CARBON‐USE STRATEGIES IN MACROALGAE: DIFFERENTIAL RESPONSES TO LOWERED PH AND IMPLICATIONS FOR OCEAN ACIDIFICATION1

Ocean acidification (OA) is a reduction in oceanic pH due to increased absorption of anthropogenically produced CO₂. This change alters the seawater concentrations of inorganic carbon species that are utilized by macroalgae for photosynthesis and calcification: CO₂ and HCO₃^? increase; CO₃^2? decreases. Two common methods of experimentally reducing seawater pH differentially alter other aspects of carbonate chemistry: the addition of CO₂ gas mimics changes predicted due to OA, while the addition of HCl results in a comparatively lower [HCO₃^?]. We measured the short‐term photosynthetic responses of five macroalgal species with various carbon‐use strategies in one of three seawater pH treatments: pH 7.5 lowered by bubbling CO₂ gas, pH 7.5 lowered by HCl, and ambient pH 7.9. There was no difference in photosynthetic rates between the CO₂, HCl, or pH 7.9 treatments for any of the species examined. However, the ability of macroalgae to raise the pH of the surrounding seawater through carbon uptake was greatest in the pH 7.5 treatments. Modeling of pH change due to carbon assimilation indicated that macroalgal species that could utilize HCO₃^? increased their use of CO₂ in the pH 7.5 treatments compared to pH 7.9 treatments. Species only capable of using CO₂ did so exclusively in all treatments. Although CO₂ is not likely to be limiting for photosynthesis for the macroalgal species examined, the diffusive uptake of CO₂ is less energetically expensive than active HCO₃^? uptake, and so HCO₃^?‐using macroalgae may benefit in future seawater with elevated CO₂.     

Photosynthetic carbon acquisition in <Emphasis Type="Italic">Sargassum henslowianum</Emphasis> (Fucales,Phaeophyta), with special reference to the comparison between the vegetative and reproductive tissues

The photosynthetic oxygen evolution characteristics were examined in both vegetative (blade) and sexual reproductive (receptacle) tissues of Sargassum henslowianum (Fucales, Phaeophyta) from the Shenao bay of Nanao Island, China, to establish the mechanism of photosynthetic acquisition of inorganic carbon (Ci) in this species. In natural seawater (pH 8.1, ca. 2.2 mM Ci), irradiance-saturated net photosynthetic rate (NPR) was greater by 25.3% in blade than receptacle, whereas dark respiratory rate (DR) was 2-fold higher in receptacle than blade. NPR at pH 8.1 was nearly saturated with the 2.2 mM Ci for both blade and receptacle. However, the values of the half-saturation constant for Ci were sharply increased at pH 9.0. NPR was significantly affected, but DR was remained unchanged, with the variation of the pH values in seawater. The data from the final pH value derived from the pH-drift experiments and the comparison between the measured and theoretically estimated photosynthetic rates suggested that both blade and receptacle were capable of acquiring HCO₃ ⁻ in seawater. The inhibitors experiments showed that a HCO₃ ⁻ dehydration mechanism mediated by external carbonic anhydrase activity occurred in both the blade and receptacle tissues of S. henslowianum. The proton buffer TRIS had no inhibitory effect on NPR at normal pH value in natural seawater (pH 8.1), but it significantly depressed NPR at pH 9.0. This suggested that proton transport occurred at the outside of the plasma membrane facilitated the operation of the carbon acquisition at pH 9.0. It was proposed that the strategy of photosynthetic carbon acquisition at higher pH would prevent the alga from the damage of over-excitation and photoinhibition in case of sunshine and calm water. We concluded that the blade and receptacle tissues of S. henslowianum have similar mechanism of acquisition of exogenous Ci from seawater to drive photosynthesis; yet they are differentiated more or less with the photosynthetic properties.     

PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON IN THE ECONOMIC BROWN ALGA,HIZIKIA FUSIFORME (SARGASSACEAE) FROM THE SOUTH CHINA SEA1

The mechanism of inorganic carbon (C_i) acquisition by the economic brown macroalga, Hizikia fusiforme (Harv.) Okamura (Sargassaceae), was investigated to characterize its photosynthetic physiology. Both intracellular and extracellular carbonic anhydrase (CA) were detected, with the external CA activity accounting for about 5% of the total. Hizikia fusiforme showed higher rates of photosynthetic oxygen evolution at alkaline pH than those theoretically derived from the rates of uncatalyzed CO₂ production from bicarbonate and exhibited a high pH compensation point (pH 9.66). The external CA inhibitor, acetazolamide, significantly depressed the photosynthetic oxygen evolution, whereas the anion‐exchanger inhibitor 4,4′‐diisothiocyano‐stilbene‐2,2′‐disulfonate had no inhibitory effect on it, implying the alga was capable of using HCO₃^? as a source of C_i for its photosynthesis via the mediation of the external CA. CO₂ concentrations in the culture media affected its photosynthetic properties. A high level of CO₂ (10,000 ppmv) resulted in a decrease in the external CA activity; however, a low CO₂ level (20 ppmv) led to no changes in the external CA activity but raised the intracellular CA activity. Parallel to the reduction in the external CA activity at the high CO₂ was a reduction in the photosynthetic CO₂ affinity. Decreased activity of the external CA in the high CO₂ grown samples led to reduced sensitiveness of photosynthesis to the addition of acetazolamide at alkaline pH. It was clearly indicated that H. fusiforme, which showed CO₂‐limited photosynthesis with the half‐saturating concentration of C_i exceeding that of seawater, did not operate active HCO₃^? uptake but used it via the extracellular CA for its photosynthetic carbon fixation.     

CO2 uptake and transport in leaf mesophyll cells

Abstract The acquisition of inorganic carbon for photosynthetic assimilation by leaf mesophyll cells and chloroplasts is discussed with particular reference to membrane permeation of CO₂ and HCO^?₃. Experimental evidence indicates that at the apoplast pH normally experienced by leaf mesophyll cells (pH 6–7) CO₂ is the principal species of inorganic carbon taken up. Uptake of HCO^?₃ may also occur under certain circumstances (i.e. pH 8.5), but its contribution to the net flux of inorganic carbon is small and HCO^?₃ uptake does not function as a CO₂-concentrating mechanism. Similarly, CO₂ rather than HCO^?₃ appears to be the species of inorganic carbon which permeates the chloroplast envelope. In contrast to many C₃ aquatic plants and C₄ plants, C₃ terrestrial plants lack specialized mechanisms for the acquisition and transport of inorganic carbon from the intercellular environment to the site of photosynthetic carboxylation, but rely upon the diffusive uptake of CO₂.     

Growth, photosynthetic properties and Rubisco activities and amounts of marine macroalgae grown under current and elevated seawater CO2 concentrations

Growth rates, photosynthetic responses and the activity, amount and CO₂ affinity of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) were determined for common marine macroalgae grown in seawater (containing 14.5 ± 2.1 µM CO₂) or CO₂‐enriched seawater (averaging 52.8 ± 19.2 µM CO₂). The algae were grown in 40 L fiberglass tanks (outdoor) for 4–15 weeks and in a field experimental setup for 5 days. Growth rates of the species studied (representing the three major divisions, i.e. Chlorophyta, Rhodophyta and Phaeophyta) were generally not significantly affected by the increased CO₂ concentrations in the seawater medium. Rubisco characteristics of algae cultivated in CO₂‐enriched seawater were similar to those of algae grown in nonenriched seawater. The lack of response of photosynthetic traits in these aquatic plants is likely to be because of the presence of CO₂ concentrating mechanisms (CCMs) which rely on HCO₃^– utilization, the inorganic carbon (Ci) form that dominates the total Ci pool available in seawater. Significant changes on the productivity of these particular marine algae species would not be anticipated when facing future increasing atmospheric CO₂ levels.     

