Accumulation Of Dissolved Inorganic Carbon Research Articles

Coupling of Carbon and Oxygen in the Pearl River Plume in Summer: Upwelling, Hypoxia, Reoxygenation and Enhanced Acidification

AbstractAcidification and hypoxia are universal environmental issues in coastal seas, especially in large river estuaries such as the Pearl River estuary. In July and August of 2015, two legs of a field survey were conducted in the Pearl River plume. Leg 1 was sampled during the influence of upwelling favorable winds, while Leg 2 was during downwelling favorable winds. During both legs, instead of the typically observed dissolved inorganic carbon (DIC) consumption and dissolved oxygen (DO) over‐saturation, upwelling‐induced high DIC (>2,000 μmol kg−1), low pH (7.7–7.8) and low DO (140–150 μmol kg−1) values were observed in surface waters at the estuary mouth and the area off Hong Kong. In the bottom waters, hypoxia, acidification (pH 7.6–7.8) and DIC accumulation (DIC addition of ∼100–180 μmol kg−1) were observed. Hypoxia was less severe during Leg 2 compared to Leg 1. The stoichiometry of oxygen depletion to DIC addition was 0.89 for bottom water, suggesting remineralization was dominated by marine sourced organic matter. However, a comparison of data from the two legs showed that the stoichiometry of oxygen consumption to DIC accumulation was significantly higher during Leg 2 (0.73 ± 0.03 for Leg 1 vs. 0.80 ± 0.05 for Leg 2), although N/P ratios were the same (13.54 ± 1.93 for Leg 1 vs. 13.51 ± 2.04 for Leg 2). This phenomenon was attributed mainly to enhanced ventilation (re‐oxygenation) under the influence of the downwelling favorable winds during Leg 2. Although ventilation relieves some hypoxia, it might enhance acidification in bottom waters after a short‐term ventilation event. The enhanced acidification after short‐term ventilation is worthy of further study considering that most hypoxia and acidification are found in shallow coastal seas.

Coastal freshening drives acidification state in Greenland fjords

Greenland's fjords and coastal waters are highly productive and sustain important fisheries. However, retreating glaciers and increasing meltwater are changing fjord circulation and biogeochemistry, which may threaten future productivity. The freshening of Greenland fjords caused by unprecedented melting of the Greenland Ice Sheet may alter carbonate chemistry in coastal waters, influencing CO2 uptake and causing biological consequences from acidification. However, few studies to date explore the current acidification state in Greenland coastal waters. Here we present the first-ever large-scale measurements of carbonate system parameters in 16 Greenlandic fjords and seek to identify the drivers of acidification state in these freshening ecosystems. Aragonite saturation state (Ω), a proxy for ocean acidification, was calculated from dissolved inorganic carbon (DIC) and total alkalinity from fjords along the east and west coast of Greenland spanning 68–75°N. Aragonite saturation was primarily >1 in the surface mixed layer. However, undersaturated—or corrosive––conditions (Ω < 1) were observed on both coasts (west: Ω = 0.28–3.11, east: Ω = 0.70–3.07), albeit at different depths. West Greenland fjords were largely corrosive at depth while undersaturation in East Greenland fjords was only observed in surface waters. This reflects a difference in the coastal boundary conditions and mechanisms driving acidification state. We suggest that advection of Sub Polar Mode Water and accumulation of DIC from organic matter decomposition drive corrosive conditions in the West, while freshwater alkalinity dilution drives acidification in the East. The presence of marine terminating glaciers also impacted local acidification states by influencing fjord circulation: upwelling driven by subglacial discharge brought corrosive bottom waters to shallower depths. Meanwhile, discharge from land terminating glaciers strengthened stratification and diluted alkalinity. Regardless of the drivers in each system, increasing freshwater discharge will likely lower carbonate saturation states and impact biotic and abiotic carbon uptake in the future.

