Photoreduction of O(2) Primes and Replaces CO(2) Assimilation

A mass spectrometer with a membrane inlet system was used to monitor directly gaseous components in a suspension of algae. Using labeled oxygen, we observed that during the first 20 seconds of illumination after a dark period, when no net O₂ evolution or CO₂ uptake was observed, O₂ evolution was normal but completely compensated by O₂ uptake. Similarly, when CO₂ uptake was totally or partially inhibited, O₂ evolution proceeded at a high (near maximal) rate. Under all conditions, O₂ uptake balanced that fraction of the O₂ evolution which could not be accounted for by CO₂ uptake.     

O(2) uptake in the light in chlamydomonas: evidence for persistent mitochondrial respiration

The nature of the process responsible for the stationary O₂ uptake occurring in the light under saturating CO₂ concentration in Chlamydomonas reinhardii has been investigated. For this purpose, a mass spectrometer with a membrane inlet system was used to measure O₂ uptake and evolution in the algal suspension. First, we observed that the O₂ uptake rate was constant (about 0.5 micromoles of O₂ per milligram chlorophyll per minute) during a light to dark transition and was not affected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Salicylhydroxamic acid had no effect on O₂ uptake in the dark or in the light, but was found to have the same inhibitory effect either in the dark or in the light when added to cyanide-treated algae. The stimulation of the O₂ uptake rate due to the uncoupling effect of carbonyl cyanide m-chlorophenylhydrazone was about the same in the dark or in the light. From these results, we conclude that mitochondrial respiration is maintained during illumination and therefore is not inhibited by high ATP levels. Another conclusion is that in conditions where photorespiration is absent, no other light-dependent O₂ uptake process occurs. If Mehler reactions are involved, in Chlamydomonas, under conditions where both photosynthetic carbon oxidation and reduction cycles cannot operate (as in cyanide-treated algae), their occurrence in photosynthesizing algae either under saturating CO₂ concentration or at the CO₂ compensation point appears very unlikely. The comparison with the situation previously reported in Scenedesmus (R. J. Radmer and B. Kok 1976 Plant Physiol 58: 336-340) suggests that different O₂ uptake processes might be present in these two algal species.     

Effect of temperature on h(2) evolution and acetylene reduction in pea nodules and in isolated bacteroids

Nitrogenase (EC 1.7.99.2) activity in pea (Pisum savitum) nodules formed after infection with Rhizobium leguminosarum (lacking uptake hydrogenase) was measured as acetylene reduction, H₂ evolution in air and H₂ evolution in Ar:O₂. With detached roots the relative efficiency, calculated from acetylene reduction, showed a decrease (from 55 to below 0%) with increasing temperature. With excised nodules and isolated bacteroids similar results were obtained. However, the relative efficiency calculated from H₂ evolution in Ar:O₂ was unaffected by temperature. Measurements on both excised nodules and isolated bacteroids showed a marked difference between acetylene reduction and H₂ evolution in Ar:O₂ with increased temperature, indicating that either acetylene reduction or H₂ evolution in Ar:O₂ are inadequate measures of nitrogenase activity at higher temperature.     

Photorespiration in Air and High CO(2)-Grown Chlorella pyrenoidosa

Oxygen inhibition of photosynthesis and CO₂ evolution during photorespiration were compared in high CO₂-grown and air-grown Chlorella pyrenoidosa, using the artificial leaf technique at pH 5.0. High CO₂ cells, in contrast to air-grown cells, exhibited a marked inhibition of photosynthesis by O₂, which appeared to be competitive and similar in magnitude to that in higher C₃ plants. With increasing time after transfer to air, the photosynthetic rate in high CO₂ cells increased while the O₂ effect declined. Photorespiration, measured as the difference between ¹⁴CO₂ and ¹²CO₂ uptake, was much greater and sensitive to O₂ in high CO₂ cells. Some CO₂ evolution was also present in air-grown algae; however, it did not appear to be sensitive to O₂. True photosynthesis was not affected by O₂ in either case. The data indicate that the difference between high CO₂ and air-grown algae could be attributed to the magnitude of CO₂ evolution. This conclusion is discussed with reference to the oxygenase reaction and the control of photorespiration in algae.     

