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.     

VARIATION IN THE OCCURRENCE OF EXTERNAL CARBONIC ANHYDRASE AMONG STRAINS OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Eleven different strains of Phaeodactylum tricornutum Bohlin were obtained from three culture collections and were examined for the presence of external and internal carbonic anhydrase (CA). Cells of all strains, grown in standing culture at alkaline pH and low, dissolved inorganic carbon had internal CA, but only eight were found to have external CA. External CA activity was reduced when cultures were bubbled with air and was completely repressed when they were grown on 5% CO₂. Expression of external CA activity appears to be regulated by CO₂ concentration in the growth medium, but within one species, there appears to be a variation in occurrence of external CA and consequently in the mode of inorganic carbon acquisition.     

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.     

Effect of dissolved inorganic carbon on the expression of carboxysomes,localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO₂+HCO _inf3 ^sup- ) in the growth medium. In cells grown under 5% CO₂ and in those bubbled with air, carboxysomes were present in low numbers (<2 · longitudinal section^-1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 l CO₂ · 1^-1 possessed many carboxysomes (>8 · longitudinal section^-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO _inf3 ^sup- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon (CO₂+HCO _inf3 ^sup- +CO _inf3 ^sup2- ) - PRK phosphoribulokinase - RuBP ribulose 1,5-bisphosphate - Rubisco LS large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase     

CO2 exchange characteristics during dark-light transitions in wild-type and mutant Chlamydomonas reinhardii cells

A burst of net CO₂ uptake was observed during the first 3–4 min after the onset of illumination in both wild-type Chlamydomonas reinhardii in which carbonic anhydrase was chemically inhibited with ethoxyzolamide and in a mutant of C. reinhardii (ca-1-12-1C) deficient in carbonic anhydrase activity. The burst was followed by a rapid decrease in the CO₂ uptake rate so that net evolution often occurred. After a 2–3 min period of CO₂ evolution, net CO₂ uptake again increased and ultimately reached a steady-state, positive rate. From [¹⁴CO₂]-tracer studies it was determined that CO₂ fixation proceeded at a nearly linear rate throughout the period of illumination. Thus, prior to reaching a steady state, there was a rapid accumulation of inorganic carbon inside the cells which apparently reached a supercritical concentration and the excess was excreted, causing a subsequent efflux of CO₂. A post illumination burst of net CO₂ efflux was also observed in ethoxyzolamide-inhibited wild type and ca-1 mutant cells, but not in the unihibited wild type. [¹⁴CO₂]-tracer experiments revealed that this burst was the result of a collapse of a large internal inorganic carbon pool at the onset of darkness rather than a photorespiratory post-illumination burst. These results indicate that upon illumination, chemical or genetic inhibition of carbonic anhydrase initially causes an accumulation of excess inroganic carbon in C. reinhardii cells, and that unknown regulatory mechanisms correct for this imbalance by first excreting the excess inorganic carbon and then, after several dampened oscillations, achieving an equilibrium between bicarbonate uptake, bicarbonate dehydration, and CO₂ fixation.     

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.     

Adaptation of Chlamydomonas reinhardtii High-CO(2)-Requiring Mutants to Limiting CO(2)

Photosynthetic characteristics of four high-CO₂-requiring mutants of Chlamydomonas reinhardtii were compared to those of wild type before and after a 24-hour exposure to limiting CO₂ concentrations. The four mutants represent two loci involved in the CO₂-concentrating system of this unicellular alga. All mutants had a lower photosynthetic affinity for inorganic carbon than did the wild type when grown at an elevated CO₂ concentration, indicating that the genetic lesion in each is expressed even at elevated CO₂ concentrations. Wild type and all four mutants exhibited adaptive responses to limiting CO₂ characteristic of the induction of the CO₂-concentrating system, resulting in an increased affinity for inorganic carbon only in wild type. Although other components of the CO₂-concentrating system were induced in these mutants, the defective component in each was sufficient to prevent any increase in the affinity for inorganic carbon. It was concluded that the genes corresponding to the ca-1 and pmp-1 loci exhibit at least partially constitutive expression and that all components of the CO₂-concentrating system may be required to significantly affect the photosynthetic affinity for inorganic carbon.     

