eCA activity in two marine diatoms (Thalassiosira pseudonana and Thalassiosira weissflogii), characterized the kinetics of this enzyme, and studied its regulation as a function of culture pH and CO2 concentration. In support of a role for eCA in CO2 supply, eCA activity specifically responded to low CO2 rather than to changes in pH or HCO3(-), and the rates of eCA activity are nearly optimal for maintaining cell surface CO2 concentrations near those in the bulk solution. Although the CO2 gradients abolished by eCA are small (less than 0.5 μm concentration difference between bulk and cell surface), CO2 uptake in these diatoms is a passive process driven by small concentration gradients. Analysis of the effects of short-term and long-term eCA inhibition on photosynthesis and growth indicates that eCA provides a small energetic benefit by reducing the surface-to-bulk CO2 gradient. Alternative roles for eCA in CO2 recovery as HCO3(-) and surface pH regulation were investigated, but eCA was found to have minimal effects on these processes.

INORGANIC CARBON UTILIZATION BY ROSS SEA PHYTOPLANKTON ACROSS NATURAL AND EXPERIMENTAL CO2 GRADIENTS1

We present results from a field study of inorganic carbon (C) acquisition by Ross Sea phytoplankton during Phaeocystis‐dominated early season blooms. Isotope disequilibrium experiments revealed that HCO3− was the primary inorganic C source for photosynthesis in all phytoplankton assemblages. From these experiments, we also derived relative enhancement factors for HCO3−/CO2 interconversion as a measure of extracellular carbonic anhydrase activity (eCA). The enhancement factors ranged from 1.0 (no apparent eCA activity) to 6.4, with an overall mean of 2.9. Additional eCA measurements, made using membrane inlet mass spectrometry (MIMS), yielded activities ranging from 2.4 to 6.9 U · [μg chl a]−1 (mean 4.1). Measurements of short‐term C‐fixation parameters revealed saturation kinetics with respect to external inorganic carbon, with a mean half‐saturation constant for inorganic carbon uptake (K1/2) of ∼380 μM. Comparison of our early springtime results with published data from late‐season Ross Sea assemblages showed that neither HCO3− utilization nor eCA activity was significantly correlated to ambient CO2 levels or phytoplankton taxonomic composition. We did, however, observe a strong negative relationship between surface water pCO2 and short‐term 14C‐fixation rates for the early season survey. Direct incubation experiments showed no statistically significant effects of pCO2 (10 to 80 Pa) on relative HCO3− utilization or eCA activity. Our results provide insight into the seasonal regulation of C uptake by Ross Sea phytoplankton across a range of pCO2 and phytoplankton taxonomic composition.

Carbon acquisition by Trichodesmium: the effect of pCO2 and diurnal changes

We investigated carbon acquisition by the N2‐fixing cyanobacterium Trichodesmium IMS101 in response to CO2 levels of 15.1, 37.5, and 101.3 Pa (equivalent to 150, 370, and 1000 ppm). In these acclimations, growth rates as well as cellular C and N contents were measured. In vivo activities of carbonic anhydrase (CA), photosynthetic O2 evolution, and CO2 and HCO3‐ fluxes were measured using membrane inlet mass spectrometry and the 14C disequilibrium technique. While no differences in growth rates were observed, elevated CO2 levels caused higher C and N quotas and stimulated photosynthesis and N2 fixation. Minimal extracellular CA (eCA) activity was observed, indicating a minor role in carbon acquisition. Rates of CO2 uptake were small relative to total inorganic carbon (Ci) fixation, whereas HCO{3 contributed more than 90% and varied only slightly over the light period and between CO2 treatments. The low eCA activity and preference for HCO3‐ were verified by the 14C disequilibrium technique. Regarding apparent affinities, half‐saturation concentrations (K1/2) for photosynthetic O2 evolution and HCO3‐ uptake changed markedly over the day and with CO2 concentration. Leakage (CO2 efflux : Ci uptake) showed pronounced diurnal changes. Our findings do not support a direct CO2 effect on the carboxylation efficiency of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RubisCO) but point to a shift in resource allocation among photosynthesis, carbon acquisition, and N2 fixation under elevated CO2 levels. The observed increase in photosynthesis and N2 fixation could have potential biogeochemical implications, as it may stimulate productivity in N‐limited oligotrophic regions and thus provide a negative feedback in rising atmospheric CO2 levels.

RESPONSES TO SOLAR UV RADIATION OF THE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE) GROWN AT DIFFERENT Zn(2+) CONCENTRATIONS(1).

Zn availability in the ocean has been suggested to limit primary production by affecting CO2 acquisition processes for photosynthesis, therefore influencing the global carbon cycle. Also, UV radiation (UVR, 280-400 nm) is known to affect primary production in different ways. It remains to be ascertained whether Zn availability and UVR can act synergistically, antagonistically, or independently on oceanic primary production. We cultured the cosmopolitan diatom Skeletonema costatum (Grev.) Cleve under different radiation treatments with or without UVR (only photosynthetically active radiation), at 0, 3, and 10 pmol · L(-1) Zn(2+) . Specific growth rate, photosynthetic carbon assimilation, external carbonic anhydrase (eCA) activity, and estimated cell abundance increased with increasing concentrations of Zn(2+) from 0 to 3 and 10 pmol · L(-1) , irrespective of the radiation treatment. Higher eCA activity was observed in the cells grown at the high level of Zn(2+) in the presence of UVR. An approximately linear relationship between μ and the daily dose of PAR was observed at 3 and 10 pmol · L(-1) Zn(2+) concentrations. However, the dependency of μ on the daily PAR dose disappeared when the cells were grown in the presence of UVR, which overall depressed both μ and photosynthetic carbon assimilation. The inhibitory effect of UVR was inversely related to Zn(2+) concentrations. The ultraviolet-B (UVB)-related inhibition of growth and photosynthesis decreased with time, reflecting a faster acclimation of the cells to UVR at replete Zn(2+) levels. Overall, growth in the presence of higher Zn(2+) concentrations reduced the sensitivity to UV radiation in Skeletonema costatum.