The Relationship between H(2) Evolution and Acetylene Reduction in Pisum sativum-Rhizobium leguminosarum Symbioses Differing in Uptake Hydrogenase Activity

Peas (Pisum sativum L.) were inoculated with strains of Rhizobium leguminosarum having different levels of uptake hydrogenase (Hup) activity and were grown in sterile Leonard jars under controlled conditions. Rates of H₂ evolution and acetylene reduction were determined for intact nodulated roots at intervals after the onset of darkness or after removal of the shoots. Hup activity was estimated using treatment plants or equivalent plants from the growth chamber, by measuring the uptake of H₂ or ³H₂ in the presence of acetylene. In all cases, the rate of H₂ evolution was a continuous function of the rate of acetylene reduction. In symbioses with no demonstrable Hup activity, H₂ evolution increased in direct proportion to acetylene reduction and the slopes were similar with the Hup⁻ strains NA502 and 128C79. Hup activity was similar in strains 128C30 and 128C52 but significantly lower in strain 128C54. With these strains, the slopes of the H₂ evolution versus acetylene reduction curves initially increased with acetylene reduction, but became constant and similar to those for the Hup⁻ strains at high rates of acetylene reduction. On these parallel portions of the curves, the decreases in H₂ evolution by Hup⁺ strains were similar in magnitude to their H₂-saturated rates of Hup activity. The curvilinear relationship between H₂ evolution and acetylene reduction for a representative Hup⁺ strain (128C52) was the same, regardless of the experimental conditions used to vary the nitrogenase activity.     

Kinetics and Apparent K(m) of Oxygen Cycle under Conditions of Limiting Carbon Dioxide Fixation

A mass spectrometer with a membrane inlet was used to monitor light-driven O₂ evolution, O₂ uptake, and CO₂ uptake in suspensions of algae (Scenedesmus obliquus). We observed the following. (a) The rate of O₂ uptake, which, in the presence of iodoacetamide, replaces the uptake of CO₂, showed a distinct plateau (V_max) beyond ~30% O₂ and was half-maximal at ~8% O₂. We concluded that this light-driven O₂ uptake process, which does not involve carbon compounds, is saturated at lower O₂ concentrations than are photorespiration and glycolate formation. (b) In the absence of inhibitor, O₂ evolution was relatively unaffected by the presence or absence of CO₂. During the course of CO₂ depletion, electron flow to CO₂ was replaced by an equivalent flow to O₂. (c) There was a distinct delay between the cessation of CO₂ uptake and the increase in O₂ uptake. We ascribe this delay to the transient utilization of another electron acceptor—possibly bicarbonate or another bound form of CO₂.     

Investigation of the H(2) Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids

The H₂-oxidizing complex in Rhizobium japonicum 122 DES bacteroids failed to catalyze, at a measurable rate, ²H¹H exchange from a mixture of ²H₂ and ¹H₂ in presence of ²H₂O and ¹H₂O, providing no evidence for reversibility of the hydrogenase reaction in vivo. In the H₂ oxidation reaction, there was no significant discrimination between ²H₂ and ¹H₂, indicating that the initial H₂-activation step in the over-all H₂ oxidation reaction is not rate-limiting. By use of improved methods, an apparent K_m for H₂ of 0.05 micromolar was determined. The H₂ oxidation reaction in bacteroids was strongly inhibited by cyanide (88% at 0.05 millimolar), theonyltrifluoroacetone, and other metal-complexing agents. Carbonyl cyanide m-chlorophenylhydrazone at 0.005 millimolar and 2,4-dinitrophenol at 0.5 millimolar inhibited H₂ oxidation and stimulated O₂ uptake. This and other evidence suggest the involvement of cytochromes and nonheme iron proteins in the pathway of electron transport from H₂ to O₂. Partial pressures of H₂ at 0.03 atmosphere and below had a pronounced inhibitory effect on endogenous respiration by bacteroid suspensions. The inhibition of CO₂ evolution by low partial pressures of H₂ suggests that H₂ utilization may result in conservation of oxidizable substrates and benefits the symbiosis under physiological conditions. Succinate, acetate, and formate at concentrations of 50 millimolar inhibited rates of H₂ uptake by 8, 29, and 25%, respectively. The inhibition by succinate was noncompetitive and that by acetate and formate was uncompetitive. A concentration of 11.6 millimolar CO₂ (initial concentration) in solution inhibited H₂ uptake by bacteroid suspensions by 18%. Further research is necessary to establish the significance of the inhibition of H₂ uptake by succinate, acetate, formate, and CO₂ in the metabolism of the H₂-uptake-positive strains of Rhizobium.     

The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.     

H2-Uptake and evolution in the unicellular cyanobacterium Chroococcidiopsis thermalis CALU 758

The unicellular cyanobacterium Chroococcidiopsis thermalis CALU 758 growing photoautotrophically synthesised a hydrogenase which catalysed an in vivo H₂ uptake in the oxyhydrogen reaction at a significant rate and showed only low level of in vitro MV-dependent H₂ evolution. The in vitro hydrogenase activity was not induced under microaerobic or nitrate-limiting conditions. Some correlation observed between the two activities indicated that the same enzyme may be involved in both H₂ uptake and H₂ evolution. Heterologous Southern hybridisations, using cyanobacterial hup and hox DNA fragments as probes, showed the presence of sequences similar to hox (encoding for a bidirectional hydrogenase) in C. thermalis CALU 758 with no indication for the presence of any sequences corresponding to an uptake hydrogenase. Further molecular experiments, using specific primers directed against different conserved regions of the large subunit (hoxH) of the bidirectional hydrogenase confirmed the presence of corresponding sequences in C. thermalis CALU 758. Low-stringency Southern hybridisations detected only one copy of hoxH within the genome of C. thermalis CALU 758.     

Active, Irreversible Accumulation of Extreme Levels of H(2)SO(4) in the Brown Alga, Desmarestia

The brown algae Desmarestia ligulata var. ligulata (Lightf.) Lamour., and D. viridis (Mull.) Lamour., accumulate H₂SO₄ until their average internal pH is 0.5 to 0.8. A related species, D. aculeata (L.) Lamour., does not accumulate acid. The H₂SO₄ accumulation is accompanied by a reduction in the K⁺ and Cl⁻ content, presumedly to maintain osmotic balance. Measurements of the membrane potential and H⁺ and SO₄²⁻ concentrations indicate that both ions are accumulated in the vacuole against their electrochemical potential gradients.

The internal pH remains constant in all three species over the growing season, despite striking changes in the algal morphology. The pH is not affected by periods of darkness of up to 34 hours. Sulfate accumulated in the vacuoles appears to be trapped there since incubation of D. ligulata for up to 10 days in sulfate-free medium resulted in little loss of either vacuolar sulfate or H⁺. Although the uptake of H₂SO₄ into the vacuole must require energy, the maintenance of the vacuolar H₂SO₄ may be due to the impermeability of the tonoplast, with little necessity for continued expenditure of energy.

     

Evolution of o(2) in brown algal chloroplasts

A method is described for the isolation of photosynthetically active chloroplasts from four species of brown algae: Fucus vesiculosis, Nereocystis luetkeana, Laminaria saccharina, and Macrocystis integrifolia. When compared to lettuce and spinach chloroplasts, the algal chloroplasts all showed lower activities for both photosystems II and I. Chloroplasts from all the plants produced H₂O₂, with photosystem I functioning as the O₂ reductant in the light. In contrast to the green plants, however, brown algal chloroplasts strongly reduced O₂ under conditions where both photosystems II and I remain active. Relative variable fluorescence values were lower both in intact plants and chloroplasts of the brown algae than for either spinach or lettuce. It is suggested that although light harvesting activities appear similar in all the plants, details of electron transport in brown algae may differ from those of green plants.     

Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli

Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H₂ to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H₂ evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H₂ evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H₂-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H₂ oxidation or H₂ evolution in whole cells, reversible H₂-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H₂ evolution mode. Our findings suggest that Hyd-2 switches between H₂-consuming and H₂-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H₂ evolution from glycerol requires reverse electron transport.     

A highly sensitive, flow through h(2) gas analyzer for use in nitrogen fixation studies

Studies of H₂ evolution by N₂ fixing systems are frequently limited by an inability to accurately measure H₂ gas concentrations of less than about 10 microliters per liter. In this study, a H₂ gas analyzer is described which is able to accurately and reproducibly detect up to 100 times lower H₂ concentrations than most thermal conductivity gas chromatographs or other conventional instruments used for the measurement of H₂ gas. This high level of sensitivity (maximum of about 0.02 microliter per liter H₂ per millivolt output) and the ability to continuously monitor H₂ concentration directly in a flowing gas stream, makes this instrument well suited for use in an open gas exchange system.

Since the sensor used in the instrument was also sensitive to other combustible gases, it was necessary to demonstrate that H₂ was the only combustible gas produced by the N₂ fixing system being studied. When an air stream was passed through a pot containing nodulated soybean (Glycine max L.) roots, gas chromatographic analysis of the effluent gas stream revealed that H₂ was the only combustible gas present. These results were supported by other studies in which no combustible gases were detected in the effluent gas stream from soybean roots nodulated with USDA 110, a Rhizobium strain which displays active uptake hydrogenase activity.

     

Diurnal variation in n(2) fixation and photosynthesis by aquatic blue-green algae

Rates of ¹⁴CO₂ fixation, O₂ evolution, and N₂ fixation (acetylene reduction) by natural populations of blue-green algae recovered from Lake Mendota were measured at frequent intervals between sunrise and sunset. Photosynthesis and N₂ fixation were depressed during midday when light intensity was greatest. As the light intensity rose, most of the algal population migrated to deeper, light-limited waters where radiation damage would be diminished. As the relative rate of N₂ fixation compared to CO₂ fixation increases with depth, it is suggested that the algae maintain balanced growth by migrating vertically via buoyancy regulation. High concentrations of dissolved O₂ in lake water may inhibit N₂ fixation by enhancing photorespiration. Several factors such as photosynthetic rate, light intensity, dissolved O₂, species composition, and vertical and horizontal migration all affect observed rates of in situ N₂ fixation.     

Varying Photoperiod, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase and CO(2) Uptake in Thalassiosira fluviatilis (Bacillariophyceae)

The purpose of this research was to test the hypothesis that acclimation of the unicellular marine alga, Thalassiosira fluviatilis Hustedt, to short photoperiods results in decreased cellular concentrations of ribulose 1,5-bisphosphate carboxylase/oxygenase and decreased rates of light-saturated CO₂ uptake. Cells were acclimated to photoperiods of 6:18, 12:12, and 18:6 h:h light:dark, and concentrations of the large subunit of the enzyme and responses of CO₂ uptake to varying irradiance were measured. Concentrations of the large subunit, which weighed approximately 50 kilodaltons, were conserved while rates of CO₂ uptake under light saturation and limitation, and cellular contents of chlorophyll a increased as photoperiod decreased. Apparently, these cells acclimate to short photoperiods by increasing rates of CO₂ uptake under saturating irradiances by increasing in vivo activation of ribulose 1,5-bisphosphate carboxylase/oxygenase. Also, chlorophyll-specific concentrations and specific activities of the enzyme appear to be lower and higher, respectively, in diatomaceous algae than in higher plants.     

