Inorganic carbon and pH effect on growth and lipid productivity of Tetraselmis suecica and Chlorella sp (Chlorophyta) grown outdoors in bag photobioreactors

There has been considerable interest in cultivation of green microalgae (Chlorophyta) as a source of lipid that can alternatively be converted to biodiesel. However, almost all mass cultures of algae are carbon-limited. Therefore, to reach a high biomass and oil productivities, the ideal selected microalgae will most likely need a source of inorganic carbon. Here, growth and lipid productivities of Tetraselmis suecica CS-187 and Chlorella sp were tested under various ranges of pH and different sources of inorganic carbon (untreated flue gas from coal-fired power plant, pure industrial CO₂, pH-adjusted using HCl and sodium bicarbonate). Biomass and lipid productivities were highest at pH 7.5 (320?±?29.9 mg biomass L^?1 day^?1and 92?±?13.1 mg lipid L^?1 day^?1) and pH 7 (407?±?5.5 mg biomass L^?1 day^?1 and 99?±?17.2 mg lipid L^?1 day^?1) for T. suecica CS-187 and Chlorella sp, respectively. In general, biomass and lipid productivities were pH 7.5?>?pH 7?>?pH 8?>?pH 6.5 and pH 7?>?pH 7.5?=?pH 8?>?pH 6.5?>?pH 6?>?pH 5.5 for T. suecica CS-187 and Chlorella sp, respectively. The effect of various inorganic carbon on growth and productivities of T. suecica (regulated at pH?=?7.5) and Chlorella sp (regulated at pH?=?7) grown in bag photobioreactors was also examined outdoor at the International Power Hazelwood, Gippsland, Victoria, Australia. The highest biomass and lipid productivities of T. suecica (51.45?±?2.67 mg biomass L^?1 day^?1 and 14.8?±?2.46 mg lipid L^?1 day^?1) and Chlorella sp (60.00?±?2.4 mg biomass L^?1 day^?1 and 13.70?±?1.35 mg lipid L^?1 day^?1) were achieved when grown using CO₂ as inorganic carbon source. No significant differences were found between CO₂ and flue gas biomass and lipid productivities. While grown using CO₂ and flue gas, biomass productivities were 10, 13 and 18 %, and 7, 14 and 19 % higher than NaHCO₃, HCl and unregulated pH for T. suecica and Chlorella sp, respectively. Addition of inorganic carbon increased specific growth rate and lipid content but reduced biomass yield and cell weight of T. suecica. Addition of inorganic carbon increased yield but did not change specific growth rate, cell weight or content of the cell weight of Chlorella sp. Both strains showed significantly higher maximum quantum yield (F_v/F_m) when grown under optimum pH.     

Utilization of gaseous emissions from a methanol plant for cultivation of <Emphasis Type="Italic">Acutodesmus obliquus</Emphasis>

This study aimed to culture the green alga Acutodesmus obliquus utilizing the gaseous emissions containing a high concentration of CO₂ (99.13 %) from a methanol plant and study the tolerance of microalgae. The effect of CO₂ concentration, aeration rate, inoculum concentration, intermittent sparging, and nitrogen sources on the growth of A. obliquus was examined. Acutodesmus obliquus also was cultivated in a 500-L pilot outdoor tubular photobioreactor (OTP) to advance the laboratory scale system to outdoor scale-up applications. The results showed that A. obliquus could tolerate high CO₂ concentrations of 50 %, and a maximum biomass of 0.935 g L^?1 (dry weight) was achieved at 20 % CO₂. An aeration rate of 500 mL min^?1, inoculum concentration (optical density at 680 nm [OD₆₈₀]?=?0.3), and intermittent sparging of 10 min per 2 h enhanced growth to the optimum and influenced culture pH and photosynthesis. Urea as a nitrogen source was shown to be more beneficial to cell growth. A urea concentration of 0.3 g L^?1 and an N/P ratio of 15 led to maximum biomass accumulation thus enhancing the gaseous emission utilization efficiency. In conclusion, this work demonstrated that gaseous emissions containing high concentration of CO₂ from a methanol plant could be directly introduced into A. obliquus cultures and that A. obliquus was suitable well for large-scale outdoor cultivation in a tubular photobiorecator.     

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.     