Hydrology influences carbon flux through metabolic pathways in the hypolimnion of a Mediterranean reservoir

Global change is modifying meteorological and hydrological factors that influence the thermal regime of water bodies. These modifications can lead to longer stratification periods with enlarged hypolimnetic anoxic periods, which can promote heterotrophic anaerobic processes and alter reservoir carbon cycling. Here, we quantified aerobic and anaerobic heterotrophic processes (aerobic respiration, denitrification, iron and manganese reduction, sulfate reduction, and methanogenesis) on dissolved inorganic carbon (DIC) production in the hypolimnion of a Mediterranean reservoir (El Gergal, Spain) under two contrasting hydrological conditions: a wet year with heavy direct rainfall and frequent water inputs from upstream reservoirs, and a dry year with scarce rainfall and negligible water inputs. During the wet year, water inputs and rainfall induced low water column thermal stability and earlier turnover. By contrast, thermal stratification was longer and more stable during the dry year. During wet conditions, we observed lower DIC accumulation in the hypolimnion, mainly due to weaker sulfate reduction and methanogenesis. By contrast, longer stratification during the dry year promoted higher hypolimnetic DIC accumulation, resulting from enhanced methanogenesis and sulfate reduction, thus increasing methane emissions and impairing reservoir water quality. Aerobic respiration, denitrification and metal reduction produced a similar amount of DIC in the hypolimnion during the two studied years. All in all, biological and geochemical (calcite dissolution) processes explained most of hypolimnetic DIC accumulation during stratification regardless of the hydrological conditions, but there is still ~ 30% of hypolimnetic DIC production that cannot be explained by the processes contemplated in this study and the assumptions made.

Large Increase in Dissolved Inorganic Carbon in the East Sea (Japan Sea) From 1999 to 2019

The East Sea (also known as the Japan Sea; hereafter, EJS) has its own deep overturning circulation, that operates over a centurial timescale compared with a millennial timescale in the ocean. This allows the EJS to be used as a natural laboratory for investigating potential future changes in the oceanic system. Dissolved inorganic carbon (DIC), total alkalinity (TA), and pH were measured in 2019 in a wide area of the EJS to investigate the characteristics and changes of the carbonate system since the last extensive survey in 1999. In the layer below ∼1,000 m, DIC and apparent oxygen utilization (AOU) was uniform implying rapid horizontal mixing within a few years. Since 1999, DIC concentration increased by ∼11 μmol kg–1 in the layer deeper than 500 m. This increase accompanied a commensurate increase in AOU with the canonical ratio of 1.3, indicating that the accumulation of DIC was supplied from organic matter decomposition. This observation is consistent with a previous study suggesting that the slowed deep water ventilation was the cause of the increase in AOU and fast acidification. In the EJS, increase in DIC from the surface water to deep waters is much higher than that in TA, which is caused by high primary productivity and export production together with low rates of CaCO3 export. Thus, the DIC/TA ratio of deep waters, an indicator of vulnerability to acidification, is high. A recently reported change in deep water ventilation, namely, re-initiation of deep water formation reaching deeper depths to the Deep Water and the Bottom Water, implies that unexpected changes in the carbonate system may be detected in the future, which needs to be further monitored.

Transport and transformation of dissolved inorganic carbon in a subtropical groundwater‑fed reservoir, south China

