MATERIALS AND METHODS
Organisms I. orientalis MF-121, an acidesalt tolerant yeast, was isolated
from an acidic hot spring (11). Saccharomyces cerevisiae kyokai 7 was obtained
from the Brewing Society of Japan (Tokyo, Japan). S. cerevisiae NBRC 1953 and
Pichia stipitis NBRC 10063 were obtained from the NITE Biological Resource
Center (Chiba, Japan).
Preparation of acid and salt mineralized media Since the nutrient
medium composed of peptone and yeast extract is expensive, a cheaper mineralbased
medium was used. Considering the simple preparation of acidified and saltrich
medium, a mineralized medium (M medium) composed of (NH4)2SO4 0.2 g,
KH2PO4 0.1 g, MgSO4$7H2O 75 mg, ZnSO4$7H2O 6.0 mg, and yeast extract 0.05 g in
100 ml distilled water was used to investigate the ethanol fermentations properties
of yeasts.Mmedium supplemented with salt (MS medium) was prepared by mixing
M medium with appropriate amounts of sodium sulfate. Acid M medium was also
prepared by mixing M medium with appropriate amounts of 2.0 M H2SO4. M
medium, salt solution, sulfuric acid solution, and sugar solution were sterilized
separately at 120C for 20 min and then mixed in order to prepare an objective
medium in a clean bench. MS media containing monosaccharides (5.0 g/100 ml) as