Differential effects of ocean acidification on carbon acquisition in two bloom‐forming dinoflagellate species

Dinoflagellates represent a cosmopolitan group of phytoplankton with the ability to form harmful algal blooms. Featuring a Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RubisCO) with very low CO₂ affinities, photosynthesis of this group may be particularly prone to carbon limitation and thus benefit from rising atmospheric CO₂ partial pressure (pCO₂) under ocean acidification (OA). Here, we investigated the consequences of OA on two bloom‐forming dinoflagellate species, the calcareous Scrippsiella trochoidea and the toxic Alexandrium tamarense. Using dilute batch incubations, we assessed growth characteristics over a range of pCO₂ (i.e. 180–1200 µatm). To understand the underlying physiology, several aspects of inorganic carbon acquisition were investigated by membrane‐inlet mass spectrometry. Our results show that both species kept growth rates constant over the tested pCO₂ range, but we observed a number of species‐specific responses. For instance, biomass production and cell size decreased in S. trochoidea, while A. tamarense was not responsive to OA in these measures. In terms of oxygen fluxes, rates of photosynthesis and respiration remained unaltered in S. trochoidea whereas respiration increased in A. tamarense under OA. Both species featured efficient carbon concentrating mechanisms (CCMs) with a CO₂‐dependent contribution of HCO₃^? uptake. In S. trochoidea, the CCM was further facilitated by exceptionally high and CO₂‐independent carbonic anhydrase activity. Comparing both species, a general trade‐off between maximum rates of photosynthesis and respective affinities is indicated. In conclusion, our results demonstrate effective CCMs in both species, yet very different strategies to adjust their carbon acquisition. This regulation in CCMs enables both species to maintain growth over a wide range of ecologically relevant pCO₂.     

The role of external carbonic anhydrase in photosynthesis during growth of the marine diatom Chaetoceros muelleri

Carbon dioxide concentrating mechanisms (CCMs) act to improve the supply of CO₂ at the active site of ribulose‐1,5‐bisphosphate carboxylase/oxygenase. There is substantial evidence that in some microalgal species CCMs involve an external carbonic anhydrase (CA_ext) and that CA_ext activity is induced by low CO₂ concentrations in the growth medium. However, much of this work has been conducted on cells adapted to air‐equilibrium concentrations of CO₂, rather than to changing CO₂ conditions caused by growing microalgal populations. We investigated the role of CA_ext in inorganic carbon (C_i) acquisition and photosynthesis at three sampling points during the growth cycle of the cosmopolitan marine diatom Chaetoceros muelleri. We observed that CA_ext activity increased with decreasing C_i, particularly CO₂, concentration, supporting the idea that CA_ext is modulated by external CO₂ concentration. Additionally, we found that the contribution of CA_ext activity to carbon acquisition for photosynthesis varies over time, increasing between the first and second sampling points before decreasing at the last sampling point, where external pH was high. Lastly, decreases in maximum quantum yield of photosystem II (F_v/F_m), chlorophyll, maximum relative electron transport rate, light harvesting efficiency (α) and maximum rates of C_i‐ saturated photosynthesis (V_max) were observed over time. Despite this decrease in photosynthetic capacity an up‐regulation of CCM activity, indicated by a decreasing half‐saturation constant for CO₂ (K_0.5CO₂), occurred over time. The flexibility of the CCM during the course of growth in C. muelleri may contribute to the reported dominance and persistence of this species in phytoplankton blooms.     

Impacts of increased atmospheric CO<Subscript>2</Subscript> concentration on photosynthesis and growth of micro- and macro-algae

