Inorganic carbon sources and biomass regulation in intensive microalgal cultures

Three freshwater and one marine algal species were grown under inorganic carbon limitation in laboratory continuous cultures. Comparisons were made between HCO₃^? alkalinity and bubbled CO₂ as carbon sources. HCO₃^? alkalinity was an excellent source of inorganic carbon below specific pH levels, but chemical precipitation at high pH placed an upper limit on productivity that was far lower than potential light-limiting levels. With bubbled CO₂ it was possible to achieve light limitation. The main factor controlling productivity was the mass flux of inorganic carbon added to the culture, which is the product of gas flow rate and influent P level. Small bubbles were more efficient than large bubbles at low gas flow rates and P levels, but led to froth flotation of algal cells and concomitant reductions in productivity at high bubble rates. At 1% CO₂ productivity was still dependent on mass fluxes of added carbon, but was independent of bubble size. At high bubble rates with 1% CO₂ narcosis was evident. Maximum yields occurred at intermediate dilution rates when inorganic carbon was supplied via bubbled gas.     

The utilization of bicarbonate ions by the macroalga Ascophyllum nodosum (L.) Le Jolis

Abstract A comparison of some of the methods used to determine whether aquatic plants have the ability to utilize bicarbonate ions as a source of inorganic carbon for photosynthesis has been applied to the intertidal macroalga Ascophyllum nodosum. These include: observing photosynthesis at a high pH (below the alga's CO₂ compensation point), pH compensation point determinations, comparing the photosynthetic characteristics at low pH (5.20) and at high pH (7.95), estimating the maximal rates at which CO₂ can diffuse through the unstirred layer and the rate at which CO₂ can be produced from bicarbonate dehydration in the unstirred layer. All indicated that Ascophyllum nodosum can use bicarbonate ions for photosynthesis, though some were not always consistent. Calculating the total inorganic carbon concentration from pH measurements and acidification CO₂ determinations revealed that the assumption that the alkalinity remains constant during pH drift experiments is not always valid.     

Measurement of carbon dioxide compensation points of freshwater algae

A technique is described for the measurement of total dissolved inorganic carbon by acid release as CO₂ followed by its conversion to methane and detection by flame ionization in a modified gas chromatograph. This method was used to determine the dissolved inorganic carbon concentration reached at compensation point when algae were allowed to photosynthesize in a closed system in a buffer at known pH, and the CO₂ compensation point was calculated from this concentration. The CO₂ compensation points of 16 freshwater algae were measured at acid and alkaline pH in air-saturated medium: at acid pH the CO₂ compensation points ranged from 4.8 to 41.5 microliters per liter while at alkaline pH they ranged from 0.2 to 7.2 microliters per liter. Removal of O₂ from the medium caused a slight lowering of compensation point at acid pH but had little effect at alkaline pH. These low, O₂-insensitive compensation points are characteristic of C₄ plants. It is suggested that these low CO₂ compensation points are maintained by an active bicarbonate uptake by algae especially at alkaline pH.     

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.     

Rising CO2 Levels Will Intensify Phytoplankton Blooms in Eutrophic and Hypertrophic Lakes

Harmful algal blooms threaten the water quality of many eutrophic and hypertrophic lakes and cause severe ecological and economic damage worldwide. Dense blooms often deplete the dissolved CO₂ concentration and raise pH. Yet, quantitative prediction of the feedbacks between phytoplankton growth, CO₂ drawdown and the inorganic carbon chemistry of aquatic ecosystems has received surprisingly little attention. Here, we develop a mathematical model to predict dynamic changes in dissolved inorganic carbon (DIC), pH and alkalinity during phytoplankton bloom development. We tested the model in chemostat experiments with the freshwater cyanobacterium Microcystis aeruginosa at different CO₂ levels. The experiments showed that dense blooms sequestered large amounts of atmospheric CO₂, not only by their own biomass production but also by inducing a high pH and alkalinity that enhanced the capacity for DIC storage in the system. We used the model to explore how phytoplankton blooms of eutrophic waters will respond to rising CO₂ levels. The model predicts that (1) dense phytoplankton blooms in low- and moderately alkaline waters can deplete the dissolved CO₂ concentration to limiting levels and raise the pH over a relatively wide range of atmospheric CO₂ conditions, (2) rising atmospheric CO₂ levels will enhance phytoplankton blooms in low- and moderately alkaline waters with high nutrient loads, and (3) above some threshold, rising atmospheric CO₂ will alleviate phytoplankton blooms from carbon limitation, resulting in less intense CO₂ depletion and a lesser increase in pH. Sensitivity analysis indicated that the model predictions were qualitatively robust. Quantitatively, the predictions were sensitive to variation in lake depth, DIC input and CO₂ gas transfer across the air-water interface, but relatively robust to variation in the carbon uptake mechanisms of phytoplankton. In total, these findings warn that rising CO₂ levels may result in a marked intensification of phytoplankton blooms in eutrophic and hypertrophic waters.     

