Effect of pH on Inorganic Carbon Uptake in Algal Cultures

Biomass production by the green algae Scenedesmus obliquus and Chlorella vulgaris in intensive laboratory continuous cultures was considerably affected by the pH at which the cultures were maintained. Carbon photoassimilation experiments revealed that pH values in the range of 8 to 9 were important for determining the free CO₂ concentrations in the medium. With higher pH values, additional pH effects were observed involving a decrease in the relative high affinity of low CO₂-adapted algae to free CO₂. The carbon uptake rate by high CO₂-adapted algae after transfer to low free CO₂ medium was characterized by a lag period of about 30 min, after which the affinity of the algae to CO₂ increased considerably. Both continuous growth and carbon uptake experiments indicated that artificially maintained high free CO₂ concentrations are recommended for maximal production in intensive outdoor algal cultures.     

Photosynthetic inorganic carbon utilization and growth of Porphyra linearis (Rhodophyta)

Photosynthetic (oxygen evolution) and growth (biomass increase) responses to ambient pH and inorganic carbon (Ci) supply were determined for Porphyralinearis grown in 0.5 L glass cylinders in the laboratory, or in 40 L fibreglass outdoor tanks with running seawater. While net photosynthetic rates were uniform at pH 6.0–8.0, dropping only at pH 8.7, growth rates were significantly affected by pH levels other than that of seawater (c. pH 8.3). In glass cylinders, weekly growth rates averaged 76% at external pH 8.0, 13% at pH 8.7 and 26% at pH 7.0. Photosynthetic O₂ evolution on a daily basis(i.e. total O₂ evolved during day time less total O₂ consumed during night time) was similar to the growth responses at all experimental pH levels, apparently due to high dark respiration rates measured at acidic pH. Weekly growth rates averaged 53% in algae grown in fibreglass tanks aerated with regular air (360 mg L_-1 CO₂) and 28% in algae grown in tanks aerated with CO₂-enriched air (750 mg L^-1 CO₂). The pH of the seawater medium in which P. linear is was grown increased slightly during the day and only rarely reached 9.0. The pH at the boundary layer of algae submerged in seawater increased in response to light reaching, about pH 8.9 within minutes, or remained unchanged for algae submerged in a CO₂-free artificial sea water medium. Photosynthesis of P. linearissaturated at Ci concentrations of seawater (K_0.5560 μM at pH 8.2) and showed low photosynthetic affinity for CO₂(K_0.5 61 μM) at pH 6.0. It is therefore concluded that P. linearisuses primarily CO₂ with HCO₃ ^- being an alternative source of Ci for photosynthesis. Its fast growth could be related to the enzyme carbonic anhydrase whose activity was detected intra- and extracellularly. This revised version was published online in August 2006 with corrections to the Cover Date.     

CO2- and Anaerobiosis-Induced Changes in Physiology and Gene Expression of Different Listeria monocytogenes Strains

Although carbon dioxide (CO₂) is known to inhibit growth of most bacteria, very little is known about the cellular response. The food-borne pathogen Listeria monocytogenes is characterized by its ability to grow in high CO₂ concentrations at refrigeration temperatures. We examined the listerial responses of different strains to growth in air, 100% N₂, and 100% CO₂. The CO₂-induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent. The acid-tolerant L. monocytogenes LO28 responded in the same manner to CO₂ as to other anaerobic, slightly acidic environments (100% N₂, pH 5.7). An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres. In contrast, the acid-sensitive L. monocytogenes strain EGD responded differently to CO₂ and N₂ at the same pH. In a separate experiment with L. monocytogenes 412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO₂-containing atmospheres. Together, our findings demonstrate that the CO₂-response is a partly strain-dependent complex mechanism. The possible links between the CO₂-dependent changes in isocitrate dehydrogenase activity, glutamate metabolism and branched fatty acid biosynthesis are discussed.     

