Responses of macroalgae to CO2 enrichment cannot be inferred solely from their inorganic carbon uptake strategy

Increased plant biomass is observed in terrestrial systems due to rising levels of atmospheric CO₂, but responses of marine macroalgae to CO₂ enrichment are unclear. The 200% increase in CO₂ by 2100 is predicted to enhance the productivity of fleshy macroalgae that acquire inorganic carbon solely as CO₂ (non‐carbon dioxide‐concentrating mechanism [CCM] species—i.e., species without a carbon dioxide‐concentrating mechanism), whereas those that additionally uptake bicarbonate (CCM species) are predicted to respond neutrally or positively depending on their affinity for bicarbonate. Previous studies, however, show that fleshy macroalgae exhibit a broad variety of responses to CO₂ enrichment and the underlying mechanisms are largely unknown. This physiological study compared the responses of a CCM species (Lomentaria australis) with a non‐CCM species (Craspedocarpus ramentaceus) to CO₂ enrichment with regards to growth, net photosynthesis, and biochemistry. Contrary to expectations, there was no enrichment effect for the non‐CCM species, whereas the CCM species had a twofold greater growth rate, likely driven by a downregulation of the energetically costly CCM(s). This saved energy was invested into new growth rather than storage lipids and fatty acids. In addition, we conducted a comprehensive literature synthesis to examine the extent to which the growth and photosynthetic responses of fleshy macroalgae to elevated CO₂ are related to their carbon acquisition strategies. Findings highlight that the responses of macroalgae to CO₂ enrichment cannot be inferred solely from their carbon uptake strategy, and targeted physiological experiments on a wider range of species are needed to better predict responses of macroalgae to future oceanic change.     

Synthetic DNA system for structure-function studies of the high affinity CO2 uptake NDH-13 protein complex in cyanobacteria

The CO₂-concentrating mechanism (CCM) in cyanobacteria supports high rates of photosynthesis by greatly increasing the concentration of CO₂ around the major carbon fixing enzyme, Rubisco. However, the CCM remains poorly understood, especially in regards to the enigmatic CO₂-hydration enzymes which couple photosynthetically generated redox energy to the hydration of CO₂ to bicarbonate. This CO₂-hydration reaction is catalysed by specialized forms of NDH-1 thylakoid membrane complexes that contain phylogenetically unique extrinsic proteins that appear to couple CO₂ hydration to NDH-1 proton pumping. The development of the first molecular genetic system to probe structure-function relationships of this important enzyme system is described. A CO₂-hydration deficient strain was constructed as a recipient for DNA constructs containing different forms of the CO₂-hydration system. This was tested by introducing a construct to an ectopic location that gives constitutive expression, rather than native inducible expression, of the ndhF3-ndhD3-cupA-cupS, (cupA operon) encoding high affinity CO₂-hydration complex, NDH-1₃. Uptake assays show the restoration of high affinity for CO₂ uptake, but demonstrate that the CupA complex can drive only modest uptake fluxes, underlining the importance of its tandem operation with the CupB-containing complex NDH-1₄, the complementary high flux, low affinity CO₂ hydration system. Experiments with the carbonic anhydrase inhibitor, ethoxyzolamide, indicate that the NDH-1₃ complex is strongly inhibited, yet the remaining NDH-1₄ activity in the wild-type is less so, suggesting structural differences between the low affinity and high affinity CO₂–hydration systems. This new construct will be an important tool to study and better understand cyanobacterial CO₂ uptake systems.     

CO<Subscript>2</Subscript>-concentrating mechanism in cyanobacterial photosynthesis: organization,physiological role,and evolutionary origin

The cellular and molecular organization of the CO₂-concentrating mechanism (CCM) of cyanobacteria is reviewed. The primary processes of uptake, translocation, and accumulation of inorganic carbon (C_i) near the active site of carbon assimilation by the enzyme ribulose-1,5-bisphosphate carboxylase in the C₃ cycle in cyanobacteria are described as one of the specialized forms of CO₂ concentration which occurs in some photoautotrophic cells. The existence of this form of CO₂ concentration expands our understanding of photosynthetic C_i assimilation. The means of supplying C_i to the C₃ cycle in cyanobacteria is not by simple diffusion into the cell, but it is the result of coordinated functions of high-affinity systems for the uptake of CO₂ and bicarbonate, as well as intracellular CO₂/HCO₃ ^? interconversions by carbonic anhydrases. These biochemical events are under genetic control, and they serve to maintain cellular homeostasis and adaptation to CO₂ limitation. Here we describe the organization of the CCM in cyanobacteria with a special focus on the CCM of relict halo- and alkaliphilic cyanobacteria of soda lakes. We also assess the role of the CCM at the levels of the organism, the biosphere, and evolution.     