Modeling of carbon dioxide mass transfer behavior in attached cultivation photobioreactor using the analysis of the pH profiles

The CO₂ mass transfer model associated with growth kinetics of microalgal biofilm in attached cultivation photobioreactor was developed and verified by using the analysis of pH profiles which were in equilibrium with inorganic carbon components concentrations (CO₂, H₂CO₃, HCO₃ ⁻ and CO₃ ²⁻) in medium. Model simulation results showed that the model well presented the biofilm growth process. The overall volumetric mass transfer coefficient of CO₂ was more influenced by CO₂ concentration in aerated gas but less by gas aeration rate and medium circulation rate. Other bio-kinetic parameters related with the microalgal biofilm such as CO₂ diffusion coefficient in biofilm, Monod maximum utilization rate of CO₂, lag phase duration of biofilm and half-saturation CO₂ concentration in the biofilm were independent on operational conditions. The pH profiles provided a way to monitor the variations of inorganic carbon concentrations of medium and to regulate the cultivation of attached microalgal biofilm by CO₂ supplement.

     

INORGANIC CARBON ACQUISITION BY CHRYSOPHYTES1

Twelve species, representing 12 families of the chrysophytes sensu lato, were tested for their ability to take up inorganic carbon. Using the pH‐drift technique, CO₂ compensation points generally varied between 1 and 20 μmol · L^?1 with a mean concentration of 5 μmol · L^?1. Neither pH nor alkalinity affected the CO₂ compensation point. The concentration of oxygen had a relatively minor effect on CO₂‐uptake kinetics, and the mean CO₂ compensation point calculated from the kinetic curves was 3.6 μmol · L^?1 at 10–15 kPa starting oxygen partial pressure and 3.8 μmol · L^?1 at atmospheric starting oxygen partial pressure (21 kPa). Similarly, uptake kinetics were not affected by alkalinity, and hence concentration of bicarbonate. Membrane inlet mass spectrometry (MIMS) in the presence and absence of acetazolamide suggested that external carbonic anhydrase in Dinobryon sertularia Ehrenb. and Synura petersenii Korschikov was either very low or absent. Rates of net HCO₃^? uptake were very low (～5% of oxygen evolution) using MIMS and decreased rather than increased with increasing HCO₃^? concentration, suggesting that it was not a real uptake. The CO₂ compensation points determined by MIMS for CO₂ uptake and oxygen evolution were similar to those determined in pH‐drift and were >1 μmol · L^?1. Overall, the results suggest that chrysophytes as a group lack a carbon‐concentrating mechanism (CCM), or an ability to make use of bicarbonate as an alternative source of inorganic carbon. The possible evolutionary and ecological consequences of this are briefly discussed.     

The use of acetic acid as a source of carbon by cultured Chondrus crispus (Gigartinales,Rhodophyta) stackhouse

When growing seaweeds in tanks, pH and carbon source supply have to be controlled in order to maximize photosynthesis. pH can be controlled either by adding various inorganic acids which requires the extra addition of carbon, or by combining pH control and carbon source with for instance CO₂ or an organic acid such as acetic acid (CH₃COOH). We have found comparable productivity of Chondrus using CO₂ or CH₃COOH in tank culture with an increase in production of 25.0 and 27.5%, respectively, over the control. Laboratory experiments showed that acetic acid enabled us to maintain a steady state total inorganic carbon in the medium, the algae displaying an active photosynthesis. Experiments using labelled acetic acid CH₃-¹⁴COOH showed that the acid molecule or at least the -¹⁴COOH group is taken up by Chondrus although the mechanism was not elucidated. Preliminary extractions with hot ethanol showed that 67.9% of the label was solubilized from labelled tissue. Few counts were found in the carrageenans (< 1 %) and between 25.6 and 45.1% were found in the cellulosic residues. Acetic acid is suggested as an interesting means of regulating the pH and adding carbon in macrophyte culture.     

