Exploitation of Dunaliella for β-carotene production

Halotolerant microalga Dunaliella, which is exploited for the production of dried biomass or cell extract, is used as a medicinal food. With the advancement in this field in recent years, the production of bio-organic compounds such as β-carotene is established in many countries. Large-scale production of β-carotene is controlled by numerous stress factors like high light intensity, high salinity, temperature and availability of nutrients. The state-of-the-art strategies in industries in closed systems under new set of inductive factors will additionally promote the ease of commercial production of β-carotene. This review mainly focuses on the different methodologies employed recently for the optimum production of β-carotene from Dunaliella species.     

Supplementation with 9-cis β-carotene-rich alga Dunaliella improves hyperglycemia and adipose tissue inflammation in diabetic mice

Several species of the alga Dunaliella contain high levels of β-carotene. Low dietary β-carotene intake is associated with type 2 diabetes. Dunaliella contains high levels of all-trans and 9-cis isomers of β-carotene and is the best known naturally occurring nutritional source of 9-cis β-carotene. Since vitamin A has been suggested to play a role in glucose and lipid metabolism, we aimed to study the effect of Dunaliella supplementation on diabetes in mice. Ten diabetic db/db mice were fed chow diet fortified with 8 % 9-cis-rich Dunaliella powder. Ten db/db and heterozygous db/+ mice each served as controls and were fed chow diet alone. The control db/db mice developed severe hyperglycemia with fasting glucose levels reaching 400 mg dL^?1. Dunaliella significantly inhibited the elevation of plasma glucose (p?=?0.007). The area under the curve of the glucose tolerance test was 24 % lower in Dunaliella-treated mice than in the control db/db mice. Triglyceride elevation was significantly lower in the Dunaliella group than in the db/db group (p?=?0.007). The mRNA levels of several pro-inflammatory genes in adipose tissue, including monocyte chemotactic protein-1, intercellular adhesion molecule, vascular adhesion molecule-1, receptor-associated protein factor 6, and p-selectin, were elevated in the db/db group as compared to the db/+, whereas their levels were significantly lower in the Dunaliella-treated group. These results suggest that 9-cis β-carotene-rich Dunaliella may inhibit diabetes in db/db mice by reducing inflammation in adipose tissue. This study also emphasizes the importance of β-carotene isomers in our diet.     

PRODUCTION AND SELECTION OF HIGH β-CAROTENE MUTANTS OF DUNALIELLA BARDAWIL (CHLOROPHYTA)1

We describe a procedure for the selection of β-carotenerich mutants of the halotolerant alga Dunaliella bardawil Ben-Amotz & Avron. Under normal growth conditions the isolated mutants had a several-fold higher content of β-carotene than the wild type. Under carotene-induction conditions, the mutants also possessed a higher β-carotene content than the wild type. Both the production rate of phytoene and the conversion rate of phytoene to lycopene and β-carotene were accelerated in the mutants. Cycloheximide, which (in the wild type) inhibits the inductive synthesis of the proteins required for β-carotene production, had a much smaller effect on β-carotene biosynthesis in the mutants. We suggest that the mutants are affected in the regulatory path, which controls the induction of high β-carotene production in Dunaliella.     

Assessment of carotenoid production by Dunaliella salina in different culture systems and operation regimes

The effect of operation regime and culture system on carotenoid productivity by the halotolerant alga Dunaliella salina has been analyzed. Operation strategies tested included batch and semi continuous regime, as well as a two-stage approach run simultaneously in both, open tanks and closed reactor. The best results were obtained with the closed tubular photobioreactor. The highest carotenoid production (328.8 mg carotenoid l⁻¹ culture per month) was achieved with this culture system operated following the two-stage strategy. Also, closed tubular photobioreactor provided the highest carotenoid contents (10% of dry weight) in Dunaliella biomass and β-carotene abundance (90% of total carotenoids) as well as the highest 9-cis to all-trans β-carotene isomer ratio (1.5 at sunrise).     