Diversity of carbon use strategies in a kelp forest community: implications for a high CO2 ocean

Mechanisms for inorganic carbon acquisition in macroalgal assemblages today could indicate how coastal ecosystems will respond to predicted changes in ocean chemistry due to elevated carbon dioxide (CO₂). We identified the proportion of noncalcifying macroalgae with particular carbon use strategies using the natural abundance of carbon isotopes and pH drift experiments in a kelp forest. We also identified all calcifying macroalgae in this system; these were the dominant component of the benthos (by % cover) at all depths and seasons while cover of noncalcareous macroalgae increased at shallower depths and during summer. All large canopy‐forming macroalgae had attributes suggestive of active uptake of inorganic carbon and the presence of a CO₂ concentration mechanism (CCM). CCM species covered, on average, 15–45% of the benthos and were most common at shallow depths and during summer. There was a high level of variability in carbon isotope discrimination within CCM species, probably a result of energetic constraints on active carbon uptake in a low light environment. Over 50% of red noncalcifying species exhibited values below ?30‰ suggesting a reliance on diffusive CO₂ uptake and no functional CCM. Non‐CCM macroalgae covered on average 0–8.9% of rock surfaces and were most common in deep, low light habitats. Elevated CO₂ has the potential to influence competition between dominant coralline species (that will be negatively affected by increased CO₂) and noncalcareous CCM macroalgae (neutral or positive effects) and relatively rare (on a % cover basis) non‐CCM species (positive effects). Responses of macroalgae to elevated CO₂ will be strongly modified by light and any responses are likely to be different at times or locations where energy constrains photosynthesis. Increased growth and competitive ability of noncalcareous macroalgae alongside negative impacts of acidification on calcifying species could have major implications for the functioning of coastal reef systems at elevated CO₂ concentrations.     

ENHANCED TRANSPORT OF INORGANIC CARBON INTO ALGAL CELLS AND ITS IMPLICATIONS FOR THE BIOLOGICAL FIXATION OF CARBON1,2

Kinetics of uptake of inorganic carbon by the freshwater green alga Chlamydomonas reinhardtii Dang. suggest that rates of fixation may be enhanced at low tensions of CO₂ by transport of bicarbonate from the cell surface to the chloroplast. Results are evaluated in the context of models that treat diffusion and reaction of dissolved inorganic carbon across a 3 dimensional finite boundary layer, and they are consistent with the claim that CO₂ alone is the substrate used during carbon fixation. An alternative hypothesis, which presumes that both CO₂ and bicarbonate are used as substrates, yields predictions which are inconsistent with the data. Instead, bicarbonate seems to act only as a vehicle for the transport of inorganic carbon into the cell, thereby adding its flux to that of CO₂, and enhancing rates of synthesis that would otherwise be restricted by uptake of CO₂ alone.     

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 Important Role of Active Site Water in the Catalytic Mechanism of Human Carbonic Anhydrase II – A Semiempirical MO Approach to the Hydration of CO2 †

The approach of CO₂ to a series of active site model complexes of human carbonic anhydrase II (HCAII) and its catalytic hydration to bicarbonate anion have been investigated using semiempirical MO theory (AM1). The results show that direct nucleophilic attack of zinc-bound hydroxide to the substrate carbon occurs in each model system. Further rearrangement of the bicarbonate complex thus formed via a rotation-like movement of the bicarbonate ligand can only be found in active site model systems that include at least one additional water molecule. Further refinement of the model complex by adding a methanol molecule to mimic Thr-199 makes this process almost activationless. The formation of the final bicarbonate complex by an internal (intramolecular) proton transfer is only possible in the simplest of all model systems, namely {[Im₃Zn(OH)]⁺·CO₂}. The energy of activation for this process, however, is 36.8 kcal·mol^–1 and thus too high for enzymatic catalysis. Therefore, we conclude that within the limitations of the model systems presented and the level of theory employed, the overall mechanism for the formation of the bicarbonate complex comprises an initial direct nucleophilic attack of zinc-bound hydroxide to carbon dioxide followed by a rotation-like rearrangement of the bicarbonate ligand via a penta-coordinate Zn²⁺ transition state structure, including the participation of an extra active site water molecule.Electronic Supplementary Material available.     

