The Role of Glycerol in the Osmotic Regulation of the Halophilic Alga Dunaliella parva

Dunaliella parva, a green halophilic alga, was found to accumulate very large amounts of intracellular glycerol. Through measurements of the intracellular volume the internal concentration of glycerol was calculated and found to be around 2.1 m in cells cultured in 1.5 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular glycerol varied accordingly, reaching its new osmotic equilibrium after about 90 minutes. 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. The above results indicate the existence of a new type of algal osmoregulation, in which the osmotic balance depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration.     

COMPARATIVE STUDIES ON RESPIRATION : IX. THE EFFECTS OF ANTAGONISTIC SALTS ON THE RESPIRATION OF ASPERGILLUS NIGER.

1. In the presence of 0.05 per cent dextrose the respiration of Aspergillus niger is increased by NaCl in concentrations of 0.25 to 0.5M, and by 0.5M CaCl₂. 2. Stronger concentrations, as 2M NaCl and 1.25M CaCl₂, decrease the respiration. The decrease in the higher concentrations is probably an osmotic effect of these salts. 3. A mixture of 19 cc. of NaCl and 1 cc. of CaCl₂ (both 0.5M) showed antagonism, in that the respiration was normal, although each salt alone caused an increase. 4. Spores of Aspergillus niger did not germinate on 0.5M NaCl (plus 0.05 per cent dextrose) while they did on 0.5M CaCl₂ (plus 0.05 per cent dextrose) and on various mixtures of the two. This shows that a substance may have different effects on respiration from those which it has upon growth.     

Determination of intracellular osmotic volume and sodium concentration in dunaliella

A new method to measure intracellular volume in Dunaliella was developed, where lithium ions are used as monitors of the extracellular volume. Li⁺ is shown to be impenetrable to the intracellular volume, insignificantly absorbed to the algae, and is rapidly and evenly distributed within the extracellular volume. The method is suggested to be free of several limitations and consistent errors present in several previously employed techniques.

Using the new technique it is shown that both Dunaliella salina and Dunaliella bardawil adjust to a constant cellular volume when grown in a medium containing salt concentrations ranging from 0.5 molar to 4 molar NaCl. That volume is 90 femtoliter per cell for D. salina and 600 femtoliter per cell for D. bardawil. Nonosmotic volume accounts for about 10% of the total cell volume.

The intracellular sodium concentration, as determined with the new technique, was under all experimental conditions tested below 100 millimolar. This was true both for cells grown on 0.5 to 4 molar NaCl, and during the osmoregulatory process. It is thus concluded that intracellular NaCl is a minor contributor to the overall intracellular osmotic pressure in Dunaliella.

     

Mechanisms of response to salinity in halotolerant microalgae

Summary A limited number of organic solutes are used by microalgae to adjust their internal osmotic pressure in response to changing external salinities. Glycerol and proline are used by the most extremely halotolerant algae. Only glycerol allows growth at salinities approaching saturation. In addition to organic osmoregulatory solutes, inorganic ions also play an important role in osmoregulation. The ability of microalgae to maintain intracellular ions at levels compatible with metabolic functions may set upper limits for their salt tolerance. Requirements for NaCl in the external medium for nutrient transport may define the lower salinity limits for growth observed for some euryhaline algae.Osmotic upshocks generally cause severe temporary inhibition of photosynthesis in euryhaline microalgae. Extensive osmotic downshocks have little effect on photosynthesis in microalgae with strong cell walls, while wall-less species appear to be more sensitive. Rapid glycerol synthesis takes place in response to increased external salinity inChlamydomonas pulsatilla both in light and dark. Starch supplies carbon for glycerol synthesis in the dark and also during the initial periods of inhibition of photosynthesis in the light. Turnover of osmoregulatory solutes such as glycerol and isofloridoside may be an important aspect of the osmoregulatory mechanism.At salinities beyond the growth limit for the green flagellateChlamydomonas pulsatilla, resting spores are formed that enable this alga to survive extreme salinities.     