Toxicity of inorganic cadmium salts to the microalga Scenedesmus armatus (Chlorophyta) with respect to medium composition, pH and CO2 concentration

Batch cultures of algae grown at low (0.1 %) and elevated (2.0 %) concentrations of CO₂, as well as in original BBM (Bold Basal Medium) and BBM modified with phosphate, EDTA and a combination of both, were exposed to cadmium (Cd(NO₃)₂·4H₂O, 3CdSO₄·8H₂O and CdCl₂·H₂O) for 24 h. Regardless of the salt applied, the concentration-dependent relationships of Cd toxicity were found to be biphasic, suggesting the different affinity of target sites to cadmium. Nominal values of EC₅₀ obtained for algae grown in original BBM and at low CO₂ were 18.0, 16.44 and 15.37 mg·dm⁻³ for cadmium nitrate, sulphate and chloride, respectively. However, it was estimated that 97 % of the free cadmium in the added salts were bound by components of original BBM such as EDTA, phosphates, chloride and sulphate. The effect of Cd-salts at concentrations corresponding to EC₅₀ values on algae were tested in media with 10-fold reduced phosphates (BBM-P), BBM depleted of EDTA (BBM-EDTA) and of both phosphates and EDTA (BBM-P-EDTA). For algae grown at low CO₂ and BBM-P, cadmium was about 25 % less toxic than those applied in original BBM. Cadmium greatly inhibited (about 85 % of the control) the growth of algae cultured in BBM-EDTA; this effect was only slightly dependent on the CO₂ concentration. Deficits of both EDTA and P led to effects similar to those brought about by the absence of EDTA only. The toxicity of cadmium depends on CO₂ concentration only when algae are grown in original BBM. The growth of algae under high CO₂ conditions was reduced considerably less (about 80% of control) compared with low CO₂ concentrations (about 50 % of control). A relationship was found between the toxicity of cadmium salts and final pH values only in variants of low-CO₂ grown algae; with an increase of medium pH the toxicity decreased. The results suggest that both growth conditions and the binding ability of the medium markedly affect the toxicity of cadmium towards microalgae.     

Measurement of carbon dioxide compensation points of freshwater algae

A technique is described for the measurement of total dissolved inorganic carbon by acid release as CO₂ followed by its conversion to methane and detection by flame ionization in a modified gas chromatograph. This method was used to determine the dissolved inorganic carbon concentration reached at compensation point when algae were allowed to photosynthesize in a closed system in a buffer at known pH, and the CO₂ compensation point was calculated from this concentration. The CO₂ compensation points of 16 freshwater algae were measured at acid and alkaline pH in air-saturated medium: at acid pH the CO₂ compensation points ranged from 4.8 to 41.5 microliters per liter while at alkaline pH they ranged from 0.2 to 7.2 microliters per liter. Removal of O₂ from the medium caused a slight lowering of compensation point at acid pH but had little effect at alkaline pH. These low, O₂-insensitive compensation points are characteristic of C₄ plants. It is suggested that these low CO₂ compensation points are maintained by an active bicarbonate uptake by algae especially at alkaline pH.     

Increased CO<Subscript>2</Subscript> and the effect of pH on growth and calcification of <Emphasis Type="Italic">Pleurochrysis carterae</Emphasis> and <Emphasis Type="Italic">Emiliania huxleyi</Emphasis> (Haptophyta) in semicontinuous cultures

The effects of changes in CO₂ and pH on biomass productivity and carbon uptake of Pleurochrysis carterae and Emiliania huxleyi in open raceway ponds and a plate photobioreactor were studied. The pH of P. carterae cultures increased during day and decreased at night, whereas the pH of E. huxleyi cultures showed no significant diurnal changes. P. carterae coccolith production occurs during the dark period, whereas in E. huxleyi, coccolith production is mainly during the day. Addition of CO₂ at constant pH (pH-stat) resulted in an increase in P. carterae biomass and coccolith productivity, while CO₂ addition lowered E. huxleyi biomass and coccolith production. Neither of these algae could grow at less than pH 7.5. Species-specific diurnal pH and pCO₂ variations could be indicative of significant differences in carbon uptake between these two species. While E. huxleyi has been suggested to be predominantly a bicarbonate user, our results indicate that P. carterae may be using CO₂ as the main C source for photosynthesis and calcification.     

Vegetative life on Venus? Or investigations with algae which grow under pure CO2 in hot acid media at elevated pressures

Experiments are described with algae grown in pure CO₂ under pressure with an acidic nutrient medium at elevated temperatures. One species found in hot-springs was observed to grow. If the planet Venus has acidic polar seas as we suggest, they may harbor photosynthetic life.This research was supported by NASA 05-007-003 and AFOSR 67-1255A.     

