Induction of intracellular carbonic anhydrases during the adaptation to low inorganic carbon concentrations in wild-type and ca-1 mutant cells of Chlamydomonas reinhardtii

Mass-spectrometric measurements of ¹⁸O exchange from ¹³C¹⁸O₂ were used to follow changes in the intracellular carbonic anhydrase (CA) activity of cells of Chlamydomonas reinhardtii Dang, wild type and the ca-1 mutant during adaptation to air. With intact cells as well as with crude homogenates total intracellular CA activity in wild-type cells increased six to tenfold within 4 h after transferring cells from 5% CO₂ (high inorganic carbon, C_i) to ambient air (air adapted). After that time the activity slowly declined to a level similar to that observed with cells which had been continuously grown in air (low-C_i grown). In the ca-1 mutant, total CA was induced to a similar extent during 4 h of adaptation; however, absolute activities were two to three times lower in ca-1 than in the wild type regardless of the CO₂ supply. When crude extracts from wild-type cells were separated into soluble and insoluble fractions, each fraction contained about half of the internal CA activity. Within 4 h of adaptation, both forms of CA activity were simultaneously enhanced by nine to tenfold, reaching levels similar to those found in low-C_igrown cells. In contrast, in the ca-1 mutant the soluble CA activity was only enhanced by about eightfold while the level of insoluble CA was very low even in low-C_i cells. After isolation of intact chloroplasts from wild-type cells and further subfractionation, around 70–80% of total chloroplastic CA activity was found to be in the insoluble fraction while 17–20% remained in the soluble fraction. Both chloroplastic CA activities were inducible within the first 4 h of adaptation to air, with each of them being eight to ten times higher than in high-C_i algae. After that time their activities were similar to the corresponding CA values in low-C_i-grown cells. In contrast, plastids from high-C_i cells of the ca-1 mutant showed 40% less insoluble-CA activity compared to the wild type and this insoluble-CA activity was not increased at all by transferring algae to air. In addition, no soluble-CA activity was detected in chloroplasts from high-C_i and air-adapted ca-1 cells. These results indicate the presence of three intracellular CA activities in high-C_i air-adapted and low-C_i cells of the wild type and that two of them are associated with the chloroplasts. All three activities are completely induced within the first 4 h of adaptation to air in wild-type cells. In contrast, it was not possible to induce any of the chloroplastic CA activities in the ca-1 mutant. The possibility that the soluble chloroplastic CA represents a pyrenoid-located CA is discussed.This work is dedicated to Professor A. Wild on the occasion of his 65th birthday     

Regulation of the low-CO2-inducible polypeptides in Chlamydomonas reinhardtii

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii Dang. when cells are transferred from high (2%) to low (0.03%) CO₂ concentrations. The synthesis of these polypeptides is correlated with the induction of the CO₂-concentrating mechanism. In this work we studied the effect of the growth conditions on the synthesis of these polypeptides with the aim of clarifying whether the induction of all three of these low-CO₂-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO₂ conditions occurred only in the light, while the 37-kDa periplasmic carbonic anhydrase (EC 4.2.1.1) was induced in light, darkness, and in both synchronous and asynchronous cultures. In addition, induction of these polypeptides appeared to be determined more by the O₂/CO₂ ratio than by the CO₂ concentrations. None of these polypeptides could be induced in either of two different mutants of C. reinhardtii, one lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and the other with inactive enzyme. Our results indicate that the 21- and 36-kDa polypeptides are regulated by a mechanism different from that controlling the 37-kDa polypeptide.Abbreviations pCA (periplasmic) carbonic anhydrase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TAP Trisacetate phosphate medium The authors thank Prof. M. Spalding (Iowa State University, USA) for providing antisera to LIP-21 and LIP-36. We thank Prof. S. Bartlett and Dr. J. Moroney (Louisiana State University, USA) for providing antibodies to C. reinhardtii, Rubisco and 37-kDa pCA, respectively. This work was supported by the Instituto Tecnologico de Canarias.     