Use of sodium bicarbonate to stimulate triacylglycerol accumulation in the chlorophyte Scenedesmus sp. and the diatom Phaeodactylum tricornutum

There is potential for algal-derived biofuel to help alleviate part of the world’s dependency on petroleum based fuels. However, research must still be done on strain selection, induction of triacylglycerol (TAG) accumulation, and fundamental algal metabolic studies, along with large-scale culturing techniques, harvesting, and biofuel/biomass processing. Here, we have advanced the knowledge on Scenedesmus sp. strain WC-1 by monitoring growth, pH, and TAG accumulation on a 14:10 light–dark cycle with atmospheric air or 5% CO₂ in air (v/v) aeration. Under ambient aeration, there was a loss of pH-induced TAG accumulation, presumably due to TAG consumption during the lower culture pH observed during dark hours (pH 9.4). Under 5% CO₂ aeration, the growth rate nearly doubled from 0.78 to 1.53 d^?1, but the pH was circumneutral (pH 6.9) and TAG accumulation was minimal. Experiments were also performed with 5% CO₂ during the exponential growth phase, which was then switched to aeration with atmospheric air when nitrate was close to depletion. These tests were run with and without the addition of 50 mM sodium bicarbonate. Cultures without added bicarbonate showed decreased growth rates with the aeration change, but there was no immediate TAG accumulation. The cultures with bicarbonate added immediately ceased cellular replication and rapid TAG accumulation was observed, as monitored by Nile Red fluorescence which has previously been correlated by gas chromatography to cellular TAG levels. Sodium bicarbonate addition (25 mM final concentration) was also tested with the marine diatom Phaeodactylum tricornutum strain Pt-1 and this organism also accumulated TAG.     

Mechanisms of inorganic carbon acquisition in two estuarine Rhodophyceans: Bostrychia scorpioides (Hudson) ex Kützing Montagne and Catenella caespitosa (Withering) L. M. Irvine

Marine macroalgae possess a range of mechanisms to increase the availability of CO₂ for fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase. Of these, possession of a periplasmic or external carbonic anhydrase and the ability to use bicarbonate ions is widely distributed. The mechanisms of carbon acquisition were studied in two estuarine red macroalgae Bostrychia scorpioides and Catenella caespitosa using a range of techniques. pH-drift and CO₂-depletion experiments at constant pH suggested that CO₂ is the main source of inorganic carbon in both species. Inhibitors indicated that internal and external carbonic anhydrase were present in both species. Inhibitors also suggested that uptake of bicarbonate is unlikely to be present (P < 0.05).     

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     

Inorganic Carbon Limitation and Chemical Composition of Two Freshwater Green Microalgae

Two freshwater chlorophytes, Chlorella vulgaris and Scenedesmus obliquus, were grown in inorganic carbon-limited continuous cultures in which HCO₃⁻ was the sole source of inorganic carbon. The response of the steady-state growth rate to the external total inorganic carbon concentration was reasonably well described by the Monod equation; however, the response to the internal nutrient concentration was only moderately well represented by the Droop equation when the internal carbon concentration was defined on a cellular basis. The Droop equation was totally inapplicable when total biomass (dry weight) was used to define internal carbon because the ratio of carbon to dry weight did not vary over the entire growth rate spectrum. In batch cultures, maximum growth rates were achieved at the CO₂ levels present in atmospheric air and at HCO₃⁻ concentrations of 3 mM. No growth was observed at 100% CO₂. Both nitrogen uptake and chlorophyll synthesis were tightly coupled to carbon assimilation, as indicated by the constant C/N and C/chlorophyll ratios found at all growth rates. The main influence of inorganic carbon limitation appears to be not on the chemical structure of the biomass, but rather on cell size; higher steady-state growth rates lead to bigger cells.     

Vertical thin-layer photoreactor for controlled cultivation of cyanobacteria

Nostoc sp. was cultivated in an air-lift reactor with continuous recirculation of the head gas phase that aerated and agitated the cyanobacterial suspension at regulated flow rates. The supply of inorganic carbon for growth was coupled with pH control, in the range of 7.7 to 8.1, by intermittent sparging of CO₂-head gas mixtures. The formation of irregular bubbles with swirling motion at the photostage of the reactor promoted efficient CO₂ transference in dense populations of Nostoc sp. (1.1 g/l) when bubbling at flow rates of 10 l/min. Biomass productivity was almost six-fold higher in the photoreactor (16.4 mg/l.h) than in a conventional system (2.8 mg/l.h). The exponential growth phase of cultures in the photoreactor amounted to 60% of the total growth period.The authors are with the Laboratorio de Alimentos, Area Microbiologia, Facultad de Quimica Bioquimica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina     

The effect of spring water on the growth of a submerged macrophyte Egeria densa

We elucidated the effect of spring water on the growth of Egeria densa Planch., a widespread submerged macrophyte in Japan. We observed the longitudinal distributions of physical (water temperature, particle diameter of the bed sediment, sediment layer thickness, etc.), chemical [pH, dissolved oxygen (DO), dissolved inorganic carbon (DIC), phosphate (PO₄-P), total nitrogen (TN), and total phosphorus (TP) content of the sediment, etc.], and biological (species composition, biomass, and growth rate) factors related to E. densa in the Kurohashi River, a spring-fed stream flowing into the Lake Biwa. It was found that E. densa growth rate from summer to autumn was negatively correlated to pH and DO, which implies that the low pH spring water increases the growth rate of the species. The growth rate was also positively correlated to the free carbon dioxide (CO₂) concentration (r = 0.67, p = 0.02). These results indicate that the low pH spring water increases E. densa growth rate by affecting free CO₂ concentration in water.     

