Zinc deficiency, carbonic anhydrase, and photosynthesis in leaves of spinach

A shortage in the zinc supply to spinach (Spinacia oleracea L.) drastically reduced carbonic anhydrase levels with little effect on net CO₂ uptake per unit leaf area, except with the most severe zinc stresses. Under these conditions, carbonic anhydrase was below 10% and photosynthesis 60 to 70% of the control levels. When photosynthesis was measured at a range of CO₂ supply levels, zinc-deficient leaves were less efficient at 300 to 350 microliters per liter CO₂ and above, but the same as controls at lower CO₂ levels. This suggests that carbonic anhydrase does not affect the diffusion of CO₂, and that the effect of zinc deficiency was on the photosynthetic process itself. Our evidence does not support the hypothesis that carbonic anhydrase has some role in facilitating the supply of CO₂ to the sites of carboxylation within the chloroplast.     

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas

Inorganic carbon (C_i) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO₂. Both air-grown cells, that have a CO₂ concentrating system, and 5% CO₂-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C_i) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO₂-grown cells also accumulated some C_i, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO₂ fixation by high CO₂-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO₂-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.     

A new tris(2-furyl) substituted pyrazolylborate ligand and its zinc complex chemistry

The new ligand hydrotris(3-(2′-furyl)-5-methylpyrazolyl)borate (Tp^Fu,Me) was prepared by the usual procedure. With zinc salts, it forms the Tp^Fu,MeZn-X complexes (X = Cl, Br, I, NCS, CH₃COO, CF₃COO). With zinc perchlorate, the bis-ligand complex Zn(Tp^Fu,Me)₂ is formed preferrably, but by carefully controlling the reaction conditions, the “enzyme model” Tp^Fu,MeZn-OH could be obtained. The latter models carbonic anhydrase by inserting CO₂ and CS₂ in methanol producing Tp^Fu,MeZn-OCOOMe and Tp^Fu,MeZn-SCSOMe. It models hydrolases by the hydrolytic cleavage of tris(p-nitrophenyl)phosphate and γ-thiobutyrolactone. It does not hydrolyse trifluoroacetamide, but instead deprotonates it, yielding Tp^Fu,MeZn-NHCOCF₃.     

Effect of enriched rhizosphere carbon dioxide on nitrate and ammonium uptake in hydroponically grown tomato plants

Previous reports have indicated positive effects of enriched rhizosphere dissolved inorganic carbon on the growth of salinity-stressed tomato (Lycopersicon esculentum L. Mill. cv. F144) plants. In the present work we tested whether a supply of CO₂ enriched air to the roots of hydroponically grown tomato plants had an effect on nitrogen uptake in these plants. Uptake was followed over periods of 6 to 12 hours and measured as the depletion of nitrogen from the nutrient solution aerated with either ambient or CO₂ enriched air. Enriched rhizosphere CO₂ treatments (5000 μmol mol^-1) increased NO₃ ^- uptake up to 30% at pH 5.8 in hydroponically grown tomato plants compared to control treatments aerated with ambient CO₂ (360 μmol mol^-1). Enriched rhizosphere CO₂ treatments had no effect on NH₃ ⁺ uptake. Acetazolamide, an inhibitor of apoplastic carbonic anhydrase, increased NO₃ ^- uptake in ambient rhizosphere CO₂ treatments, but had no effect on NO₃ ^- uptake in enriched rhizosphere CO₂ treatments. Ethoxyzolamide, an inhibitor of both cytoplasmic and extracellular carbonic anhydrase, decreased NO₃ ^- uptake in ambient rhizosphere CO₂ treatments. In contrast, a CO₂ enriched rhizosphere increased NO₃ ^- uptake with ethoxyzolamide, although not to the same extent as in plants without ethoxyzolamide. It is suggested that a supply of enriched CO₂ to the rhizosphere influenced NO₃ ^- uptake through the formation of increased amounts of HCO₃ ^- in the cytosol. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of Extracellular Carbonic Anhydrase of Chlamydomonas reinhardtii