Recombinant thermoactive phosphoenolpyruvate carboxylase (PEPC) from Thermosynechococcus elongatus and its coupling with mesophilic/thermophilic bacterial carbonic anhydrases (CAs) for the conversion of CO2 to oxaloacetate

With the continuous increase of atmospheric CO₂ in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO₂ into useful chemicals could be a good strategy to both decreasing the CO₂ concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO₂ mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO₂ to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was constituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobacterium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic anhydrases (CAs), for converting CO₂ into oxaloacetate, a compound of potential utility in industrial processes. The catalytic procedure is in two steps: the conversion of CO₂ into bicarbonate by CA, followed by the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of oxaloacetate (OAA). All tested CAs, belonging to α-, β-, and γ-CA classes, were able to increase OAA production compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested in OAA production was in good agreement with the kinetic parameters for the CO₂ hydration reaction of these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was achieved even if the two enzymes were exposed to temperatures up to 60 °C, suggesting a possible role of the two coupled enzymes in biotechnological processes.     

PHOTOSYNTHETIC INORGANIC CARBON ACQUISITION IN AN ACID‐TOLERANT,FREE‐LIVING SPECIES OF COCCOMYXA (CHLOROPHYTA)1

The processes of CO₂ acquisition were characterized for the acid‐tolerant, free‐living chlorophyte alga, CPCC 508. rDNA data indicate an affiliation to the genus Coccomyxa, but distinct from other known members of the genus. The alga grows over a wide range of pH from 3.0 to 9.0. External carbonic anhydrase (CA) was detected in cells grown above pH 5, with the activity increasing marginally from pH 7 to 9, but most of the CA activity was internal. The capacity for HCO₃^? uptake of cells treated with the CA inhibitor acetazolamide (AZA), was investigated by comparing the calculated rate of uncatalyzed CO₂ formation with the rate of photosynthesis. Active bicarbonate transport occurred in cells grown in media above pH 7.0. Monitoring CO₂ uptake and O₂ evolution by membrane‐inlet mass spectrometry demonstrated that air‐grown cells reduced the CO₂ concentration in the medium to an equilibrium concentration of 15 μM, but AZA‐treated cells caused a drop in extracellular CO₂ concentration to a compensation concentration of 27 μM at pH 8.0. CO₂‐pulsing experiments with cells in the light indicated that the cells do not actively take up CO₂. An internal pool of unfixed inorganic carbon was not detected at the CO₂ compensation concentration, probably because of the lack of active CO₂ uptake, but was detectable at times before compensation point was reached. These results indicate that this free‐living Coccomyxa possesses a CO₂‐concentrating mechanism (CCM) due to an active bicarbonate‐uptake system, unlike the Coccomyxa sp. occurring in symbiotic association with lichens.     

LCIB in the Chlamydomonas CO2-concentrating mechanism

The CO₂-concentrating mechanism confers microalgae a versatile and efficient strategy for adapting to a wide range of environmental CO₂ concentrations. LCIB, which has been demonstrated as a key player in the eukaryotic algal CO₂-concentrating mechanism (CCM), is a novel protein in Chlamydomonas lacking any recognizable domain or motif, and its exact function in the CCM has not been clearly defined. The unique air-dier growth phenotype and photosynthetic characteristics in the LCIB mutants, and re-localization of LCIB between different subcellular locations in response to different levels of CO₂, have indicated that the function of LCIB is closely associated with a distinct low CO₂ acclimation state. Here, we review physiological and molecular evidence linking LCIB with inorganic carbon accumulation in the CCM and discuss the proposed function of LCIB in several inorganic carbon uptake/accumulation pathways. Several new molecular characteristics of LCIB also are presented.     

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.     