Inorganic-carbon uptake by a small-celled strain of Stichococcus bacillaris

Air-grown cells of a marine, small-celled (2 m diameter) strain of Stichococcus bacillaris contained appreciable carbonic-anhydrase activity but this was repressed when cells were grown on air enriched with 5% (v/v) CO₂. Assay of carbonic-anhydrase activity using intact cells and cell extracts showed all activity was intracellular in this Stichococcus strain. Measurement of inorganic-carbon-dependent photosynthetic O₂ evolution at pH 5.0, where CO₂ is the predominant form of inorganic carbon, showed that the concentration of inorganic carbon required for half-maximal rate of photosynthetic O₂ evolution [K_0.5(CO₂)] was 4.0 M for both air- and CO₂-grown cells. At pH 8.3 the K_0.5(CO₂) was 0.3 mM for air-grown and 0.6 mM for CO₂-grown cells. Sodium ions did not enhance bicarbonate utilization. Measurement of the internal inorganic-carbon pool (HCO ₃ ^– +CO₂) by the silicone-oil-layer centrifugal filtering technique showed that air- and CO₂-grown cells were able to concentrate inorganic carbon up to 20-fold in relation to the external medium at pH 5.0 but not at pH 8.3. In this alga the high affinity for CO₂ and inorganic-carbon accumulation in CO₂- and air-grown cells results from active CO₂ transport that is not dependent on carbonic-anhydrase activity.Abbreviation Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

Effects of salinity on the CO<Subscript>2</Subscript> permeation across lipid bilayer for microalgae biofixation: a molecular dynamics study

The continuous threat of increasing CO₂ concentration in the atmosphere has altered the carbon balance of our planet causing global climate change. Biological fixation of atmospheric CO₂ by unicellular microorganisms such as microalgae is a promising technology pursued extensively by researchers as a means for carbon capture. The study aimed to provide an atomic level of study that will demonstrate the effect of the salinity on the mechanism of CO₂ absorption across microalgae lipid bilayer. Molecular dynamics simulations were utilized to calculate the free energies of CO₂ molecule as it permeates inside the microalgae cell. In thermodynamics, the transport process of a molecule can be demonstrated through its free energy gradient. Thus, calculating the free energies of CO₂ molecule across microalgae lipid bilayer can elucidate the mechanisms of permeation processes. Four microalgae lipid bilayer structures were constructed that contains 128-DPPC (dipalmitoylphosphatidylcholine) lipid bilayer with 3640 water molecules with different NaCl concentrations: 0, 3, 13, and 19 NaCl molecules which correspond to a salinity level of 0, 50, 200, and 300 mM, respectively. The cavity insertion Widom method was used to calculate the free energy of CO₂ molecule along the lipid bilayer. The results demonstrated that the salinity does not affect the free energies significantly, thus, it does not hamper CO₂ transport across microalgae lipid membrane.     

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     

Effects of carbon and nitrogen sources on fatty acid contents and composition in the green microalga, Chlorella sp. 227

In order to investigate and generalize the effects of carbon and nitrogen sources on the growth of and lipid production in Chlorella sp. 227, several nutritional combinations consisting of different carbon and nitrogen sources and concentrations were given to the media for cultivation of Chlorella sp. 227, respectively. The growth rate and lipid content were affected largely by concentration rather than by sources. The maximum specific growth was negatively affected by low concentrations of carbon and nitrogen. There is a maximum allowable inorganic carbon concentration (less than 500~1,000 mM bicarbonate) in autotrophic culture, but the maximum lipid content per gram dry cell weight (g DCW) was little affected by the concentration of inorganic carbon within the concentration. The lipid content per g DCW was increased when the microalga was cultured with the addition of glucose and bicarbonate (mixotrophic) at a fixed nitrogen concentration and with the lowest nitrogen concentration (0.2 mM), relatively. Considering that lipid contents per g DCW increased in those conditions, it suggests that a high ratio of carbon to nitrogen in culture media promotes lipid accumulation in the cells. Interestingly, a significant increase of the oleic acid amount to total fatty acids was observed in those conditions. These results showed the possibility to induce lipid production of high quality and content per g DCW by modifying the cultivation conditions.     