On the mechanism of salt tolerance

As glycerol was suggested as an osmotic agent in the salt tolerantDebaryomyces hansenii the concentrations of total, intracellular, and extracellular glycerol produced by this yeast was followed during growth in 4 mM, 0.68 M, and 2.7 M NaCl media. The total amount of glycerol was not directly proportional to biomass production but to the cultural salinity with maximum concentrations just prior to or at the beginning of the stationary phase. In all cultures the cells lost some glycerol to the media, at 2.7 M NaCl the extracellular glycerol even amounted maximally to 80% of the total. A distinct maximum of intracellular glycerol, related to dry weight or cell number, appeared during the log phase at all NaCl concentrations. As the intracellular calculated glycerol concentrations amounted to 0.2 M, 0.8 M, and 2.6 M in late log phase cells at 4 mM, 0.68 M, and 2.7 M NaCl, respectively, whereas the corresponding analysed values for the glycerol concentrations of the media were 0.7 mM, 2.5 mM, and 3.0 mM, glycerol contributes to the osmotic balance of the cells.During the course of growth all cultures showed a decreasing heat production related to cell substance produced, most pronounced at 2.7 M NaCl. At 2.7 M NaCl the total heat production amounted to-1690 kJ per mole glucose consumed in contrast to-1200 and-1130 kJ at 4 mM and 0.68 M NaCl, respectively. TheY _m -values were of an inverse order, being 129, 120, and 93 at 4 mM, 0.68 M, and 2.7 M NaCl, respectively.     

Physiological characterization and stress-induced metabolic responses of Dunaliella salina isolated from salt pan

A Dunaliella strain was isolated from salt crystals obtained from experimental salt farm of the institute (latitude 21.46 N, longitude 72.11 degrees E). The comparative homology study of amplified molecular signature 18S rRNA, proves the isolated strain as D. salina. The growth pattern and metabolic responses such as proline, glycine betaine, glycerol, total protein and total sugar content to different salinity (from 0.5 to 5.5 M NaCl) were studied. The optimum growth was observed at 1.0 M NaCl and thereafter it started to decline. Maximum growth was obtained on 17th day of inoculation in all salt concentrations except 0.5 M NaCl, whereas maximum growth was observed on 13th day. There were no significant differences (P < 0.01) in chlorophyll a/b contents (1.0-1.16 +/- 0.05 mug chl. a and 0.2-0.29 +/- 0.01 mug chl. b per 10(6) cells) up to 2.0 M NaCl, however at 3.0 M NaCl a significant increase (2.5 +/- 0.12 mug chl. a and 0.84 +/- 0.4 mug chl. b per 10(6) cells) was observed which declined again at 5.5 M NaCl concentration (2.0 +/- 0.1 mug chl. a and 0.52 +/- 0.03 mug chl. b per 10(6) cells). Stress metabolites such as proline, glycine betaine, glycerol and total sugar content increased concomitantly with salt concentration. Maximum increase in proline (1.4 +/- 0.07 mug), glycine betaine (5.7 +/- 0.28 mug), glycerol (3.7 +/- 0.18 ml) and total sugar (250 +/- 12.5 mug) per 10(5) cells was observed in 5.5 M NaCl. A decrease in total protein with reference to 0.5 M NaCl was observed up to 3.0 M NaCl, however, a significant increase (P < 0.01) was observed at 5.5 M NaCl (0.19 +/- 0.01 mug per 10(5) cells). Inductive coupled plasma (ICP) analysis shows that intracellular Na(+) remained unchanged up to 2.0 M NaCl concentration and thereafter a significant increase was observed. No relevant increase in the intracellular level of K(+) and Mg(++) was observed with increasing salt concentration. Evaluation of physiological and metabolic attributes of Dunaliella salina can be used to explore its biotechnological and industrial potential.     

P and C-NMR Studies of the Phosphorus and Carbon Metabolites in the Halotolerant Alga, Dunaliella salina

The intracellular phosphorus and carbon metabolites in the halotolerant alga Dunaliella salina adapted to different salinities were monitored in living cells by ³¹P- and ¹³C-nuclear magnetic resonance (NMR) spectroscopy. The ¹³C-NMR studies showed that the composition of the visible intracellular carbon metabolites other than glycerol is not significantly affected by the salinity of the growth medium. The T₁ relaxation rates of the ¹³C-glycerol signals in intact cells were enhanced with increasing salinity of the growth medium, in parallel to the expected increase in the intracellular viscosity due to the increase in intracellular glycerol. The ³¹P-NMR studies showed that cells adapted to the various salinities contained inorganic phosphate, phosphomonoesters, high energy phosphate compounds, and long chain polyphosphates. In addition, cells grown in media containing up to 1 molar NaCl contained tripolyphosphates. The tripolyphosphate content was also controlled by the availability of inorganic phosphate during cell growth. Phosphate-depleted D. salina contained no detectable tripolyphosphate signal. Excess phosphate, however, did not result in the appearance of tripolyphosphate in ³¹P-NMR spectra of cells adapted to high (>1.5 molar NaCl) salinites.     

Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina

In studies of the outstanding salt tolerance of the unicellular green alga Dunaliella salina, we isolated a cDNA for a salt-inducible mRNA encoding a protein homologous to plant beta-ketoacyl-coenzyme A (CoA) synthases (Kcs). These microsomal enzymes catalyze the condensation of malonyl-CoA with acyl-CoA, the first and rate-limiting step in fatty acid elongation. Kcs activity, localized to a D. salina microsomal fraction, increased in cells transferred from 0.5 to 3.5 M NaCl, as did the level of the kcs mRNA. The function of the kcs gene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing the kcs cDNA. The effect of salinity on kcs expression in D. salina suggested the possibility that salt adaptation entailed modifications in the fatty acid composition of algal membranes. Lipid analyses indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 M NaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5 M salt. Thus, the salt-inducible Kcs, jointly with fatty acid desaturases, may play a role in adapting intracellular membrane compartments to function in the high internal glycerol concentrations balancing the external osmotic pressure.     

The Adaptation of Dunaliella to Widely-Differing Salt Concentrations

Methods are described for adapting pure cell-lines of Dunaliella,a green unicellular alga, to grow at concentrations between0.5 M and 3.5 M NaCl. It is shown that, provided large abruptchanges are avoided, cells of the same cell-line can becomeadapted to grow over this wide range of salt concentration.Once adapted, cultures are able to continue growing at a steadyrate for many generations provided that the salt concentrationremains constant. Viability tests performed after abrupt changesin salt concentration have shown that survival is higher afterdownward than after upward changes (3.5 M to 0.5 M NaCl: 20%;0.5 M to 3.5 M NaCl: 0.01% in D. parva 19/9). When the changein concentration from 0.5 M to 3.5 M NaCl takes place over aperiod of 48 h viability approaches 100%. The time needed for100% survival for a downward change over the same range is only1.5 h. There is no evidence for a genetic difference betweencell-lines adapted to particular concentrations of salt andit is concluded that the so-called ‘halophilic’and ‘halotolerant’ strains are interchangeable.It seems likely that the difference between the two types isa matter of gene expression. Key words: Dunaliella, salt concentration, viability     

Effect of salinity on the lipid and fatty acid composition of the halophyteNavicula sp.: potential in mariculture

Navicula sp. (cf.N. tenelloides) was isolated from a salt marsh in Kuwait. The alga grew best with 0.5M NaCl, but abundant growth still occurred up to 2.5M NaCl. The total lipid content and the carotene to chlorophyll ratio of the cells increased with increasing salinity of the medium from 0.5 to 1.7M NaCl, but declined with 2.5M NaCl. Irrespective of the medium salinity, the major lipid class was that of triacylglycerols. The predominant fatty acids in the total lipids of cells grown at different NaCl concentrations were palmitic (16:0) and palmitoleic (16:1) acids; eicosapentaenoic acid (20:5) made up 8–9% of the total fatty acids. The fatty acid composition of the individual lipid classes of cells grown at different salinities is described. The highest concentration of 20:5 occurred in monogalactosyldiacylglycerols and digalactosyldiacylglycerols. In view of the rather small size of this diatom, its halotolerance and its fair content of 20:5, it is suggested as a potential food source for the mariculture industry.     