The possible roles of algae in restricting the increase in atmospheric CO2 and global temperature

ABSTRACT

Anthropogenic inputs are increasing the CO₂ content of the atmosphere, and the CO₂ and total inorganic C in the surface ocean and, to a lesser degree, the deep ocean. The greenhouse effect of the increased CO₂ (and, to a lesser extent, other greenhouse gases) is very probably the major cause of present global warming. The warming increases temperature of the atmosphere and the surface ocean to a greater extent than the deep ocean, with shoaling of the thermocline, decreasing nutrient flux to the surface ocean where there is greater mean photosynthetic photon flux density. These global changes influence algae in nature. However, it is clear that algae are important, via the biological pump, in decreasing the steady state atmospheric and ocean surface CO₂, and thus decreasing radiative forcing, a reduction enhanced by algal increases in albedo. As well as these natural processes there are possibilities that algae can, with human intervention, partly offset the increase in atmospheric CO₂. One possibility is to grow algae as sources of fuel for transport, in principle providing an energy source that is close to CO₂-neutral. The other possibility is to increase the role of algae in sequestering CO₂ as organic C over periods of hundreds or more years in the deep ocean and marine sediments and/or increasing albedo and decreasing radiative forcing of temperature. There are problems, currently unresolved, in the economically viable production of algal biofuels without carbon trading subsidies. Enhanced algal CO₂ sequestration also has costs, both in resource input (phosphorus (P) from high P content rocks, a limited resource with a competing use as an agricultural fertilizer) and adverse environmental effects. For example, ocean anoxic zones producing N₂O and increased algal production of short-lived halocarbons by algae that both, through breakdown, destroy O₃ and increase UV flux to the Earth’s surface.     

FORECASTED CO2 MODIFIES THE INFLUENCE OF LIGHT IN SHAPING SUBTIDAL HABITAT1

Some abiotic conditions are well known to play disproportionately large roles in shaping contemporary assemblages, yet their roles may not continue to have similar magnitudes of effect into the future. We tested whether forecasted levels of CO₂ could alter the strength of influence of an abiotic factor (i.e., light intensity) well known for its strength of influence on the subtidal ecology of photosynthetic organisms. We investigated these dynamics in two subtidal algal species that form contrasting associations with kelp forests, one negatively associated with kelp canopies (turf‐forming brown algae, Feldmannia spp.) and the other positively associated with kelp as understory (calcifying red crustose algae, Lithophyllum sp.). Using an experimental approach, we assessed the independent and combined effects of [CO₂] (control and elevated) and light (shade, low ultraviolet B [UVB], full light) on growth, recruitment, and relative electron transport rate (rETR). Under control [CO₂], the effects of light corresponded to the relative light environments of the two groups of algae. The influence of light on the percentage cover and biomass of understory crusts was substantially reduced under elevated [CO₂], which caused crusts to grow less. While elevated [CO₂] had the opposite effect of positively influencing turf cover and biomass, it had the same effect of reducing the structuring effects of light and UVB. Hence, if we are to predict the ecological consequences of future CO₂ conditions, the role of contemporary processes cannot be assumed to produce similar effects relative to other processes, which will change with a changing climate.     

Coralline algal structure is more sensitive to rate,rather than the magnitude,of ocean acidification

Marine pCO₂ enrichment via ocean acidification (OA), upwelling and release from carbon capture and storage (CCS) facilities is projected to have devastating impacts on marine biomineralisers and the services they provide. However, empirical studies using stable endpoint pCO₂ concentrations find species exhibit variable biological and geochemical responses rather than the expected negative patterns. In addition, the carbonate chemistry of many marine systems is now being observed to be more variable than previously thought. To underpin more robust projections of future OA impacts on marine biomineralisers and their role in ecosystem service provision, we investigate coralline algal responses to realistically variable scenarios of marine pCO₂ enrichment. Coralline algae are important in ecosystem function; providing habitats and nursery areas, hosting high biodiversity, stabilizing reef structures and contributing to the carbon cycle. Red coralline marine algae were exposed for 80 days to one of three pH treatments: (i) current pH (control); (ii) low pH (7.7) representing OA change; and (iii) an abrupt drop to low pH (7.7) representing the higher rates of pH change observed at natural vent systems, in areas of upwelling and during CCS releases. We demonstrate that red coralline algae respond differently to the rate and the magnitude of pH change induced by pCO₂ enrichment. At low pH, coralline algae survived by increasing their calcification rates. However, when the change to low pH occurred at a fast rate we detected, using Raman spectroscopy, weaknesses in the calcite skeleton, with evidence of dissolution and molecular positional disorder. This suggests that, while coralline algae will continue to calcify, they may be structurally weakened, putting at risk the ecosystem services they provide. Notwithstanding evolutionary adaptation, the ability of coralline algae to cope with OA may thus be determined primarily by the rate, rather than magnitude, at which pCO₂ enrichment occurs.     

