Isolation of dihydroxyacetone phosphate reductase from dunaliella chloroplasts and comparison with isozymes from spinach leaves

A dihydroxyacetone phosphate (DHAP) reductase has been isolated in 50% yield from Dunaliella tertiolecta by rapid chromatography on diethylaminoethyl cellulose. The activity was located in the chloroplasts. The enzyme was cold labile, but if stored with 2 molar glycerol, most of the activity was restored at 30°C after 20 minutes. The spinach (Spinacia oleracea L.) reductase isoforms were not activated by heat treatment. Whereas the spinach chloroplast DHAP reductase isoform was stimulated by leaf thioredoxin, the enzyme from Dunaliella was stimulated by reduced Escherichia coli thioredoxin. The reductase from Dunaliella was insensitive to surfactants, whereas the higher plant reductases were completely inhibited by traces of detergents. The partially purified, cold-inactivated reductase from Dunaliella was reactivated and stimulated by 25 millimolar Mg²⁺ or by 250 millimolar salts, such as NaCl or KCl, which inhibited the spinach chloroplast enzyme. Phosphate at 3 to 10 millimolar severely inhibited the algal enzyme, whereas phosphate stimulated the isoform in spinach chloroplasts. Phosphate inhibition of the algal reductase was partially reversed by the addition of NaCl or MgCl₂ and totally by both. In the presence of 10 millimolar phosphate, 25 millimolar MgCl₂, and 100 millimolar NaCl, reduced thioredoxin causes a further twofold stimulation of the algal enzyme. The Dunaliella reductase utilized either NADH or NADPH with the same pH maximum at about 7.0. The apparent K_m (NADH) was 74 micromolar and K_m (NADPH) was 81 micromolar. Apparent V_max was 1100 μmoles DHAP reduced per hour per milligram chlorophyll for NADH, but due to NADH inhibition highest measured values were 350 to 400. The DHAP reductase from spinach chloroplasts exhibited little activity with NADPH above pH 7.0. Thus, the spinach chloroplast enzyme appears to use NADH in vivo, whereas the chloroplast enzyme from Dunaliella or the cytosolic isozyme from spinach may utilize either nucleotide.     

ADAPTATION OF THE UNICELLULAR ALGA DUNALIELLA PARVA TO A SALINE ENVIRONMENT1

The holophilic alga Dunaliella parva produces glycerol as a major product of photosynthetic ¹⁴CO₂ incorporation and accumulates very large amounts of intracellular glycerol. A method was adopted for the determination of the cell water space based on the distribution of ¹⁴C sorbitol and ³H₂O between the cells and the medium. Using these measurements the internal concentration of glycerol was found to be isoomotic with that of the medium over a broad range of 0.6 to 2.1 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular water content immediately varied so as to keep an osmotic equilibrium between the cells and the medium. During the following 90 min under metabolic conditions, glycerol content changed until a new level was reached. Since no leakage of intracellular glycerol was observed above 0.6 m NaCl, these alterations in glycerol content are interpreted as due to metabolic formation and degradation of intracellular glycerol. Determination of the glycerol sensitivity of enzymic and photosynthetic reactions of cell-free preparations from D. parva showed a broad range of tolerance to high concentrations of glycerol. These results indicate that osmoregulation in Dunaliella depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration. A proposed scheme of glycerol synthesis in Dunaliella is suggested.     

The microbiology and biogeochemistry of the Dead Sea

The Dead Sea is a hypersaline water body. Its total dissolved salts content is on the average 322.6 gm/liter. The dominant cation is Mg (40.7 gm/liter), followed by Na (39.2 gm/liter), Ca (17 gm/liter) and K (7 gm/liter). The major anion is Cl (212 gm/liter), followed by Br (5 gm/liter); SO₄ and HCO₃, are very minor. The lake contains a limited variety of microorganisms and no higher organisms. The number of recorded species is very low, but the total biomass is reasonably high (about 10⁵ bacteria/ml and 10⁴ algal cells/ml). The indigenous flora is comprised mainly of obligate halophylic bacteria, such as the pink, pleomorphicHalobacterium sp., aSarcina-like coccus, and the facultative halophilic green alga,Dunaliella. Sulfate reducers can be isolated from bottom sediments. Recently a unique obligate magnesiophile bacteria was isolated from Dead Sea sediment. Several of the Dead Sea organisms possess unusual properties. TheHalobacterium sp. has extremely high intercellular K⁺ concentration (up to 4.8M) and extraordinary specificity for K⁺ over Na. TheDunaliella has very high intracellular concentration of glycerol (up to 2.1M). The microorganisms exert marked influence on some biogeochemical processes occurring in the lake, such as the control of the sulfur cycle and the formation and diagenesis of organic matter in the sediments. The Dead Sea is an excellent example of the development of two different mechanisms for adjusting to a hostile environment. The algae adjust to the high salinity by developing a mechanism for the exclusion of salts from the intracellular fluid and using glycerol for osmotic regulation. On the other hand, the bacteria adapt to the environment by adjusting their internal inorganic ionic strength, but not composition, to that of the medium. The problem of population dynamics and limiting factors for algal and bacterial productivity are discussed in view of the total absence of zooplankton and other consumers other than bacteria.     