Carbon Oxysulfide Inhibition of the CO(2)-Concentrating Process of Unicellular Green Algae

Carbonyl sulfide (COS), a substrate for carbonic anhydrase, inhibited alkalization of the medium, O₂ evolution, dissolved inorganic carbon accumulation, and photosynthetic CO₂ fixation at pH 7 or higher by five species of unicellular green algae that had been air-adapted for forming a CO₂-concentrating process. This COS inhibition can be attributed to inhibition of external HCO₃⁻ conversion to CO₂ and OH⁻ by the carbonic anhydrase component of an active CO₂ pump. At a low pH of 5 to 6, COS stimulated O₂ evolution during photosynthesis by algae with low CO₂ in the media without alkalization of the media. This is attributed to some COS hydrolysis by carbonic anhydrase to CO₂. Although COS had less effect on HCO₃⁻ accumulation at pH 9 by a HCO₃⁻ pump in Scenedesmus, COS reduced O₂ evolution probably by inhibiting internal carbonic anhydrases. Because COS is hydrolyzed to CO₂ and H₂S, its inhibition of the CO₂ pump activity and photosynthesis is not accurate, when measured by O₂ evolution, by NaH¹⁴CO₃ accumulation, or by ¹⁴CO₂ fixation.     

Photosynthetic H<Subscript>2</Subscript> metabolism in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> (unicellular green algae)

Unicellular green algae have the ability to operate in two distinctly different environments (aerobic and anaerobic), and to photosynthetically generate molecular hydrogen (H₂). A recently developed metabolic protocol in the green alga Chlamydomonas reinhardtii permitted separation of photosynthetic O₂-evolution and carbon accumulation from anaerobic consumption of cellular metabolites and concomitant photosynthetic H₂-evolution. The H₂ evolution process was induced upon sulfate nutrient deprivation of the cells, which reversibly inhibits photosystem-II and O₂-evolution in their chloroplast. In the absence of O₂, and in order to generate ATP, green algae resorted to anaerobic photosynthetic metabolism, evolved H₂ in the light and consumed endogenous substrate. This study summarizes recent advances on green algal hydrogen metabolism and discusses avenues of research for the further development of this method. Included is the mechanism of a substantial tenfold starch accumulation in the cells, observed promptly upon S-deprivation, and the regulated starch and protein catabolism during the subsequent H₂-evolution. Also discussed is the function of a chloroplast envelope-localized sulfate permease, and the photosynthesis–respiration relationship in green algae as potential tools by which to stabilize and enhance H₂ metabolism. In addition to potential practical applications of H₂, approaches discussed in this work are beginning to address the biochemistry of anaerobic H₂ photoproduction, its genes, proteins, regulation, and communication with other metabolic pathways in microalgae. Photosynthetic H₂ production by green algae may hold the promise of generating a renewable fuel from nature’s most plentiful resources, sunlight and water. The process potentially concerns global warming and the question of energy supply and demand.     

Photosynthetic o(2) exchange kinetics in isolated soybean cells

Light-dependent O₂ exchange was measured in intact, isolated soybean (Glycine max. var. Williams) cells using isotopically labeled O₂ and a mass spectrometer. The dependence of O₂ exchange on O₂ and CO₂ was investigated at high light in coupled and uncoupled cells. With coupled cells at high O₂, O₂ evolution followed similar kinetics at high and low CO₂. Steady-state rates of O₂ uptake were insignificant at high CO₂, but progressively increased with decreasing CO₂. At low CO₂, steady-state rates of O₂ uptake were 50% to 70% of the maximum CO₂-supported rates of O₂ evolution. These high rates of O₂ uptake exceeded the maximum rate of O₂ reduction determined in uncoupled cells, suggesting the occurrence of another light-induced O₂-uptake process (i.e. photorespiration).