Marine photosynthesis drives the oceanic biological CO₂ pump to absorb CO₂ from the atmosphere, which sinks more than one third of the industry-originated CO₂ into the ocean. The increasing atmospheric CO₂ and subsequent rise of pCO₂ in seawater, which alters the carbonate system and related chemical reactions and results in lower pH and higher HCO₃ ⁻ concentration, affect photosynthetic CO₂ fixation processes of phytoplanktonic and macroalgal species in direct and/or indirect ways. Although many unicellular and multicellular species can operate CO₂-concentrating mechanisms (CCMs) to utilize the large HCO₃ ⁻ pool in seawater, enriched CO₂ up to several times the present atmospheric level has been shown to enhance photosynthesis and growth of both phytoplanktonic and macro-species that have less capacity of CCMs. Even for species that operate active CCMs and those whose photosynthesis is not limited by CO₂ in seawater, increased CO₂ levels can down-regulate their CCMs and therefore enhance their growth under light-limiting conditions (at higher CO₂ levels, less light energy is required to drive CCM). Altered physiological performances under high-CO₂ conditions may cause genetic alteration in view of adaptation over long time scale. Marine algae may adapt to a high CO₂ oceanic environment so that the evolved communities in future are likely to be genetically different from the contemporary communities. However, most of the previous studies have been carried out under indoor conditions without considering the acidifying effects on seawater by increased CO₂ and other interacting environmental factors, and little has been documented so far to explain how physiology of marine primary producers performs in a high-CO₂ and low-pH ocean.     

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.     

The physiological response of marine diatoms to ocean acidification: differential roles of seawater pCO2 and pH

Although increasing the pCO₂ for diatoms will presumably down‐regulate the CO₂‐concentrating mechanism (CCM) to save energy for growth, different species have been reported to respond differently to ocean acidification (OA). To better understand their growth responses to OA, we acclimated the diatoms Thalassiosira pseudonana, Phaeodactylum tricornutum, and Chaetoceros muelleri to ambient (pCO₂ 400 μatm, pH 8.1), carbonated (pCO₂ 800 μatm, pH 8.1), acidified (pCO₂ 400 μatm, pH 7.8), and OA (pCO₂ 800 μatm, pH 7.8) conditions and investigated how seawater pCO₂ and pH affect their CCMs, photosynthesis, and respiration both individually and jointly. In all three diatoms, carbonation down‐regulated the CCMs, while acidification increased both the photosynthetic carbon fixation rate and the fraction of CO₂ as the inorganic carbon source. The positive OA effect on photosynthetic carbon fixation was more pronounced in C. muelleri, which had a relatively lower photosynthetic affinity for CO₂, than in either T. pseudonana or P. tricornutum. In response to OA, T. pseudonana increased respiration for active disposal of H⁺ to maintain its intracellular pH, whereas P. tricornutum and C. muelleri retained their respiration rate but lowered the intracellular pH to maintain the cross‐membrane electrochemical gradient for H⁺ efflux. As the net result of changes in photosynthesis and respiration, growth enhancement to OA of the three diatoms followed the order of C. muelleri > P. tricornutum > T. pseudonana. This study demonstrates that elucidating the separate and joint impacts of increased pCO₂ and decreased pH aids the mechanistic understanding of OA effects on diatoms in the future, acidified oceans.     

Inorganic carbon acquisition by eukaryotic algae: four current questions

The phylogenetically and morphologically diverse eukaryotic algae are typically oxygenic photolithotrophs. They have a diversity of incompletely understood mechanisms of inorganic carbon acquisition: this article reviews four areas where investigations continue. The first topic is diffusive CO₂ entry. Most eukaryotic algae, like all cyanobacteria, have inorganic carbon concentrating mechanisms (CCMs). The ancestral condition was presumably the absence of a CCM, i.e. diffusive CO₂ entry, as found in a small minority of eukaryotic algae today; however, it is likely that, as is found in several cases, this condition is due to a loss of a CCM. There are a number of algae which are in various respects intermediate between diffusive CO₂ entry and occurrence of a CCM: further study is needed on this aspect. A second topic is the nature of cyanelles and their role in inorganic carbon assimilation. The cyanelles (plastids) of the euglyphid amoeba Paulinella have been acquired relatively recently by endosymbiosis with genetic integration of an α-cyanobacterium with a Form 1A Rubisco. The α-carboxysomes in the cyanelles are presumably involved in a CCM, but further investigation is needed.Also called cyanelles are the plastids of glaucocystophycean algae, but is it now clear that these were derived from the β-cyanobacterial ancestor of all plastids other than that of Paulinella. The resemblances of the central body of the cyanelles of glaucocystophycean algae to carboxysomes may not reflect derivation from cyanobacterial β-carboxysomes; although it is clear that these algae have CCMs but these are now well characterized. The other two topics concern CCMs in other eukaryotic algae; these CCMs arose polyphyletically and independently of the cyanobacterial CCMs. It is generally believed that eukaryotic algal, like cyanobacterial, CCMs are based on active transport of an inorganic carbon species and/or protons, and they have C₃ biochemistry. This is the case for the organism considered as the third topic, i.e. Chlamydomonas reinhardtii, the eukaryotic alga with the best understood CCM. This CCM involves HCO₃ ^? conversion to CO₂ in the thylakoid lumen so the external inorganic carbon must cross four membranes in series with a final CO₂ effux from the thylakoid. More remains to be investigated about this CCM. The final topic is that of the occurrence of C₄-like metabolism in the CCMs of marine diatoms. Different conclusions have been reached depending on the organism investigated and the techniques used, and several aspects require further study.     