Most reservoirs in subtropical areas experience periodic variations in the thermal structure of their water columns, with times of strong thermal stratification being succeeded by periods of mixing, over the course of the year. Understanding of the transport and transformation of dissolved inorganic carbon over such thermal cycles in artificial reservoirs remains poor. To address this problem, this study examined the spatiotemporal behavior of dissolved inorganic carbon (DIC), the partial pressure of CO2 (pCO2), carbon isotope ratios (δ13CDIC), and CO2 emission (FCO2), from 2014 to 2018 in a subtropical, groundwater-fed reservoir in southern China. It was found that CO2 emissions during mixing periods are much higher than in thermally stratified periods (particularly during transition from stratified to mixing) as a result of upwelling and release of dissolved CO2 (CO2aq) accumulated in the hypolimnion. CO2 emission fluxes at the water-gas interface accounted for only a small proportion of the DIC in the reservoir. The relationships between of DIC and δ13CDIC displayed two distinct modes, due to spatial differences in water depths and to strong thermal stratification during warmer seasons: (1) DIC concentrations increase and δ13CDIC values decrease from epilimnion to hypolimnion, and (2) δ13CDIC values decrease with increasing DIC concentrations but δ13CDIC is progressively enriched near the bottom during periods of thermal stratification. In addition, this study found three distinct processes of DIC accumulation and consumption in the reservoir: (1) DIC accumulated in the hypolimnion during thermal stratification periods, due to carbon retention but (2) DIC was substantially consumed in the epilimnion during such periods, and (3) average DIC concentrations and pCO2 increased significantly from upstream to downstream along the reservoir, while average δ13CDIC values became lighter. These results highlight that carbon behavior in groundwater‑fed reservoirs is often controlled by a combination of biogeochemical processes and seasonal variations in thermal structure. Sampling and monitoring strategies should consider these factors in order to accurately estimate carbon budgets in reservoirs, lakes or ponds.

Seasonal Carbon Dynamics in the Near‐Global Ocean

The seasonal cycle represents one of the largest signals of dissolved inorganic carbon (DIC) in the ocean, yet these seasonal variations are not well established at a global scale. Here, we present the Mapped Observation‐Based Oceanic DIC (MOBO‐DIC) product, a monthly DIC climatology developed based on the DIC measurements from GLODAPv2.2019 and a two‐step neural network method to interpolate and map the measurements. MOBO‐DIC extends from the surface down to 2,000 m and from 65°N to 65°S. We find the largest seasonal amplitudes of surface DIC in the northern high‐latitude Pacific (∼30 to &gt;50 μmol kg−1). Surface DIC maxima occur in hemispheric spring and minima in fall, driven by the input of DIC into the upper ocean by mixing during winter, and net community production (NCP) driven drawdown of DIC over summer. The seasonal pattern seen at the surface extends to a nodal depth of &lt;50 m in the tropics and several hundred meters in the subtropics. Below the nodal depth, the seasonal cycle of DIC has the opposite phase, primarily owing to the seasonal accumulation of DIC stemming from the remineralization of sinking organic matter. The well‐captured seasonal drawdown of DIC in the mid‐latitudes (23° to 65°) allows us to estimate the spring‐to‐fall NCP in this region. We find a spatially relatively uniform spring‐to‐fall NCP of 1.9 ± 1.3 mol C m−2 yr−1, which sums to 3.9 ± 2.7 Pg C yr−1 over this region. This corresponds to a global spring‐to‐fall NCP of 8.2 ± 5.6 Pg C yr−1.

Transport, anoxia and end-product accumulation control carbon dioxide and methane production and release in peat soils

Anaerobic respiration and methanogenesis have been found to slow-down in water saturated peat soils with accumulation of metabolic end-products, i.e. dissolved inorganic carbon (DIC) and methane (CH4), due to a lack of solute and gas transport. So far it is not well understood how solute and gas transport may control this effect. We conducted a column experiment with homogenized ombrotrophic peat over a period of 300 days at 20 °C. We specifically evaluated the effects of diffusive flux as control, downward advective water flux, intensified ebullition by conduit gas transport and diffusive oxygen supply on controlling anaerobic decomposition rates and carbon (C) turnover. To simulate advective flux, water and solutes were recirculated downward through the column after stripping of dissolved gases. We analyzed DIC and CH4 concentrations, production rates and fluxes, gas filled porosity, oxygen profiles (O2) and microbial C biomass over time. DIC residence time thereby served as proxy to characterize transport. A slowdown of anaerobic respiration and methanogenesis evolved with the accumulation of the end-products DIC and CH4 and set in after 150 days. This slow-down was accompanied by a decrease in the distribution of microbial biomass C with depths. Anaerobic DIC and CH4 production rates were fastest close to the water table and sharply slowed with depth. Accumulation of DIC and CH4 in the homogeneous peat material throughout the column decreased decomposition constants from about 10−5 near the surface to 10−9 year−1 deeper in the profile. Advective water transport extended the zone of active methanogenesis compared to a diffusive system; experimental enhancement of ebullition had little or no effect as well as strictly anoxic conditions. DIC residence time was negatively correlated to anaerobic respiration suggesting this parameter to be a predictor of anaerobic peat decomposition in peatlands. Overall, this study suggests that burial of peat and accumulation of metabolic end-products effectively slows decomposition and that this effect needs to be considered to explain peat accumulation and the response of peat mineralization rates to changes in environmental conditions.