Glycerol fomation inDunaliella cells under non-growing conditions with hypertonic lithium or magnesium media

Glycerol formation ofDunaliella cells in non-growing media was investigated.Dunaliella tertiolecta andD. bioculata grew well in a NaCl medium but not at all in a LiCl or a MgCl₂ medium. When the cells originally suspended in a medium containing 0.5 M NaCl were transferred to media which contained one of 1 M NaCl, 1 M LiCl or 0.7 M MgCl₂, the intracellular glycerol content increased.D. tertiolecta cultured in either a 1 M LiCl or a 0.7 M MgCl₂ medium did not multiply, but maintained abilities to evolve O₂ in the light and absorb O₂ in thedark even after about a 5 day culture. From these results, it can be concluded that the halotolerance ofDunaliella to different kinds of salts is not directly related to osmoregulation by the glycerol formation.     

Immobilized Clostridium acetobutylicum P262 Mutants for Solvent Production

The production of acetone, butanol, and ethanol by two immobilized, sporulation-deficient (spo) Clostridium acetobutylicum P262 mutants which were held in the solventogenic phase was investigated. The spoA2 mutant, which was an early-sporulation mutant and did not form a forespore septum, produced higher solvent yields than did the spoB mutant which was a late-sporulation mutant and was blocked at a stage after forespore septum formation. The spoA2 mutant was also granulose and capsule negative. In a conventional batch fermentation, the wild-type strain produced 15.44 g of solvents per liter after 50 h at a productivity of 7.41 g of solvents per liter per day. The spoA2 mutant produced 15.42 g of solvents per liter at a productivity of 72.4 g of solvents per liter per day, with a retention time of 2.4 h in a continuous immobilized cell system employing a fluidized bed reactor. This represents a major advance, since the immobilization of wild-type cells showed similar increases in productivity but a ca. fivefold reduction in final product concentrations.     

Construction and Characterization of a 1,3-Propanediol Operon

The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. These genes were reconfigured into an operon containing dhaB followed by dhaT under the control of a single promoter. The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose, Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter. The fermentation had two distinct phases. In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate. In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B₁₂, which must be provided in E. coli fermentations. However, the amount of coenzyme B₁₂ needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations. 1,3-PD is a useful intermediate in the production of polyesters. The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.     

The size and turnover of the glycerol pool in Dunaliella

Abstract The rate of incorporation of ¹⁴C from CO₂ into glycerol, and the amount of glycerol in cells of Dunaliella tertiolecta was determined at constant salinity at two representative concentrations of NaCl. From these data, the rate of synthesis and turnover of the glycerol pool was determined. The half-time for turnover of the glycerol pool was of the order of 1 h in 170 mol m^?3 NaCl and almost 6h for 700 mol m^?3 NaCl. These results indicate that turnover of the glycerol pool in Dunaliella is relatively slow under steady-state conditions. Synthesis and dissimilation of glycerol do not apparently constitute a metabolic cycle in the conventional sense. Rather, glycerol metabolism resembles that of the storage polysaccharides which arc commonly produced and degraded by different pathways.     

Salt-Induced Metabolic Changes in Dunaliella salina

An increase of medium NaCl concentration induces Dunaliella cells to evolve O₂ photosynthetically even in the absence of CO₂. This NaCl-induced O₂ evolution may reflect the induced conversion of reserve carbohydrate to glycerol. The quantum yield for the NaCl-induced O₂ evolution, in the absence of CO₂, is 1.5-fold higher than that obtained for CO₂ fixation. Since the synthesis of glycerol from reserve carbohydrate in the absence of CO₂ requires only 0.5 ATP/NADPH, whereas photosynthesis requires at least 1.3 ATP/NADPH, it is concluded that the ATP/2e⁻ ratio coupled to NADP reduction in Dunaliella is lower than required for CO₂ fixation.     