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.     

Carbon acquisition by diatoms

Diatoms are responsible for up to 40% of primary productivity in the ocean, and complete genome sequences are available for two species. However, there are very significant gaps in our understanding of how diatoms take up and assimilate inorganic C. Diatom plastids originate from secondary endosymbiosis with a red alga and their Form ID Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase) from horizontal gene transfer, which means that embryophyte paradigms can only give general guidance as to their C acquisition mechanisms. Although diatom Rubiscos have relatively high CO₂ affinity and CO₂/O₂ selectivity, the low diffusion coefficient for CO₂ in water has the potential to restrict the rate of photosynthesis. Diatoms growing in their natural aquatic habitats operate inorganic C concentrating mechanisms (CCMs), which provide a steady-state CO₂ concentration around Rubisco higher than that in the medium. How these CCMs work is still a matter of debate. However, it is known that both CO₂ and HCO₃⁻ are taken up, and an obvious but as yet unproven possibility is that active transport of these species across the plasmalemma and/or the four-membrane plastid envelope is the basis of the CCM. In one marine diatom there is evidence of C₄-like biochemistry which could act as, or be part of, a CCM. Alternative mechanisms which have not been eliminated include the production of CO₂ from HCO₃⁻ at low pH maintained by a H⁺ pump, in a compartment close to that containing Rubisco.     

Utilization of inorganic carbon in the edible cyanobacterium Ge-Xian-Mi (Nostoc) and its role in alleviating photo-inhibition

The present work investigated the inorganic carbon (C_i) uptake, fluorescence quenching and photo‐inhibition of the edible cyanobacterium Ge‐Xian‐Mi (Nostoc) to obtain an insight into the role of CO₂ concentrating mechanism (CCM) operation in alleviating photo‐inhibition. Ge‐Xian‐Mi used HCO₃^– in addition to CO₂ for its photosynthesis and oxygen evolution was greater than the theoretical rates of CO₂ production derived from uncatalysed dehydration of HCO₃^–. Multiple transporters for CO₂ and HCO₃^– operated in air‐grown Ge‐Xian‐Mi. Na⁺‐dependent HCO₃^– transport was the primary mode of active C_i uptake and contributed 53–62% of net photosynthetic activity at 250 µmol L^?1 KHCO₃ and pH 8.0. However, the CO₂‐uptake systems and Na⁺‐independent HCO₃^– transport played minor roles in Ge‐Xian‐Mi and supported, respectively, 39 and 8% of net photosynthetic activity. The steady‐state fluorescence decreased and the photochemical quenching increased in response to the transport‐mediated accumulation of intracellular C_i. Inorganic carbon transport was a major factor in facilitating quenching during the initial stage and the initial rate of fluorescence quenching in the presence of iodoacetamide, an inhibitor of CO₂ fixation, was 88% of control. Both the initial rate and extent of fluorescence quenching increased with increasing external dissolved inorganic carbon (DIC) and saturated at higher than 200 µmol L^?1 HCO₃^–. The operation of the CCM in Ge‐Xian‐Mi served as a means of diminishing photodynamic damage by dissipating excess light energy and higher external DIC in the range of 100–10000 µmol L^?1 KHCO₃ was associated with more severe photo‐inhibition under strong irradiance.     