PHOTOSYNTHETIC PROPERTIES OF PROTOPLASTS,AS COMPARED WITH THALLI,OF ULVA FASCIATA (CHLOROPHYTA)1

Protoplasts were prepared from Ulva fasciata Delile, and their photosynthetic performance was measured and compared with that of thalli discs. These protoplasts maintained maximal rates of photosynthesis as high as those of thalli (up to 300 μmol O₂·mg chlorophyll^?1·h^?1) for several hours after preparation and were therefore considered suitable for kinetic studies of inorganic carbon utilization. The photosynthetic K_1/2(inorganic carbon) at pH 6.1 was 3.8 μM and increased to 67, 158, and 1410 μM at the pH values 7.0, 7.9, and 8.9, respectively. Compared with these protoplasts, thalli had a much lower affinity for CO₂ but approximately the same affinity for HCO₃^?. Comparisons between rates of photosynthesis and the spontaneous dehydration of HCO₃^? (at 50 μM inorganic carbon) revealed that photosynthesis of both protoplasts (which lacked apparent activity of extracellular/surface-bound carbonic anhydrase) and thalli (which were only 25% inhibited by the external carbonic anhydrase inhibitor acetazolamide) could not be supported by CO₂ formation in the medium at the higher pH values, indicating HCO₃^? uptake. Since both protoplasts and thalli were sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulfonate, we suggest that HCO₃^? transport was facilitated by the membrane-located anion exchange protein recently reported to function in certain Ulva thalli. These findings suggest that the presence of a cell wall may constitute a diffusion barrier for CO₂, but not for HCO₃^?, utilization under natural seawater conditions.     

UPTAKE OF INORGANIC CARBON BY CLADOPHORA GLOMERATA (CHLOROPHYTA) FROM THE BALTIC SEA1

Carbon uptake in the green macroalga Cladophora glomerata (L.) Kütz. from the brackish Baltic Sea was studied by recording changes in pH, alkalinity, and inorganic carbon concentration of the seawater medium during photosynthesis. The use of specific inhibitors identified three uptake mechanisms: 1) dehydration of HCO₃ ^? into CO₂ by periplasmic carbonic anhydrase, followed by diffusion of CO₂ into the cell; 2) direct uptake of HCO₃ ^? via a 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonate‐sensitive mechanism; and 3) uptake of inorganic carbon by the involvement of a vanadate‐sensitive P‐type H ⁺ ‐ATPase (proton pump). A decrease in the alkalinity of the seawater medium during carbon uptake, except when treated with vanadate, indicated a net uptake of the ionic species contributing to alkalinity (i.e. HCO₃ ^? , CO₃^{2 ?} , and OH ^? ) from the medium, where OH ^? influx is equivalent to H ⁺ efflux. This would suggest that the proton pump is involved in HCO₃ ^? transport. We also show that the proton pump can be induced by carbon limitation. The inducibility of carbon uptake in C. glomerata may partly explain why this species is so successful in the upper littoral zone of the Baltic Sea. Usually, carbon limitation is not a problem in the upper littoral of the sea. However, it may occur frequently within dense Cladophora belts with high photosynthetic rates that create high pH and low carbon concentrations in the alga's microenvironment.     

Photosynthesis of Ulva sp. II. utilization of CO2 and HCO−3 when submerged

The photosynthetic capacity of submerged Ulva sp. when utilizing CO₂ and HCO^?₃ as exogenous carbon forms has been investigated and compared with ambient carbon concentrations in sea water. Saturating concentrations of HCO^{? ₃} and CO₂ were 1200 and 100 μM, respectively at saturating light, and photosynthetic rates under such conditions averaged 700 μmolO₂·gDW^?1 ·h^?1. The HCO^?₃ concentration of sea water (≈2500μM), was thus found to be saturating for photosynthesis of Ulva. At the CO₂ concentration of sea water (≈ 10 μM), the contribution of this carbon form to photosynthesis could be 27% at the most. Under conditions of slow water movement, the relative importance of CO₂ utilization would probably be minimized in favour of HCO^?₃ utilization. It is concluded that HCO^?₃ uptake is not limiting photosynthesis for Ulva under natural conditions.     

Carbon limitation enhances CO2 concentrating mechanism but reduces trichome size in Arthrospira platensis (cyanobacterium)

Arthrospira species grow well under highly enriched inorganic carbon concentrations, but little is known on the effects of inorganic carbon (Ci) limitation on its physiological performance. When Arthrospira platensis D-0083 was grown in a modified medium without NaHCO₃ under ambient air of 380 ppm CO₂, its trichomes became disassembled while the growth and photosynthetic rates were severely reduced. Phycocyanin and allophycocyanin contents decreased but the carotenoid content increased under the Ci limitation. Compared with the cells grown in Zarrouk medium, the trichomes grown under the Ci limitation increased their photosynthetic apparent affinity for Ci by about 14 times but photochemical quenching capacity was reduced. It appeared that A. platensis increased its CO₂ concentrating mechanism by inducing HCO₃ ^? transporters and reducing the trichome size which increased filamentous surface to volume ratio.     