Effect of temperature on glycerol retention in the halotolerant algae dunaliella and asteromonas

Algae of the genera Dunaliella and Asteromonas can maintain extremely high concentration gradients (>10⁴) of glycerol between the intracellular space and the medium. This unique ability is highly temperature-dependent. Treating the algae for several minutes at temperatures exceeding 60 C causes complete release of all the internally held glycerol; 50% release occurs around 50 C, but essentially none is released below 40 C. A similar behavior was observed in several species of Dunaliella, and one of Asteromonas and is independent of the salt concentration of the medium. The underlying mechanism may involve a temperature-dependent conformational transition of a component of the cellular membrane which is essential for glycerol impermeability.     

Control of Carbon and Electron Flow in Clostridium acetobutylicum Fermentations: Utilization of Carbon Monoxide to Inhibit Hydrogen Production and to Enhance Butanol Yields

Extracts prepared from non-solvent-producing cells of Clostridium acetobutylicum contained methyl viologen-linked hydrogenase activity (20 U/mg of protein at 37°C) but did not display carbon monoxide dehydrogenase activity. CO addition readily inhibited the hydrogenase activity of cell extracts or of viable metabolizing cells. Increasing the partial pressure of CO (2 to 10%) in unshaken anaerobic culture tube headspaces significantly inhibited (90% inhibition at 10% CO) both growth and hydrogen production by C. acetobutylicum. Growth was not sensitive to low partial pressures of CO (i.e., up to 15%) in pH-controlled fermentors (pH 4.5) that were continuously gassed and mixed. CO addition dramatically altered the glucose fermentation balance of C. acetobutylicum by diverting carbon and electrons away from H₂, CO₂, acetate, and butyrate production and towards production of ethanol and butanol. The butanol concentration was increased from 65 to 106 mM and the butanol productivity (i.e., the ratio of butanol produced/total acids and solvents produced) was increased by 31% when glucose fermentations maintained at pH 4.5 were continuously gassed with 85% N₂-15% CO versus N₂ alone. The results are discussed in terms of metabolic regulation of C. acetobutylicum saccharide fermentations to achieve maximal butanol or solvent yield.     

Two isozymes of dihydroxyacetone phosphate reductase in dunaliella

Two isoforms of dihydroxyacetone phosphate reductase were present in Dunaliella tertiolecta. The major form was located in the chloroplast and the minor form in the cytosol. The chloroplastic reductase eluted first from a DEAE cellulose column followed immediately by the cytosolic form. Both forms were unstable and cold labile. Addition of 5 millimolar dithiothreitol helped to stabilize the enzymes. The cytosolic isoform of DHAP reductase was detected only if the cells were in an active log phase of growth. Then its activity was 20 to 30% of the total reductase activity. When cell cultures entered late log phase of growth the activity of the cytosolic form of the enzyme disappeared, but the chloroplastic form remained. The cytosolic DHAP reductase from Dunaliella has some properties similar to the cytosolic isoform from spinach leaves. Detergents inhibited both enzymes. However, neither form of the algal dihydroxyacetone phosphate reductase was stimulated by fructose 2,6-bisphosphate. In Dunaliella the properties of the chloroplastic form were those expected for glycerol production for osmoregulation, whereas the cytosolic form, like the reductases in leaves, is more likely involved in glycerol phosphate formation for lipid synthesis.     

Effect of pH, light intensity, salt and nitrogen concentrations on growth and β-carotene accumulation by a new isolate of Dunaliella sp

Summary In the present study Dunaliella sp. that could grow in the Johnson medium was isolated from hypersaline Lake Tuz and its β-carotene production was studied in a batch system, in order to determine the optimal conditions required for the highest β-carotene accumulation. In the experiments with light intensity, the cell numbers and β-carotene content were maximum at pH 9, with 20% of NaCl concentration and 48 kerg cm⁻² s⁻¹ light intensity. At this light intensity, the β-carotene content of Dunaliella sp. ranged between 0.177 and 1.095 mg/ml for the culture broth and 0.119 to 0.261 ng/cell on a per cell basis under the nitrogen limitation. At the end of the experiments, the maximum β-carotene accumulation and the cell number were obtained at pH 7, 5 mM NaNO₃ and 20% NaCl concentrations as 0.261 ng/cell, 4.2×10⁶ cell/ml, respectively.     