INDUCIBLE MECHANISMS FOR HCO3– UTILIZATION AND REPRESSION OF PHOTORESPIRATION IN PROTOPLASTS AND THALLI OF THREE SPECIES OF ULVA (CHLOROPHYTA)1

Thalli of Ulva reticulata Forskaal, Ulva rigida C. Ag., and Ulva pulchra Jaasund were incubated at different concentrations of dissolved CO₂. Incubation at a high CO₂ concentration resulted in decreased oxygen evolution rate and lower affinity for inorganic carbon at high pH conditions, i.e. the ability to use HCO₃^– as a carbon source was reduced. This effect was reversible, and plants regained this HCO₃^– uptake capacity when transferred to air concentrations of CO₂. The phytosynthetic oxygen evolution rate of plants grown at high CO₂ concentration was reduced by high O₂ concentrations, whereas thalli and protoplasts from cultures grown at air concentration were not affected. This is interpreted as a deactivation of the carbon-concentrating mechanism during conditions of high CO₂ resulting in high photorespiration when plants are exposed to high O₂ concentrations. Protoplasts were not affected by high O₂ to the same extent and were not able to utilize HCO₃^– from the medium. The algae were able to grow at very low CO₂ concentrations, but growth was suppressed when an inhibitor of external carbonic anhydrase was present. Assay of carbonic anhydrase activities showed that external and internal CA activities were lower in plants grown at a high CO₂ concentration compared to plants grown at a low concentration of CO₂. Possible mechanisms for HCO₃^– utilization in these Ulva species are discussed.     

Chloroplast pH values and buffer capacities in darkened leaves as revealed by CO2 solubilization in vivo

Leaves of the C₃ plants Brassica oleracea L., Datura suaveolens Humb. & Bonpl. ex Willd., Helianthus annuus L. and Nicotiana tabacum L. with open stomata were exposed in a leaf chamber in the dark to CO₂ concentrations varying from 1 to 20% in air. When they were transferred back into CO₂-free air, CO₂ was rapidly released. It originated from dissolved CO₂ and from the bicarbonate in the chloroplast stroma, since vacuoles are acidic and chloroplasts contain carbonic anhydrase which rapidly liberates CO₂ from bicarbonate. The data were fitted to a model which accounts for the CO₂/bicarbonate equilibrium in buffers with different CO₂ concentrations and initial pH values. From this, pH values and CO₂-dependent pH changes in the chloroplast stroma were calculated. The full range of external CO₂ concentration caused acidic shifts up to 1 pH unit. The best fits of the data points were obtained with stromal buffer concentrations ranging from 45 to 65 mM and stromal pH values at low CO₂ between 7.5 and 7.9. Calculated buffer capacities ranged from 23 to 31 mM H⁺ per pH unit. The work shows that measurements of solubilized CO₂ are useful to investigate proton buffering and pH regulation in the chloroplast stroma of intact leaves.Abbreviation Chl chlorophyll This work was supported by Sonderforschungsbereich 251 of the University of Würzburg and the Volkswagenstiftung grant I-67762. We are very grateful to Dr. V. Oja for helpful advice.     