The effect of gradual increase in salinity on the biomass productivity and biochemical composition of several marine,halotolerant<Emphasis Type="Bold">,</Emphasis> and halophilic microalgae

Open ponds are the preferred cultivation system for large-scale microalgal biomass production. To be more sustainable, commercial scale biomass production should rely on seawater, as freshwater is a limiting resource, especially in places with high irradiance. If seawater is used for both pond fill and evaporative volume makeup, salinity of the growth media will rise over time. It is not possible for any species to achieve optimum growth over the whole saline spectrum (from seawater salinity level up to salt saturation state). In this study, we investigated the effects of gradual salinity increase (between 35 and 233 ppt) on biomass productivity and biochemical composition (lipid and carbohydrate) of six marine, two halotolerant, and a halophilic microalgae. A gradual and slow stepped salinity increase was found to expand the salinity tolerance range of tested species. A gradual reduction in biomass productivity and maximum photochemical efficiency was observed as a consequence of increased salinity in all tested species. Among the marine microalgae, Tetraselmis showed highest biomass productivity (32 mg L^?1 day^?1) with widest salinity tolerance range (35 to 109 ppt). Halotolerant Amphora and Navicula were able to grow from 35 ppt to 129 ppt salinity. Halophilic Dunaliella was the only species capable of growing between 35 and 233 ppt and showed highest lipid content (56.2%) among all tested species. This study showed that it should be possible to maintain high biomass in open outdoor cultivation utilizing seawater by growing Tetraselmis, Amphora, and Dunaliella one after another as salinity increases in the cultivation system.     

Production kinetics of β-carotene from Planococcus sp. TRC1 with concomitant bioconversion of industrial solid waste into crystalline cellulose rich biomass

Cost effective bioprocessing of nutraceuticals in present global scenario is a matter of concern. This study explored Paper mill sludge (PMS) and sugarcane bagasse (SCB) as inexpensive substrate for Planococcus sp. TRC1 mediated valuable β-carotene production and residual treated biomass as value added crystalline cellulose source simultaneously. Both biomass supported significant bacterial growth reaching highest yield 38.54 ± 1.4 mg/g on PMS (36 h) and 47.13 ± 1.9 mg/g (48 h) on SCB in solid state fermentation. Luedeking-Piret model revealed growth associated production with α and much lower β values of 5.18 and 0.24 for PMS and 4.5 and 0.165 for SCB. Cost analysis exhibited decrementation of pigment cost/mg by 84 % compared to synthetic media. Optimum production conditions were 30 °C temperature, pH 7, 10 % inoculum and initial moisture content 80 % (PMS) and 85 % (SCB). TLC (R_f = 0.9), HPLC (RT = 7.646) and lambda max (465 nm) confirmed pigment’s β-carotene nature with significant antioxidant and antimicrobial activity. It showed stability at varied temperature, pH and light conditions along with negligible phytotoxicity on Vigna radiata. Planococcus sp. TRC1 delignified PMS (41 %) and SCB (38 %) and FT-IR, FESEM and XRD suggested crystalline nature of residual cellulose rich fraction shedding light on a biorefinery approach for valorization of industrial solid wastes.     

Correlation between lipid and carotenoid synthesis in torularhodin-producing <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

The oleaginous red yeast Rhodotorula glutinis produces carotenoid pigments, especially torularhodin and β-carotene, in significant amounts. We have analyzed in detail carotenoid and lipid biosynthesis in a torularhodin-producing strain of R. glutinis cultivated at different carbon:nitrogen (C/N) ratios (20:1, 50:1, 70:1, and 100:1). When the strain was cultivated in media with low C/N ratios (20:1 and 50:1), glucose was completely utilized and carotenoid formation was stimulated. Maximum pigment production reached 12.9 mg/L of medium and 2.3 mg/g of biomass at the C/N ratio of 20:1. It was noted that β-carotene synthesis was prominent when glucose was present in the medium. However, glucose exhaustion in the media at C/N ratios of 20:1 and 50:1 was closely accompanied by the predominant formation of torularhodin. The growth of R. glutinis in media with C/N ratios of 70:1 and 100:1 favored lipid accumulation in the cells but carotenoid biosynthesis was reduced. In addition, glucose consumption was linked to a rapid decrease in oleic acid levels in the total intracellular lipids. The kinetic analysis clearly indicated a correlation between oleic acid levels in total lipids and torularhodin accumulation in the cells. The results may suggest that acetyl-CoA formed from oleic acid degradation is metabolized through the mevalonate/isoprenoid/carotenoid pathways directly to torularhodin.     

