UPTAKE,EFFLUX, AND PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON BY THE MARINE EUSTIGMATOPHYTE NANNOCHLOROPSIS SP.1

Uptake, efflux and utilization of inorganic carbon were investigated in the marine eustigmatophyte Nannochloropsis sp. grown under an air level of CO₂. Maximal photosynthetic rate was hardly affected by raising the pH porn 5.0 to 9.0. The apparent photosynthetic affinity for dissolved inorganic carbon (DIC) was 35 μM DIC between pH 6.5 to 9.0, but increased approximately threefold at pH 5.0 suggesting that HCO₃- was the main DIC species used from the medium. No external carbonic anhydrase (CA) activity could be detected by the pH drift method. However, application of ethoxyzolamide (an inhibitor of CA) resulted an a significant inhibition of photosynthetic O₂ evolution and carbon utilization, suggesting involvement of internal CA or CA-like activity in DIC utilization. Under high light conditions, the rate of HCO₃^? uptake and its internal conversion to CO₂ apparently exceeded the rate of carbon fixation, resulting in a large leak of CO₂ from the cells to the external medium. When the cells were exposed to low DIC concentrations, the ratio of internal to external DIC concentration was about eight. On the other hand, in the presence of 2 mM DIC, conditions prevailing in the marine environment, the internal concentration of DIC was only 50% higher than the external one.     

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.     

PHOTOSYNTHETIC INORGANIC CARBON ACQUISITION IN AN ACID‐TOLERANT,FREE‐LIVING SPECIES OF COCCOMYXA (CHLOROPHYTA)1

The processes of CO₂ acquisition were characterized for the acid‐tolerant, free‐living chlorophyte alga, CPCC 508. rDNA data indicate an affiliation to the genus Coccomyxa, but distinct from other known members of the genus. The alga grows over a wide range of pH from 3.0 to 9.0. External carbonic anhydrase (CA) was detected in cells grown above pH 5, with the activity increasing marginally from pH 7 to 9, but most of the CA activity was internal. The capacity for HCO₃^? uptake of cells treated with the CA inhibitor acetazolamide (AZA), was investigated by comparing the calculated rate of uncatalyzed CO₂ formation with the rate of photosynthesis. Active bicarbonate transport occurred in cells grown in media above pH 7.0. Monitoring CO₂ uptake and O₂ evolution by membrane‐inlet mass spectrometry demonstrated that air‐grown cells reduced the CO₂ concentration in the medium to an equilibrium concentration of 15 μM, but AZA‐treated cells caused a drop in extracellular CO₂ concentration to a compensation concentration of 27 μM at pH 8.0. CO₂‐pulsing experiments with cells in the light indicated that the cells do not actively take up CO₂. An internal pool of unfixed inorganic carbon was not detected at the CO₂ compensation concentration, probably because of the lack of active CO₂ uptake, but was detectable at times before compensation point was reached. These results indicate that this free‐living Coccomyxa possesses a CO₂‐concentrating mechanism (CCM) due to an active bicarbonate‐uptake system, unlike the Coccomyxa sp. occurring in symbiotic association with lichens.     

