Yeasts have been known to giow at the expense of n-paraffins for a number of years (TAUSSON 1939). It is generally accepted that the alkane is mainly metabolized through the corresponding primary alcohol, aldehyde and fatty acid. The enzymes involved in this pathway are adaptative or constitutive depending upon the microorganism studied (VAN DER LINDEN and THIJSSE
1956).Candida lipolytica is one of the most extensively investigated hydrocarbon assimilating microorganisms. Whether the enzymes, by which the yeast oxidizes n-hexadecane into palmitic acid, are constitutive to the microbial cell or formed adaptatively upon exposure to the substrate, has remained open to question, and was investigated by respirometric experiments in a WARBURG apparatus using resting cells of C. lipolytica grown on 1 yo n-hexadecane, cetylalcohol, palmitaldehyde, palmitic acid or glucose.
1969 373-380
JIuterial and methodsCultures: Candida Zipolytica, strain 599 from the Centraal Bureau voor Schimmelkulturen (C. B. S., Delft, Neth.) was maintained, grown on either 1% n-hexadecane or glucose, and harvested in the middle of its logarithmic phase of growth, as described earlier (NYNS et al. 1967). The same procedure was used when cetylalcohol, palmitaldehyde or palmitic acid served as sole source of carbon and energy. I n these cases, 100 ml of the liquid growth medium were prepared as follows: 1 g of the carbon source and 89 ml distilled water were steam-sterilized a t 120 "C for 20 min. A tenfold concentrated aqueous solution of Yeast Nitrogen Base (DIFCO) was filter-sterilized through a membrane of 0.22 p diameter (Millipore, Bedford, Mass.). 10 ml of this solution were aseptically added to the sterilized medium.The shaft of an Ultra-Turrax (type TP 18/2, JANKE and KUNKEL, Staufen i. Br., Germ.) was sterilized with ethanol and rinsed with sterile distilled water. The growth medium was aseptically homogenized a t top speed (12,000 rpm) for 1 min. The microbial population was estimated by counting appropriate dilutions of the cell suspension using a THOMAS haemacytometer. Cell clusters were homogenized with Tween 80 (NYKS et al. 1967).The cell crop was washed and the dry weight of the washed cell suspension was determined as reported earlier (NYNS et al. 1968). FRITSCHE (1966) could not use C. Zipolytica grown on n-alkanes for WARBURG respirometric studies, because the endogenous respiration was too high. ROBINSON (1964) reduced the endogenous respiration by starvation in distilled water for 2 hr. a t room temperature. In the case of C . lipolytica, it was necessary t o incubate the yeast cells in a carbonfree nutrient medium for 24 hr. before the endogenous respiration substantially decreased (NYNS et UZ. 1968). Whenever n-hexadecane, cetylalcohol, palmitaldehyde or palmitic acid served as the carbon source, the cells were starved in this manner. Before starvation, hydrocarbon grown cells contained up to 1/3 of their weight of adsorbed