We have examined the induction of carbonic anhydrase activity in Chlamydomonas reinhardtii and have identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photoautotrophically on 5% CO₂, but its synthesis was induced under low concentrations of CO₂ (air levels of CO₂). In CW-15, a mutant of C. reinhardtii which lacks a cell wall, between 80 and 90% of the carbonic anhydrase activity of air-adapted cells was present in the growth medium. Furthermore, between 80 and 90% of the carbonic anhydrase is released if wild type cells are treated with autolysin, a hydrolytic enzyme responsible for cell wall degradation during mating of C. reinhardtii. These data extend the work of Kimpel, Togasaki, Miyachi (1983 Plant Cell Physiol 24: 255-259) and indicate that the bulk of the carbonic anhydrase is located either in the periplasmic space or is loosely bound to the algal cell wall. The polypeptide associated with carbonic anhydrase activity has a molecular weight of approximately 37,000. Several lines of evidence indicate that this polypeptide is responsible for carbonic anhydrase activity: (a) it appears following the transfer of C. reinhardtii from growth on 5% CO₂ to growth on air levels of CO₂, (b) it is located in the periplasmic space or associated with the cell wall, like the bulk of the carbonic anhydrase activity, (c) it binds dansylamide, an inhibitor of the enzyme which fluoresces upon illumination with ultraviolet light, (d) antibodies which inhibit carbonic anhydrase activity only cross-react with this 37,000 dalton species.     

Bicarbonate-Reversible and Irreversible Inhibition of Photosystem II by Monovalent Anions

We tested a number of inhibitory monovalent anions for their primary site of action on photosystem II(PSII) in chloroplasts. We find that the inhibitory effects of F⁻, HCO₂⁻, NO₂⁻, NO₃⁻, and CH₃CO₂⁻ are all reversed by addition of a high concentration of HCO₃⁻. This class of anions competitively inhibits H¹⁴CO₃⁻ binding to PSII. All of those anions tested reduced H¹⁴CO₃⁻ binding more in the light than in the dark. We conclude that the primary inhibitory site of action of a number of monovalent anions is at the HCO₃⁻ binding site(s) on the PSII complex. The carbonic anhydrase inhibitor gold cyanide, and also azide, inhibit PSII but at a site other than the HCO₃⁻ binding site. We suggest that the unique ability of HCO₃⁻ to reverse the effects of inhibitory anions reflects its singular ability to act as a proton donor/acceptor at the anion binding site. A similar role has been proposed for non-substrate-bound HCO₃⁻ on carbonic anhydrase by Yeagle et al. (1975 Proc Natl Acad Sci USA 72: 454-458).     

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii

Mass spectrometric measurements of dissolved free ¹³CO₂ were used to monitor CO₂ uptake by air grown (low CO₂) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the ¹³CO₂ concentration in the medium to close to zero. Similar results were obtained with low CO₂ cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO₂ uptake revealed that the removal of CO₂ from the medium in the light was due to selective and active CO₂ transport rather than uptake of total dissolved inorganic carbon. In the light, low CO₂ cells and protoplasts incubated with carbonic anhydrase took up CO₂ at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO₂ uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO₂ cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO₂ compensation point. During the linear uptake of CO₂, low CO₂ cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O₂ evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O₂ evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO₂ uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO₂ had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O₂ evolution. These results show low CO₂ cells of Chlamydomonas are able to transport both CO₂ and HCO₃⁻ but CO₂ is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO₂ from the dehydration of HCO₃⁻.     

Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO₂ is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO₂ required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO₂ fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K_0.5(CO₂) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO₂ is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO₂ from HCO₃⁻ at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO₃⁻ transport for internal accumulation might occur at the level of the chloroplast envelope.     

Purification and properties of the carbonic anhydrase of Rhodospirillum rubrum

The carbonic anhydrase (EC 4.2.1.1) of Rhodospirillum rubrum has been purified to apparent homogeneity and some of its properties have been determined. The enzyme was cytoplasmic and was found only in photosynthetically grown cells. It had a molecular weight of about 28,000, and was apparently composed of two equal subunits. The amino acid composition was similar to that of other reported carbonic anhydrases except that the R. rubrum enzyme contained no arginine. The isoelectric point of the enzyme was 6.2 and the pH optimum was 7.5. It required Zn(II) for stability and enzymatic activity. The K _m(CO₂) was 80 mM. Typical carbonic anhydrase inhibition patterns were found with the R. rubrum enzyme. Strong acetazolamide and sulfanilamide inhibition confirmed the importance of Zn(II) for enzymatic activity as did the anionic inhibitors iodide, and azide. Other inhibitors indicated that histidine, sulfhydryl, lysine and serine residues were important for enzymatic activity.Abbreviation CA carbonic anhydrase In memory of R. Y. Stanier     