INORGANIC CARBON UTILIZATION BY ROSS SEA PHYTOPLANKTON ACROSS NATURAL AND EXPERIMENTAL CO2 GRADIENTS1

We present results from a field study of inorganic carbon (C) acquisition by Ross Sea phytoplankton during Phaeocystis‐dominated early season blooms. Isotope disequilibrium experiments revealed that HCO₃^? was the primary inorganic C source for photosynthesis in all phytoplankton assemblages. From these experiments, we also derived relative enhancement factors for HCO₃^?/CO₂ interconversion as a measure of extracellular carbonic anhydrase activity (eCA). The enhancement factors ranged from 1.0 (no apparent eCA activity) to 6.4, with an overall mean of 2.9. Additional eCA measurements, made using membrane inlet mass spectrometry (MIMS), yielded activities ranging from 2.4 to 6.9 U · [μg chl a]^?1 (mean 4.1). Measurements of short‐term C‐fixation parameters revealed saturation kinetics with respect to external inorganic carbon, with a mean half‐saturation constant for inorganic carbon uptake (K_1/2) of ～380 μM. Comparison of our early springtime results with published data from late‐season Ross Sea assemblages showed that neither HCO₃^? utilization nor eCA activity was significantly correlated to ambient CO₂ levels or phytoplankton taxonomic composition. We did, however, observe a strong negative relationship between surface water pCO₂ and short‐term ¹⁴C‐fixation rates for the early season survey. Direct incubation experiments showed no statistically significant effects of pCO₂ (10 to 80 Pa) on relative HCO₃^? utilization or eCA activity. Our results provide insight into the seasonal regulation of C uptake by Ross Sea phytoplankton across a range of pCO₂ and phytoplankton taxonomic composition.     

Introducing an algal carbon‐concentrating mechanism into higher plants: location and incorporation of key components

Many eukaryotic green algae possess biophysical carbon‐concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO₂ concentrations. They are thus an attractive option for improving productivity in higher plants. In this study, the intracellular locations of ten CCM components in the unicellular green alga Chlamydomonas reinhardtii were confirmed. When expressed in tobacco, all of these components except chloroplastic carbonic anhydrases CAH3 and CAH6 had the same intracellular locations as in Chlamydomonas. CAH6 could be directed to the chloroplast by fusion to an Arabidopsis chloroplast transit peptide. Similarly, the putative inorganic carbon (Ci) transporter LCI1 was directed to the chloroplast from its native location on the plasma membrane. CCP1 and CCP2 proteins, putative Ci transporters previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas and tobacco, suggesting that the algal CCM model requires expansion to include a role for mitochondria. For the Ci transporters LCIA and HLA3, membrane location and Ci transport capacity were confirmed by heterologous expression and H¹⁴CO₃^‐ uptake assays in Xenopus oocytes. Both were expressed in Arabidopsis resulting in growth comparable with that of wild‐type plants. We conclude that CCM components from Chlamydomonas can be expressed both transiently (in tobacco) and stably (in Arabidopsis) and retargeted to appropriate locations in higher plant cells. As expression of individual Ci transporters did not enhance Arabidopsis growth, stacking of further CCM components will probably be required to achieve a significant increase in photosynthetic efficiency in this species.     