Cytoplasmic Membrane Changes during Adaptation of the Fresh Water Cyanobacterium Synechococcus 6311 to Salinity

In this investigation, changes were characterized in cell structure and cytoplasmic membrane organization that occur when the freshwater cyanobacterium Synechococcus 6311 is transferred from `low salt' (0.03 molar NaCl) to `high salt' (0.5 molar NaCl) media (i.e. sea water concentration). Cells were examined at several time points after the imposition of the salt stress and compared to control cells, in thin sections and freeze fracture electron microscopy, and by flow cytometry. One minute after exposure to high salt, i.e. `salt shock,' virtually all intracellular granules disappeared, the density of the cytoplasm decreased, and the appearance of DNA material was changed. Glycogen and other granules, however, reappeared by 4 hours after salt exposure. The organization of the cytoplasmic membrane undergoes major reorganization following salt shock. Freeze-fracture electron microscopy showed that small intramembrane particles (diameters 7.5 and 8.5 nanometers) are reduced in number by two- to fivefold, whereas large particles, (diameters 14.5 and 17.5 nanometers) increase two- to fourfold in frequency, compared to control cells grown in low salt medium. The changes in particle size distribution suggest synthesis of new membrane proteins, in agreement with the known increases in respiration, cytochrome oxidase, and sodium proton exchange activity of the cytoplasmic membrane.     

Na-NMR Studies of the Intracellular Sodium Ion Concentration in the Halotolerant Alga Dunaliella salina

The Intracellular Na⁺ concentration in the halotolerant alga Dunaliella salina was measured in intact cells by ²³Na-NMR spectroscopy, utilizing the dysprosium tripolyphosphate complex as a sodium shift reagent, and was found to be 88 ± 28 millimolar. Intracellular sodium ion content and intracellular volume were the same, within the experimental error, in cells adapted to grow in media containing between 0.1 and 4.0 molar NaCl. These values assume extracellular and intracellular NMR visibilities of the ²³Na nuclei of 100 and 40%, respectively. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the growth medium, in parallel to the intracellular osmosity due to the presence of glycerol, indicating that Na⁺ ions and glycerol are codistribbuted within the cell volume.     

Determination of intracellular volume and internal solute concentrations of the green alga Chlorococcum submarinum

A method is described for measuring the cell volume of the unicellular green alga Chlorococcum submarinum, which depends on measurements of bromide concentration before and after disruption of the cells by ammonium hydroxide. Simultaneous equations are derived, which along with direct determination of cell water weight, allow the calculation of the intracellular volume in three different ways. The volumes calculated are in agreement indicating the validity of the method. The cell volumes and internal concentrations of glycerol, proline, potassium and sodium were determined for algae adapted to three salinities, 0.1, 0.5 and 1.0 M NaCl. The results showed that glycerol was the major internal solute and that the total measured solutes balanced the external osmotic pressure at all three salinities.Abbreviations DMSO dimethyl sulphoxide - Hepes N-[2-hydroxyethyl]piperazine-N-2-ethane sulfonic acid - TCA trichloroacetic acid - Tris tris[hydroxymethyl]aminoethane     

On the mechanism of salt tolerance. Production of glycerol and heat during growth of Debaryomyces hansenii.

As glycerol was suggested as an osmotic agent in the salt tolerant Debaryomyces hansenii the concentrations of total, intracellular, and extracellular glycerol produced by this yeast was followed during growth in 4 mM, 0.68 M, and 2.7 M NaCl media. The total amount of glycerol was not directly proportional to biomass production but to the cultural salinity with maximum concentrations just prior to or at the beginning of the stationary phase. In all cultures the cells lost some glycerol to the media, at 2.7 M NaCl the extracellular glycerol even amounted maximally to 80% of the total. A distinct maximum of intracellular glycerol, related to dry weight or cell number, appeared during the log phase at all NaCl concentrations. As the intracellular calculated glycerol concentrations amounted to 0.2 M, 0.8 M, and 2.6 M in late log phase cells at 4mM, 0.68 M, and 2.7 M NaCl, respectively, whereas the corresponding analysed values for the glycerol concentrations of the media were 0.7 mM, 2.5 mM, and 3.0 mM, glycerol contributes to the osmotic balance of the cells. During the course of growth all cultures showed a decreasing heat production related to cell substance produced, most pronounced at 2.7 M NaCl. At 2.7 M NaCl the total heat production amounted to--1690 kJ per mole glucose consumed in contrast to--1200 and--1130 kJ at 4 mM and 0.68 M NaCl, respectively. The Ym-values were of an inverse order, being 129, 120, and 93 at 4 mM, 0.68 M, and 2.7 M NaCl respectively.     