Agro-food wastes utilization by Blakeslea trispora for carotenoids production

The all-trans-β-carotene is a natural pigment used in various industrial fields (food, cosmetics, pharmaceuticals, etc) and possesses the higher provitamin A activity, in respect to other carotenoids. All-trans-β-carotene is produced industrially by chemical and biotechnological means. For β-carotene biotechnological production in industrial scale mated cultures of Blakeslea trispora, a heterothallic fungus, are mainly used. Despite the intense research for β-carotene production by B. trispora, natural substrate utilization has not been extensively studied. Solid agro-food wastes such as cabbage, watermelon husk and peach peels from northern Greece as main carbon source into submerged B. trispora cultures for carotenoids production, was examined. The media containing only the agro-food waste (2-4) gave a biomass accumulation 7.77 ± 0.4 g/L, while a reference medium 1 with glucose (10 g/L) gave 4.65 ± 0.21 g/L. In another experiments series agro-food wastes were used with corn steep liquor and thiamine (media 6-8), giving a biomass accumulation and total carotenoid volumetric production 10.2 ± 2.41 g/L and 230.49 ± 22.97 mg/L, respectively. These are the higher values reported for solid wastes so far in respect to those obtained from a synthetic medium, with higher glucose concentration of 50 g/L where the correspondent values were 9.41 ± 1.18 g/L and 45.63 mg/L respectively. The results support that B. trispora is able to utilize, almost equivalently, different origin agro-food wastes for carotenoids production. Furthermore, β-carotene percentage in all examined cases was over 76%, as it was estimated by HPLC analysis, suggesting that these agro food wastes may be used for high purity, large scale β carotene production.     

DNA fingerprinting differentiation between β-carotene hyperproducer strains of Dunaliella from around the world

Background

Dunaliella salina is the most important species of the genus for β-carotene production. Several investigations have demonstrated that D. salina produces more than 10% dry weight of pigment and that the species grows in salt saturated lagoons. High plasticity in the green stage and the almost indistinguishable differences in the red phase make identification and differentiation of species and ecotypes very difficult and time consuming.

Results

In this work, we applied our intron-sizing method to compare the 18S rDNA fingerprint between D. salina (CCAP 19/18), D. salina/bardawil (UTEX LB2538) and β-carotene hyperproducing strains of Dunaliella isolated from salt saturated lagoons in Baja, Mexico. All hyperproducer strains reached β-carotene levels of about 10 pg/cell. Optical microscopy did not allow to differentiate between these Dunaliella strains; however, 18S rDNA fingerprinting methodology allowed us to differentiate D. salina from D. salina/bardawil.

Conclusion

In Baja Mexico we found D. salina and D. salina/bardawil species by using intron-sizing-method. The National Center for Biotechnology Information (NCBI) Dunaliella 18S rDNA gene sequences were analyzed with our methodology and extraordinary correlation was found with experimental results.     

Enhanced production of biomass and lipids by supplying CO2 in marine microalga Dunaliella sp.

Non-food-based biofuel feedstocks are in high demand worldwide. Among the various feedstocks, microalgae are the most promising feedstock for mitigating atmospheric CO₂ and producing biodiesel. In this study, various concentrations of CO₂, from 0.03 to 12%, were used to investigate their effect on the cell growth, biomass and lipid production and fatty acid composition of Dunaliella sp. in a closed photobioreactor. The results showed that the highest biomass and total lipids, 521 mg/L/d and 40 mg/L/d, respectively, were produced with 5% CO₂ aeration during the logarithmic growth phase. The oleic acid (18:1n9c) and elaidic acid (18:1n9t) contents were increased approximately two fold. The physiological responses of Dunaliella sp. at 10% CO₂ were similar to those at 5% CO₂. Therefore, the present results suggest that 5–10% is a suitable CO₂ concentration range for Dunaliella sp. growth to mitigate atmospheric CO₂ and increase biofuel production.     