EFFECT OF CARBONIC ANHYDRASE INHIBITORS ON THE INORGANIC CARBON UPTAKE BY PHYTOPLANKTON NATURAL ASSEMBLAGES1

The role of carbonic anhydrase (CA) in inorganic carbon acquisition (dissolved inorganic carbon, DIC) was examined in Alboran Sea phytoplankton assemblages. The study area was characterized by a relatively high variability in nutrient concentration and in abundance and taxonomic composition of phytoplankton. Therefore, the relationship between environmental variability and capacity for using HCO₃^? via external CA (eCA) was examined. Acetazolamide (AZ, an inhibitor of eCA) inhibited the primary productivity (PP) in 50% of the samples, with inhibition percentages ranging from 13% to 60%. The AZ effect was more prominent in the samples that exhibited PP >1 mg C · m^?3 · h^?1, indicating that the contribution of eCA to the DIC photosynthetic flux was irrelevant at low PP. The inhibition of primary productivity by AZ was significantly correlated to the abundance of diatoms. However, there was no a relationship between AZ effect and CO₂ partial pressure (pCO₂) or nutrient concentration, indicating that the variability in the PP percentage supported by eCA was mainly due to differences in taxonomic composition of the phytoplankton assemblages. Ethoxyzolamide (EZ, an inhibitor of both external and internal CA) affected 13 of 14 analyzed samples, with PP inhibition percentages varying from 50% to 95%. The effects of AZ and EZ were partially reversed by doubling DIC concentration. These results imply that CA activity (external and/or internal) was involved in inorganic carbon acquisition in most the samples. However, EZ effect was not correlated with pCO₂ or taxonomic composition of the phytoplankton.     

Uptake and utilization of inorganic carbon by cyanobacteria

In the cyanobacteria, mechanisms exist that allow photosynthetic CO₂ reduction to proceed efficiently even at very low levels of inorganic carbon. These inducible, active transport mechanisms enable the cyanobacteria to accumulate large internal concentrations of inorganic carbon that may be up to 1000-fold higher than the external concentration. As a result, the external concentration of inorganic carbon required to saturate cyanobacterial photosynthesis in vivo is orders of magnitude lower than that required to saturate the principal enzyme (ribulose bisphosphate carboxylase) involved in the fixation reactions. Since CO₂ is the substrate for carbon fixation, the cyanobacteria somehow perform the neat trick of concentrating this small, membrane permeable molecule at the site of CO₂ fixation. In this review, we will describe the biochemical and physiological experiments that have outlined the phenomenon of inorganic carbon accumulation, relate more recent genetic and molecular biological observations that attempt to define the constituents involved in this process, and discuss a speculative theory that suggests a unified view of inorganic carbon utilization by the cyanobacteria.Abbreviations C_i Inorganic carbon - H-cells Cells grown under high CO₂ - L-cells Cells grown under low CO₂ - RuBP Ribulose-1,5-bisphosphate - WT Wild type     

Effect of external pH on inorganic carbon assimilation in unicellular marine green algae

Carbonic anhydrase (CA) induction has been studied in three marine green algae under acidic (pH 4.5) or alkaline (pH 8.0) conditions. An inhibition of the induction of the external CA in acidic conditions, similar to that observed in some freshwater green algae, could be observed in only Chlorella saccharophila. In the two other species, Chlorococcum littorale and Stichococcus bacillaris, no significant difference in CA induction was found under two pH conditions. The exact function of the external CA of C. saccharophila remains unclear, since cells grown under acidic conditions (under which this enzyme is repressed) possess the same abilities to use inorganic carbon (Ci) as alkaline‐grown cells. Internal pH values were not modified by the pH of the medium used to cultivate C. saccharophila. Regardless of the growth conditions, activities related to carbon fixation, that is, photosynthetic oxygen evolution, Ci uptake and assimilation were enhanced when the measurements were performed at acidic pH. This indicates that this marine alga is able to use CO₂ more efficiently than HCO₃^?. No evidence could be found for a specific Ci uptake and assimilation system in the acid‐grown cells.     

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii

The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO₂ assimilation was determined by CO₂ exchange studies at three, limiting CO₂ concentrations with a ca-1 mutant of Chlamydomonas reinhardiii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO₂ assimilation did not respond to the varying photon flux densities because of CO₂ limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100 mol photons m^-2 s^-1, indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO₂ assimilation responded to external CO₂ concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO₂ into the cells supplies most of the CO₂ for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO₂ concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO₂ exchange method to determine whether inorganic carbon accumulation and photosynthetic CO₂ assimilation compete for energy at low photon flux densities proved inconclusive.     

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.     