Low-CO2-inducible protein synthesis in the green alga Dunaliella tertiolecta

In the green marine alga Dunaliella tertiolecta, a CO₂-concentrating mechanism is induced when the cells are grown under low-CO₂ conditions (0.03% CO₂). To identify proteins induced under low-CO₂ conditions the cells were labelled with ³⁵SO₄ ^2–, and seven polypeptides with molecular weights of 45, 47, 49, 55, 60, 68 and 100 kDa were detected. The induction of these polypeptides was observed when cells grown in high CO₂ (5% CO₂ in air) were switched to low CO₂, but only while the cultures were growing in light. Immunoblot analysis of total cell protein against pea chloroplastic carbonic anhydrase polyclonal antibodies showed immunoreactive 30-kDa bands in both high- and low-CO₂-grown cells and an aditional 49-kDa band exclusively in low-CO₂-grown cells. The 30-kDa protein was shown to be located in the chloroplast. Western blot analysis of the plasmamembrane fraction against corn plasma-membrane AT-Pase polyclonal antibodies showed 60-kDa bands in both high- and low-CO₂ cell types as well as an immunoreactive 100-kDa band occurring only in low-CO₂-grown cells. These results suggest that there are two distinct forms of both carbonic anhydrase and plasma-membrane ATPase, and that one form of each of them can be regulated by the CO₂ concentration.Abbreviations CA carbonic anhydrase - DIC dissolved inorganic carbon (CO₂+ HCO₃ ^–) - CCM CO₂-concentrating mechanism - low CO₂ air containing 0.03% CO₂ - high CO₂ air supplemented with 5% CO₂ (v/v) We thank Prof. John Coleman for providing antibodies raised against pea chloroplast CA, Dr. James V. Moroney for providing antibodies raised against the 37-kDa periplasmic carbonic anhydrase of CO₂ Chlamydomonas reinhardtii, and Prof. Leonard T. Robert for a gift of corn plasma-membrane 100-kDa ATPase antibodies. We thank Dr. Jeanine Olsen (University of Groningen, the Netherlands) for style comments. This work was supported by the Institute Tecnológico de Canarias (Spain).     

Atherosclerosis: the carbonic anhydrase, carbon dioxide, calcium concerted theory

Atherosclerosis is a dynamic multifaceted disease which affects the aorta and its major branches, characterized by the presence of lesions called atheromatous plaques. The plaque is a focal thickening of the intima caused by proliferation of smooth muscle cells, and the deposition of cholesterol, other lipids, hydroxyapatite and fibrous connective tissue. It is proposed that the determinant step of the process which leads to the disease atherosclerosis is the calcium precipitation which traps cholesterol in the plaque precursor matrix which contains lipoproteins, calcium carbonate, hydroxyapapatite, triglycerides, albumin, calmodulin and other proteins. The bear, a species which does not contract the disease is used as an example in support of the hypothesis. The bear's ability to regulate calcium levels and the regulation of acid base balance via regulation of carbon dioxide levels permits the control of the determinant step of plaque formation, that is calcification of the plaque.     

Isolation of cDNA clones of genes induced upon transfer of Chlamydomonas reinhardtii cells to low CO2

Unicellular algae grow well under limiting CO₂ conditions, aided by a carbon concentrating mechanism (CCM). In C. reinhardtii, this mechanism is inducible and is present only in cells grown under low CO₂ conditions. We constructed a cDNA library from cells adapting to low CO₂, and screened the library for cDNAs specific to low CO₂-adapting cells. Six classes of low CO₂-inducible clones were identified. One class of clone, reported here, represents a novel gene associated with adaptation of cells to air. A second class of clones corresponds to the air-inducible periplasmic carbonic anhydrase I (CAH1). These clones represent genes that respond to the level of CO₂ in the environment.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

The CO2 permeability of the plasma membrane of Chlamydomonas reinhardtii: mass-spectrometric 18O-exchange measurements from 13C18O2 in suspensions of carbonic anhydrase-loaded plasma-membrane vesicles