Enhanced growth of the red algaPorphyra yezoensis Ueda in high CO2 concentrations

Leafy thalli of the red algaPorphyra yezoensis Ueda, initiated from conchospores released from free-living conchocelis, were cultured using aeration with high CO₂. It was found that the higher the CO₂ concentration, the faster the growth of the thalli. Aeration with elevated CO₂ lowered pH in dark, but raised pH remarkably in light with the thalli, because the photosynthetic conversion of HCO ₃ ^? to OH^? and CO₂ proceeded much faster than the dissociation of hydrated CO₂ releasing H⁺. Photosynthesis of the alga was found to be enhanced in the seawater of elevated dissolved inorganic carbon (DIC, CO₂ + HCO ₃ ^? + CO ₃ ^? ). It is concluded that the increased pH in the light resulted in the increase of DIC in the culture media, thus enhancing photosynthesis and growth. The relevance of the results to removal of atmospheric CO₂ by marine algae is discussed.     

Inorganic Carbon Source for Photosynthesis in the Seagrass Thalassia hemprichii (Ehrenb.) Aschers

Photosynthetic carbon uptake of the tropical seagrass Thalassia hemprichii (Ehrenb.) Aschers was studied by several methods. Photosynthesis in buffered seawater in media in the range of pH 6 to pH 9 showed an exponentially increasing rate with decreasing pH, thus indicating that free CO₂ was a photosynthetic substrate. However, these experiments were unable to determine whether photosynthesis at alkaline pH also contained some component due to HCO₃⁻ uptake. This aspect was further investigated by studying photosynthetic rates in a number of media of varying pH (7.8-8.61) and total inorganic carbon (0.75-13.17 millimolar). In these media, photosynthetic rate was correlated with free CO₂ concentration and was independent of the HCO₃⁻ concentration in the medium. Short time-course experiments were conducted during equilibration of free CO₂ and HCO₃⁻ after injection of ¹⁴C labeled solution at acid or alkaline pH. High initial photosynthetic rates were observed when acidic solutions (largely free CO₂) were used but not with alkaline solutions. The concentration of free CO₂ was found to be a limiting factor for photosynthesis in this plant.     

ACQUISITION OF INORGANIC CARBON BY THE MARINE HAPTOPHYTE ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE)1

Inorganic carbon acquisition has been investigated in the marine haptophyte Isochrysis galbana. External carbonic anhydrase (CA) was present in air‐grown (0.034% CO₂) cells but completely repressed in high (3%) CO₂‐grown cells. External CA was not inhibited by 1.0 mM acetazolamide. The capacity of cells to take up bicarbonate was examined by comparing the rate of photosynthetic O₂ evolution with the calculated rate of spontaneous CO₂ supply; at pH 8.2 the rates of O₂ evolution exceeded the CO₂ supply rate 14‐fold, indicating that this alga was able to take up HCO₃ ^? . Monitoring CO₂ concentrations by mass spectrometry showed that suspensions of high CO₂‐grown cells caused a rapid drop in the extracellular CO₂ in the light and addition of bovine CA raised the CO₂ concentration by restoring the HCO₃ ^? ‐CO₂ equilibrium, indicating that cells were maintaining the CO₂ in the medium below its equilibrium value during photosynthesis. A rapid increase in extracellular CO₂ concentration occurred on darkening the cells, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. Active CO₂ uptake was blocked by the photosynthetic electron transport inhibitor 3‐(3′,4′‐dichlorphenyl)‐1,1‐dimethylurea, indicating that CO₂ transport was supported by photosynthetic reactions. These results demonstrate that this species has the capacity to take up HCO₃ ^? and CO₂ actively as sources of substrate for photosynthesis and that inorganic carbon transport is not repressed by growth on high CO₂, although external CA expression is regulated by CO₂ concentration.     