The active uptake of carbon dioxide by the marine diatoms Phaeodactylum ticornutum and Cyclotella sp.

Mass spectromelry has been used to investigate the uptake of CO₂ by two marine diatoms, Phaeodactylum tricornutum and Cyclotella sp. The time course of CO₂ formation in the dark after addition of 100 mmol m^?3 dissolved inorganic carbon (DIC) to cell suspensions showed that external carbonic anhydrase (CA) was not present in cells of P. tricornutum but was present in Cyclotella sp. In the absence of external CA, or when it was inhibited by 5 mmol m^?3 acetazolamide, cells of both species preincubated with 100 mmol m^?3 DIG rapidly depleted almost all of the free CO₂ (3·2mmol m^?31 at pH7·5) from the suspending medium within seconds of illumination and prior to the onset of steady-state photosynthesis. Addition of bovine CA quickly restored the HCO₃^?–CO₂ equilibrium in the medium, indicating that the initial depletion of CO₂ resulted from the selective uptake of CO₂ rather than uptake of all DIG species. Transfer of cells to the dark caused a rapid increase in the CO₂ concentration in the medium, largely as a result of the efflux of unfixed inorganic carbon from the cells. The measured CO₂ uptake rates for both species accounted for 50% of the total DIG uptake at HCO₃^?–CO₂ equilibrium, indicating that HCOHCO₃^? was also being taken up. These results indicate that both Phaeodactylum tricornutum and Cyclotella sp. have the capacity to transport CO₂ actively against concentration and pH gradients.     

A NEW METHOD FOR ESTIMATING EXTERNAL,CARBONIC ANHYDRASE ACTMTY IN MACROALGAE1,2

The effects of external carbonic anhydrase (CA, E.C. 4.2.1.1) on HCO^?₃and CO₂use under disequilibnum conditions were examined in 14 species of macroalgae. CO₂ was added to the algae in synthetic seawater free from inorganic carbon and buffered to pH 8.7. This resulted in a transient O₂ evolution, which was similar for most of the species tested: an initial rapid increase in the rate was followed by a gradual decrease, approaching a steady state within 10 min. The initial high rate of O₂evolution was attributed to diffusive entry of CO₂into the cells and the steady state to use of HCO^?₃. An enhancement of CO₂diffusive entry was obtained with acetazolamide, an inhibitor of external CA activity. Using this enhancement, a variable was developed to quantify the degree to which CO₂entry into the cell was prevented by the external CA. This variable, CA (%), was used as a measure of the external CA activity of the alga. Comparative measurements of the external CA activity using a potentiomtric method revealed that the new method was able to detect low levels of external CA activity, where the potentiometric method failed. These two different methods could be used together to increase the reliability of the measurements. The new method was useful for red and brown macroalgae and for those green macroalgae that lacked direct HCO₃uptake. Thus prominent external CA activity was found for Valonia utricularis (green), Halopteris scoparia (brown), and Osmunda pinnatifida (red), where the potentiometric method failed, or nearly failed, to indicate any external CA activity. Direct uptake of HCO^?₃interfered with the new method and the degree of this interference was dependent on the magnitude of the uptake.     

Regulation of the mechanism for HCO 3 − use by the inorganic carbon level in Porphyra leucosticta Thur. in Le Jolis (Rhodophyta)