CARBON‐USE STRATEGIES IN MACROALGAE: DIFFERENTIAL RESPONSES TO LOWERED PH AND IMPLICATIONS FOR OCEAN ACIDIFICATION1

Ocean acidification (OA) is a reduction in oceanic pH due to increased absorption of anthropogenically produced CO₂. This change alters the seawater concentrations of inorganic carbon species that are utilized by macroalgae for photosynthesis and calcification: CO₂ and HCO₃^? increase; CO₃^2? decreases. Two common methods of experimentally reducing seawater pH differentially alter other aspects of carbonate chemistry: the addition of CO₂ gas mimics changes predicted due to OA, while the addition of HCl results in a comparatively lower [HCO₃^?]. We measured the short‐term photosynthetic responses of five macroalgal species with various carbon‐use strategies in one of three seawater pH treatments: pH 7.5 lowered by bubbling CO₂ gas, pH 7.5 lowered by HCl, and ambient pH 7.9. There was no difference in photosynthetic rates between the CO₂, HCl, or pH 7.9 treatments for any of the species examined. However, the ability of macroalgae to raise the pH of the surrounding seawater through carbon uptake was greatest in the pH 7.5 treatments. Modeling of pH change due to carbon assimilation indicated that macroalgal species that could utilize HCO₃^? increased their use of CO₂ in the pH 7.5 treatments compared to pH 7.9 treatments. Species only capable of using CO₂ did so exclusively in all treatments. Although CO₂ is not likely to be limiting for photosynthesis for the macroalgal species examined, the diffusive uptake of CO₂ is less energetically expensive than active HCO₃^? uptake, and so HCO₃^?‐using macroalgae may benefit in future seawater with elevated CO₂.     

Energy crosstalk between photosynthesis and the algal CO2-concentrating mechanisms

Microalgal photosynthesis is responsible for nearly half of the CO₂ annually captured by Earth’s ecosystems. In aquatic environments where the CO₂ availability is low, the CO₂-fixing efficiency of microalgae greatly relies on mechanisms – called CO_2-concentrating mechanisms (CCMs) – for concentrating CO₂ at the catalytic site of the CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the transport of inorganic carbon (C_i) across membrane bilayers against a concentration gradient consumes part of the chemical energy generated by photosynthesis, the bioenergetics and cellular mechanisms involved are only beginning to be elucidated. Here, we review the current knowledge relating to the energy requirement of CCMs in the light of recent advances in photosynthesis regulatory mechanisms and the spatial organization of CCM components.     

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales,Phaeophyceae) under variable pH

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO₃^?) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO₃^? by the surface‐bound enzyme carbonic anhydrase (CA_ext). Here, we examined other putative HCO₃^? uptake mechanisms in M. pyrifera under pH_T 9.00 (HCO₃^?: CO₂ = 940:1) and pH_T 7.65 (HCO₃^?: CO₂ = 51:1). Rates of photosynthesis, and internal CA (CA_int) and CA_ext activity were measured following the application of AZ which inhibits CA_ext, and DIDS which inhibits a different HCO₃^? uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO₃^? uptake by M. pyrifera is via an AE protein, regardless of the HCO₃^?: CO₂ ratio, with CA_ext making little contribution. Inhibiting the AE protein led to a 55%–65% decrease in photosynthetic rates. Inhibiting both the AE protein and CA_ext at pH_T 9.00 led to 80%–100% inhibition of photosynthesis, whereas at pH_T 7.65, passive CO₂ diffusion supported 33% of photosynthesis. CA_int was active at pH_T 7.65 and 9.00, and activity was always higher than CA_ext, because of its role in dehydrating HCO₃^? to supply CO₂ to RuBisCO. Interestingly, the main mechanism of HCO₃^? uptake in M. pyrifera was different than that in other Laminariales studied (CA_ext‐catalyzed reaction) and we suggest that species‐specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO₃^?:CO₂ due to ocean acidification.     

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.     

The CO2‐concentrating mechanism in the bloom‐forming cyanobacterium Microcystis aeruginosa (Cyanophyceae) and effects of UVB radiation on its operation1