The Influence of CO2 Concentration and pH on Two Chlorella Species Grown in Continuous Light

Both Chlorella pyrenoidosa and Chlorella vulgaris grow equally well at 20°C aerated with ordinary air or mixtures of air with 5 or 12 per cent CO₂ (5 klux continuous light). Whereas C. vulgaris relatively rapidly adapts to a higher CO₂ tension, adaptation takes about 24 hours for C. pyrenoidosa. In Chlorella vulgaris pH in the range 3.6–7.6 has no apparent influence on the rate of photosynthesis in experiments having a duration of two hours. This is true both for algae grown aerated by ordinary air and for algae grown with a mixture of 5 per cent CO₂ in air. The adaptation time must be short. In Chlorella pyrenoidosa the same is found for algae in ordinary air, whereas an influence of pH is seen in some experiments where the aeration was by 5 per cent CO₂ in air. As is to be expected, the rate of photosynthesis in C. pyrenoidosa during the first two hours is very much influenced by the concentration of free CO₂. The highest rate is found at the CO₂ concentration at which the algae had been growing previously. The influence on the rate of photosynthesis in C. vulgaris is very much less, although in principle the same. The investigation of the corresponding influence on the rate of respiration is complicated by considerable variation from one series to another. In C. vulgaris this is particularly of importance. In C. pyrenoidosa, the highest rate of respiration is generally found at the CO₂-concentration at which the alga had been growing before the experiment. It seems probable that variations between similar series is due to the fact that the algae were grown in continuous light but with dilution with fresh culture medium when the optical density had reached a certain magnitude. Algae grown in this way are neither synchronized nor non-synchronized.Our thanks are due to the Danish State Research Foundation for financial support.     

The response of the filamentous cyanobacterium Spirulina platensis to salt stress

The responses of the filamentous cyanobacterium Spirulina platensis to increased NaCl concentrations (0.25–1.0 M) in addition to the concentration of sodium in the growth medium were studied. A two stage response to the salt stress was observed. This consisted of a relatively short shock stage, followed by adaptation process. It was shown that upon exposure to high salt concentrations of 0.5 M and above, immediate inhibition of photosynthesis and respiration, and complete cessation of growth occurred. After a time lag, the energy-yielding processes exhibited restored activity. At 0.5 and 1.0M NaCl photosynthesis reached 80% and 50% that of the control, while respiration was enhanced by 140 and 200%, respectively. The time lags were longer when the cells were exposed to higher NaCl concentrations. The resumption of growth and the establishment of new steady state growth rates were found to be correlated to the recovery in respiration. The relationship between the growth rates after adaptation and the increased NaCl concentrations was found to be inversely linear. The cellular sodium content was maintained at a constant low level, regardless of the external NaCl concentration, while potassium content declined linearly vs. the external NaCl concentration. The carbohydrate content of the cells rose exponentially with the increase in NaCl concentration.Publication No. 34 from the Micro-Algal Biotechnology Lab.     

Photosynthetic response of Chlamydomonas reinhardtii to short-term storage on ice in darkness. Dependence of the response on growth conditions

The effect of storage of the unicellular green alga Chlamydomonas reinhardtii (strain 137+) in the pelleted state in darkness on ice (0.2–0.5°C) (further simply “SPDI-treatment”) on its photosynthetic and respiratory activities was studied. To this end, the steady-state rates of O₂ exchange in darkness (dark respiration) and under saturating light (apparent photosynthesis) as well as the induction periods (IP) of apparent photosynthesis were measured at 25°C in the SPDI-untreated and SPDI-treated for the period from ～0.5 to ～30 h algal cells. In contrast to expectations, the SPDI-treatment consistently affected the rate and IP of photosynthesis depending on the physiological state of C. reinhardtii. Dark respiration was affected by the SPDI-treatment as well. However, in absolute values the respiratory changes were much less than the photosynthetic ones, and they were insufficiently reproducible. The SPDI-treatment affected photosynthesis most significantly in high-CO₂-grown cells (cells grown at 5% CO₂ in white light). The rate of photosynthesis in these cells declined quasi-exponentially as a function of time during the SPDI-treatment with a t _1/2 ～1.5 h and finally became by about 60% lower than that before the SPDI-treatment. This decline of photosynthesis was accompanied by continuous and essential increase in the photosynthetic IP. The SPDI-induced photosynthetic changes in high-CO₂-grown cells resulted from the firm disfunction of the photosynthetic apparatus. After switch from growth at 5% CO₂ in white light to growth at ～0.03% CO₂ (air) in white, blue, or red light, the alga gradually transited to a physiological state, in which the negative effects of the SPDI-treatment on the rate and IP of photosynthesis became weak and absent, respectively. Remarkably, this transition was faster in blue (≤5 h) than in white and red light (>10 h). Similar changes in the response of the alga to the SPDI-treatment occurred when high-CO₂-grown cells (5% CO₂, white light, 26°C) were incubated in darkness (air, 24–26°C) for 20–25 h. The results of study were analyzed in the light of literature data relating to the effects of CO₂ concentration, darkness, and light quality on carbohydrates in plant organisms. The analysis led to suggestion that there is connection between the negative effect of the SPDI-treatment on C. reinhardtii and nonstructural carbohydrates presented in the alga: the more carbohydrates contain the alga, the more extensive inactivation of the photosynthetic apparatus occurs in it during its storage in the dense (pelleted) state in darkness on ice.     