Comparison and Analysis of Zinc and Cobalt-Based Systems as Catalytic Entities for the Hydration of Carbon Dioxide

In nature, the zinc metalloenzyme carbonic anhydrase II (CAII) efficiently catalyzes the conversion of carbon dioxide (CO₂) to bicarbonate under physiological conditions. Many research efforts have been directed towards the development of small molecule mimetics that can facilitate this process and thus have a beneficial environmental impact, but these efforts have met very limited success. Herein, we undertook quantum mechanical calculations of four mimetics, 1,5,9-triazacyclododedacane, 1,4,7,10-tetraazacyclododedacane, tris(4,5-dimethyl-2-imidazolyl)phosphine, and tris(2-benzimidazolylmethyl)amine, in their complexed form either with the Zn²⁺ or the Co²⁺ ion and studied their reaction coordinate for CO₂ hydration. These calculations demonstrated that the ability of the complex to maintain a tetrahedral geometry and bind bicarbonate in a unidentate manner were vital for the hydration reaction to proceed favorably. Furthermore, these calculations show that the catalytic activity of the examined zinc complexes was insensitive to coordination states for zinc, while coordination states above four were found to have an unfavorable effect on product release for the cobalt counterparts.     

Biochemical characterization of the δ-carbonic anhydrase from the marine diatom Thalassiosira weissflogii,TweCA

Abstract

Diatom genome sequences clearly reveal the presence of different systems for HCO₃^? uptake. Carbon-concentrating mechanisms (CCM) based on HCO₃^? transport and a plastid-localized carbonic anhydrase (CA, EC 4.2.1.1) appear to be more probable than the others because CAs have been identified in the genome of many diatoms. CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate. Five genetically distinct classes of CAs exist, α-, β-, γ-, δ- and ζ and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the δ-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO₂ hydration and its inhibition profile with sulfonamide/sulfamate and anions have also been investigated. Here, we report the detailed biochemical characterization and chemico-physical properties of the δ-CA of T. weissflogii. The δ-CA encoding gene was cloned and expressed in Artic Express cells and the recombinant protein purified to homogeneity. Interesting to note that TweCA has no intrinsic esterase activity with 4-nitrophenyl acetate (pNpA) as substrate although the phylogenetic analysis showed that δ-CAs are closer to the α-CAs than to the other classes of such enzymes.     

Strategies of dissolved inorganic carbon use in macroalgae across a gradient of terrestrial influence: implications for the Great Barrier Reef in the context of ocean acidification

Macroalgae are generally used as indicators of coral reef status; thus, understanding the drivers and mechanisms leading to increased macroalgal abundance are of critical importance. Ocean acidification (OA) due to elevated carbon dioxide (CO₂) concentrations has been suggested to stimulate macroalgal growth and abundance on reefs. However, little is known about the physiological mechanisms by which reef macroalgae use CO₂ from the bulk seawater for photosynthesis [i.e., (1) direct uptake of bicarbonate (HCO₃ ^?) and/or CO₂ by means of carbon concentrating mechanisms (CCM) and (2) the diffusive uptake of CO₂], which species could benefit from increased CO₂ or which habitats may be more susceptible to acidification-induced algal proliferations. Here, we provide the first quantitative examination of CO₂-use strategies in coral reef macroalgae and provide information on how the proportion of species and the proportional abundance of species utilising each of the carbon acquisition strategies varies across a gradient of terrestrial influence (from inshore to offshore reefs) in the Great Barrier Reef (GBR). Four macroalgal groups were identified based on their carbon uptake strategies: (1) CCM-only (HCO₃ ^? only users); (2) CCM-HCO₃ ^?/CO₂ (active uptake HCO₃ ^? and/or CO₂ use); (3) Non-CCM species (those relying on diffusive CO₂ uptake); and (4) Calcifiers. δ¹³C values of macroalgae, confirmed by pH drift assays, show that diffusive CO₂ use is more prevalent in deeper waters, possibly due to low light availability that limits activity of CCMs. Inshore shallow reefs had a higher proportion of CCM-only species, while reefs further away from terrestrial influence and exposed to better water quality had a higher number of non-CCM species than inshore and mid-shelf reefs. As non-CCM macroalgae are more responsive to increased seawater CO₂ and OA, reef slopes of the outer reefs are probably the habitats most vulnerable to the impacts of OA. Our results suggest a potentially important role of carbon physiology in structuring macroalgal communities in the GBR.     

