The possibility of HC03-transport in the blue-green alga (cyanobac- It is generally accepted that various algal species are capable of transporting the bicarbonate ion across the cell membrane for use in photosynthesis (25), but substantive evidence is lacking in most cases. Raven (25) suggested that if the rate of photosynthesis by an alga is markedly higher at an alkaline pH, for a given CO2 concentration, than at a more acid pH it can be concluded that the bicarbonate ion crosses the cell membrane and contributes C for photosynthesis. By this criterion, Raven (24) case does not rest solely upon a comparison of photosynthetic rates at acid and alkaline pH values. Lucas (16) has shown that C. corallina at pH 9.0 has a rate of photosynthesis higher than can be supported solely by the fixation of CO2 derived from the spontaneous dehydration of HCO3 in the external medium. It is probable that these cells take up HC03-in exchange for OH-(generated by photosynthetic fixation of the transported HCO3) on distinct ion carriers in the cell membrane (17). Tailing (30) has shown that the algae Microcystis aeruginosa and Ceratium hirudinella both have photosynthetic rates at pH values of 10 or greater that are in excess of the rates that could be supported by the spontaneous dehydration of HC03-in the medium. Transport of HCO3-across cell membranes is thus indicated.In an interesting series of experiments Badger et al. (2) have shown that both Chlamydomonas reinhardtii and Anabaena variabilis, when grown at low ambient CO2 concentrations, can substantially accumulate inorganic C. This accumulation is not the result of a more alkaline intracellular pH relative to the external medium (2) and a "C02-concentrating mechanism" is indicated, presumably across the cell membrane.We have been studying the photosynthetic carbon metabolism of the blue-green alga Coccochloris peniocystis (7,14). This alga has been placed in the genus Synechococcus by Stanier et al. (29), along with other blue-green algae with cylindrical cells that undergo repeated binary fission in a single plane, which frequently results (as with C. peniocystis) in the formation of short chains of cells. Recently, Birmingham and Colman (4) have shown that C. peniocystis, at pH 7.9 and a low inorganic C concentration, has a rate of photosynthesis 5-fold that is supportable by CO2 production from the spontaneous dehydration of HCO3 in the medium. In this paper we report further investigations of HCO3-transport in C. peniocystis. MATERIALS AND METHODSOrganism and Growth Conditions. C. peniocystis Kutz (1548) was obtained as an axenic culture from the algal collection at Indiana University, Bloomington, and was cultured as previously described (21). The cell density at harvest was such that the Chl content was 6-1 1 ,ug/ml. Cells were harvested by centrifugation at 6,0)0g at 25 C and were then washed with the solution to be used in the subsequent experiment (usually 20 mm K2HPO4 phosphate adjusted to pH 8.0 with 1 N NaOH).

Eukaryotic microalgae have developed CO2concentrating mechanisms to maximise the concentration of CO2 at the active site of Rubisco in response to the low CO2 concentrations in the external aquatic medium. In these organisms, the modes of inorganic carbon (Ci) uptake are diverse, ranging from diffusive CO2 uptake to the active transport of HCO3 -and CO2 and many have an external carbonic anhydrase to facilitate HCO3- use. There is unequivocal evidence for the mechanisms of Ci uptake in only about 25 species of microalgae of the chlorophyte, haptophyte, rhodophyte, diatom, and eustigmatophyte groups. Most of these species take up both CO2 and HCO3-, but the rates of uptake of each of these substrates varies with the algal species. A few species take up only one of the two forms of Ci, an adaptation that is not necessarily correlated with their ecological distribution. Evidence is presented for the active uptake of HCO3- and CO2 in two marine haptophytes,Isochrysis galbana Parke and Dicrateria inornata Parke, and for active transport of CO2 but lack of HCO3- uptake in two marine dinoflagellates, Amphidinium carteraeHulburt and Heterocapsa oceanica Stein.