Phylogenetic position of Dunaliella acidophila (Chlorophyceae) based on ITS and rbcL sequences

Dunaliella acidophila is one of the most extreme acidophiles on earth and is able to survive in highly acidic habitats. This characteristic has made this organism the universal model for the study of abiotic stress. Although D. acidophila is currently circumscribed to the subgenus Pascheria within Dunaliella Teodoresco (Chlorophyceae), its taxonomic position has stirred controversy. The comparison of D. acidophila CCAP19/35 internal transcribed spacers (including ITS2 secondary structure analysis) and RuBisCo large subunit (rbcL) sequences with other Dunaliella species confirms that D. acidophila should maintain its phylogenetic position within the genus Dunaliella, suggesting its inclusion within the subgenus Dunaliella. Furthermore, the ITS1 and ITS2 data revealed that D. acidophila was highly divergent from the other freshwater species assessed, D. lateralis, with which it barely shares a 56.8% similarity.     

Control of CO2 fixation during the induction period. The role of thiol-mediated enzyme activation in the alga,Dunaliella

(1) Illumination of the unicellular green alga, Dunaliella, produced a 2–3-fold enhancement of ATPase activity in subsequently lysed algae. Using the inhibitor, tentoxin, it was shown that this light-induced activity, but not the light-independent activity, was attributable to the chloroplast coupling factor, CF₁. (1) A 4–5-fold increase in fructose-1,6-bisphosphatase activity was measured in Dunaliella lysed subsequent to illumination. (3) Experiments with methyl viologen demonstrated that both light-induced CF₁-ATPase and fructose-1,6-bisphosphatase activities were due to thiol-modulation of the enzymes by the algal thioredoxin system. (4) The light-induced increase in fructose-1,6-bisphosphatase activity could be simulated by incubation of intact algae in the dark with dithiothreitol. This thiol-induced increase in enzyme activity was accompanied by a decrease in the induction period of CO₂-dependent O₂ evolution upon subsequent measurement. (5) The kinetics of induction of both enzyme activities were very similar to the kinetics of induction of CO₂-dependent O₂ evolution in Dunaliella. As the light intensity was increased to 180 W · m² the steady-state enzyme activities increased in parallel with the rate of CO₂-dependent O₂ evolution. (6) The results are consistent with the imposition of a kinetic restraint on CO₂ fixation by the extent of enzyme activation under certain conditions in Dunaliella.     

Ion and Glycerol Concentrations in 12 Isolates of Dunaliella

Ginzburg, M., and Ginzburg, B. Z., 1985. Ion and glycerol concentrationsin 12 isolates of Dunaliella.—J. exp. Bot. 36: 1064–1074. Twelve isolates of Dunaliella with average cell volumes rangingfrom 50 to 1400x10^–18 m³ were grown in batch culture at0.5 M or 2.0 M NaCl. Glycerol and ions (Na⁺, K⁺, Mg²⁺, CI^–,phosphate) were measured in log-phase cultures. The contentsof Mg²⁺, K⁺ and phosphate per cell were found to be a functionof cell-volume. Cell glycerol, Na⁺ and Cl^– were functionsof cell-volume and of the NaCl concentration in the medium.Solute concentrations were calculated from the measured cell-volumesand from the ³H₂O content of pellets corrected for intercellularspace using Blue Dextran. Cell glycerol was found to accountfor about one-half of the expected osmolarity, the remainderbeing largely accounted for by Na⁺ and CI^–. Key words: —Dunaliella, isolates, glycerol, ion concentrations     

Biosynthetic Pathways of Glycerol Accumulation under Salt Stress in Aspergillus nidulans

Redkar, R. J., Locy. R. D., and Singh, N. K. 1995. Biosynthetic pathways of glycerol accumulation under salt stress in Aspergillus nidulans. Experimental Mycology 19, 241-246. A culture of Aspergillus nidulans (FGSC 359) was gradually adapted for growth in media containing up to 2 M NaCl or was exposed to a salt shock with 2 M NaCl. The intracellular glycerol level increased by about 7.9-fold in salt-adapted and 2.4-fold in salt-shocked cultures when compared to the unadapted culture. The biosynthetic pathway involved in the accumulation of glycerol was investigated under long-term salt adaptation and short-term salt shock. Glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was induced 1.4-fold in salt-shocked but not in salt-adapted cultures. An alternate enzymatic pathway involving glycerol dehydrogenase (NADP⁺-dependent) utilizing dihydroxyacetone (DHA) and/or DL-glyceraldehyde (DL-GAD) was induced by NaCl. DHA-dependent glycerol dehydrogenase activity was induced about 6.3-fold in salt adapted and 1.35-fold in salt-shocked cultures, while DL-GAD-dependent activity was induced about 6.1-fold in salt-adapted and 1.2-fold in salt shocked cultures. However, the level of glycerol dehydrogenase activity with DL-GAD as substrate was 7% of the DHA-dependent activity. We conclude that a salt-inducible NADP⁺-dependent glycerol dehydrogenase activity electrophoretically indistinguishable from previously described glycerol dehydrogenase I results in glycerol accumulation in salt-stressed A. nidulans.     

