Leishmania donovani: correlation among assays of amastigote viability

The viability of Leishmania donovani amastigotes was evaluated using two in vitro assays: the initiation of differentiation of viable cells toward the promastigote stage in tissue culture Medium 199 and the staining of nonviable amastigotes with erythrosin B. The results of these assays correlated with those of a previously described in vivo assay in which viability was related to the minimum number of amastigotes in mouse liver following intravenous injection. Results indicate that erythrosin B staining should be routinely used to evaluate viability of amastigote populations, but that the initiation of differentiation of amastigotes is a more sensitive assay. For best results with the latter method, it is suggested that no more than 1.2 × 10⁷ amastigotes in 1 ml of Medium 199 be incubated at 25 C in 5% CO₂ in air. The data presented indicate that routine manipulations may affect amastigote viability.     

A micromethod for the quantitative determination of the viability of Candida albicans hyphae

A micromethod for the quantitative determination of the viability of Candida albicans hypae was devised which takes advantage of the dimorphic nature of C. albicans which grows exclusively in the yeast form when incubated aerobically on Sabouraud dextrose agar at 30°C. When tested by thisd method, all viable, C. albicans hyphae were recognized as microcolonies consisting of one hypha surrounded by several yeast form progeny. In contrast to this, no yeast form progeny emerged from nonviable hypae. By counting appropriate total numbers (200–400) of microcolony-forming hypae and infertile hyphae, it was possible to determine the ratio of viable to nonviable cells in a given hyphal suspension. This micromethod may be used for quantitative assessment of the candidacidal effects of various antimycotic agents or phagocytes C. albicans hyphae whose viability could not have been determined by the conventional plating technique because of the species' high propensity to clump.     

Carbon allocation and element composition in four Chlamydomonas mutants defective in genes related to the CO2 concentrating mechanism

Four mutants of Chlamydomonas reinhardtii with defects in different components of the CO₂ concentrating mechanism (CCM) or in Rubisco activase were grown autotrophically at high pCO₂ and then transferred to low pCO₂, in order to study the role of different components of the CCM on carbon allocation and elemental composition. To study carbon allocation, we measured the relative size of the main organic pools by Fourier Transform Infrared spectroscopy. Total reflection X-ray fluorescence was used to analyze the elemental composition of algal cells. Our data show that although the organic pools increased their size at high CO₂ in all strains, their stoichiometry was highly homeostatic, i.e., the ratios between carbohydrates and proteins, lipid and proteins, and carbohydrates and lipids, did not change significantly. The only exception was the wild-type 137c, in which proteins decreased relative to carbohydrates and lipids, when the cells were transferred to low CO₂. It is noticeable that the two wild types used in this study responded differently to the transition from high to low CO₂. Malfunctions of the CCM influenced the concentration of several elements, somewhat altering cell elemental stoichiometry: especially the C/P and N/P ratios changed appreciably in almost all strains as a function of the growth CO₂ concentration, except in 137c and the Rubisco activase mutant rca1. In strain cia3, defective in the lumenal carbonic anhydrase (CA), the cell quotas of P, S, Ca, Mn, Fe, and Zn were about 5-fold higher at low CO₂ than at high CO₂. A Principle Components Analysis showed that, mostly because of its elemental composition, cia3 behaved in a substantially different way from all other strains, at low CO₂. The lumenal CA thus plays a crucial role, not only for the correct functioning of the CCM, but also for element utilization. Not surprisingly, growth at high CO₂ attenuated differences among strains.     

Comparative Analysis and Limitations of Ethidium Monoazide and Propidium Monoazide Treatments for the Differentiation of Viable and Nonviable Campylobacter Cells

The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (C_T) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R²) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 μg/ml) and PMA (51.10 μg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>10⁴) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.     