The capacity for HCO₃ ⁻ use by Porphyra leucosticta Thur. in Le Jolis grown at different concentrations of inorganic carbon (C_i) was investigated. The use of HCO₃ ⁻ at alkaline pH by P. leucosticta was␣demonstrated by comparing the O₂ evolution rates measured with the O₂ evolution rates theoretically supported by the CO₂ spontaneously formed from HCO₃ ⁻ . Both external and internal carbonic anhydrase (CA; EC 4.2.1.1) were implied in HCO₃ ⁻ use during photosynthesis because O₂ evolution rates and the increasing pH during photosynthesis were inhibited in the presence of azetazolamide and ethoxyzolamide (inhibitors for external and total CA respectively). Both external and internal CA were regulated by the C_i level at which the algae were grown. A high C_i level produced a reduction in total CA activity and a low C_i level produced an increase in total CA activity. In contrast, external CA was increased at low C_i although it was not affected at high C_i . Parallel to the reduction in total CA activity at high C_i is a reduction in the affinity for C_i, as estimated from photosynthesis versus C_i curves, was found. However, there was no evident relationship between external CA activity and the capacity for HCO₃ ⁻ use because the presence of external CA became redundant when P. leucosticta was cultivated at high C_i. Our results suggest that the system for HCO₃ ⁻ use in P. leucosticta is composed of different elements that can be activated or inactivated separately. Two complementary hypotheses are postulated: (i) internal CA is an absolute requirement for a functioning C_i-accumulation mechanism; (ii) there is a CO₂ transporter that works in association with external CA. Received: 20 April 1996 / Accepted: 5 August 1996     

INDUCIBLE MECHANISMS FOR HCO3– UTILIZATION AND REPRESSION OF PHOTORESPIRATION IN PROTOPLASTS AND THALLI OF THREE SPECIES OF ULVA (CHLOROPHYTA)1

Thalli of Ulva reticulata Forskaal, Ulva rigida C. Ag., and Ulva pulchra Jaasund were incubated at different concentrations of dissolved CO₂. Incubation at a high CO₂ concentration resulted in decreased oxygen evolution rate and lower affinity for inorganic carbon at high pH conditions, i.e. the ability to use HCO₃^– as a carbon source was reduced. This effect was reversible, and plants regained this HCO₃^– uptake capacity when transferred to air concentrations of CO₂. The phytosynthetic oxygen evolution rate of plants grown at high CO₂ concentration was reduced by high O₂ concentrations, whereas thalli and protoplasts from cultures grown at air concentration were not affected. This is interpreted as a deactivation of the carbon-concentrating mechanism during conditions of high CO₂ resulting in high photorespiration when plants are exposed to high O₂ concentrations. Protoplasts were not affected by high O₂ to the same extent and were not able to utilize HCO₃^– from the medium. The algae were able to grow at very low CO₂ concentrations, but growth was suppressed when an inhibitor of external carbonic anhydrase was present. Assay of carbonic anhydrase activities showed that external and internal CA activities were lower in plants grown at a high CO₂ concentration compared to plants grown at a low concentration of CO₂. Possible mechanisms for HCO₃^– utilization in these Ulva species are discussed.     

Photosynthetic inorganic carbon uptake and accumulation in two marine diatoms

Some physiological characteristics of photosynthetic inorganic carbon uptake have been examined in the marine diatoms Phaeodactylum tricornutum and Cyclotella sp. Both species demonstrated a high affinity for inorganic carbon in photosynthesis at pH7.5, having K_1/2(CO₂) in the range 1.0 to 4.0mmol m^?3 and O_2? and temperature-insensitive CO₂ compensation concentrations in the range 10.8 to 17.6 cm³ m^?3. Intracellular accumulation of inorganic carbon was found to occur in the light; at an external pH of 7.5 the concentration in P. tricornutum was twice, and that in Cyclotella 3.5 times, the concentration in the suspending medium. Carbonic anhydrase (CA) was detected in intact Cyclotella cells but not in P. tricornutum, although internal CA was detected in both species. The rates of photosynthesis at pH 8.0 of P. tricornutum cells and Cyclotella cells treated with 0.1 mol m^?3 acetazolamide, a CA inhibitor, were 1.5- to 5-fold the rate of CO₂ supply, indicating that both species have the capacity to take up HCO₃^? as a source of substrate for photosynthesis. No Na⁺ dependence for HCO₃^? could be detected in either species. These results indicate that these two marine diatoms have the capacity to accumulate inorganic carbon in the light as a consequence, in part, of the active uptake of bicarbonate.     