Assessing seasonal changes in carbonate parameters across small spatial gradients in the Northeastern Chukchi Sea

Observations of the marine carbonate system were made in 2010 in the northeastern Chukchi Sea to constrain the seasonal progression of carbonate mineral saturation states (Ω) throughout the water column and determine the air–sea flux of carbon dioxide (CO2). As sea ice retreats from the Chukchi Shelf, primary production consumes dissolved inorganic carbon (DIC) in the euphotic zone causing pH and carbonate mineral saturation states to increase. Throughout the summer and early autumn months of 2010, saturation states for calcite and aragonite ranged from 2.5 to 4.0 and 1.5 to 2.5, respectively, well about the saturation horizon of 1.0. Much of the organic matter produced during the bloom was vertically exported from the relatively small study area leading to an uptake of CO2 from the atmosphere of at least 340,000kg-C. The exported organic matter settled near the bottom and was remineralized back into DIC, causing concentrations to increase sharply, particularly in autumn months, driving down pH to as low as 7.75 and suppressing the concentrations of important carbonate minerals to the point that aragonite became undersaturated. The data showed a definitive seasonal progression of this process with aragonite becoming partially undersaturated along the bottom in September, and broadly undersaturated in October. While carbonate saturation states would naturally be suppressed by the high rates of export production and the accumulation of DIC near the bottom, the penetration of anthropogenic CO2 into the water column (ocean acidification) has caused these observed undersaturations, which will likely expand as CO2 levels in the atmosphere continue to rise in the coming decades.

Export of inorganic carbon from two Southeast Asian mangrove forests to adjacent estuaries as estimated by the stable isotope composition of dissolved inorganic carbon

The influence of mangrove forests on the dynamics of dissolved inorganic carbon (DIC) in tropical estuaries was estimated quantitatively using newly developed isotope (δ13C) mass balance models that take into account both the input of DIC and the air‐water exchange of CO2. To this aim, the concentration and δ13C of DIC were determined across the salinity gradient of two river estuaries facing the Andaman Sea. The longitudinal distribution of DIC could be explained by conservative mixing of the river water and seawater DIC in the low‐discharge period (March 2006), while a net accumulation of DIC up to 190 μmol L−1 was observed in the high‐discharge period (December 2006). δ13CDIC values were generally lower than expected for the mixing of the river water and seawater DIC, due to the 13C‐depleted DIC inputs from the riverside mangroves. The concentration of mangrove‐derived DIC in the estuarine waters was estimated by the proposed models to be as much as 856 μmol L−1, and was higher during the low‐discharge period. This suggested that the mangroves exported much higher levels of DIC to the estuaries than indicated by the net accumulation of DIC. Our results confirm that mangroves function as an effective CO2 pump that takes CO2 from the atmosphere and releases it into estuarine waters. This study illustrates that δ13CDIC is a sensitive and quantitative indicator for DIC emission to the sea from coastal wetlands including mangroves.

Geochemical controls on anaerobic organic matter decomposition in a northern peatland