Regulation of carbon metabolic fluxes in response to CO<Subscript>2</Subscript> supplementation in phototrophic <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>: a cytomic and biochemical study

As one of the promising species of microalgae for biofuel production, Chlorella vulgaris CS-42 was cultivated phototrophically in two cylindrical photobioreactors with aeration of 5 % (v/v) CO₂ or air for 13 days to evaluate the effects of CO₂ supplementation on biomass, CO₂ fixation performance, and biochemical content. Significant increases of specific growth rate and total carbon content in biomass resulting in a higher CO₂ fixation rate were found with 5 % CO₂. The maximum biomass concentration, carbohydrate and fatty acid contents with 5 % CO₂ were significantly higher than those with air, while carbohydrate biosynthesis was most affected as compared to other biochemical components. Cytomic analysis revealed a rapid accumulation of neutral lipid in the late growth phase with more lipid bodies visualized by confocal laser scanning microscopy (CLSM), when nitrate consumption was accelerated with CO₂ supplementation. Gas chromatography mass spectrometry (GC-MS) analysis indicated that 5 % CO₂ favored the formation of C18:2, which led to a decrease in the degree of lipid unsaturation (DLU). These results proved that CO₂ supplementation was one of the most efficient methods to significantly prompt the growth of microalgae and increase the C/N ratio in the medium, which in turn regulated the carbon metabolic flux to enhance neutral lipid and fatty acid production in C. vulgaris.     

On-line stable isotope gas exchange reveals an inducible but leaky carbon concentrating mechanism in Nannochloropsis salina

Carbon concentrating mechanisms (CCMs) are common among microalgae, but their regulation and even existence in some of the most promising biofuel production strains is poorly understood. This is partly because screening for new strains does not commonly include assessment of CCM function or regulation despite its fundamental role in primary carbon metabolism. In addition, the inducible nature of many microalgal CCMs means that environmental conditions should be considered when assessing CCM function and its potential impact on biofuels. In this study, we address the effect of environmental conditions by combining novel, high frequency, on-line ¹³CO₂ gas exchange screen with microscope-based lipid characterization to assess CCM function in Nannochloropsis salina and its interaction with lipid production. Regulation of CCM function was explored by changing the concentration of CO₂ provided to continuous cultures in airlift bioreactors where cell density was kept constant across conditions by controlling the rate of media supply. Our isotopic gas exchange results were consistent with N. salina having an inducible “pump-leak” style CCM similar to that of Nannochloropsis gaditana. Though cells grew faster at high CO₂ and had higher rates of net CO₂ uptake, we did not observe significant differences in lipid content between conditions. Since the rate of CO₂ supply was much higher for the high CO₂ conditions, we calculated that growing cells bubbled with low CO₂ is about 40 % more efficient for carbon capture than bubbling with high CO₂. We attribute this higher efficiency to the activity of a CCM under low CO₂ conditions.     

Acclimation of photosynthesis to supersaturated CO2 in aquatic plant bicarbonate users

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Characteristics of light-dependent inorganic carbon uptake by isolated spinach chloroplasts

The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach (Spinacia oleracea L.) chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5- to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5°C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [¹⁴C]dimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and ¹⁴CO₂ bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO₂ in the chloroplast.     

In vitro and in vivo analyses of the role of the carboxysomal β-type carbonic anhydrase of the cyanobacterium Synechococcus elongatus in carboxylation of ribulose-1,5-bisphosphate

The carboxylase activities of crude carboxysome preparations obtained from the wild-type Synechococcus elongatus strain PCC 7942 strain and the mutant defective in the carboxysomal carbonic anhydrase (CA) were compared. The carboxylation reaction required high concentrations of bicarbonate and was not even saturated at 50 mM bicarbonate. With the initial concentrations of 50 mM and 25 mM for bicarbonate and ribulose-1,5-bisphosphate (RuBP), respectively, the initial rate of RuBP carboxylation by the mutant carboxysome (0.22 μmol mg^?1 protein min^?1) was only 30 % of that observed for the wild-type carboxysomes (0.71 μmol mg^?1 protein min^?1), indicating the importance of the presence of CA in efficient catalysis by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the mutant defective in the ccmLMNO genes, which lacks the carboxysome structure, could grow under aeration with 2 % (v/v) CO₂ in air, the mutant defective in ccaA as well as ccmLMNO required 5 % (v/v) CO₂ for growth, indicating that the cytoplasmically localized CcaA helped utilization of CO₂ by the cytoplasmically localized Rubisco by counteracting the action of the CO₂ hydration mechanism. The results predict that overexpression of Rubisco would hardly enhance CO₂ fixation by the cyanobacterium at CO₂ levels lower than 5 %, unless Rubisco is properly organized into carboxysomes.     

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.