Synthesis and characterization of a series of diphenyldipyrazolylmethane complexes with zinc(II)

The zinc(II) coordination chemistry of a series of diphenyldipyrazolylmethane ligands was explored using ¹H NMR and single crystal X-ray diffraction. Unsubstituted diphenyldipyrazolylmethane (dpdpm), diphenylbis(3-methylpyrazolyl)methane (dpdp′m), and diphenylbis(3,5-dimethylpyrazolyl)methane (dpdp″m) were reacted with Zn(NO₃)₂ to afford Zn(dpdpm)(NO₃)₂, Zn(dpdp′m)(NO₃)₂ and Zn(Pz″)₂(NO₃)₂ where Pz″ = 3,5-dimethylpyrazole, respectively. All attempts to isolate Zn(dpdp″m)(NO₃)₂ with the intact dpdp″m ligand were unsuccessful due to decomposition of the ligand. These bidentate ligands support the formation of 1:1 ligand to metal complexes and structurally model the two histidine coordination mode common in zinc proteins.     

Identification and localization of a thylakoid-bound carbonic anhydrase from the green algae Tetraedron minimum (Chlorophyta) and Chlamydomonas noctigama (Chlorophyta)

In order to broaden our understanding of the eukaryotic CO₂-concentrating mechanism the occurrence and localization of a thylakoid-associated carbonic anhydrase (EC 4.2.1.1) were studied in the green algae Tetraedron minimum and Chlamydomonas noctigama. Both algae induce a CO₂-concentrating mechanism when grown under limiting CO₂ conditions. Using mass-spectrometric measurements of ¹⁸O exchange from doubly labelled CO₂, the presence of a thylakoid-associated carbonic anhydrase was confirmed for both species. From purified thylakoid membranes, photosystem I (PSI), photosystem II (PSII) and the light-harvesting complex of the photosynthetic apparatus were isolated by mild detergent gel. The protein fractions were identified by 77 K fluorescence spectroscopy and immunological studies. A polypeptide was found to immunoreact with an antibody raised against thylakoid carbonic anhydrase (CAH3) from Chlamydomonas reinhardtii. It was found that this polypeptide was mainly associated with PSII, although a certain proportion was also connected to light harvesting complex II. This was confirmed by activity measurements of carbonic anhydrase in isolated bands extracted from the mild detergent gel. The thylakoid carbonic anhydrase isolated from T. minimum had an isoelectric point between 5.4 and 4.8. Together the results are consistent with the hypothesis that thylakoid carbonic anhydrase resides within the lumen where it is associated with the PSII complex. Received: 13 May 2000 / Accepted: 16 August 2000     

Carbonic anhydrase activity in acetate grown Methanosarcina barkeri

Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K _i=0.5 mM), by azide (apparent K _i=1 mM), and by cyanide (apparent K _i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H₂/CO₂ grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was induced suggesting a function of this enzyme in acetate fermentation to CO₂ and CH₄. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO₂ with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein).     

Basic muscle protein,a third genetic locus isoenzyme of carbonic anhydrase?

Rabbit muscle cytosol extract contains a basic protein which represents about 2% of the total cytosol protein. It contains zinc in a 1:1 stoichiometric ratio, based on a molecular weight of 30,000, and it catalyzes the hydration of CO₂. It is immunochemically distinct from the high and low activity forms of rabbit blood carbonic anhydrase. It has comparatively poor activity as an esterase, and about 20% of the CO₂ hydratase activity of the rabbit blood low activity carbonic anhydrase. This CO₂ hydratase activity is not inhibited by acetazolamide at concentrations which totally inhibit the activity of the blood carbonic anhydrases. The evidence obtained to date, though circumstantial, suggests that this basic metalloprotein is a carbonic anhydrase derived from a third genetic locus with properties considerably different from those of the mammalian carbonic anhydrases heretofore identified.     