Characterization of an evolved carotenoids hyper-producer of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> through bioreactor parameter optimization and Raman spectroscopy

An evolutionary engineering approach for enhancing heterologous carotenoids production in an engineered Saccharomyces cerevisiae strain was used previously to isolate several carotenoids hyper-producers from the evolved populations. β-Carotene production was characterized in the parental and one of the evolved carotenoids hyper-producers (SM14) using bench-top bioreactors to assess the impact of pH, aeration, and media composition on β-carotene production levels. The results show that with maintaining a low pH and increasing the carbon-to-nitrogen ratio (C:N) from 8.8 to 50 in standard YNB medium, a higher β-carotene production level at 25.52 ± 2.15 mg β-carotene g^?1 (dry cell weight) in the carotenoids hyper-producer was obtained. The increase in C:N ratio also significantly increased carotenoids production in the parental strain by 298 % [from 5.68 ± 1.24 to 22.58 ± 0.11 mg β-carotene g^?1 (dcw)]. In this study, it was shown that Raman spectroscopy is capable of monitoring β-carotene production in these cultures. Raman spectroscopy is adaptable to large-scale fermentations and can give results in near real-time. Furthermore, we found that Raman spectroscopy was also able to measure the relative lipid compositions and protein content of the parental and SM14 strains at two different C:N ratios in the bioreactor. The Raman analysis showed a higher total fatty acid content in the SM14 compared with the parental strain and that an increased C:N ratio resulted in significant increase in total fatty acid content of both strains. The data suggest a positive correlation between the yield of β-carotene per biomass and total fatty acid content of the cell.     

Canthaxanthin production with modified Mucor circinelloides strains

Canthaxanthin is a natural diketo derivative of β-carotene primarily used by the food and feed industries. Mucor circinelloides is a β-carotene-accumulating zygomycete fungus and one of the model organisms to study the carotenoid biosynthesis in fungi. In this study, the β-carotene ketolase gene (crtW) of the marine bacterium Paracoccus sp. N81106 fused with fungal promoter and terminator regions was integrated into the M. circinelloides genome to construct stable canthaxanthin-producing strains. Different transformation methods including polyethylene glycol-mediated transformation with linear DNA fragments, restriction enzyme-mediated integration and Agrobacterium tumefaciens-mediated transformation were tested to integrate the crtW gene into the Mucor genome. Mitotic stability, site of integration and copy number of the transferred genes were analysed in the transformants, and several stable strains containing the crtW gene in high copy number were isolated. Carotenoid composition of selected transformants and effect of culturing conditions, such as temperature, carbon sources and application of certain additives in the culturing media, on their carotenoid content were analysed. Canthaxanthin-producing transformants were able to survive at higher growth temperature than the untransformed strain, maybe due to the effect of canthaxanthin on the membrane fluidity and integrity. With the application of glucose, trehalose, dihydroxyacetone and l-aspartic acid as sole carbon sources in minimal medium, the crtW-expressing M. circinelloides strain, MS12+pCA8lf/1, produced more than 200 μg/g (dry mass) of canthaxanthin.     

Carotenoids of cryptophyceae

The carotenoids of selected Cryptophyceae, Rhodomonas D3 and Cryptomonas ovata, have been examined by methods including HPLC, mass spectrometry ¹H NMR and circular dichroism. $\text{3′R,6′R-}\text{Chirality}$ has been assigned to monadoxanthin from ¹H NMR and CD data; β,?-carotene possessed the common $\text{6′R-}\text{chirality}$. The quantitative distribution pattern of carotenoids in Cryptophyceae established here and previously, totalling five species' is discussed in chemosystematic context. β,?-Carotene (3–8% of total) is the major carotene, accompanied by ?,?-carotene (0.2%), β,β-carotene (0–1%) and lycopene (0-trace). Zeaxanthin (2%) was identified in C. ovate. The diacetylenic alloxanthin is the major carotenoid (70–88%), and the monoacetylenic crocoxanthin (5–15%) and monadoxanthin (0–16%) less abundant. No epoxidic or allenic carotenoids could be detected. The biosynthetic precursor of acetylenic carotenoids in this primitive algal class is discussed. The significance of Cryptophyceae in the marine food chain is commented on, using alloxanthin as an indicator.     