The effects of pH and dissolved inorganic carbon on external carbonic anhydrase activity in Chlorella saccharophila

External carbonic anhydrase (CA) activity in Chlorella saccharophila is suppressed by growth at high dissolved inorganic carbon and at acid pH. External CA activity was shown to be suppressed by growth at pHs below 7.0, with total repression at pH5.0. Growth in the presence of the buffer 3-[N-Morpholino]propane-sulphonic acid (MOPS) between pH 7 and 8 suppressed CA activity. Cells grown at pH8.0 aerated at 6 dm³ h^?1 exhibited external CA activity of 5 units mg^?1 Chl once the dissolved inorganic carbon (DIC) was reduced to 300 mmol m^?3, and this increased to 30 units mg^?1 Chl over a period of 3d while the DIC dropped to 30mmol m^?3. Cells aerated at 180 dm³ h^?1 showed a similar trend in CA activity, although the onset was delayed by 1 d and the DIC did not drop below 300 mmol m^?3. Cells grown at pH 7.8 near an air equilibrium DIC of 300 mmol m^?3had no detectable external CA activity. It is probable that it is the CO² supply to the cell, and not total DIC or HCO^?₃ which controls external CA activity. Cells grown at pH 5.0 had no detectable activity, although they reduced the CO² concentration to 0.6 mmol m^?3. The loss of CA upon transfer of air-grown cells to 10 mmol mol^?1 CO² took place over 48 h and was light dependent, while the loss upon transfer from alkaline pH to acid pH look place over 12 h and was independent of light. The effects of pH are independent of the response to CO₂.     

Growth and Photosynthesis of the Cyanobacterium Synechococcus leopoliensis in HCO(3)-Limited Chemostats

Synechococcus leopoliensis was grown in HCO₃⁻-limited chemostats. Growth at 50% the maximum rate occurred when the inorganic carbon concentration was 10 to 15 micromolar (or 5.6 to 8.4 nanomolar CO₂). The O₂ to CO₂ ratios during growth were as high as 192,000 to 1. At growth rates below 80% the maximum rate, essentially all the supplied inorganic carbon was converted to organic carbon, and the cells were carbon limited. Carbon-limited cells used HCO₃⁻ rather than CO₂ for growth. They also exhibited a very high photosynthetic affinity for inorganic carbon in short-term experiments. Cells growing at greater than 80% maximum growth rate, in the presence of high dissolved inorganic carbon, were termed carbon sufficient. These cells had photosynthetic affinities that were about 1000-fold lower than HCO₃⁻-limited cells and also had a reduced capacity for HCO₃⁻ transport. HCO₃⁻-limited cells are reminiscent of the air-grown cells of batch culture studies while the carbon sufficient cells are reminiscent of high-CO₂ grown cells. However, the low affinity cells of the present study were growing at CO₂ concentrations less than air saturation. This suggests that supranormal levels of CO₂ not required to induce the physiological changes usually ascribed to high CO₂ cells.     

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     

Characterisation of carbon dioxide and bicarbonate transport during steady-state photosynthesis in the marine cyanobacterium Synechococcus strain PCC7002

Net O₂ evolution, gross CO₂ uptake and net HCO _inf3 ^su– uptake during steady-state photosynthesis were investigated by a recently developed mass-spectrometric technique for disequilibrium flux analysis with cells of the marine cyanobacterium Synechococcus PCC7002 grown at different CO₂ concentrations. Regardless of the CO₂ concentration during growth, all cells had the capacity to transport both CO₂ and HCO _inf3 ^su– ; however, the activity of HCO _inf3 ^su– transport was more than twofold higher than CO₂ transport even in cyanobacteria grown at high concentration of inorganic carbon (C_i = CO₂ + HCO _inf3 ^su– ). In low-C_i cells, the affinities of CO₂ and HCO _inf3 ^su– transport for their substrates were about 5 (CO₂ uptake) and 10 (HCO _inf3 ^su– uptake) times higher than in high-C_i cells, while air-grown cells formed an intermediate state. For the same cells, the intracellular accumulated C_i pool reached 18, 32 and 55 mM in high-C_i, air-grown and low-C_i cells, respectively, when measured at 1 mM external C_i. Photosynthetic O₂ evolution, maximal CO₂ and HCO _inf3 ^su– transport activities, and consequently their relative contribution to photosynthesis, were largely unaffected by the CO₂ provided during growth. When the cells were adapted to freshwater medium, results similar to those for artificial seawater were obtained for all CO₂ concentrations. Transport studies with high-C_i cells revealed that CO₂ and HCO _inf3 ^su– uptake were equally inhibited when CO₂ fixation was reduced by the addition of glycolaldehyde. In contrast, in low-C_i cells steady-state CO₂ transport was preferably reduced by the same inhibitor. The inhibitor of carbonic anhydrase ethoxyzolamide inhibited both CO₂ and HCO _inf3 ^su– uptake as well as O₂ evolution in both cell types. In high-C_i cells, the degree of inhibition was similar for HCO _inf3 ^su– transport and O₂ evolution with 50% inhibition occurring at around 1 mM ethoxyzolamide. However, the uptake of CO₂ was much more sensitive to the inhibitor than HCO _inf3 ^su– transport, with an apparent I₅₀ value of around 250 M ethoxyzolamide for CO₂ uptake. The implications of our results are discussed with respect to C_i utilisation in the marine Synechococcus strain.Abbreviations Chl chlorophyll - C_i inorganic carbon (CO₂ + HCO _inf3 ^su– ) - CA carbonic anhydrase - CCM CO₂-concentrating mechanism - EZA ethoxyzolamide - GA glycolaldehyde - K_1/2 concentration required for half-maximal response - Rubisco ribulose-1,5,-bisphosphate carboxylase-oxygenase D.S. is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft (D.F.G.). In addition, we are grateful to Donald A. Bryant, Department of Molecular and Cell Biology and Center of Biomolecular Structure Function, Pennsylvania State University, USA, for sending us the wild-type strain of Synechococcus PCC7002.     