The unicellular green alga Chlamydomonas reinhardtii possesses a CO₂-concentrating mechanism. In order to measure the CO₂ permeability coefficients of the plasma membranes (PMs), carbonic anhydrase (CA) loaded vesicles were isolated from C. reinhardtii grown either in air enriched with 50 mL CO₂ · L?1} (high-C_i cells) or in ambient air (350 μL CO₂ · L?1}; low-C_i cells). Marker-enzyme measurements indicated less than 1% contamination with thylakoid and mitochondrial membranes, and that more than 90% of the PMs from high and low-C_i cells were orientated right-side-out. The PMs appeared to be sealed as judged from the ability of vesicles to accumulate [¹⁴C]acetate along a proton gradient for at least 10 min. Carbonic anhydrase-loaded PMs from high and low-C_i cells of C. reinhardtii were used to measure the exchange of ¹⁸O between doubly labelled CO₂ (¹³C¹⁸O₂) and H₂O in stirred suspensions by mass spectrometry. Analysis of the kinetics of the ¹⁸O depletion from ¹³C¹⁸O₂ in the external medium provides a powerful tool to study CO₂ diffusion across the PM to the active site of CA which catalyses ¹⁸O exchange only inside the vesicles but not in the external medium (Silverman et al., 1976, J Biol Chem 251: 4428–4435). The activity of CA within loaded PM vesicles was sufficient to speed-up the ¹⁸O loss to H₂O to 45360–128800 times the uncatalysed rate, depending on the efficiency of CA-loading and PM isolation. From the ¹⁸O-depletion kinetics performed at pH 7.3 and 7.8, CO₂ permeability coefficients of 0.76 and 1.49·10?3} cm·s?1}, respectively, were calculated for high C_i cells. The corresponding values for low-C_i cells were 1.21 and 1.8·10?3} cm·s?1}. The implications of the similar and rather high CO₂ permeability coefficients (low CO₂ resistance) in high and low-C_i cells for the CO_i-concentrating mechanism of C. reinhardtii are discussed.     

Carboxysomal carbonic anhydrases: Structure and role in microbial CO2 fixation

Cyanobacteria and some chemoautotrophic bacteria are able to grow in environments with limiting CO₂ concentrations by employing a CO₂-concentrating mechanism (CCM) that allows them to accumulate inorganic carbon in their cytoplasm to concentrations several orders of magnitude higher than that on the outside. The final step of this process takes place in polyhedral protein microcompartments known as carboxysomes, which contain the majority of the CO₂-fixing enzyme, RubisCO. The efficiency of CO₂ fixation by the sequestered RubisCO is enhanced by co-localization with a specialized carbonic anhydrase that catalyzes dehydration of the cytoplasmic bicarbonate and ensures saturation of RubisCO with its substrate, CO₂. There are two genetically distinct carboxysome types that differ in their protein composition and in the carbonic anhydrase(s) they employ. Here we review the existing information concerning the genomics, structure and enzymology of these uniquely adapted carbonic anhydrases, which are of fundamental importance in the global carbon cycle.     

Characterization of carbonic anhydrase from diversified genus for biomimetic carbon-dioxide sequestration

Diversified group of bacteria were screened for carbonic anhydrase (CA) activity. Significant CA activity was found in crude enzyme extracts of Enterobacter and Aeromonas isolates while minimal or negligible CA activity was observed in case of Shigella and Klebsiella spp. Optimization and characterization study of potent CA producing isolates revealed that the maximum enzyme activity of 3.86 EU/ml was observed in E. taylorae and the optimum pH range for enzyme stability was found to be 7.5–9.0 along with an optimum temperature range of 35–50 °C. The molecular mass of CA was 29-kDa indicating α-type with periplasmic and cytosolic location. Present investigation for the first time reports CA in diversified genus and optimized parameters for enhanced production of CA in Enterobacter sp. & Aeromonas sp. from fresh water bodies that inturn lay down grounds for exploitation of CA from E. taylorae as an efficient catalyst for CO₂ sequestration within a bioreactor.     

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii

The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO₂ assimilation was determined by CO₂ exchange studies at three, limiting CO₂ concentrations with a ca-1 mutant of Chlamydomonas reinhardiii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO₂ assimilation did not respond to the varying photon flux densities because of CO₂ limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100 mol photons m^-2 s^-1, indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO₂ assimilation responded to external CO₂ concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO₂ into the cells supplies most of the CO₂ for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO₂ concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO₂ exchange method to determine whether inorganic carbon accumulation and photosynthetic CO₂ assimilation compete for energy at low photon flux densities proved inconclusive.     