Physiological properties of photosynthesis were determined in the marine diatom, Phaeodactylum tricornutum UTEX640, during acclimation from 5% CO 2 to air and related to H 2 CO 3 dissociation kinetics and equilibria in artificial seawater. The concentration of dissolved inorganic carbon at half maximum rate of photosynthesis (K 0·5 [DIC]) value in high CO 2 -grown cells was 1009 mmol m -3 but was reduced three-fold by the addition of bovine carbonic anhydrase (CA), whereas in air-grown cells K 0·5 [DIC] was 71 mmol m -3 , irrespective of the presence of CA. The maximum rate of photosynthesis (P max ) values varied between 300 and 500 mmol O 2 mg Chl -1 h -1 regardless of growth pCO 2 . Bicarbonate dehydration kinetics in artificial seawater were re-examined to evaluate the direct HCO 3 -uptake as a substrate for photosynthesis. The uncatalysed CO 2 formation rate in artificial seawater of 31·65°/ oo of salinity at pH 8·2 and 25°C was found to be 0·6 mmol m -3 min -1 at 100 mmol m -3 DIC, which is 53·5 and 7·3 times slower than the rates of photosynthesis exhibited in air-and high CO 2 -grown cells, respectively. These data indicate that even high CO 2 -grown cells of P. tricornutum can take up both CO 2 and HCO 3 -as substrates for photosynthesis and HCO 3 -use improves dramatically when the cells are grown in air. Detailed time courses were obtained of changes in affinity for DIC during the acclimation of high CO 2 -grown cells to air. The development of high-affinity photosynthesis started after a 2-5 h lag period, followed by a steady increase over the next 15 h. This acclimation time course is the slowest to be described so far. High CO 2 -grown cells were transferred to controlled DIC conditions, at which the concentrations of each DIC species could be defined, and were allowed to acclimate for more than 36 h. The

The blue-green alga Coccochlorispeniocystis photosynthesizes optimally over the pH range of 7.0 to 10.0, but the 02-evolution rate is inhibited below pH 7.0 and ceases below pH 5.25. Measurement of the inorganic carbon pool in this alga in the light, using the silicone-fluid filtration technique demonstrated that the rate of accumulation of dissolved inorganic carbon remained relatively constant over a wide pH range. At external dissolved inorganic carbon concentrations of 0.56 to 0.89 millimolar the internal concentration after 30 seconds illumination was greater than 3.5 millimolar over the entire pH range. Intracellular pH measured in the light using I14C15,5-dimethyloxazolidine-2,4dione and I'4Cimethyla-mine dropped from pH 7.6 at an external pH of 7.0 to pH 6.6 at an external pH of 5.25. Above an external pH of 7.0 the intracellular pH rose gradually to pH 7.9 at an external pH 10.0. Ribulose-1,5-bisphosphate carboxylase activity of cell-free algal extracts exhibited optimal activity at pH 7.5 to 7.8 but was inactive below pH 6.5. It is suggested that the inability of Coccochioris to maintain its intracellular pH when in an acidic environment restricts its photosynthetic capacity by a direct pH effect on the principal CO2 fixing enzyme.Blue-green algae generally have been found in alkaline natural waters, and many species in laboratory culture exhibit high rates of growth and photosynthesis only at an alkaline pH (16). Conversely, most blue-green algae are unable to grow or photosynthesize in an acidic environment.As the HC03-ion is the predominant species of DIC2 at pH values in the range of 7.0 to 10.0 (5), the capacity of these algae to grow in an alkaline environment suggests that they are capable of assimilating HC03-as a substrate for photosynthetic carbon fixation. Previous studies in this laboratory have shown that the blue-green alga Coccochloris is indeed capable of transporting HC03-at an alkaline pH (8,19,20). Other reports indicate that the result of this transport is the formation of a large, internal, inorganic carbon pool prior to photosynthetic carbon fixation (2,19). This study is an examination of the effect of external pH on photosynthesis and the accumulation of inorganic carbon in the blue-green alga Coccochloris peniocystis and a hypothesis is proposed to explain the apparent inhibition of photosynthesis at an acid pH.

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 K1/2(CO2) in the range 1.0 to 4.0mmol m−3 and O2− 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.

A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO 2 -grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO 2 conditions. Ethoxyzolamide (0.4 mm), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mm acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to ␤-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO 2 -binding site appeared to be unique among the ␤-CAs so far reported.

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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.