Physiological behavior of Scenedesmus sp. during exposure to elevated levels of Cu and Zn and after withdrawal of metal stress

Summary. A 48 h exposure of Scenedesmus sp. to sublethal concentrations of Cu (2.5 and 10 μM) and Zn (5 and 25 μM) caused a concentration-dependent inhibition of growth, photosynthesis, respiration, NO₃ ⁻ uptake, and nitrate reductase (EC 1.6.6.1) activity, and a reduction in protein, carbohydrate, and photosynthetic-pigment levels with a concomitant increase in intracellular levels of the test metals. After exposure, algal cells were transferred to the basal medium without the excess level of test metals, to study the recovery of various processes. The growth of the test algae had not recovered up to 12 h after transfer to the basal medium, but some physiological parameters such as photosynthesis and respiration recovered within 6 h. The quicker recovery of photosynthesis and respiration might be used as acclimatory responses as they prepare a background for the recovery of other parameters, including growth, of the test alga by generating energy, forming photosynthate, and establishing the usual catabolism to attain normal conditions. Most of the processes recovered completely or almost completely after being stressed with 2.5 μM Cu or 5 μM Zn. However, the maintenance of a relatively high level of Cu and Zn in the cells previously exposed to 10 μM Cu and 25 μM Zn slowed down the recovery of different processes, which did not fully recover even at the end of the experiment after 96 h. The present study demonstrates that a chain of metabolic events, beginning with respiration and photosynthesis and continuing with assimilation and uptake of nutrients and subsequent restoration of other metabolic processes, is involved in the recovery of the algae from Cu and Zn stress. Each studied parameter seems to play an important role in balancing the cellular homeostasis during recovery from metal stress. Correspondence and reprints: Department of Bioscience and Biotechnology, Banasthali Vidyapith, Banasthali 304 022, Rajasthan, India.     

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 salt relations of marine and halophilic species of the unicellular green alga,Dunaliella

1. Comparisons were made of the effects of salt on the exponential growth rates of two unicellular algae,Dunaliella tertiolecta (marine) andDunaliella viridis (halophilic).
2. The algae contained glycerol in amounts which varied directly with the salt concentration of the growth media. The highest measured glycerol content ofD. tertiolecta was approximately equivalent to 1.4 molal and occurred in algae grown in 1.36 M sodium chloride. The highest glycerol content measured inD. viridis was approximately equivalent to 4.4 molal and occurred in algae grown in 4.25 M sodium chloride. Lower concentrations of free glucose, which varied inversely with extracellular salt concentration, were also detected.
3. It is inferred that Na⁺ is effectively excluded from the two algae. There was some evidence of a moderate uptake of K⁺.
4. Comparisons were made of erude preparations of the glucose-6-phosphate dehydrogenase and an NADP-specific glycerol dehydrogenase from each species and of the effects of salt and glycerol on the activities of these enzymes. It is concluded that the different salt tolerances of the two algae cannot be explained by generalized differences between their enzyme proteins.
5. Although intracellular glycerol must necessarily contribute to the osmotic status of the algae, its primary function in influencing their salt relations is considered to be that of a compatible solute, whereby glycerol maintains enzyme activity under conditions of high extracellular salt concentration and hence low (thermodynamic) water activity.