Influence of stress factors on growth and pigment production in three Dunaliella species cultivated outdoors in flat-plate photobioreactors

Abstract

Growth and production of carotenoid in three Dunaliella species (Dunaliella salina (Dunal) Teodoresco, Dunaliella bardawil Ben-Amotz & Avron and Dunaliella sp.) were investigated using flat-plate photobioreactors in outdoor conditions with two optical paths (3?cm and 5?cm). The experiment was conducted in duplicate and lasted four weeks during which light intensity, temperature, pH and optical density were checked daily. The pigment production (total carotenoid and chlorophyll a) was monitored every two days. To induce an additional stress besides temperature and light intensity, two different salt concentrations were used, i.e. 6% and 8% NaCl. The highest growth in all treatment groups was noticed for Dunaliella sp. followed by D. bardawil and D. salina. D. salina produced a higher content of carotenoid concentrations corresponding to 5?cm/8% and 5?cm/6% groups; 779.102?±?0.434?μg.mL^?1 and 694.326?±?0.098?μg.mL^?1 were registered at the end of the experiment. The same species had also greater content of β-carotene.     

Evaluation of halophilic microalgae isolated from Rabigh Red Sea coastal area for biodiesel production: Screening and biochemical studies

In the present study, different water samples from Red Sea coastal area at Rabigh city, Saudi Arabia were studied for their dominant algal species. Microalgal isolation was carried out based on dilution method and morphologically examined using F/2 as a growth medium. Dry weight and main biochemical composition (protein, carbohydrates, lipids) of all species were performed at the end of the growth, and biodiesel characteristics were estimated. Nannochloropsis sp., Dunaliella sp., Tetraselmis sp., Prorocentrum sp., Chlorella sp., Nitzschia sp., Coscinodiscus sp., and Navicula sp. were the most dominant species in the collected water samples and were used for further evaluation. Nannochloropsis sp. surpassed all other isolates in concern of biomass production with the maximum recorded dry weight of 0.89 g L^?1, followed by Dunaliella sp. (0.69 g L^?1). The highest crude protein content was observed in Nitzschia sp. (38.21%) and Dunaliella sp. (18.01%), while Nannochloropsis sp. showed 13.38%, with the lowest recorded lipid content in Coscinodiscus sp. (10.09%). Based on the growth, lipid content, and biodiesel characteristics, the present study suggested Dunaliella sp. and Nitzschia sp. as promising candidates for further large-scale biodiesel production.     

Isolation and characterization of Dunaliella salina from Thailand

Screening of Dunaliella was performed in four provinces along the coast of the Gulf of Thailand (Chanthaburi, Chon Buri, Chachoengsao and Samut Songkhram) and in Nakhorn Ratchasima province in the north-east. Six clones of D. salina were isolated from salt fields in Samut-Songkhram province. Clone DS91008 produced the highest carotenoid content of 80.3 pg cell^-1, in modified J/1 medium at 30% NaCl under continuous illumination at 270 μmol m^-2 s^-1.     

Sodium Chloride Reduces Production of Curvacin A, a Bacteriocin Produced by Lactobacillus curvatus Strain LTH 1174, Originating from Fermented Sausage

Lactobacillus curvatus LTH 1174, a strain originating in fermented sausage, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of salt (sodium chloride) were investigated in vitro during laboratory fermentations using modified MRS medium. A model was set up to describe the effects of different NaCl concentrations on microbial behavior. Both cell growth and bacteriocin activity were affected by changes in the salt concentration. Sodium chloride clearly slowed down the growth of L. curvatus LTH 1174, but more importantly, it had a detrimental effect on specific curvacin A production (k_B) and hence on overall bacteriocin activity. Even a low salt concentration (2%, wt/vol) decreased bacteriocin production, while growth was unaffected at this concentration. The inhibitory effect of NaCl was mainly due to its role as an a_w-lowering agent. Further, it was clear that salt interfered with bacteriocin induction. Additionally, when 6% (wt/vol) sodium chloride was added, the minimum biomass concentration necessary to start the production of curvacin A (X_B) was 0.90 g (cell dry mass) per liter. Addition of the cell-free culture supernatant or a protein solution as a source of induction factor resulted in a decrease in X_B, an increase in k_B, and hence an increase in the maximum attainable bacteriocin activity.