Formation of esters of yeast. I. The production of ethyl acetate by standing surface cultures of Hansenula anomala.

It has been known for some time (Hansen, 1891) that organic esters may be produced in small amounts by some microorganisms as byproducts in their utilization of organic compounds. A detailed review of the literature to 1922 on aroma producing microorganisms is given by Omelianski (1923). The taxonomy of the yeast genus Hansenula, which contains species producing the greatest yields of ester, is discussed by Bedford (1942) and Gray (1949). The early observations on the physiology of ester formation were reviewed by Tabachnick (1950). While our studies of ester formation by yeast, begun in 1946, were in progress, several papers appeared on the physiology of ester formation by H. anomala. Gray (1949), working with standing surface cultures of H. anomala (Hansen) in a glucose medium, showed: (1) that ester was produced under conditions of limited aeration from some unknown anaerobically produced intermediate and (2) that the ester was utilized as rapidly as it was produced. Although the ester had been identified by its odor as ethyl acetate (Takahashi and Sato, 1911; Bedford, 1942), Gray was the first to identify it as ethyl acetate. Davies et al. (1951) presented additional proof that the ester is ethyl acetate. Peel (1951), using cell suspensions of H. anomala in an ethanol, acetic acid medium, found ester formation to be an aerobic process with an optimum pH of 4.5 to 5. In ethanol alone the pH optimum was difficult to define but appeared to range from 2.5 to 3. Peel also showed with growing cells and cell suspensions that the amount of ester formed is too large to be accounted for by the reversal of a simple esterase reaction, indicating a more exergonic mechanism of ester formation. In the following report on growing cultures of 'Present address: Division of Microbiology, Albert Einstein Medical Center, Northern Division, Philadelphia 41, Pennsylvania. 1 H. anomala, some of the observations recorded by Gray (1949), Davies et al. (1951), and Peel (1951) are confirmed. These workers identified the ester indirectly; we isolated and purified sufficient ester to determine both physical and chemical properties. In addition, it will be shown that ethyl acetate is not produced directly from glucose but is formed as the result of an aerobic utilization of ethanol accumulated in the fermentation of glucose. The influence of several environmental factors on ester production will be discussed.