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.     

INORGANIC CARBON TRANSPORT IN RELATION TO CULTURE AGE AND INORGANIC CARBON CONCENTRATION IN A HIGH-CALCIFYING STRAIN OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

The relationships among inorganic carbon transport, bicarbonate availability, intracellular pH, and culture age were investigated in high-calcifying cultures of Emiliania huxleyi (Lohmann) Hay & Mohler. Measurement of inorganic carbon transport by the silicone-oil centrifugation technique demonstrated that gadolinium, a potential Ca²⁺ channel inhibitor, blocked intracellular inorganic carbon uptake and photosynthetic ¹⁴CO₂₊ fixation in exponential-phase cells. In stationary-phase cells, the intracellular inorganic carbon concentration was unaffected by gadolinium. Gadolinium was also used to investigate the link between bicarbonate and Ca²⁺ transport in high-calcifying cells of E. huxleyi. Bicarbonate availability had significant and rapid effects on pH_i in exponential- but not in stationary-phase cells. 4′, 4′-Diisothiocyanostilbene-2,2′-disulfonic acid did not block bicarbonate uptake from the external medium by exponential-phase cells. Inorganic carbon utilization by exponential- and stationary-phase cells of Emiliania huxleyi was investigated using a pH drift technique in a closed system. Light-dependent alkalization of the medium by stationary-phase cells resulted in a final pH of 10.1 and was inhibited by dextran-bound sulphonamide, an inhibitor of external carbonic anhydrase. Exponential-phase cells did not generate a pH drift. Overall, the results suggest that for high-calcifying cultures of E. huxleyi the predominant pathway of inorganic carbon utilization differs in exponential and stationary phase cells of the same culture.     

Inorganic Carbon Limitation and Chemical Composition of Two Freshwater Green Microalgae

Two freshwater chlorophytes, Chlorella vulgaris and Scenedesmus obliquus, were grown in inorganic carbon-limited continuous cultures in which HCO₃⁻ was the sole source of inorganic carbon. The response of the steady-state growth rate to the external total inorganic carbon concentration was reasonably well described by the Monod equation; however, the response to the internal nutrient concentration was only moderately well represented by the Droop equation when the internal carbon concentration was defined on a cellular basis. The Droop equation was totally inapplicable when total biomass (dry weight) was used to define internal carbon because the ratio of carbon to dry weight did not vary over the entire growth rate spectrum. In batch cultures, maximum growth rates were achieved at the CO₂ levels present in atmospheric air and at HCO₃⁻ concentrations of 3 mM. No growth was observed at 100% CO₂. Both nitrogen uptake and chlorophyll synthesis were tightly coupled to carbon assimilation, as indicated by the constant C/N and C/chlorophyll ratios found at all growth rates. The main influence of inorganic carbon limitation appears to be not on the chemical structure of the biomass, but rather on cell size; higher steady-state growth rates lead to bigger cells.     

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.     

CO2‐CONCENTRATING MECHANISMS OF THE POTENTIALLY TOXIC DINOFLAGELLATE PROTOCERATIUM RETICULATUM (DINOPHYCEAE,GONYAULACALES)1