SHORT‐ AND LONG‐TERM EFFECTS OF ELEVATED CO2 ON PHOTOSYNTHESIS AND RESPIRATION IN THE MARINE MACROALGA HIZIKIA FUSIFORMIS (SARGASSACEAE,PHAEOPHYTA) GROWN AT LOW AND HIGH N SUPPLIES1

The short‐term and long‐term effects of elevated CO₂ on photosynthesis and respiration were examined in cultures of the marine brown macroalga Hizikia fusiformis (Harv.) Okamura grown under ambient (375 μL · L^?1) and elevated (700 μL · L^?1) CO₂ concentrations and at low and high N availability. Short‐term exposure to CO₂ enrichment stimulated photosynthesis, and this stimulation was maintained with prolonged growth at elevated CO₂, regardless of the N levels in culture, indicating no down‐regulation of photosynthesis with prolonged growth at elevated CO₂. However, the photosynthetic rate of low‐N‐grown H. fusiformis was more responsive to CO₂ enrichment than that of high‐N‐grown algae. Elevation of CO₂ concentration increased the value of K_1/2(Ci) (the half‐saturation constant) for photosynthesis, whereas high N supply lowered it. Neither short‐term nor long‐term CO₂ enrichment had inhibitory effects on respiration rate, irrespective of the N supply, under which the algae were grown. Under high‐N growth, the Q₁₀ value of respiration was higher in the elevated‐CO₂‐grown algae than the ambient‐CO₂‐grown algae. Either short‐ or long‐term exposure to CO₂ enrichment decreased respiration as a proportion of gross photosynthesis (Pg) in low‐N‐grown H. fusiformis. It was proposed that in a future world of higher atmospheric CO₂ concentration and simultaneous coastal eutrophication, the respiratory carbon flux would be more sensitive to changing temperature.     

Combined effects of ocean acidification and solar UV radiation on photosynthesis,growth, pigmentation and calcification of the coralline alga Corallina sessilis (Rhodophyta)

Previous studies have shown that increasing atmospheric CO₂ concentrations affect calcification in some planktonic and macroalgal calcifiers due to the changed carbonate chemistry of seawater. However, little is known regarding how calcifying algae respond to solar UV radiation (UVR, UVA+UVB, 280–400 nm). UVR may act synergistically, antagonistically or independently with ocean acidification (high CO₂/low pH of seawater) to affect their calcification processes. We cultured the articulated coralline alga Corallina sessilis Yendo at 380 ppmv (low) and 1000 ppmv (high) CO₂ levels while exposing the alga to solar radiation treatments with or without UVR. The presence of UVR inhibited the growth, photosynthetic O₂ evolution and calcification rates by13%, 6% and 3% in the low and by 47%, 20% and 8% in the high CO₂ concentrations, respectively, reflecting a synergistic effect of CO₂ enrichment with UVR. UVR induced significant decline of pH in the CO₂‐enriched cultures. The contents of key photosynthetic pigments, chlorophyll a and phycobiliproteins decreased, while UV‐absorptivity increased under the high pCO₂/low pH condition. Nevertheless, UV‐induced inhibition of photosynthesis increased when the ratio of particulate inorganic carbon/particulate organic carbon decreased under the influence of CO₂‐acidified seawater, suggesting that the calcified layer played a UV‐protective role. Both UVA and UVB negatively impacted photosynthesis and calcification, but the inhibition caused by UVB was about 2.5–2.6 times that caused by UVA. The results imply that coralline algae suffer from more damage caused by UVB as they calcify less and less with progressing ocean acidification.     

Vertical tubular reactor for microalgae cultivation

Summary Vertical glass tubular reactors, 5 cm in diameter and 2.35 m high, were used to grow several species of cyanobacteria, green algae, and diatoms. The reactors were gassed with an air/CO₂ mixture, to supply CO₂, remove O₂, and provide mixing. Most of the 10 strains tested had productivities similar to those observed with mechanically mixed reactors. The advantages of the vertical tubular reactors are their high surface to volume ratios, low shear forces, low cost, absence of wall growth, high CO₂ use efficiency, and the ability to use sunlight.     