Utilization of inorganic carbon in the edible cyanobacterium Ge-Xian-Mi (Nostoc) and its role in alleviating photo-inhibition

The present work investigated the inorganic carbon (C_i) uptake, fluorescence quenching and photo‐inhibition of the edible cyanobacterium Ge‐Xian‐Mi (Nostoc) to obtain an insight into the role of CO₂ concentrating mechanism (CCM) operation in alleviating photo‐inhibition. Ge‐Xian‐Mi used HCO₃^– in addition to CO₂ for its photosynthesis and oxygen evolution was greater than the theoretical rates of CO₂ production derived from uncatalysed dehydration of HCO₃^–. Multiple transporters for CO₂ and HCO₃^– operated in air‐grown Ge‐Xian‐Mi. Na⁺‐dependent HCO₃^– transport was the primary mode of active C_i uptake and contributed 53–62% of net photosynthetic activity at 250 µmol L^?1 KHCO₃ and pH 8.0. However, the CO₂‐uptake systems and Na⁺‐independent HCO₃^– transport played minor roles in Ge‐Xian‐Mi and supported, respectively, 39 and 8% of net photosynthetic activity. The steady‐state fluorescence decreased and the photochemical quenching increased in response to the transport‐mediated accumulation of intracellular C_i. Inorganic carbon transport was a major factor in facilitating quenching during the initial stage and the initial rate of fluorescence quenching in the presence of iodoacetamide, an inhibitor of CO₂ fixation, was 88% of control. Both the initial rate and extent of fluorescence quenching increased with increasing external dissolved inorganic carbon (DIC) and saturated at higher than 200 µmol L^?1 HCO₃^–. The operation of the CCM in Ge‐Xian‐Mi served as a means of diminishing photodynamic damage by dissipating excess light energy and higher external DIC in the range of 100–10000 µmol L^?1 KHCO₃ was associated with more severe photo‐inhibition under strong irradiance.     

SOURCES OF INORGANIC CARBON FOR PHOTOSYNTHESIS BY THREE SPECIES OF MARINE DIATOM1

The utilization of inorganic carbon by three species of marine diatom, Skeletonema costatum (Grev.) Cleve. Ditylum brightwellii (West) Grun., and Chaetoceros calcitrans Paulsen was investigated using an inorganic carbon isotopic disequilibnum technique and inorganic carbon dose-response curves. Stable carbon isotope data of the diatoms are also presented. Observed rates of photosynthetic oxygen evolution were greater than could be accounted for by the theoretical rate of CO₂ supply from the uncatalyzed dehydration of HCO₃^? in the external medium, suggesting use of HCO₃^? as an inorganic carbon source. Data from the isotopic disequilibrium experiment demonstrate the use of both HCO₃^? and CO₂ for photosynthesis. Carbon isotope discrimination values support the use of HCO₃^? by the diatoms.     

Characterisation of CO2 and HCO3 − uptake in the cyanobacterium Synechocystis sp. PCC6803

The availability of a complete genome database for the cyanobacterium Synechocystissp. PCC6803 (glucose-tolerant strain) has raised expectations that this organism would become a reference strain for work aimed at understanding the CO₂-concentrating mechanism (CCM) in cyanobacteria. However, the amount of physiological data available has been relatively limited. In this report we provide data on the relative contributions of net HCO₃ ⁻ uptake and CO₂ uptake under steady state photosynthetic conditions. Cells were compared after growth at high CO₂ (2% v/v in air) or limiting CO₂ conditions (20 ppm CO₂). Synechocystishas a very high dependence on net HCO₃ ⁻ uptake at low to medium concentrations of inorganic carbon (Ci). At high Ci concentrations net CO₂ uptake became more important but did not contribute more than 40% to the rate of photosynthetic O₂ evolution. The data also confirm that high Ci cells of Synechocystissp. PCC6803 possess a strong capacity for net HCO₃ ⁻ uptake under steady state photosynthetic conditions. Time course experiments show that induction of maximal Ci uptake capacity on a shift from high CO₂ to low CO₂ conditions was near completion by four hours. By contrast, relaxation of the induced state on return of cells to high CO₂, takes in excess of 230 h. Experiments were conducted to determine if Synechocystissp. PCC6803 is able to exhibit a `fast induction' response under severe Ci limitation and whether glucose was capable of causing a rapid inactivation in Ci uptake capacity. Clear evidence for either response was not found. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effects of severe carbon limitation on the green seaweed,Ulva conglobata (Chlorophyta)