High internal resistance to CO2 uptake by submerged macrophytes that use HCO3 −: measurements in air,nitrogen and helium

Previous studies have shown that in water the affinity of submerged macrophytes for CO₂ is higher for species restricted to CO₂ than for species with an additional ability to use bicarbonate. We measured slopes of CO₂ uptake versus CO₂ concentration in the gas phase in air, nitrogen and helium for pairs of species, having or lacking the ability to use bicarbonate, but with similar leaf morphology. For species restricted to CO₂, the slope in nitrogen and helium was 1.5 times and 3.2 times greater than in air. The increased slope in nitrogen results from a suppression of photorespiration. The further increase in helium reflects the increased rate of diffusion of CO₂ and shows that, even in gas, external diffusion through the boundary layer is a significant hindrance to CO₂ uptake. In contrast, in species able to use bicarbonate, the uptake slope was not affected by gas composition, suggesting that photorespiration is absent or photorespired CO₂ is efficiently trapped and that internal resistance is high relative to external resistance. Elodea canadensis specimens grown under high concentrations of CO₂ de-regulated their ability to use bicarbonate, and slopes of CO₂ uptake in helium were significantly greater than in air or nitrogen. Overall, these results are consistent with the notion that while a high affinity for CO₂ will maximise carbon uptake in species restricted to CO₂, for species able to use bicarbonate, a high internal resistance would reduce loss of CO₂ and help maintain high concentrations of CO₂ at the site of fixation. This revised version was published online in August 2006 with corrections to the Cover Date.     

A modified pH drift assay for inorganic carbon accumulation and external carbonic anhydrase activity in microalgae

Threat of global warming due to carbon dioxide (CO₂) emissions has stimulated research into carbon sequestration and emissions reduction technologies. Alkaline scrubbing allows CO₂ to be captured as bicarbonate, which can be photochemically fixed by microalgae. The carbon concentrating mechanism (CCM), of which external carbonic anhydrase is a key component, allows organisms to utilise this bicarbonate. In order to select a suitable strain for this application, a screening tool is required. The current method for determining carbonic anhydrase activity, the Wilbur and Anderson assay, was found to be unsuitable as a screening tool as the associated error was unacceptably large and tests on whole cells were inconclusive. This paper presents the development of a new, whole cell assay to measure inorganic carbon uptake and external carbonic anhydrase activity, based on classical pH drift experiments. Spirulina platensis was successfully used to develop a correlation between the specific carbon uptake (C) and the specific pH change (dpH). The relationship is described by the following: C[mmol C (g dry algae)^?1?h^?1]?=?0.064?×?(dpH). Inhibitor and salt dissociation tests validated the activity and presence of external carbonic anhydrase and allowed correlation between the Wilbur and Anderson assay and the new whole cell assay. Screening tests were conducted on S. platensis, Scenedesmus sp., Chlorella vulgaris and Dunaliella salina that were found to have carbon uptake rates of 5.76, 5.86, 3.86 and 2.15 mmol C (g dry algae)^?1?h^?1, respectively. These results corresponded to the species' known bicarbonate utilisation abilities and validated the use of the assay as a screening tool.     

SOURCE OF INORGANIC CARBON FOR PHOTOSYNTHESIS IN TWO MARINE DINOFLAGELLATES1

Inorganic carbon uptake was investigated in two marine dinoflagellates, Amphidinium carterae Hulburt and Heterocapsa oceanica Stein. Mass spectrometric and potentiometric assays indicated that both species lacked external carbonic anhydrase (CA). The presence of internal CA was demonstrated by potentiometric assay and by the inhibition of photosynthesis upon the addition of 500 μM ethoxyzolamide a membrane‐permeable inhibitor of CA. The capacity for bicarbonate transport was investigated by comparing the calculated rate of spontaneous CO₂ formation at pH 8.2 and 25°C with the rate of photosynthesis after the addition of 100 μM NaHCO₃. Both species appeared to have a very limited capacity for direct bicarbonate uptake. Monitoring of CO₂ and O₂ fluxes in both species by mass spectrometry demonstrated a rapid uptake of CO₂ on illumination, to concentrations below the CO₂ equilibrium concentration, indicating an effective selective uptake of CO₂. This dependence of photosynthesis on free CO₂ alone suggests that these species are CO₂ limited in their natural environment because the CO₂ concentration of seawater is very low.     

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.