The relationships between relative growth rate,meristematic potential and compensatory growth of semiarid-land shrubs

The submersed macrophyte Vallisneria americana was grown for seven weeks in a greenhouse to test for differences in the ability of three different sediments to support growth stimulation in response to CO₂ enrichment at low pH. Plants accumulated 21- to 24-fold greater biomass at 10 × ambient CO₂ concentrations than at ambient CO₂ on all sediments. At both CO₂ levels, plants grown on sediment from an acidified lake accumulated ca. 81%, and those grown on oligotrophic lake sediment ca. 47% as much biomass as plants grown on alkaline lake sediment. Despite striking CO₂ and sediment effects on biomass accumulation, there was no significant interaction (using log-transformed data) between CO₂ and sediment effects, indicating that all sediments allowed similar proportionate growth responses to CO₂ enrichment. Plants grown on the less fertile sediments showed greater relative allocation to horizontal versus vertical growth by producing more rosette-bearing stolons in relation to plant height (leaf length) than plants grown on relatively fertile, alkaline lake sediment. Tissue analysis suggested that sediment effects on Vallisneria growth could be attributed neither to mineral putrient (nitrogen and phosphorus) limitation nor to aluminum toxicity in these low pH treatments. In any case, CO₂ availability can be an important regulator of submersed macrophyte growth at low pH on a variety of sediment types, including those from oligotrophic and acidic lakes.     

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.     

Two Systems for Concentrating CO(2) and Bicarbonate during Photosynthesis by Scenedesmus

Scenedesmus cells grown on high CO₂, when adapted to air levels of CO₂ for 4 to 6 hours in the light, formed two concentrating processes for dissolved inorganic carbon: one for utilizing CO₂ from medium of pH 5 to 8 and one for bicarbonate accumulation from medium of pH 7 to 11. Similar results were obtained with assays by photosynthetic O₂ evolution or by accumulation of dissolved inorganic carbon inside the cells. The CO₂ pump with K_0.5 for O₂ evolution of less than 5 micromolar CO₂ was similar to that previously studied with other green algae such as Chlamydomonas and was accompanied by plasmalemma carbonic anhydrase formation. The HCO₃⁻ concentrating process between pH 8 to 10 lowered the K_0.5 (DIC) from 7300 micromolar HCO₃⁻ in high CO₂ grown Scenedesmus to 10 micromolar in air-adapted cells. The HCO₃⁻ pump was inhibited by vanadate (K_i of 150 micromolar), as if it involved an ATPase linked HCO₃⁻ transporter. The CO₂ pump was formed on low CO₂ by high-CO₂ grown cells in growth medium within 4 to 6 hours in the light. The alkaline HCO₃⁻ pump was partially activated on low CO₂ within 2 hours in the light or after 8 hours in the dark. Full activation of the HCO₃⁻ pump at pH 9 had requirements similar to the activation of the CO₂ pump. Air-grown or air-adapted cells at pH 7.2 or 9 accumulated in one minute 1 to 2 millimolar inorganic carbon in the light or 0.44 millimolar in the dark from 150 micromolar in the media, whereas CO₂-grown cells did not accumulate inorganic carbon. A general scheme for concentrating dissolved inorganic carbon by unicellular green algae utilizes a vanadate-sensitive transporter at the chloroplast envelope for the CO₂ pump and in some algae an additional vanadate-sensitive plasmalemma HCO₃⁻ transporter for a HCO₃⁻ pump.     

CO2 reduction by intact chloroplasts under a diminished proton gradient

9-Aminoacridine has been used to monitor the intrathylakoid pH of photo-synthetically competent intact chloroplasts. Values obtained from 9-aminoacridine accumulation in the chloroplasts must be corrected for light-dependent binding of 9-aminoacridine to the thylakoid membranes. During nitrite reduction by intact chloroplasts, the intrathylakoid proton concentration increased. It decreased somewhat during CO₂ reduction. However, low concentrations of uncoupling amines such as NH₃ or cyclohexylamine, which rapidly penetrated the chloroplast envelope and decreased the intrathylakoid proton concentration, failed to reduce, and actually stimulated, rates of CO₂-dependent oxygen evolution even under rate-limiting light. In contrast, low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or nigericin, which inhibited CO₂ reduction, even appeared to increase the intrathylakoid proton concentration. As indicated by measurements of the 515 nm signal of the chloroplasts, the light-induced membrane potential was not much affected by low concentrations of the uncoupling amines, but was decreased by FCCP and by high concentrations of the amines. Even in the presence of high concentrations of NH₄Cl, ATP/ADP ratios of illuminated chloroplasts remained far above the ratios observed in the dark. In contrast, low concentrations of FCCP were sufficient to reduce ATP/ADP ratios to the dark value even under high intensity illumination. The observations are difficult to explain within the framework of the chemiosmotic hypothesis as presently discussed.