Complementation analysis of the inorganic carbon concentrating mechanism of Chlamydomonas reinhardtii

Summary Six independently isolated mutants of Chlamydomonas reinhardtii that require elevated CO₂ for photoautotrophic growth were tested by complementation analysis. These mutants are likely to be defective in some aspect of the algal concentrating mechanism for inorganic carbon as they exhibit CO₂ fixation and inorganic carbon accumulation properties different from the wild-type. Four of the six mutants defined a single complementation group and appear to be defective in an intracellular carbonic anhydrase. The other two mutations represent two additional complementation groups.Abbreviations HS high salt medium which has 13 mM phosphate at pH 6.8 - HSA high salt plus 36 mM acetate medium - YA high salt medium with 4 g yeast extract per L and 36mM acetate - Arg arginine - cia^- CO₂ accumulation mutants that cannot grow on low CO₂ - C_i inorganic carbon (CO₂+HCO _- ³ ) - CA carbonic anhydrase - mt mating type Supported in part by the McKnight Foundation and by NSF grant PCM 8005917 and published as journal article 11924 from the Michigan State Agriculatural Experiment Station     

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.     

Inorganic carbon acquisition in the acid‐tolerant alga Chlorella kessleri

The ability of the freshwater alga, Chlorella kessleri, to maintain a carbon concentrating mechanism when grown at acid pH was investigated. The alga grows over the pH range 4.0–9.0 and was found to take up bicarbonate and CO₂ actively when grown at pH 6.0. However, when grown at acid pH (below 5.5), it does not have active CO₂ uptake. The acidotolerant species maintained an internal pH of 6.1–7.5 over the external pH range 4.5–7.5, thus the pH difference between the cell interior and the external medium was large enough to allow for the diffusive uptake of CO₂ at acid external pH. Mass spectrometric monitoring of O₂ and CO₂ fluxes by suspensions of C. kessleri, grown at acid pH, and maintained at pH 7.5 showed that the rates of O₂ evolution did not exceed those of CO₂ uptake. The final CO₂ compensation concentrations of 14.0–17.7 µM reached by photosynthetic cells were above the CO₂ equilibrium concentration in the external medium, indicating a lack of active CO₂ uptake at acid pH. Chlorella kessleri accumulated CO₂ with internal concentrations that were 9.9, 18.7 and 22.7‐fold that of the external medium for cells grown, respectively, at pH 4.5, 5.0 and 5.5. The ability of C. kessleri cells to accumulate high intracellular concentrations of inorganic carbon at acid pH would provide a sufficiently high concentration of CO₂ at the active site of Rubisco thus allowing the alga to maintain growth rates similar to those at alkaline pH.     

Inorganic carbon uptake by an Antarctic sea-ice diatom,Nitzschia frigida

There have been no studies to date on the mechanisms of inorganic carbon acquisition by Antarctic microalgae. Consequently, we have examined inorganic carbon (DIC) use inNitzschia frigida, a diatom typical of the Antarctic bottom-ice community. The K_0.5(CO₂) of photosynthesis in this organism was estimated to be 1.09 μM at pH 7.5. The internal concentration of DIC was approximately 4050 μM at an external [DIC] of 45 μM. At air-equilibration levels of inorganic carbon this would be sufficient for a ten-fold accumulation ratio of CO₂. Cells ofN. frigida are capable of carbon-dependent photosynthesis at rates that exceed that expected from uncatalysed CO₂ supply to the cell. About 25% of the total carbonic anhydrase activity appears to be associated with the cell surface inN. frigida. These results support the hypothesis thatN. frigida, like many microalgae from temperate waters, has an active carbon-concentrating mechanism, associated with the ability to utilize external HCO ₃ ⁻ for photosynthesis.     

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.     

Circadian rhythm and fast responses to blue light of photosynthesis in Ectocarpus (Phaeophyta,Ectocarpales)

