Sterols of Chlorella. II. The Occurrence of an Unusual Sterol Mixture in Chlorella vulgaris

The first identification of a sterol in Chlorella was by Klosty and Bergmann (5) in 1952 when they isolated ergosterol from Chlorella pyrenoidosa. More recently, Otsuka (6) identified ergosterol and a A5 sterol in Chlorella ellipsoidea. Ergosterol was the principal component of the mixture. Recent work in this laboratory has been centered about the identification of sterols from other species of Chlorella. A mixture of the A5 sterols, 'poriferasterol, clionasterol, and 22-dihydrobrassicasterol was 'found (7) in C. ellipsoidea and C. saccharophila, but no ergosterol was detected. Chlorella vulgaris was shown to contain chondrillasterol as its prin,cipal sterol component, and although 2 other sterols were present in significant quantities, they were not isolated. Since plant sterols are known to have an effect on animal metajbolism, and algae are being considered as future sources of food, it is important that we know the sterol composition of these algae. This paper describes the isolation and identification of each of the 2 unknown sterols occurring in C. vulgaris. iCells of Chlorella vzlgaris Beyer., Emerson's strain, were grown heterotrophically on 'basal inorganic medium containing 0.5 % glucose in 15-liter carboys equipped with bubbling tubes for air (3). The cells were harvested in a Sharples Super Centrifuge and freeze-dried before extraction. Average yield was 3 grams dry weight per liter. Lipid material was extracted from the cells with acetone in a soxhlet apparatus, saponified under nitrogen, and the nonsaponifiable matter extracted with ether in a liquidliquid extraction apparatus. The non-saponifiable lipid was fractionated as described by Hef'tmann et al. (3) on Woelm Grade III neutral alumina. The fraction containing sterols was acetylated and rechromatographed under the same conditions described above. The fraction containing the mixture of sterol acetates was su'bjected to column chromatography on Anasil B which was added to the 3 cm X 40 cm column in a slurry of n-hexane. The sterol acetates were added to the column in a minimum amount of