Complementation analysis of the inorganic carbon concentrating mechanism of Chlamydomonas reinhardtii

Summary Six independently isolated mutants of Chlamydomonas reinhardtii that require elevated CO₂ for photoautotrophic growth were tested by complementation analysis. These mutants are likely to be defective in some aspect of the algal concentrating mechanism for inorganic carbon as they exhibit CO₂ fixation and inorganic carbon accumulation properties different from the wild-type. Four of the six mutants defined a single complementation group and appear to be defective in an intracellular carbonic anhydrase. The other two mutations represent two additional complementation groups.Abbreviations HS high salt medium which has 13 mM phosphate at pH 6.8 - HSA high salt plus 36 mM acetate medium - YA high salt medium with 4 g yeast extract per L and 36mM acetate - Arg arginine - cia^- CO₂ accumulation mutants that cannot grow on low CO₂ - C_i inorganic carbon (CO₂+HCO _- ³ ) - CA carbonic anhydrase - mt mating type Supported in part by the McKnight Foundation and by NSF grant PCM 8005917 and published as journal article 11924 from the Michigan State Agriculatural Experiment Station     

Effect of dissolved inorganic carbon on oxygen evolution and uptake by Chlamydomonas reinhardtii suspensions adapted to ambient and CO2-enriched air

Mass spectrometric measurements of ¹⁶O₂ and ¹⁸O₂ isotopes were used to compare the rates of gross O₂ evolution (E₀), O₂ uptake (U₀) and net O₂ evolution (NET) in relation to different concentrations of dissolved inorganic carbon (DIC) by Chlamydomonas reinhardtii cells grown in air (air-grown), in air enriched with 5% CO₂ (CO₂-grown) and by cells grown in 5% CO₂ and then adapted to air for 6h (air-adapted).At a photon fluence rate (PFR) saturating for photosynthesis (700 mol photons m^-2 s^-1), pH=7.0 and 28°C, U₀ equalled E₀ at the DIC compensation point which was 10M DIC for CO₂-grown and zero for air-grown cells. Both E₀ and U₀ were strongly dependent on DIC and reached DIC saturation at 480 M and 70 M for CO₂-grown and air-grown algae respectively. U₀ increased from DIC compensation to DIC saturation. The U₀ values were about 40 (CO₂-grown), 165 (air-adapted) and 60 mol O₂ mg Chl^-1 h^-1 (air-grown). Above DIC compensation the U₀/E₀ ratios of air-adapted and air-grown algae were always higher than those of CO₂-grown cells. These differences in O₂ exchange between CO₂- and air-grown algae seem to be inducable since air-adapted algae respond similarly to air-grown cells.For all algae, the rates of dark respiratory O₂ uptake measured 5 min after darkening were considerably lower than the rates of O₂ uptake just before darkening. The contribution of dark respiration, photorespiration and the Mehler reaction to U₀ is discussed and the energy requirement of the inducable CO₂/HCO₃ ^- concentrating mechanism present in air-adapted and air-grown C. reinhardtii cells is considered.Abbreviations DIC dissolved inorganic carbon - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - E₀ rate of photosynthetic gross O₂ evolution - PCO photosynthetic carbon oxidation - PFR photon fluence rate - PS I photosystem I - PS II photosystem II - U₀ rate of O₂ uptake in the light - MS mass spectrometer     

Very high light resistant mutants of Chlamydomonas reinhardtii: Responses of Photosystem II,nonphotochemical quenching and xanthophyll pigments to light and CO2