The bloom‐forming cyanobacterium Microcystis aeruginosa (Kütz.) Kütz. 854 was cultured with 1.05 W · m^?2 ultraviolet‐B radiation (UVBR) for 3 h every day, and the CO₂‐concentrating mechanism (CCM) within this species as well as effects of UVBR on its operation were investigated. Microcystis aeruginosa 854 possessed at least three inorganic carbon transport systems and could utilize external HCO₃^? and CO₂ for its photosynthesis. The maximum photosynthetic rate was approximately the same, but the apparent affinity for dissolved inorganic carbon was significantly decreased from 74.7 μmol · L^?1 in the control to 34.7 μmol · L^?1 in UVBR‐treated cells. At 150 μmol · L^?1 KHCO₃ and pH 8.0, Na⁺‐dependent HCO₃^? transport contributed 43.4%–40.2% to the photosynthesis in the control and 34.5%–31.9% in UVBR‐treated cells. However, the contribution of Na⁺‐independent HCO₃^? transport increased from 8.7% in the control to 18.3% in UVBR‐treated cells. The contribution of CO₂‐uptake systems showed little difference: 47.9%–51.0% in the control and 49.8%–47.2% in UVBR‐treated cells. Thus, the rate of total inorganic carbon uptake was only marginally affected, although UVBR had a differential effect on various inorganic carbon transporters. However, the number of carboxysomes in UVBR‐treated cells was significantly decreased compared to that in the control.     

LCI1, a Chlamydomonas reinhardtii plasma membrane protein,functions in active CO2 uptake under low CO2

In response to high CO₂ environmental variability, green algae, such as Chlamydomonas reinhardtii, have evolved multiple physiological states dictated by external CO₂ concentration. Genetic and physiological studies demonstrated that at least three CO₂ physiological states, a high CO₂ (0.5–5% CO₂), a low CO₂ (0.03–0.4% CO₂) and a very low CO₂ (< 0.02% CO₂) state, exist in Chlamydomonas. To acclimate in the low and very low CO₂ states, Chlamydomonas induces a sophisticated strategy known as a CO₂‐concentrating mechanism (CCM) that enables proliferation and survival in these unfavorable CO₂ environments. Active uptake of C_i from the environment is a fundamental aspect in the Chlamydomonas CCM, and consists of CO₂ and HCO₃^– uptake systems that play distinct roles in low and very low CO₂ acclimation states. LCI1, a putative plasma membrane C_i transporter, has been linked through conditional overexpression to active C_i uptake. However, both the role of LCI1 in various CO₂ acclimation states and the species of C_i, HCO₃^– or CO₂, that LCI1 transports remain obscure. Here we report the impact of an LCI1 loss‐of‐function mutant on growth and photosynthesis in different genetic backgrounds at multiple pH values. These studies show that LCI1 appears to be associated with active CO₂ uptake in low CO₂, especially above air‐level CO₂, and that any LCI1 role in very low CO₂ is minimal.     

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.     

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.     

Microsensor studies on Padina from a natural CO2 seep: implications of morphology on acclimation to low pH

Low seawater pH can be harmful to many calcifying marine organisms, but the calcifying macroalgae Padina spp. flourish at natural submarine carbon dioxide seeps where seawater pH is low. We show that the microenvironment created by the rolled thallus margin of Padina australis facilitates supersaturation of CaCO₃ and calcifi‐cation via photosynthesis‐induced elevated pH. Using microsensors to investigate oxygen and pH dynamics in the microenvironment of P. australis at a shallow CO₂ seep, we found that, under saturating light, the pH inside the microenvironment (pH_ME) was higher than the external seawater (pH_SW) at all pH_SW levels investigated, and the difference (i.e., pH_ME ? pH_SW) increased with decreasing pH_SW (0.9 units at pH_SW 7.0). Gross photosynthesis (P_g) inside the microenvironment increased with decreasing pH_SW, but algae from the control site reached a threshold at pH 6.5. Seep algae showed no pH threshold with respect to P_g within the pH_SW range investigated. The external carbonic anhydrase (CA) inhibitor, acetazolamide, strongly inhibited P_g of P. australis at pH_SW 8.2, but the effect was diminished under low pH_SW (6.4–7.5), suggesting a greater dependence on membrane‐bound CA for the dehydration of HCO₃^? ions during dissolved inorganic carbon uptake at the higher pH_SW. In comparison, a calcifying green alga, Halimeda cuneata f. digitata, was not inhibited by AZ, suggesting efficient bicarbonate transport. The ability of P. australis to elevate pH_ME at the site of calcification and its strong dependence on CA may explain why it can thrive at low pH_SW.