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The mechanism of inorganic carbon (Ci) uptake was examined in the marine green microalgae Stichococcus cylindricus and Stichococcus minor. External carbonic anhydrase (CA) activity was not detected in either species, by potentiometric assay or by mass spectrometry. Photosynthetic characteristics of Ci uptake indicate that both species have high apparent affinity for CO2 with a low K1/2(CO2) of about 10 µm. The O2 evolution rates in light exceeded the spontaneous CO2 formation rate by 2.5-fold in both species, which thus have active bicarbonate uptake. Mass spectrometric monitoring of CO2 and O2 fluxes showed that rates of O2 evolution exceeded those of CO2 depletion by about three- and twofold in S. minor and S. cylindricus, respectively, and also showed, in cells photosynthesizing at pH 8.2, a rapid depletion of CO2 upon illumination to a CO2 compensation concentration of 15.42 and 12.03 µm in S. minor and S. cylindricus, respectively. Both species also exhibit active CO2 uptake: addition of bovine CA at CO2 compensation concentration caused a rapid rise in CO2 as the CO2–HCO3- equilibrium was restored. Accumulation of unfixed Ci by cells at pH 8.2 was calculated to be 84.33 mm in S. cylindricus, and 30.37 mm in S. minor to give internal accumulations of 23- and 8-fold, respectively, compared to the external Ci concentration.

CO(2)-insensitive mutants of the green alga Chlorella ellipsoidea were previously shown to be unable to repress an inorganic carbon-concentrating mechanism (CCM) when grown under 5% CO(2). When air-grown, wild-type (WT) cells were transferred to 5% CO(2), an abrupt drop of P(max) to 43% the original level of air-grown cells was observed within the initial 12 h. Photosynthetic affinities of WT cells to dissolved inorganic carbon (DIC) were maintained at high levels for the initial 4 d of acclimation, and then decreased gradually to lower levels over the next 6 d. In contrast to WT cells, the CO(2)-insensitive mutant, ENU16, exhibited a constant P(max) at maximum levels and a low K(1/2)[DIC] throughout the acclimation period. The rapid P(max) drop within 12 h of acclimation in WT cells was significantly reduced by treatment with 0.5 mm of 6-ethoxybenzothiazole-2-sulphonamide (EZA), a specific membrane-permeable inhibitor of carbonic anhydrase (CA), suggesting the participation of internal CAs in the temporary drop in P(max) in WT cells. WT and ENU16 cells were grown in controlled equilibrium [CO(2)], and the photosynthetic rate of each acclimated cell type was measured under equilibrated growth [DIC] conditions. In WT cells acclimated to 0.14-0.4% [CO(2)], K(1/2)[DIC] values increased as [CO(2)] increased, and the photosynthetic rates at growth DIC conditions were shown to decrease to about 70% the P(max) level in this intermediate [CO(2)] range. Such decreases in the net photosynthetic rates were not observed in ENU16. These results suggest that algal primary production could be depressed significantly under moderately enriched CO(2) conditions as a result of acquiring intermediate affinities for DIC because of their sensitive responses to changes in the ambient [CO(2)].

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 μmol 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‰ 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 K 0.5 [DIC] values in acclimated cells appeared to be correlated only with [CO 2(aq) ] in the medium but not to HCO 3 - , CO 3 2- , total [DIC] or the pH of the medium and indicate that the critical signal regulating the affinity of cells for DIC in the marine diatom, P. tricornutum, is [CO 2(aq) ] in the medium.

Induction of the carbon concentrating mechanism (CCM) has been investigated during the acclimation of 5% CO 2 -grown Chlamydomonas reinhardtii 2137 mt + cells to well-defined dissolved inorganic carbon (C i ) limited conditions. The CCM components investigated were active HCO 3 - transport, active CO 2 transport and extracellular carbonic anhydrase (CA ext ) activity. The CA ext activity increased 10-fold within 6 h of acclimation to 0.035% CO 2 and there was a further slight increase over the next 18 h. The CA ext activity also increased substantially after an 8 h lag period during acclimation to air in darkness. Active CO 2 and HCO 3 - uptake by C. reinhardtii cells were induced within 2 h of acclimation to air, but active CO 2 transport was induced prior to active HCO 3 - transport. Similar results were obtained during acclimation to air in darkness. The critical C i concentrations effecting the induction of active C i transport and CA ext activity were determined by allowing cells to acclimate to various inflow CO 2 concentrations in the range 0.035-0.84% at constant pH. The total C i concentration eliciting the induction and repression of active C i transport was higher during acclimation at pH 7.5 than at pH 5.5, but the external CO 2 concentration was the same at both pHs of acclimation. The concentration of external CO 2 required for the full induction and repression of C; transport and CA ext activity were 10 and 100 μM, respectively. The induction of CA ext and active C i transport are not correlated temporally, but are regulated by the same critical CO 2 concentration in the medium.