We have isolated very high light resistant nuclear mutants (VHL ^R) in Chlamydomonas reinhardtii, that grow in 1500–2000 mol photons m^–2 s^–1 (VHL) lethal to wildtype. Four nonallelic mutants have been characterized in terms of Photosystem II (PS II) function, nonphotochemical quenching (NPQ) and xanthophyll pigments in relation to acclimation and survival under light stress. In one class of VHL ^R mutants isolated from wild type (S4 and S9), VHL resistance was accompanied by slower PS II electron transfer, reduced connectivity between PS II centers and decreased PS II efficiency. These lesions in PS II function were already present in the herbicide resistant D1 mutant A251L (L ^*) from which another class of VHL ^R mutants (L4 and L30) were isolated, confirming that optimal PS II function was not critical for survival in very high light. Survival of all four VHL ^R mutants was independent of CO₂ availability, whereas photoprotective processes were not. The de-epoxidation state (DPS) of the xanthophyll cycle pigments in high light (HL, 600 mol photons m^–2 s^–1) was strongly depressed when all genotypes were grown in 5% CO₂. In S4 and S9 grown in air under HL and VHL, high DPS was well correlated with high NPQ. However when the same genotypes were grown in 5% CO₂, high DPS did not result in high NPQ, probably because high photosynthetic rates decreased thylakoid pH. Although high NPQ lowered the reduction state of PS II in air compared to 5% CO₂ at HL in wildtype, S4 and S9, this did not occur during growth of S4 and S9 in VHL. L ^* and VHL ^R mutants L4 and L30, also showed high DPS with low NPQ when grown air or 5% CO₂, possibly because they were unable to maintain sufficiently high pH due to constitutively impaired PS II electron transport. Although dissipation of excess photon energy through NPQ may contribute to VHL resistance, there is little evidence that the different genes conferring the VHL ^R phenotype affect this form of photoprotection. Rather, the decline of chlorophyll per biomass in all VHL ^R mutants grown under VHL suggests these genes may be involved in regulating antenna components and photosystem stoichiometries.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effect of O2 and CO2 on net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii during dark-light-dark transitions

Net CO₂ exchange was monitored through a dark-light-dark transition, under 2% and 21% O₂ in the presence and absence of CO₂, in Chlamydomonas reinhardtii wild type and the high-CO₂-requiring mutant ca-1-12-1C. Upon illumination at 350 l/l CO₂, ca-1-12-1C cell exhibited a large decrease in net CO₂ uptake following an initial surge of CO₂ uptake. Net CO₂ uptake subsequently attained a steady-state rate substantially lower than the maximum. A large, O₂-enchanced post-illumination burst of CO₂ efflux was observed after a 10-min illumination period, corresponding to a minimum in the net CO₂ uptake rate. A smaller, but O₂-insensitive post-illumination burst was observed following a 30-min illumination period, when net CO₂ uptake was at a steady-state rate. These post-illumination bursts appeared to reflect the release of an intracellular pool of inorganic carbon, which was much larger following the initial surge of net CO₂ uptake than during the subsequent steady-state CO₂ uptake period.With the mutant in CO₂-free gas, O₂-stimulated, net CO₂ efflux was observed in the light, and a small, O₂-dependent post-illumination burst was observed. With wild-type cells no CO₂ efflux was observed in the light in CO₂-free gas under either 2% or 21% O₂, but a small, O₂-dependent post-illumination burst was observed. These results were interpreted as indicating that photorespiratory rates were similar in the mutant and wild-type cells in the absence of CO₂, but that the wild-type cells were better able to scavenge the photorespiratory CO₂.     

The thylakoid carbonic anhydrase associated with photosystem II is the component of inorganic carbon accumulating system in cells of halo- and alkaliphilic cyanobacterium Rhabdoderma lineare

The organization of carbonic anhydrase (CA) system in halo- and alkaliphilic cyanobacterium Rhabdoderma lineare was studied by Western blot analysis and immunocytochemical electron microscopy. The presence of putative extracellular α-CA of 60 kDa in the glycocalyx, forming a tight sheath around the cell, and of two intracellular β-CA is reported. We show for the first time that the β-CA of 60 kDa is expressed constitutively and associated with polypeptides of photosystem II (β-CA-PS II). Another soluble β-CA of 25 kDa was induced in low-bicarbonate medium. Induction of synthesis of the latter β-CA was accompanied by an increase in the intracellular pool of inorganic carbon, which suggests an important role of this enzyme in the functioning of a CO₂-concentrating mechanism.     