Low inorganic carbon (Ci) concentrations in seawater are usually an important factor controlling photosynthesis and growth of seaweeds. The green seaweed, Ulva conglobata Kjellm, collected from a rock pool in a middle intertidal zone located at Nanao Island, Shantou, China, were cultured under low Ci level for several days, to examine the effect of severe carbon limitation on photosynthesis. The rather high pH compensation points obtained from the pH-drift experiments indicated that U. conglobata was capable of acquiring HCO₃ ^? from surrounding seawater as its Ci source for photosynthesis. However, thalli of U. conglobata cultured in Ci-starved seawater exhibited a decline of biomass, showing that the realistic photosynthetic carbon gain could not compensate for the respiratory carbon consumption in the thalli under severe Ci limitation during laboratory culture. Compared with ambient Ci conditions, the culture under severe Ci limitation significantly had an increased pigment content, but a lower maximum quantum yield and photosynthetic electron transport rate. Additionally, the maximum carbon-saturating photosynthesis rate and the apparent photosynthetic conductance of U. conglobata thalli increased in cultures with severe Ci limitation compared with ambient Ci in low N-grown thalli. The results suggest that under severe Ci limitation, U. conglobata thalli increased capacities of both light absorption processes and carbon fixation pathways.     

A chloroplast pump model for the CO2 concentrating mechanism in the diatom Phaeodactylum tricornutum

Prior analysis of inorganic carbon (C_i) fluxes in the diatom Phaeodactylum tricornutum has indicated that transport of C_i into the chloroplast from the cytoplasm is the major C_i flux in the cell and the primary driving force for the CO₂ concentrating mechanism (CCM). This flux drives the accumulation of C_i in the chloroplast stroma and generates a CO₂ deficit in the cytoplasm, inducing CO₂ influx into the cell. Here, the “chloroplast pump” model of the CCM in P. tricornutum is formalized and its consistency with data on CO₂ and HCO₃ ^? uptake rates, carbonic anhydrase (CA) activity, intracellular C_i concentration, intracellular pH, and RubisCO characteristics is assessed. The chloroplast pump model can account for the major features of the data. Analysis of photosynthetic and C_i uptake rates as a function of external C_i concentration shows that the model has the most difficulty obtaining sufficiently low cytoplasmic CO₂ concentrations to support observed CO₂ uptake rates at low external C_i concentrations and achieving high rates of photosynthesis. There are multiple ways in which model parameters can be varied, within a plausible range, to match measured rates of photosynthesis and CO₂ uptake. To increase CO₂ uptake rates, CA activity can be increased, kinetic characteristics of the putative chloroplast pump can be enhanced to increase HCO₃ ^? export, or the cytoplasmic pH can be raised. To increase the photosynthetic rate, the permeability of the pyrenoid to CO₂ can be reduced or RubisCO content can be increased.     

Photosynthesis and inorganic carbon acquisition in the cyanobacterium Chlorogloeopsis sp. ATCC 27193

The ability of the morphologically complex cyanobacterium Chlorogloeopsis sp. ATCC 27193 to actively transport and accumulate inorganic carbon (C₁= CO₂+ HCO₃^?+ CO₃^2?) for photosynthetic CO₂ fixation was investigated. Mass-spectrometric assays revealed that Chlorogloeopsis cells grown under C₁ limitation rapidly took up CO₂ from the medium in a light-dependent reaction which was independent of CO₂ fixation. Ethoxyzolamide, a carbonic anhydrase (CA) inhibitor, inhibited CO₂ transport. Since electrometric and mass-spectrometric assays did not detect the presence of a periplasmic CA, it is suggested that CO₂ transport was mediated by a CA-like activity which converted CO₂ to HCO₃^? during passage across the membrane. Radiochemical assays, using H¹⁴CO₃ as substrate, showed that C₃-limited cells also had a high affinity (K_0.5 HCO₃^?= 37 μM), Na⁺-independent HCO₃^? uptake mechanism. HCO₃^?uptake was light dependent and occurred against its electrochemical potential indicating a carrier-mediated, active transport process. The rate of Na⁺-independent HCO₃^? transport was sufficient to account for the steady state rate of CO₂ fixation. Although not absolutely required. Na⁺ did specifically enhance the rate of HCO₃^? transport by up to 2-fold, but had no effect on the apparent affinity of the transport system for HCO₃^? Combined CO₂ and HCO₃^? transport resulted in C₁ accumulation as high as 25 mM and in excess of 300 times the external concentration. The C₁ pool was the source of CO₂ for photo-synthetic fixation and was generated, presumably, by the dehydration of HCO₃^? catalyzed by an intracellular CA. The collective evidence indicates that Chlorogloeopsis has a physiologically functional CO₂-concentrating mechanism which is essential for photosynthesis.     