The decomposition of deep peat deposits controls the long‐term carbon balance of peatlands but is poorly understood with respect to rates and controls. To rectify this deficiency, we estimated in situ dissolved inorganic carbon (DIC) and methane (CH4) production rates from a beaver pond to a central bog dome and related them to organic matter properties, Gibbs free energies of respiration, and δ13C values of DIC and CH4. DIC and CH4 production decreased from maxima of ~10 nmol cm‐3 d‐1 near the water table to values &lt;0.1 nmol cm‐3 d‐1 at depths &gt;1 m, and there was little differentiation among sites. Deeper into the peat, we measured an accumulation of DIC, CH4, and dissolved organic matter (DOM) enriched in aromatic and phenolic moieties, which resulted from the slowness of diffusive vertical pore‐water movement. Lack of transport may have slowed decomposition in two ways: (1) Aromatic and phenolic DOM moieties accumulated, while the release of carbohydrate‐rich DOM from peat was apparently impeded. (2) The accumulation of DIC and CH4 reduced Gibbs free energy of acetoclastic methanogenesis toward a critical threshold value of ‐25 to ‐20 kJ mol‐1 CH4. Hydrogenotrophic methanogenesis was energetically more favorable and generally dominated according to an isotopic fractionation between CO2 and CH4 of 1.053 to 1.076, but it was apparently impeded by some other factor. We conclude that lateral homogeneity and slowness of decomposition in geologically sealed deep peat deposits are assisted by a lack of solute transport, which facilitates the formation of deep peat deposits over millennia.

Winter respiration of allochthonous and autochthonous organic carbon in a subarctic clear‐water lake

We studied a small subarctic lake to assess the magnitude of winter respiration and the organic carbon (OC) source for this respiration. The concentration and stable isotopic composition (δ13C) of dissolved inorganic carbon (DIC) accumulating in the lake water under ice was analyzed over one winter (7 months). The DIC concentration increased and the δ13C of DIC decreased over time, with the greatest changes at the lake bottom. Winter respiration was 26% of annual respiration in the lake. Keeling plot analysis demonstrated that the δ13C of respired DIC varied spatially, high δ13C values occurring at shallow (2.5 m, 221.7‰%) compared with intermediate (4 m, 225.1‰) and deep (6 m, 227.8‰) locations in the lake. The variation in the δ13C of respired DIC was related to the variation in the δ13C of the sediments between locations, suggesting that sediment OC supported much of the winter respiration and that the dominant OC source for respiration was OC from benthic algae at shallow locations and settled OC, of predominately terrestrial origin, at deep locations. The respiration of OC from benthic algae constituted 55% of the winter respiration, equaling 54% of the primary production by benthic algae the previous summer. The study indicates the importance of temporal and spatial variation in respiration for the metabolism and net DIC production in unproductive high‐latitude lakes; both allochthonous and autochthonous carbon can contribute to winter DIC accumulation and, consequently, to spring CO2 emissions from lakes.

Respiration of allochthonous organic carbon in unproductive forest lakes determined by the Keeling plot method

We carried out short‐term (2 d) experiments in nine unproductive lakes in northern Sweden in order to investigate organic carbon sources supporting lake water respiration. Surface water was incubated in gas‐tight bottles in the dark, and the concentration and isotopic composition (δ13C) of dissolved inorganic carbon (DIC) were measured at the start and end of the incubations. Keeling plot analyses revealed that the δ13C of the respired carbon was between ‐28.4% and ‐30.6% in the lakes and that the respired carbon was mainly of allochthonous organic carbon (AlloOC) origin. The respiration of AlloOC corresponded well with metabolic imbalances indicated by negative net ecosystem production (NEP) values in the lake waters. Keeling plot analysis of DIC accumulating in the hypolimnion of two lakes during summer stratification showed δ13C values of around ‐26.6% for excess DIC, implying that the accumulation of DIC was mainly derived from respiration of AlloOC. Our data provide direct evidence that net heterotrophy of these lakes is caused by input and respiration of AlloOC. We conclude that the Keeling plot method is a powerful technique that enables characterization and quantification of the organic carbon sources contributing to respiration in aquatic systems.