Role of external carbonic anhydrase in light-dependent alkalization byFucus serratus L. andLaminaria saccharina (L.) Lamour. (Phaeophyta)

It has been proposed that many marine macroalgae are able to utilize HCO ₃ ^– for photosynthesis and growth, and that energy-dependent ion pumping is involved in this process. We have therefore studied the light-dependent alkalization of the surrounding medium by two species of marine macroscopic brown algae,Fucus serratus L. andLaminaria saccharina (L.) Lamour. with the aim of investigating the role of extracellular carbonic anhydrase (EC 4.2.1.1.) in the assimilation of inorganic carbon from the seawater medium. In particular, the influence of membrane-impermeable or slowly permeable carbonic-anhydrase inhibitors on the rate of alkalization of the seawater has been investigated. Inhibition of the alkalization rate occurred in both species at an alkaline pH (pH 8.0) but no inhibition was observed at an acidic pH (pH 6.0). The alkalization was found to be light-dependent and inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and, thus, correlated with photosynthesis. Alkalization by macroalgae has previously been shown to be proportional to inorganiccarbon uptake. We suggest that alkalization of the medium at alkaline pH in both of the species examined is mainly the consequence of an extracellular reaction. The reaction is catalyzed by extracellular carbonic anhydrase which converts HCO ₃ ^– to OH^– and CO₂; CO₂ is then taken up through the plasmalemma. However, we do not exclude the involvement of other mechanisms of inorganic-carbon uptake.Abbreviations AZ acetazolamide - CA carbonic anhydrase - CAext extracellular carbonic anhydrase - C_i inorganic carbon - DBS dextran-bound sulfonamide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PPFD photosynthetic photon flux density This study was carried out with financial support by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Trygger's Fund for Scientific Research (Sweden), SJFR (Swedish Council for Forestry and Agricultural Research) and CICYT (Spain). Z. Ramazanov is an invited professor of Ministerio de Educación y Ciencia, Spain.     

Effect of oxygen on the growth (yield) of Chlorella vulgaris

The growth yield of Chlorella vulgaris, Y _kJ defined as g cells harvested per kJ of light energy absorbed by the cells, was assessed in a turbidostat culture by varying CO₂ and O₂ partial pressures ( and ). The value of Y _kJ ranged from 3.1×10^-3 to 5.0×10^-3 g cells/kJ under light-limited conditions [ = 1.02.4%, = 065%; total pressure of gas (composed of CO₂, O₂ and N₂)=1 atm]. In the light-limited environment, the algal specific growth rate deteriorated appreciably with the increase of . The deterioration accounts for the above range of Y _kJ observed. The growth inhibition due to oxygen that was defined by subtracting from 1.0 the ratio of at given values of to that at = 0% extended from 0.07–0.30 (7–30%). However, glycolate could not be detected in the turbidostat culture. Isotopic experiments on the specific rate of ¹⁴CO₂ uptake also revealed that the inhibition due to oxygen was from 22–38% when was varied from 0 to nearly 100%. These effects of oxygen were discussed, referring to the activity of ribulose-1,5-bisphosphate carboxylase that is inhibited competitively by oxygen.Non-Standard Abbreviations INH isonicotinic acid hydrazide - PPO 2,5-diphenyloxazole - DCMU 3-(-3,4-dichlorophenyl)-1,1-dimetylurea - CA carbonic anhydrase - RuP₂ ribulose-1,5-bisphosphate     

Inhibition of the growth of two blue-green algae species (Microsystis aruginosa and Anabaena spiroides) by acidification treatments using carbon dioxide

The effect of pH adjusted by aeration with carbon dioxide (CO₂) on the growth of two species of blue-green algae, Microcystis aeruginosa and Anabaena spiroides, was investigated. Three conditions (pH 5.5, 6.0 and 6.5) were found to have significant inhibitory effects on the growth of the two algae species when acidification treatment was conducted during the logarithmic phase. Differences in the inhibition effect of acidification existed between the two species algae. The tolerance of M. aeruginosa to these conditions was also investigated. The results indicated that M. aeruginosa was inhibited significantly, but not dead at pH 6.5, whereas death occurred at pH 5.5 and 6.0. The greatest inhibitory effect of acidification treatment conducted during the stable breeding phase of M. aeruginosa occurred at pH 5.5, while no inhibitory effect was found at pH 6.5.