Internal Inorganic Carbon Pool of Chlamydomonas reinhardtii: EVIDENCE FOR A CARBON DIOXIDE-CONCENTRATING MECHANISM

The external inorganic carbon pool (CO₂ + HCO₃⁻) was measured in both high and low CO₂-grown cells of Chlamydomonas reinhardtii, using a silicone oil layer centrifugal filtering technique. The average internal pH values were measured for each cell type using [¹⁴C]dimethyloxazolidinedione, and the internal inorganic carbon pools were recalculated on a free CO₂ basis. These measurements indicated that low CO₂-grown cells were able to concentrate CO₂ up to 40-fold in relation to the external medium. Low and high CO₂-grown cells differed in their photosynthetic affinity for external CO₂. These differences could be most readily explained as being due to the relative CO₂-concentrating capacity of each cell type. This physiological adaptation appeared to be based on changes in the abilities of the cells actively to accumulate inorganic carbon using an energy-dependent transport system.     

Regulation of Caffeate Respiration in the Acetogenic Bacterium Acetobacterium woodii

The anaerobic acetogenic bacterium Acetobacterium woodii can conserve energy by oxidation of various substrates coupled to either carbonate or caffeate respiration. We used a cell suspension system to study the regulation and kinetics of induction of caffeate respiration. After addition of caffeate to suspensions of fructose-grown cells, there was a lag phase of about 90 min before caffeate reduction commenced. However, in the presence of tetracycline caffeate was not reduced, indicating that de novo protein synthesis is required for the ability to respire caffeate. Induction also took place in the presence of CO₂, and once a culture was induced, caffeate and CO₂ were used simultaneously as electron acceptors. Induction of caffeate reduction was also observed with H₂ plus CO₂ as the substrate, but the lag phase was much longer. Again, caffeate and CO₂ were used simultaneously as electron acceptors. In contrast, during oxidation of methyl groups derived from methanol or betaine, acetogenesis was the preferred energy-conserving pathway, and caffeate reduction started only after acetogenesis was completed. The differential flow of reductants was also observed with suspensions of resting cells in which caffeate reduction was induced prior to harvest of the cells. These cell suspensions utilized caffeate and CO₂ simultaneously with fructose or hydrogen as electron donors, but CO₂ was preferred over caffeate during methyl group oxidation. Caffeate-induced resting cells could reduce caffeate and also p-coumarate or ferulate with hydrogen as the electron donor. p-Coumarate or ferulate also served as an inducer for caffeate reduction. Interestingly, caffeate-induced cells reduced ferulate in the absence of an external reductant, indicating that caffeate also induces the enzymes required for oxidation of the methyl group of ferulate.     

Hypercapnia slows down proliferation and apoptosis of human bone marrow promyeloblasts

Stem cells are being applied in increasingly diverse fields of research and therapy; as such, growing and culturing them in scalable quantities would be a huge advantage for all concerned. Gas mixtures containing 5 % CO₂ are a typical concentration for the in vitro culturing of cells. The effect of varying the CO₂ concentration on promyeloblast KG-1a cells was investigated in this paper. KG-1a cells are characterized by high expression of CD34 surface antigen, which is an important clinical surface marker for human hematopoietic stem cells (HSCs) transplantation. KG-1a cells were cultured in three CO₂ concentrations (1, 5 and 15 %). Cells were batch-cultured and analyzed daily for viability, size, morphology, proliferation, and apoptosis using flow cytometry. No considerable differences were noted in KG-1a cell morphological properties at all three CO₂ levels as they retained their myeloblast appearance. Calculated population doubling time increased with an increase in CO₂ concentration. Enhanced cell proliferation was seen in cells cultured in hypercapnic conditions, in contrast to significantly decreased proliferation in hypocapnic populations. Flow cytometry analysis revealed that apoptosis was significantly (p = 0.0032) delayed in hypercapnic cultures, in parallel to accelerated apoptosis in hypocapnic ones. These results, which to the best of our knowledge are novel, suggest that elevated levels of CO₂ are favored for the enhanced proliferation of bone marrow (BM) progenitor cells such as HSCs.     