Photosynthesis of Ectocarpus siliculosus (Dillwyn) Lyngb. under continuous saturating red irradiation follows a circadian rhythm. Blue-light pulses rapidly stimulate photosynthesis with high effectiveness in the troughs of this rhythm but the effectiveness of such pulses is much lower at its peaks. In an attempt to understand how blue light and the rhythm affected photosynthesis, the effects of inorganic carbon on photosynthetic light saturation curves were studied under different irradiation conditions. The circadian rhythm of photosynthesis was apparent only at irradiances which were not limiting for photosynthesis. The same was found for blue-light-stimulated photosynthesis, although stimulation was observed also under very low red-light irradiances after a period of adaptation, provided that the inorganic-carbon concentration was not in excess. Double-reciprocal plots of light-saturated photosynthetic rates versus the concentration of total inorganic carbon (up to 10 mM total inorganic carbon) were linear and had a common constant for half-saturation (3.6 mM at pH 8) at both the troughs and the peaks of the rhythm and before and after blue-light pulses. Only at very low carbon concentrations was a clear deviation found from these lines for photosynthesis at the rhythm maxima (red and blue light), which indicated that the strong carbon limitation specifically affected photosynthesis at the peak phases of the rhythm. Very high inorganic carbon concentrations (20 mM) in the medium diminished the responses to blue light, although they did not fully abolish them. The kinetics of the stimulation indicate that the rate of photosynthesis is affected by two blue-light-dependent components with different time courses of induction and decay. The faster component seemed to be at least partially suppressed at red-light irradiances which were not saturating for photosynthesis. Lowering the pH of the medium had the same effects as an increase of the carbon concentration to levels of approx. 10 mM. This indicates that Ectocarpus takes up free CO₂ only and not bicarbonate, although additional physiological mechanisms may enhance the availability of CO₂.Abbreviation TIC total inorganic carbon     

ATP-dependent carbon transport in perfused Chara cells

Carbon transport across the plasma membrane, and carbon fixation were measured in perfused Chara internodal cells. These parameters were measured in external media of pH 5·5 and pH 8·5, where CO₂ and HCO₃^- are, respectively, the predominant carbon species in both light and dark conditions. Cells perfused with medium containing ATP could utilize both CO₂ and HCO₃^- from the external medium in the light. Photosynthetic carbon fixation activity was always higher at pH 5·5 than at pH 8·5. When cells were perfused either with medium containing hexokinase and 2-deoxyglucose to deplete ATP from the cytosol (HK medium) or with medium containing vanadate, a specific inhibitor of the plasma membrane H⁺_-ATPase (V medium), photosynthetic carbon fixation was strongly inhibited at both pH 5·5 and 8·5. Perfusion of cells with medium containing pyruvate kinase and phosphoenolpyruvate (PEP) to maximally activate the H⁺_-ATPase (PK medium), stimulated the photosynthetic carbon fixation activities. Oxygen evolution of isolated chloroplasts and the carbon fixation of cells supplied ¹⁴C intracellularly were not inhibited by perfusion media containing either hexokinase and 2-deoxyglucose or vanadate. The results indicate that Chara cells possess CO₂ and HCO₃^- transport systems energized by ATP and sensitive to vanadate in the light. In the dark, intact cells also fix carbon. By contrast, in cells perfused with medium containing ATP, no carbon fixation was detected in 1 mol m ^-3 total dissolved inorganic carbon (TDIC) at pH 8·5. By increasing TDIC to 10 mol m^-3, dark fixation became detectable, although it was still lower than that of intact cells at 1mol m^-3 TDIC. Addition of PEP or PEP and PEP carboxylase to the perfusion media significantly increased the dark-carbon fixation. Perfusion with vanadate had no effect on the dark-carbon fixation.     

Effect of CO2 enhancement in an outdoor algal production system usingTetraselmis

One of the objectives of microalgal culture is to provide reliable production technology for important live aquaculture feed organisms. Presented here are the results of experiments designed to provide a better understanding of the relationship between inorganic carbon availability and algal production.Our results suggest that through additions of CO₂ gas we were able to maintain sufficient dissolved carbon to stabilize outdoor algal cultures. Increases in the rate of addition of CO₂ increased levels of dissolved CO₂, total dissolved inorganic carbon (CO₂), and decreased pH in the growth medium. This translated into improved buffering capacity of the culture medium and higher growth rate. A minimum of 2.4 mM CO₂ was found necessary to maintain a maximal growth rate of 0.7 doublings/day. We also found that the increased productivity more than offsets the cost of adding the CO₂.     