Rates of O₂ exchange in uncoupled cells were half-saturated at 7% to 8% O₂. Initial rates (during induction) of O₂ exchange in uninhibited cells were also half-saturated at 7% to 8% O₂. In contrast, steady-state rates of O₂ evolution and O₂ uptake (at low CO₂) were half-saturated at 18% to 20% O₂. O₂ uptake was significantly suppressed in the presence of nitrate, suggesting that nitrate and/or nitrite can compete with O₂ for photoreductant.

These results suggest that two mechanisms (O₂ reduction and photorespiration) are responsible for the light-dependent O₂ uptake observed in uninhibited cells under CO₂-limiting conditions. The relative contribution of each process to the rate of O₂ uptake appears to be dependent on the O₂ level. At high O₂ concentrations (≥40%), photorespiration is the major O₂-consuming process. At lower (ambient) O₂ concentrations (≤20%), O₂ reduction accounts for a significant portion of the total light-dependent O₂ uptake.

     

Purification and properties of the H2-oxidizing (uptake) hydrogenase of the N2-fixing anaerobe Clostridium pasteurianum W5

Clostridium pasteurianum has two distinct hydrogenases, the bidirectional hydrogenase and the H₂-oxidizing (uptake) hydrogenase. The H₂-oxidizing hydrogenase has been purified (up to 970-fold) to a specific activity of 17,600 μmol H₂ oxidized/min·mg protein (5 mM methylene blue) or 3.5 μmol H₂ produced/min·mg protein (1 mM methyl viologen). The uptake hydrogenase has a M_r of 53,000 (one polypeptide chain). Depending upon how protein was measured, the Fe and S⁼ contents (gatom/mol) were 4.7 and 5.2 (by the dye-binding assay) or 7.2 and 8.0 (by the Lowry method). Both reduced and oxidized forms of the enzyme gave electron paramagnetic resonance signals. The activation energy for H₂-production and H₂-oxidation by the uptake hydrogenase was 59.1 and 31.2 kJ/mol, respectively. In the exponential phase of growth, the ratio of uptake hydrogenase/bidirectional hydrogenase in NH₃-grown cells was much lower than that in N₂-fixing cells.     

Salicylhydroxamic Acid (SHAM) Inhibition of the Dissolved Inorganic Carbon Concentrating Process in Unicellular Green Algae

Rates of photosynthetic O₂ evolution, for measuring K_0.5(CO₂ + HCO₃⁻) at pH 7, upon addition of 50 micromolar HCO₃⁻ to air-adapted Chlamydomonas, Dunaliella, or Scenedesmus cells, were inhibited up to 90% by the addition of 1.5 to 4.0 millimolar salicylhydroxamic acid (SHAM) to the aqueous medium. The apparent K₁(SHAM) for Chlamydomonas cells was about 2.5 millimolar, but due to low solubility in water effective concentrations would be lower. Salicylhydroxamic acid did not inhibit oxygen evolution or accumulation of bicarbonate by Scenedesmus cells between pH 8 to 11 or by isolated intact chloroplasts from Dunaliella. Thus, salicylhydroxamic acid appears to inhibit CO₂ uptake, whereas previous results indicate that vanadate inhibits bicarbonate uptake. These conclusions were confirmed by three test procedures with three air-adapted algae at pH 7. Salicylhydroxamic acid inhibited the cellular accumulation of dissolved inorganic carbon, the rate of photosynthetic O₂ evolution dependent on low levels of dissolved inorganic carbon (50 micromolar Na-HCO₃), and the rate of ¹⁴CO₂ fixation with 100 micromolar [¹⁴C] HCO₃⁻. Salicylhydroxamic acid inhibition of O₂ evolution and ¹⁴CO₂-fixation was reversed by higher levels of NaHCO₃. Thus, salicylhydroxamic acid inhibition was apparently not affecting steps of photosynthesis other than CO₂ accumulation. Although salicylhydroxamic acid is an inhibitor of alternative respiration in algae, it is not known whether the two processes are related.