The low CO₂ concentration in seawater poses severe restrictions on photosynthesis, especially on those species with form II RUBISCO. We found that the potentially toxic dinoflagellate Protoceratium reticulatum Clap. et J. Lachm. possesses a form II RUBISCO. To cast some light on the mechanisms this organism undergoes to cope with low CO₂ availability, we compared cells grown at atmospheric (370 ppm) and high (5000 ppm) CO₂ concentrations, with respect to a number of physiological parameters related to dissolved inorganic carbon (DIC) acquisition and assimilation. The photosynthetic affinity for DIC was about one order of magnitude lower in cells cultivated at high [CO₂]. End‐point pH‐drift experiments suggest that P. reticulatum was not able to efficiently use HCO₃^? under our growth conditions. Only internal carbonic anhydrase (CA) activity was detected, and its activity decreased by about 60% in cells cultured at high [CO₂]. Antibodies raised against a variety of algal CAs were used for Western blot analysis: P. reticulatum extracts only cross‐reacted with anti‐ß‐CA sera, and the amount of immunoreactive protein decreased in cells grown at high [CO₂]. No pyrenoids were observed under all growth conditions. Our data indicate that P. reticulatum has an inducible carbon‐concentrating mechanism (CCM) that operates in the absence of pyrenoids and with little intracellular CO₂ accumulation. Calculations on the impact of the CA activity to photosynthetic growth [CO₂] suggest that it is an essential component of the CCM of P. reticulatum and is necessary to sustain the photosynthetic rates observed at ambient CO₂.     

RESPONSES OF THE ACIDOPHILIC ALGA EUGLENA MUTABILIS (EUGLENOPHYCEAE) TO CARBON ENRICHMENT AT pH 31

A defined medium (MAM) simulating acid mine drainage waters was developed which supported reproducible growth rates of three axenic strains of Euglena mutabilis Schmitz. Growth responses to various pHs and carbon sources were examined under defined culture conditions. A lab strain and two 5eld isolates, tested over pH range 1.5-9.0, grew best under acidic conditions (pH < 5.5) with highest growth rates at pH 3-4. Photoauxotrophic growth rates of all strains at pH 3 were improved significantly over unstirred batch controls by bubbling with air and even more by enrichment with 5% CO₂ in air. These results confirmed inorganic carbon limitation in batch culture. Organic carbon substrates were tested as possible carbon supplements in batch culture at pH 3. None of the strains survived in the dark on any of the twenty organic sources added. In the light, the lab strain exhibited some photoheterotrophic growth potential on glucose, sucrose, ethanol, and amino acids but growth was inhibited by acetate. Field strains showed little or no growth improvement with any organic substrate addition. Under simultaneous enrichment with acetate and 5% CO₂ acetate continued to be inhibitory. Simultaneous enrichment with glucose and 5% CO₂ gave higher yields of the lab strain than with CO₂ alone but did not enhance growth of the field strain. We conclude that E. mutabilis is an acidophilic photoauxotroph which appears unable to use organic carbon supplements for growth even under conditions of carbon limitation.     

Inorganic Carbon Uptake by Chlamydomonas reinhardtii

The rates of CO₂-dependent O₂ evolution by Chlamydomonas reinhardtii, grown with either air levels of CO₂ or air with 5% CO₂, were measured at varying external pH. Over a pH range of 4.5 to 8.5, the external concentration of CO₂ required for half-maximal rates of photosynthesis was constant, averaging 25 micromolar for cells grown with 5% CO₂. This is consistent with the hypothesis that these cells take up CO₂ but not HCO₃⁻ from the medium and that their CO₂ requirement for photosynthesis reflects the K_m(CO₂) of ribulose bisphosphate carboxylase. Over a pH range of 4.5 to 9.5, cells grown with air required an external CO₂ concentration of only 0.4 to 3 micromolar for half-maximal rates of photosynthesis, consistent with a mechanism to accumulate external inorganic carbon in these cells. Air-grown cells can utilize external inorganic carbon efficiently even at pH 4.5 where the HCO₃⁻ concentration is very low (40 nanomolar). However, at high external pH, where HCO₃⁻ predominates, these cells cannot accumulate inorganic carbon as efficiently and require higher concentrations of NaHCO₃ to maintain their photosynthetic activity. These results imply that, at the plasma membrane, CO₂ is the permeant inorganic carbon species in air-grown cells as well as in cells grown on 5% CO₂. If active HCO₃⁻ accumulation is a step in CO₂ concentration by air-grown Chlamydomonas, it probably takes place in internal compartments of the cell and not at the plasmalemma.