Accumulation of α-tocopherol and β-carotene in <Emphasis Type="Italic">Euglena gracilis</Emphasis> Cells Under Autotrophic and Mixotrophic Culture Conditions

The aim of the work was to find the mode of cultivation of unicellular flagellate Euglena gracilis, favorable for the simultaneous accumulation of α-tocopherol and β-carotene. Cells were grown either in photoautotrophic or photoheterotrophic conditions in the presence of 100 mM ethanol (variant Et) or 40 mM glutamate (variant Gt), or their combination (variant EtGt). The exogenous substrates significantly stimulated light-dependent growth of E. gracilis. The largest increase of biomass was recorded on the 20th day in the variant EtGt and exceeded the autotrophic control by 7 times. The content of β-carotene and chlorophyll (Chl) per cell in mixotrophic cultures exceeded the control by 2–3 and 1.6–2 times, respectively. At the same time, α-tocopherol accumulation in autotrophic cells was greater than in the cells of mixotrophic cultures by 2–7 times. Total yield of tocopherol per unit volume of culture medium, which depended not only on its intracellular content, but also on the amount of accumulated biomass was highest in EtGt variant. A correlation between the accumulation of the antioxidants and the equilibrium concentration of oxygen in the growth medium, which depended on the intensities of photosynthesis and respiration has been analyzed.     

Efficient production of retinol in Yarrowia lipolytica by increasing stability using antioxidant and detergent extraction

The demand for bio-based retinol (vitamin A) is currently increasing, however its instability represents a major bottleneck in microbial production. Here, we developed an efficient method to selectively produce retinol in Yarrowia lipolytica. The β-carotene 15,15′-dioxygenase (BCO) cleaves β-carotene into retinal, which is reduced to retinol by retinol dehydrogenase (RDH). Therefore, to produce retinol, we first generated β-carotene-producing strain based on a high-lipid-producer via overexpressing genes including heterologous β-carotene biosynthetic genes, GGS1^F43I mutant of endogenous geranylgeranyl pyrophosphate synthase isolated by directed evolution, and FAD1 encoding flavin adenine dinucleotide synthetase, while deleting several genes previously known to be beneficial for carotenoid production. To produce retinol, 11 copies of BCO gene from marine bacterium 66A03 (Mb.Blh) were integrated into the rDNA sites of the β-carotene overproducer. The resulting strain produced more retinol than retinal, suggesting strong endogenous promiscuous RDH activity in Y. lipolytica. The introduction of Mb.Blh led to a considerable reduction in β-carotene level, but less than 5% of the consumed β-carotene could be detected in the form of retinal or retinol, implying severe degradation of the produced retinoids. However, addition of the antioxidant butylated hydroxytoluene (BHT) led to a >20-fold increase in retinol production, suggesting oxidative damage is the main cause of intracellular retinol degradation. Overexpression of GSH2 encoding glutathione synthetase further improved retinol production. Raman imaging revealed co-localization of retinol with lipid droplets, and extraction of retinol using Tween 80 was effective in improving retinol production. By combining BHT treatment and extraction using Tween 80, the final strain CJ2104 produced 4.86 g/L retinol and 0.26 g/L retinal in fed-batch fermentation in a 5-L bioreactor, which is the highest retinol production titer ever reported. This study demonstrates that Y. lipolytica is a suitable host for the industrial production of bio-based retinol.     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

Production of 6-kestose by the filamentous fungus Gliocladium virens as affected by sucrose concentration