Glycolaldehyde Inhibits CO(2) Fixation in the Cyanobacterium Synechococcus UTEX 625 without Inhibiting the Accumulation of Inorganic Carbon or the Associated Quenching of Chlorophyll a Fluorescence

When studying active CO₂ and HCO₃⁻ transport by cyanobacteria, it is often useful to be able to inhibit concomitant CO₂ fixation. We have found that glycolaldehyde was an efficient inhibitor of photosynthetic CO₂ fixation in Synechococcus UTEX 625. Glycolaldehyde did not inhibit inorganic carbon accumulation due to either active CO₂ or HCO₃⁻ transport. When glycolaldehyde (10 millimolar) was added to rapidly photosynthesizing cells, CO₂ fixation was stopped within 15 seconds. The quenching of chlorophyll a fluorescence remained high (≤ 82% control) when CO₂ fixation was completely blocked by glycolaldehyde. This quenching was relieved upon the addition of a glucose oxidase oxygentrap. This is consistent with our previous finding that q-quenching in the absence of CO₂ fixation was due to O₂ photoreduction. Photosynthetic CO₂ fixation was also inhibited by d,l,-glyceraldehyde but a sixfold higher concentration was required. Glycolaldehyde acted much more rapidly than iodoacetamide (15 seconds versus 300 seconds) and did not cause the onset of net O₂ evolution often observed with iodoacetamide. Glycolaldehyde will be a useful inhibitor when it is required to study CO₂ and HCO₃⁻ transport without the complication of concomitant CO₂ fixation.     

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.     

Massive light-dependent cycling of inorganic carbon between oxygenic photosynthetic microorganisms and their surroundings

Membrane inlet mass spectrometry indicated massive light-dependent cycling of inorganic carbon between the medium and the cells of various phytoplankton species representing the main groups of aquatic primary producers. These included diatoms, symbiotic and free living dinoflagellates, a coccolithophorid, a green alga and filamentous and single cell cyanobacteria. These organisms could maintain an ambient CO₂ concentration substantially above or below that expected at chemical equilibrium with HCO₃ ⁻. The coccolithophorid Emiliania huxleyishifted from net CO₂ uptake to net CO₂ efflux with rising light intensity. Differing responses of CO₂ uptake and CO₂ fixation to changing light intensity supported the notion that these two processes are not compulsorily linked. Simultaneous measurements of CO₂ and O₂ exchange and of the fluorescence parameters in Synechococcus sp. strain PCC 7942, showed that CO₂ uptake can serve as a sensitive probe of the energy status of the photosynthetic reaction centers. However, during transitions in light intensity, changes in CO₂ uptake did not accord with those expected from fluorescence change. Quantification of the net fluxes of CO₂, HCO₃ ⁻ and of photosynthesis at steady-state revealed that substantial HCO₃ ⁻ efflux accompanied CO₂ uptake and fixation in the case of `CO<sub>2</sub> users'. On the other hand, `HCO₃ ⁻ users' were characterized by a rate of net CO₂ uptake below that of CO₂ fixation. The results support the notion that entities associated with the CCM function not only in raising the CO₂ concentration at the site of Rubisco; they may also serve as a means of diminishing photodynamic damage by dissipating excess light energy. This revised version was published online in August 2006 with corrections to the Cover Date.     