Dissolved inorganic carbon concentration mechanism in Chlamydomonas moewusii

The dissolved inorganic carbon concentrating mechanism(s) of Chlamydomonas moewusii CC 55 was compared with C. reinhardtii strain 137. C. moewusii is similar to C. reinhardtii with respect to maximal rates of photosynthetic oxygen evolution, CO 2 fixation, respiration, and the ability to efficiently concentrate inorganic carbon. C. moewusii has a low, but measurable amount of external carbonic anhydrase (CA) that was not inhibited by acetazolamide (AZ), an inhibitor of periplasmic carbonic anhydrase (pCA) in C. reinhardtii. The K 0.5(CO 2) for air-grown C. moewusii is about 1 μM and the algal cells accumulated dissolved inorganic carbon (DIC) to a level of about 1 mM in 60 s. AZ did not inhibit CO 2 fixation and the DIC accumulation by air-grown cells of C. moewusii. The K 0.5(CO 2) for both species remains constant from pH 6.5 to 9.5 while K 0.5(HCO 3 -) increased logarithmically, which indicates that CO 2 is the apparent inorganic carbon species that enters the cells in both algae. Antiserum prepared against the 37 kDa peptide of pCA from C. reinhardtii was immunoreactive with polypeptides of 26, 28, and 32 kDa in C. moewusii. The periplasmic carbonic anhydrase (pCA) activity is a part of the dissolved inorganic carbon concentrating mechanism in C. reinhardtii, but C moewusii accomplished inorganic carbon accumulation without an AZ-sensitive pCA.

Oxygen inhibition of dissolved inorganic carbon uptake in unicellular green algae

SUMMARY Unicellular green algae have a dissolved inorganic carbon (DIC) concentrating mechanism, commonly known as the DIC pump, to concentrate inorganic carbon into cells and chloroplasts. The DIC pump activity is normally measured as the K0.5(DIC) that equals the CO2 plus HCO3- concentration at a cited pH at which the rate of DIC-dependent photosynthetic O2 evolution is half-maximal, or by the amount of intra-cellular DIC accumulation in 15–60 s, using a limited amount of NaH14CO3, measured by the silicone oil cen-trifugation technique. The dissolved oxygen in the assay inhibits or reduces the DIC uptake by the cells of unicellular green algae Chlamydomonas reinhardtii Dangeard, strain 137 and in a cell wall-less marine algae Dunaliella tertiolecta Butcher. The algal cells concentrated the highest amount of DIC when little or no oxygen was present in the assay medium. The results suggest that the amount of O2 and DIC must be carefully monitored before DIC-pump assay.

Extracellular carbonic anhydrase facilitates carbon dioxide availability for photosynthesis in the marine dinoflagellate prorocentrum micans

This study investigated inorganic carbon accumulation in relation to photosynthesis in the marine dinoflagellate Prorocentrum micans. Measurement of the internal inorganic carbon pool showed a 10-fold accumulation in relation to external dissolved inorganic carbon (DIC). Dextran-bound sulfonamide (DBS), which inhibited extracellular carbonic anhydrase, caused more than 95% inhibition of DIC accumulation and photosynthesis. We used real-time imaging of living cells with confocal laser scanning microscopy and a fluorescent pH indicator dye to measure transient pH changes in relation to inorganic carbon availability. When steady-state photosynthesizing cells were DIC limited, the chloroplast pH decreased from 8.3 to 6.9 and cytosolic pH decreased from 7.7 to 7.1. Re-addition of HCO3- led to a rapid re-establishment of the steady-state pH values abolished by DBS. The addition of DBS to photosynthesizing cells under steady-state conditions resulted in a transient increase in intracellular pH, with photosynthesis maintained for 6 s, the amount of time needed for depletion of the intracellular inorganic carbon pool. These results demonstrate the key role of extracellular carbonic anhydrase in facilitating the availability of CO2 at the exofacial surface of the plasma membrane necessary to maintain the photosynthetic rate. The need for a CO2-concentrating mechanism at ambient CO2 concentrations may reflect the difference in the specificity factor of ribulose-1,5 bisphosphate carboxylase/oxygenase in dinoflagellates compared with other algal phyla.