The effects of heavy metal salts on the phytohormonal status and sex expression in marijuana

We studied the effects of heavy metal salts (Pb(NO₃)₂, CuSO₄, and ZnSO₄) on phytohormonal status and sex expression in various cultivars of marijuana (Cannabis sativa L.), a dioecious plant, grown on Knop nutrient medium. Pb(NO₃)₂ and ZnSO₄ were added to the medium at the concentration of 10⁻⁹ M, and CuSO₄, at the concentration of 10⁻¹⁰ M. Plant were grown under controlled conditions at luminescent illumination, 22–24°C, and 80% humidity. The contents of GA and zeatin were determined by HPLC. Copper and zinc salts induced plant feminization, and this effect was coupled with zeatin accumulation. Lead salts favored plant masculinization coupled with GA accumulation. Thus, a shift in sex expression in marijuana plants was correlated with the heavy metal action on the balance of phytohormones, GA and zeatin.     

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.     

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).     

Is carbonic anhydrase activity of photosystem II required for its maximum electron transport rate?

It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO₃^?/CO₂ as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α?carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α?carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO₃^?/CO₂. Addition of exogenous HCO₃^? or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII.     

Carbonic anhydrases in plants and algae

Carbonic anhydrases catalyse the reversible hydration of CO₂, increasing the interconversion between CO₂ and HCO₃⁻ + H⁺ in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated α-, β-and γ-CA. Animals have only the α-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C₃ dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases.     

Influencing factors and formation mechanism of CaCO3 precipitation induced by microbial carbonic anhydrase

Microbial carbonic anhydrase promotes carbonate deposition, which is important in the formation and evolution of global carbon cycle and geological processes. A kind of bacteria producing extracellular carbonic anhydrase was selected to study the effects of temperature, pH value and Ca²⁺ concentration on bacterial growth, carbonic anhydrase activity and calcification rate in this paper. The results showed that the activity of carbonic anhydrase at 30 °C was the highest, which was beneficial to the calcification reaction, calcification rate of CaCO₃ was the fastest in alkaline environment with the initial pH value of 9.0. When the Ca2+ concentration was 60 mM, compared with other Ca²⁺ concentration, CA bacteria could grow and reproduce best, and the activity of bacteria was the highest, too low Ca²⁺ concentration would affect the generation of CaCO₃, while too high Ca²⁺ concentration would seriously affect the growth of bacteria and reduce the calcification rate. Finally, the mechanism of CaCO₃ precipitation induced by microbial carbonic anhydrase was studied. Carbonic anhydrase can accelerate the hydration of CO₂ into HCO₃⁻, and react with OH⁻ and Ca²⁺ to form CaCO₃ precipitation in alkaline environment and in the presence of calcium source.     

Biochemical Modulation by Carbon and Nitrogen Addition in Cultures of <Emphasis Type="Italic">Dictyota menstrualis</Emphasis> (Dictyotales,Phaeophyceae) to Generate Oil-based Bioproducts

Dictyota menstrualis (Hoyt) Schnetter, Hörning & Weber-Peukert (Dictyotales, Phaeophyceae) was studied for the production of oil-based bioproducts and co-products. Experiments were performed to evaluate the effect of carbon dioxide (CO₂) concentration, under nitrogen (NO₃ ^?) limiting and saturation conditions, on growth rate (GR), photosynthesis, as well as nitrate reductase (NR), carbonic anhydrase (CA), and Rubisco activities. In addition, the biochemical composition of D. menstrualis under these conditions was estimated. GR, protein content, and N content in D. menstrualis were higher in treatments containing NO₃ ^?, irrespective of CO₂ addition. However, when CO₂ was added to medium saturated with NO₃ ^?, values of maximum photosynthesis, Rubisco, and NR activity, as well as total soluble carbohydrates and lipids, were increased. CA activity did not vary under the different treatments. The fatty acid profile of D. menstrualis was characterized by a high content of polyunsaturated fatty acids, especially the omega-3 fatty acids, making it a possible candidate for nutraceutical use. In addition, this species presented high GR, photosynthetic rate, and fatty acid content, highlighting its economic importance and the possibility of different biotechnological applications.