The filamentous fungus Gliocladium virens is able to produce fructooligosaccharides (FOS), fructose-containing sugars, used as functional ingredients to improve nutritional and technological properties of foods. In this work we evaluated FOS production by G. virens when grown in a wide range of sucrose concentrations (10–400 g l^?1). High sucrose concentrations increased both biomass and FOS production, including 6-kestose, a trisaccharide comprising β (2 → 6) linked fructosyl units, with enhanced stability and prebiotic activity when compared to the typical FOS β (2 → 1) linked. The highest 6-kestose yield (3 g l^?1) was achieved in media containing 150 g l^?1 sucrose after 4–5 days of culture, production being 90% greater than in media containing 10, 30, or 50 g l^?1 sucrose. After 5 days, FOS production declined markedly, following complete sucrose depletion in the medium. Although most of the β-fructofuranosidases preferentially catalyze sucrose hydrolysis, FOS production in G. virens grown in high sucrose concentration, might be attributed to a reverse hydrolysis by these enzymes. In conclusion, high sucrose concentrations increase growth of G. virens whilst 6-kestose accumulation in the medium seems to be controlled both by specific properties of β-fructofuranosidases and on the sucrose concentration.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.     

A pH control strategy for increased β-carotene production during batch fermentation by recombinant industrial wine yeast

The effects of different pH values, ranging from 4.0 to 7.0, on cell growth and β-carotene production by recombinant industrial wine yeast Saccharomyces cerevisiae T73-63 in a synthetic grape juice medium was investigated. Based on the kinetic analysis of the batch fermentation process, a two-stage pH control strategy was developed in which the pH was maintained at 7.0 for the first 24 h and then shifted to 5.0 after 24 h. Using this strategy, the highest β-carotene production (50.39 mg/l) and the formation rate (1.40 mg/l/h) were increased by 19.1% and 18.6%, respectively, compared to the maximum values of constant pH fermentation. The oxidative stress during β-carotene production was also determined in terms of the catalase (CAT) and superoxide dismutase (SOD) activities. Oxidative stress appears to be induced by the lowering of pH as indicated by the increase in activities of CAT and SOD due to pH shift from pH 7.0 to pH 5.0. Pre-treating cells with ascorbic acid (an antioxidative agent) reversed the improvement of β-carotene production while addition of H₂O₂ enhanced it. Considering that induction of oxidative stress is associated with increased β-carotene production, it was concluded that the enhancement of β-carotene production by the low-pH strategy involved the induction of oxidative stress.     

ACCUMULATION OF β-CAROTENE IN HALOTOLERANT ALGE: PURIFICATION AND CHARACTERIZATION OF β-CAROTENE-RICH GLOBULES FROM DUNALIELLA BARDAWIL (CHLOROPHYCEAE)1

Dunaliella bardawil Ben-Amotz & Avron, but not most other Dunaliella species, has a unique property of being able to accumulate, in addition to glycerol, large amounts of β-carotene when cultivated under appropriate conditions. These include high light intensity, a high sodium chloride concentration, nitrate deficiency and extreme temperatures. Under conditions of maximal carotene accumulation D. bardawil contains at least 8% of its dry weight as β-carotene while D. salina grown under similar conditions contains only about 0.3%. Electron micrographs of D. bardawil grown under conditions of high β-carotene accumulation show many β-carotene containing globules located in the interthylakoid spaces of the chloroplast. The same algae grown under conditions where β-carotene does not accumulate, contain few to no β-carotene globules. The β-carotene-rich globules were released from the algae into an aqueous medium by a two-stage osmotic shock technique and further purified by centrifugal ion on 10% sucrose. The isolated purified globules were shown by electron microscopy to be free of significant contamination and composed of membrane-free osmiophilic droplets with an average diameter of 150 nm. Reversed phase high performance liquid chromatography of a total pigment extract of the cells revealed the presence of β-carotene as the major pigment, together with chlorophylls a and b, α-carotene and the xanthophylls lutein, neoxauthin and zeaxanthin. β-Carotene accounted for essentially all the pigment in the purified globules. Analysis of the algal and globule β-carotene fractions by HPLC showed that the β-carotene was composed of approximately equal amounts of all-trans β-carotene and of its 9-cis isomer. Intact D. bardawil cells contained on a dry weight basis about 30% glycerol, 30% protein, 18% lipid, 11% carbohydrate, 9%β-carotene and 1% chlorophyll. The β-carotene globules were composed of practically only neutral lipids, more than half of which was β-carotene. It is suggested that the β-carotene globules may serve to protect D. bardawil against injury by the high intensity irradiation to which this alga is usually exposed in nature.