Evaluation of Fourier-transform infrared spectroscopy for data capture in predictive microbiology

Chemical changes in the medium, induced by the fermentative species Lactobacillus plantarum and Lactobacillus brevis and by the enzymatic action of a proteolytic, spoilage species, Yarrowia lipolytica, were analysed using Fourier-transform i.r. spectroscopy (FTIR). Changes in the absorbance data over time could be modelled using one of the more current predictive, mathematical models of microbial growth, such as the Gompertz equation. Moreover, a linear correlation between FTIR data (expressed as absorbance of some selected peaks) and viability data (expressed as log₁₀ c.f.u./g or ml) was observed during the fermentation process, both for L. plantarum and L. brevis.     

A 36 Kilodalton Limiting-CO(2) Induced Polypeptide of Chlamydomonas Is Distinct from the 37 Kilodalton Periplasmic Carbonic Anhydrase

Chlamydomonas reinhardtii possesses a CO₂-concentrating mechanism, induced by limiting CO₂, which involves active transport and accumulation of inorganic carbon within the cell. Synthesis of several proteins is induced by limiting CO₂, but, of those, only periplasmic carbonic anhydrase has an identified function in the system. No proteins involved in active transport have yet been identified, but induced, membrane-associated polypeptides, such as the 36 kilodalton polypeptide focused on in this paper, would seem to be candidates for such involvement. The 36 kilodalton polypeptide was shown to be synthesized de novo upon transfer of cells to limiting CO₂. It was purified using SDS-PAGE and used to produce polyclonal antibodies. Antibodies were used to confirm the air-specific nature of the polypeptide, its strict association with membrane fractions, and the time course of its induction. Using the antibodies, a single, 36 kilodalton polypeptide was found to be specifically immunoprecipitated from in vitro translation products of poly(A⁺) RNA from cells only after exposure to limiting CO₂. The absence of translatable mRNA for this polypeptide in CO₂-enriched cells indicated that regulation occurs at the level of message abundance. The antibodies were also used to demonstrate the distinction between the limiting-CO₂ induced 36 kilodalton polypeptide and the similarly sized, limiting-CO₂ induced periplasmic carbonic anhydrase.     

Rapid isolation of mesophyll cells from leaves of soybean for photosynthetic studies

Mesophyll cells were rapidly isolated from soybean (Glycine max [L.]) leaves using a combined Macerase enzyme-stirring technique. About 50% to 70% of the leaf cells on a chlorophyll basis from 3 grams of leaves could be isolated in 15 minutes. The cells obtained by this method were capable of high rates of photosynthesis even after storage in the dark for periods of up to 9 hours. The CO₂-saturated rate of photosynthesis increased from 5 μm CO₂/mg Chl·hour at 5 C to 170 μm CO₂/mg Chl·hour at 40 C. At atmospheric CO₂ concentration, the rate varied from 5 to 55 μm CO₂/mg Chl·hour over this temperature range. The reduced temperature response of photosynthesis at low CO₂ concentration was due to an increased Km(CO₂) of the cells with increasing temperature. The products of photosynthesis in the isolated cells were similar to the products of leaf photosynthesis.     

Photorespiration in Air and High CO(2)-Grown Chlorella pyrenoidosa

Oxygen inhibition of photosynthesis and CO₂ evolution during photorespiration were compared in high CO₂-grown and air-grown Chlorella pyrenoidosa, using the artificial leaf technique at pH 5.0. High CO₂ cells, in contrast to air-grown cells, exhibited a marked inhibition of photosynthesis by O₂, which appeared to be competitive and similar in magnitude to that in higher C₃ plants. With increasing time after transfer to air, the photosynthetic rate in high CO₂ cells increased while the O₂ effect declined. Photorespiration, measured as the difference between ¹⁴CO₂ and ¹²CO₂ uptake, was much greater and sensitive to O₂ in high CO₂ cells. Some CO₂ evolution was also present in air-grown algae; however, it did not appear to be sensitive to O₂. True photosynthesis was not affected by O₂ in either case. The data indicate that the difference between high CO₂ and air-grown algae could be attributed to the magnitude of CO₂ evolution. This conclusion is discussed with reference to the oxygenase reaction and the control of photorespiration in algae.     