Inorganic carbon transport across cell compartments of the halotolerant alga Dunaliella salina

The inorganic carbon (C_i) accumulation and the intracellular location of carbonic anhydrase (CA, EC 4.2.1.1) in the halotolerant unicellular alga Dunaliella salina have been investigated. The rate of HCO₃ -dependent O₂ evolution was determined by growth conditions. Algae grown under high CO₂ conditions (5% CO₂ in air, v/v; high C_i cells) had a very low affinity for HCO₃^? at pH 7.0 and 8.2, whereas algae grown under low CO₂ conditions (0.03% CO₂ in air; low C_i cells) showed a high affinity for HCO₃^? at both pH values and were sensitive to Dextran-bound sulfonamide (DBS), an inhibitor of extracellular CA. The photosynthetic rate or HCO₄^? dependent O₂ evolution was always higher at pH 7.0 than at pH 8.2. Ethoxyzolamide (EZ), an inhibitor of total (extacellular plus intracellular) CA activity, strongly inhibited photosynthesis at both pH values. During adaptation from high to low CO₂ conditions CA activity increased in chloroplasts in a process dependent on the novo protein synthesis. Carbonic anhydrase activity was found in the supernatant and pellet fractions of chloroplast homogenates. The rate of photosynthesis of chloroplasts from low C_i cells was higher at pH 7.0 than at pH 8.2. The alkalinization of the growth medium, which took place only in the presence of C_i, was partially inhibited by DBS and completely by EZ. We suggest that in D. salina CO₂ is the general form of C_i transported across the plasma membrane and the chloroplast envelope and that bicarbonate enters the cell mainly, although not entirely, by an ‘indirect’ mechanism after dehydration to CO₂.     

EFFECTS OF CO2 ENRICHMENT ON THE BLOOM‐FORMING CYANOBACTERIUM MICROCYSTIS AERUGINOSA (CYANOPHYCEAE): PHYSIOLOGICAL RESPONSES AND RELATIONSHIPS WITH THE AVAILABILITY OF DISSOLVED INORGANIC CARBON1

Microcystis aeruginosa Kütz. 7820 was cultured at 350 and 700 μL·L ^{? 1} CO₂ to assess the impacts of doubled atmospheric CO₂ concentration on this bloom‐forming cyanobacterium. Doubling of CO₂ concentration in the airflow enhanced its growth by 52%–77%, with pH values decreased and dissolved inorganic carbon (DIC) increased in the medium. Photosynthetic efficiencies and dark respiratory rates expressed per unit chl a tended to increase with the doubling of CO₂. However, saturating irradiances for photosynthesis and light‐saturated photosynthetic rates normalized to cell number tended to decrease with the increase of DIC in the medium. Doubling of CO₂ concentration in the airflow had less effect on DIC‐saturated photosynthetic rates and apparent photosynthetic affinities for DIC. In the exponential phase, CO₂ and HCO₃ ^? levels in the medium were higher than those required to saturate photosynthesis. Cultures with surface aeration were DIC limited in the stationary phase. The rate of CO₂ dissolution into the liquid increased proportionally when CO₂ in air was raised from 350 to 700 μL·L ^{? 1}, thus increasing the availability of DIC in the medium and enhancing the rate of photosynthesis. Doubled CO₂ could enhance CO₂ dissolution, lower pH values, and influence the ionization fractions of various DIC species even when the photosynthesis was not DIC limited. Consequently, HCO₃ ^? concentrations in cultures were significantly higher than in controls, and the photosynthetic energy cost for the operation of CO₂ concentrating mechanism might decrease.