Inorganic carbon uptake and intracellular assimilation by subarctic Pacific phytoplankton assemblages

We report the results of inorganic carbon (C) uptake experiments and activity measurements for carbonic anhydrase, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), and phosphoenolpyruvate carboxylase (PEPC) in offshore and coastal regions of the eastern subarctic Pacific Ocean. HCO3- was the dominant source of inorganic C taken up by phytoplankton at all sampling locations, accounting for ∼60−90% of total C uptake. The uptake of HCO3- occurred primarily through a direct transport system, while indirect HCO3− use, mediated by extracellular carbonic anhydrase (eCA), played a minor role in the C uptake system. Direct HCO3- transport and eCA activity were not related to ambient nutrient or CO2 concentrations or to phytoplankton biomass (chlorophyll a [Chl a]) or primary productivity. There was significant variability in the biomass-normalized activities of Rubisco, PEPC, and total (intracellular and extracellular) carbonic anhydrase. The activities of all of the enzymes measured exhibited significant correlations with both CO2 concentrations and Chl a. PEPC activity averaged 20% of Rubisco activity (range 0.5-110%), and the PEPC : Rubisco ratio was positively correlated with CO2 concentrations and negatively correlated with Chl a. Carbonic anhydrase activity was strongly anticorrelated with CO2 and positively correlated with Chl a. The results provide evidence for the importance of CO2-regulated carbon concentrating mechanisms in marine waters.

Carbon acquisition of bloom‐forming marine phytoplankton

Carbon acquisition in relation to CO2 supply was investigated in three marine bloom‐forming microalgae, the diatom Skeletonema costatum, the flagellate Phaeocystis globosa, and the coccolithophorid Emiliania huxleyi. In vivo activities of extracellular (eCA) and intracellular (iCA) carbonic anhydrase activity, photosynthetic O2 evolution, CO2 and HCO3− uptake rates were measured by membrane inlet mass spectrometry in cells acclimated to pCO2 levels of 36, 180, 360, and 1,800 ppmv. Large differences were obtained between species both with regard to the efficiency and regulation of carbon acquisition. While eCA activity increased with decreasing CO2 concentration in S. costatum and P. globosa, consistently low values were obtained for E. huxleyi. No clear trends with pCO2 were observed in iCA activity for any of the species tested. Half saturation concentrations (K1/2) for photosynthetic O2 evolution, which were highest for E. huxleyi and lowest for S. costatum, generally decreased with decreasing CO2 concentration. In contrast, K1/2 values for P. globosa remained unaffected by pCO2 of the incubation. CO2 and HCO3− were taken up simultaneously by all species. The relative contribution of HCO3− to total carbon uptake generally increased with decreasing CO2, yet strongly differed between species. Whereas K1/2 for CO2 and HCO3− uptake was lowest at the lowest pCO2 for S. costatum and E. huxleyi, it did not change as a function of pCO2 in P. globosa. The observed taxon‐specific differences in CO2 sensitivity, if representative for the natural environment, suggest that changes in CO2 availability may influence phytoplankton species succession and distribution. By modifying the relative contribution of different functional groups, e.g., diatomaceous versus calcareous phytoplankton, to the overall primary production this could potentially affect marine biogeochemical cycling and air—sea gas exchange.

CO2 and HCO3 ߚ uptake in marine diatoms acclimated to different CO2 concentrations

Rates of cellular uptake of CO2 and HCO3− during steadyߚstate photosynthesis were measured in the marine diatoms Thalassiosira weissflogii and Phaeodactylum tricornutum, acclimated to CO2 partial pressures of 36, 180, 360, and 1,800 ppmv. In addition, in vivo activity of extracellular (eCA) and intracellular (iCA) carbonic anhydrase was determined in relation to CO2 availability. Both species responded to diminishing CO2 supply with an increase in eCA and iCA activity. In P. tricornutum, eCA activity was close to the detection limit at higher CO2 concentrations. Simultaneous uptake of CO2 and HCO3− was observed in both diatoms. At airߚequilibrated CO2 levels (360 ppmv), T. weissflogii took up CO2 and HCO3− at approximately the same rate, whereas CO2 uptake exceeded HCO3− uptake by a factor of two in P. tricornutum. In both diatoms, CO2 :HCO3− uptake ratios progressively decreased with decreasing CO2 concentration, whereas substrate affinities of CO2 and HCO3− uptake increased. Halfߚsaturation concentrations were always &lt;=5 mM CO2 for CO2 uptake and &lt;700 mM HCO3− for HCO3− uptake. Our results indicate the presence of highly efficient uptake systems for CO2 and HCO3− in both diatoms at concentrations typically encountered in ocean surface waters and the ability to adjust uptake rates to a wide range of inorganic carbon supply.