Maintenance of High Photosynthetic Rates in Mesophyll Cells Isolated from Papaver somniferum

The establishment and maintenance of high rates of photosynthetic CO₂ incorporation in mesophyll cells of Papaver somniferum (opium poppy) depend on a regime of dark and light periods immediately following isolation, as well as carefully adjusted conditions of isolation. Analysis of the incorporation pattern of ¹⁴CO₂ by the isolated cells indicates an initial “stress-response” period of approximately 20 hours characterized by increased respiratory-type metabolism and diminished photosynthesis. Under the favorable regime, this period is followed by rapid recovery and the reinstatement of a metabolic state strikingly similar to that of intact leaves in which the initial rate of CO₂ incorporation is between 110 and 175 μmoles CO₂ fixed per mg chlorophyll per hour. The photosynthetic viability of these cells can be maintained for up to 80 hours.     

Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans

Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-¹⁴C] pyruvate, and chemically synthesized O-[methyl-¹⁴C]vanillate were used to further investigate the catabolism of one-carbon (C₁) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO₂ was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO₂ readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO₂ and pyruvate predominantly labeled the carboxyl group of acetate. No CO₂ was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate.     

Gas Exchange Characteristics of the Submerged Aquatic Crassulacean Acid Metabolism Plant, Isoetes howellii

The submerged aquatic plant Isoetes howellii Engelmann possesses Crassulacean acid metabolism (CAM) comparable to that known from terrestrial CAM plants. Infrared gas analysis of submerged leaves showed Isoetes was capable of net CO₂ uptake in both light and dark. CO₂ uptake rates were a function of CO₂ levels in the medium. At 2,500 microliters CO₂ per liter (gas phase, equivalent to 1.79 milligrams per liter aqueous phase), Isoetes leaves showed continuous uptake in both the light and dark. At this CO₂ level, photosynthetic rates were light saturated at about 10% full sunlight and were about 3-fold greater than dark CO₂ uptake rates. In the dark, CO₂ uptake rates were also a function of length of time in the night period. Measurements of dark CO₂ uptake showed that, at both 2,500 and 500 microliters CO₂ per liter, rates declined during the night period. At the higher CO₂ level, dark CO₂ uptake rates at 0600 h were 75% less than at 1800 h. At 500 microliters CO₂ per liter, net CO₂ uptake in the dark at 1800 h was replaced by net CO₂ evolution in the dark at 0600 h. At both CO₂ levels, the overnight decline in net CO₂ uptake was marked by periodic bursts of accelerated CO₂ uptake. CO₂ uptake in the light was similar at 1% and 21% O₂, and this held for leaves intact as well as leaves split longitudinally. Estimating the contribution of light versus dark CO₂ uptake to the total carbon gain is complicated by the diurnal flux in CO₂ availability under field conditions.     

Photosynthetic oxygen exchange in isolated cells and chloroplasts of c(3) plants

Photosynthetic O₂-production and photorespiratory O₂-uptake were measured, using stable isotope techniques, in isolated intact leaf cells of the C₃ plant Xanthium strumarium L., and isolated intact chloroplasts of Spinacia oleracea L (var. Yates 102). Considerable light dependent O₂-uptake was observed in both systems, a proportion of which could be suppressed by CO₂ (63% suppression in chloroplasts by 50 micromolar CO₂, 58% in cells by 100 micromolar CO₂ and 250 micromolar O₂). At low O₂, O₂-uptake was CO₂ insensitive. At high CO₂ up to 19% of total electron flow was to O₂ in cells and up to 14% in chloroplasts. O₂-uptake showed inhibition by KCN (61% in cells, 35% in chloroplasts by 0.2 millimolar KCN). O₂-uptake half saturated at 75 to 85 micromolar O₂ in cells and 50 to 65 micromolar O₂ in chloroplasts, at low CO₂. The results are discussed in terms of the RuP₂-oxygenase reaction and direct photoreduction of O₂ via a Mehler reaction.     