TRANSPORT OF INORGANIC CARBON AND THE ‘CO2 CONCENTRATING MECHANISM’ IN CHLORELLA EMERSONII (CHLOROPHYCEAE)1

Chlorella emersonii Shihira et Krauss var. emersonii exhibits ‘C₄-like’ gas exchange characteristics when grown at air levels of CO₂, but is ‘C₃-like’ when grown with extra CO₂. The total inorganic carbon concentration, and the free CO₂ concentration, averaged over the cell interior are higher in air-adapted cells than can be accounted for by passive CO₂ equilibration from the medium and the mean intracellular pH value. The ‘extra’ inorganic C in the air-grown cells probably cannot all be accounted for in terms of binding to proteins and requires an active transport process to account for it. The electrical potential of the cell interior becomes more negative when the ‘CO₂ concentrating mechanism’ is operative; this is most readily explained if the active step in inorganic C accumulation is primary active uniport of HCO₃^?. Since the ‘CO₂ concentrating mechanism’ can operate when CO₂ is the species crossing the outer permeation barrier, it is suggested that the site of active HCO₃^? transport in Chlorella (and other eukaryotes) is the chloroplast envelope, and the plasmalemma in cyanobacteria. This scheme explains the obligatory role of the de-repressed carbonic anhydrase in C₄-like photosynthesis in algae, but some other data support an explanation of C₄-like photosynthesis in terms of special properties of carbonic anhydrase as a carbon donor to RuBP carboxylase-oxygenase.     

HCO3− and CO2 leakage from Synechococcus UTEX 625

The leakage of various inorganic carbon species from air-grown cells of Synechococcus UTEX 625 was investigated after a light to dark transition or during a light period using a mass spectrometer under a wide variety of experimental conditions. Total inorganic carbon efflux and CO₂ efflux during the initial period of darkness were measured with or without carbonic anhydrase in the reaction medium respectively. The HCO₃^? efflux after a light to dark transition was estimated by difference. Carbon dioxide efflux in the light was measured by inhibiting CO₂ transport with either Na₂S or COS₃ or quenching the ¹³C inorganic carbon transport by the addition of ¹²C inorganic carbon in excess. In cells in which CO₂ fixation was inhibited, when only the HCO₃^? transport system was fully operative, CO₂ effluxed continuously during the light period at a rate equal to about 25% of that in darkness. When only the CO₂ transport system was operative, HCO₃^? effluxed during the light period. The difference between the light and dark efflux rates was consistent with a 0.6 unit decrease in the intracellular pH upon darkening the cells. The permeabilities of the cell for CO₂ (2.94 ± 0.14 ± 10^?8ms^?1; mean ± SE, n=137) and HCO₃^? (1.4–1.7 ± 10^?9 ms^?1) were calculated.     

Photosynthesis in Ulva fasciata: V. Evidence for an Inorganic Carbon Concentrating System, and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase CO(2) Kinetics

Evidence of an inorganic carbon concentrating system in a marine macroalga is provided here. Based on an O₂ technique, supported by determinations of inorganic carbon concentrations, of experimental media (as well as compensation points) using infrared gas analysis, it was found that Ulva fasciata maintained intracellular inorganic carbon levels of 2.3 to 6.0 millimolar at bulk medium concentrations ranging from 0.02 to 1.5 millimolar. Bicarbonate seemed to be the preferred carbon form taken up at all inorganic carbon levels. It was found that ribulose-1,5-bisphosphate carboxylase/oxygenase from Ulva had a K_m(CO₂) of 70 micromolar and saturated at about 250 micromolar CO₂. Assuming a cytoplasmic pH of 7.2 (as measured for another Ulva species, P Lundberg et al. [1988] Plant Physiol 89: 1380-1387), and given the high activity of internal carbonic anhydrase (S Beer, A Israel [1990] Plant Cell Environ [in press]) and the here measured internal inorganic carbon level, it was concluded that internal CO₂ in Ulva could, at ambient external inorganic carbon concentrations, be maintained at a high enough level to saturate ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation. It is suggested that this suppresses photorespiration and optimizes net photosynthetic production in an alga representing a large group of marine plants faced with limiting external CO₂ concentrations in nature.     

Uptake of inorganic carbon by isolated chloroplasts from air-adapted dunaliella

Neither Dunaliella cells grown with 5% CO₂ nor their isolated chloroplasts had a CO₂ concentrating mechanism. These cells primarily utilized CO₂ from the medium because the K_(0.5) (HCO₃⁻) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K_(0.5) CO₂ was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO₂ concentrating mechanism was present that decreased the K_0.5 (CO₂) to about 0.5 micromolar and K_0.5 (HCO₃⁻) to 11 micromolar at pH 8. These K_0.5 values suggest that air-adapted cells preferentially concentrated CO₂ but could also use HCO₃⁻ from the medium. Chloroplasts isolated from air-adapted cells had a K_(0.5) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO₂. Chloroplasts from air-adapted cells, but not CO₂-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO₂ concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO₂ concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO₂ concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.