Active uptake of inorganic carbon byChlorella saccharophilais not repressed by growth in high CO2

Regulation of transport of dissolved inorganic carbon (DIC) in response to CO 2 concentration in the external medium has been compared in two closely-related green algae, Chlorella ellipsoidea and Chlorella saccharophila. C. ellipsoidea, when grown in high CO 2 , had reduced activities of both CO 2 and HCO 3 - transport and DIC transport activities were increased after the cells had acclimated to air. However, high CO 2 -grown C. saccharophila had a comparable level of photosynthetic affinity for DIC to that of air-grown C. ellipsoidea and this was accompanied by a capacity to accumulate high internal concentrations of DIC. The high photosynthetic affinity and the high intracellular DIC accumulation did not change in cells grown in air except that the occurrence of external carbonic anhydrase (CA) in air-grown C. saccharophila stimulated the intracellular DIC accumulation in the absence of added CA. These data indicate that active DIC transport is constitutively expressed in C. saccharophila, presumably because this alga is insensitive to the repressive effect of high CO 2 on DIC transport. This strongly supports the existence of a direct sensing mechanism for external CO 2 in Chlorella species, but also indicates that external CA is regulated independently of DIC transport in Chlorella species.

Induction of Inorganic Carbon Accumulation in the Unicellular Green Algae Scenedesmus obliquus and Chlamydomonas reinhardtii

The induction of a dissolved inorganic carbon (DIC) accumulating mechanism in the two algal species Scenedesmus obliquus (WT) and Chlamydomonas reinhardtii (137 c+) was physiologically characterized by monitoring DIC uptake kinetics at a low and constant DIC concentration (120-140 micromolar), after transfer from high-DIC culturing conditions. A potentiometric titration method was used to measure and calculate algal DIC uptake. Full acclimation to low-DIC conditions was obtained within a period of 90 min, after which time the DIC uptake had been increased 7 to 10 times. Experiments were also conducted in the presence of inhibitors against DIC accumulation. The inhibitor of extracellular carbonic anhydrase (CA), acetazolamide (50 micromolar), inhibited the adaptation partly, while the inhibitor of both extra- and intracellular CA, ethoxyzolamide (50 micromolar) totally inhibited the acclimation. Cycloheximide (10 micrograms per milliliter), which inhibits protein synthesis on cytoplasmic ribosomes, and vanadate (180 micromolar), which inhibits the plasmamembrane bound ATPase, also inhibited the acclimation totally. These results taken together suggest that the algae are dependent on intracellular CA, plasmamembrane bound ATPase, and de novo protein synthesis for DIC accumulation. Also, these components are more important than extracellular CA for the overall function of the DIC-accumulating mechanism.

The Role of External Carbonic Anhydrase in Inorganic Carbon Acquisition by Chlamydomonas reinhardii at Alkaline pH

The role of external carbonic anhydrase in inorganic carbon acquisition and photosynthesis by Chlamydomonas reinhardii at alkaline pH (8.0) was studied. Acetazolamide (50 micromolar) completely inhibited external carbonic anhydrase (CA) activity as determined from isotopic disequilibrium experiments. Under these conditions, photosynthetic rates at low dissolved inorganic carbon (DIC) were far greater than could be maintained by CO(2) supplied from the spontaneous dehydration of HCO(3) (-) thereby showing that C. reinhardii has the ability to utilize exogenous HCO(3) (-). Acetazolamide increased the concentration of DIC required to half-saturate photosynthesis from 38 to 80 micromolar, while it did not affect the maximum photosynthetic rate. External CA activity was also removed from the cell-wall-less mutant (CW-15) by washing. This had no effect on the photosynthetic kinetics of the algae while the addition of acetazolamide to washed cells (CW-15) increased the K((1/2)) (DIC) from 38 to 80 micromolar. Acetazolamide also caused a buildup of the inorganic carbon pool upon NaHCO(3) addition, indicating that this compound partially inhibited internal CA activity. The effects of acetazolamide on the photosynthetic kinetics of C. reinhardii are likely due to the inhibition of internal rather than a consequence of the inhibition of external CA. Further analysis of the isotopic disequilibrium experiments at saturating concentration of DIC provided evidence consistent with active CO(2) transport by C. reinhardii. The observation that C. reinhardii has the ability to take up both CO(2) and bicarbonate throws into question the role of external CA in the accumulation of DIC in this alga.