Structural evaluation of sugar cane bagasse steam pretreated in the presence of CO2 and SO2

Background

Previous studies on the use of SO₂ and CO₂ as impregnating agent for sugar cane bagasse steam treatment showed comparative and promising results concerning the cellulose enzymatic hydrolysis and the low formation of the inhibitors furfural and hydroxymethylfurfural for the use of CO₂ at 205°C/15 min or SO₂ at 190°C/5 min. In the present study sugar cane bagasse materials pretreated as aforementioned were analyzed by scanning and transmission electron microscopy (SEM and TEM), X-Ray Diffraction (XRD) and Infrared (FTIR spectroscopy) aiming a better understanding of the structural and chemical changes undergone by the pretreated materials.

Results

SEM and TEM data showed that the structural modifications undergone by the pretreatment with CO₂ were less pronounced in comparison to that using SO_2, which can be directly related to the combined severity of each pretreatment. According to XRD data, untreated bagasse showed, as expected, a lower crystallinity index (CI = 48.0%) when compared to pretreated samples with SO₂ (CI = 65.5%) or CO₂ (CI = 56.4%), due to the hemicellulose removal of 68.3% and 40.5%, respectively. FTIR spectroscopy supported SEM, TEM and XRD results, revealing a more extensive action of SO₂.

Conclusions

The SEM, TEM, XRD and FTIR spectroscopy techniques used in this work contributed to structural and chemical analysis of the untreated and pretreated bagasse. The images from SEM and TEM can be related to the severity of SO₂ pretreatment, which is almost twice higher. The crystallinity index values obtained from XRD showed that pretreated materials have higher values when compared with untreated material, due to the partial removal of hemicellulose after pretreatment. FTIR spectroscopy supported SEM, TEM and XRD results. CO₂ can actually be used as impregnating agent for steam pretreatment, although the present study confirmed a more extensive action of SO₂.     

Carbon Dioxide and Nisin Act Synergistically on Listeria monocytogenes

This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nis^r) cells grown in broth at 4°C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nis^r cells. Wild-type cells grown in 100% CO₂ were two to five times longer than cells grown in air. Nisin (2.5 μg/ml) did not decrease the viability of Nis^r cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO₂. There was a quantifiable synergistic action between nisin and CO₂ in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 μg of nisin per ml increased from 3.1 to 12.5 μg/ml over 35 days, but this increase was markedly delayed for cultures in CO₂. This synergism between nisin and CO₂ was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO₂ and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO₂ atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4°C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30°C. Cells grown in the presence of 100% CO₂ and those grown at 4°C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO₂ is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.     

In situ observation of a cell adhesion and metabolism using surface infrared spectroscopy

In this study, we report on an in situ monitoring system of living cultured cells using infrared absorption spectroscopy in the geometry of multiple internal reflections (MIR-IRAS). In order to observe living cultured cells, the temperature in the sample chamber of a FT-IR spectrometer was maintained at 37 °C and a humidified gas mixture containing 5% CO₂ was introduced into the sample chamber. Human breast cell line MCF-7 cultured on Si MIR prisms were placed in the sample chamber and infrared spectra of MCF-7 cells were collected for 5 h. It was found that the adhesion and metabolism of MCF-7 cells could be monitored by the absorption intensity of amide-II protein band (1,545 cm⁻¹) and also by the absorption intensities of CH _x bands (2,700–3,100 cm⁻¹). These results suggest that our system is useful for a nondestructive and non-label monitoring of cell viability. Our method based on infrared absorption spectroscopy has a potential for bioscreening application.     

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas

Inorganic carbon (C_i) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO₂. Both air-grown cells, that have a CO₂ concentrating system, and 5% CO₂-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C_i) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO₂-grown cells also accumulated some C_i, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO₂ fixation by high CO₂-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO₂-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.     

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.