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

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Abstract
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.