自养微生物同化CO_2的分子生态研究及同化碳在土壤中的转化

大气中CO2浓度持续升高和全球气候变暖是亟待解决的重大环境问题。自养微生物在环境中广泛分布,能直接参与CO2的同化,因此研究自养微生物同化CO2的分子生态学机制具有重大的科学意义。以往对自养微生物的研究多针对基因组DNA,从DNA水平揭示了不同生态系统中碳同化自养微生物的种群结构和多样性,但这些微生物在生态系统中的具体功能有待进一步的研究。近年来,随着转录组学研究技术和稳定同位素探针技术(SIP)的发展,自养微生物同化CO2的生态机理研究不断深入,这些研究明确揭示了碳同化自养微生物是河流、湖泊和海洋生态系统中CO2固定作用的驱动者,并新发现了一些具有CO2同化功能的微生物群落。基于国内外有关研究进展,从DNA和RNA水平上对自养微生物同化CO2的分子机理以及稳定同位素探针技术(SIP)在碳同化微生物研究中的应用进行了分析和总结,初步展望了RNA-SIP技术在陆地生态系统碳同化微生物分子生态学研究中的前景。同时,探讨了陆地生态系统同化碳的转化和稳定性机理,以期为深入了解生态系统碳循环过程和应对气候变化提供理论依据。     

Chlorophyll a Fluorescence Yield as a Monitor of Both Active CO(2) and HCO(3) Transport by the Cyanobacterium Synechococcus UTEX 625

Simultaneous measurements have been made of inorganic carbon accumulation (by mass spectrometry) and chlorophyll a fluorescence yield of the cyanobacterium Synechococcus UTEX 625. The accumulation of inorganic carbon by the cells was accompanied by a substantial quenching of chlorophyll a fluorescence. The quenching occurred even when CO₂ fixation was inhibited by iodoacetamide and whether the accumulation of inorganic carbon resulted from either active CO₂ or HCO₃⁻ transport. Measurement of chlorophyll a fluorescence yield of cyanobacteria may prove to be a rapid and convenient means of screening for mutants of inorganic carbon accumulation.     

Carbon Concentration Mechanism(s) in Unicellular Green Algae and Cyanobacteria

Unicellular green algae and cyanobacteria have mechanism(s) to actively concentrate dissolved inorganic carbon (DIC) into the cells, only if they are grown with air levels of CO₂. The DIC concentration mechanisms are environmental adaptations to actively transport and accumulate inorganic carbon into the chloroplasts of green algae or into the carboxysomes of cyanobacteria. The current working model of cyanobacterial carbon concentration mechanism consists of at least two basic components: an active C_i transport system and a Rubisco-rich polyhedral carboxysome. In case of unicellular green algae, the working model for DIC concentration mechanism includes several isoforms of carbonic anhydrase (CA), and ATPase driven active bicarbonate transporters at the plasmalemma and at the inner chloroplast envelopes. In the past twenty years, significant progress has been made in isolating and characterizing the isoforms of carbonic anhydrase. However, active transporters are yet to be characterized. This mini-review summarizes the current status of research on DIC-pumps including its significance and possible application to increase the productivity of plants of economic importance.     

Variations in short term products of inorganic carbon fixation in exponential and stationary phase cultures of Aphanocapsa 6308

Aphanocapsa 6308 metabolizes both NaHCO₃ and Na₂CO₃. The short term incorporation (5-s) metabolic pattern and the patterns of incorporation of bicarbonate for exponential versus stationary phase cultures differ, however. Cells were equilibrated for 10 min in air and distilled water prior to injection of either NaH¹⁴CO₃ at pH 8.0, or Na₂ ¹⁴CO₃ at pH 11.0. Hot ethanol extracts were analyzed via paper chromatography and autoradiography for products of CO₂ fixation. At 5 s, malate (51.5%) predominates slightly as a primary bicarbonate fixation product over 3-phosphoglycerate (40.3%); 3-phosphoglycerate is the primary product of carbonate fixation. At 60 s, the carbonate and bicarbonate labelling patterns are similar. Cells in stationary phase fix in 5 s a greater proportion of bicarbonate into malate (36% vs. 14% for 3-phosphoglycerate) than do cells in exponential growth. Likewise, 60 s incorporations show a large amount of bicarbonate fixed into aspartate (30.9%) in stationary phase cells over that of exponential phase (11.6%). These data suggest an operative C₄ pathway for purposes not related to carbohydrate synthesis but rather as compensation for the incomplete tricarboxylic acid cycle in cyanobacteria. The enhancement of both aspartate fixation and CO₂ fixation into citrulline in stationary phase correlates with an increase in cyanophycin granule production which requires both aspartate and arginine.Nonstandard Abbreviations 3-PGA 3-phosphoglyceric acid - TCA tricarboxylic acid