Since it was confirmed that ethanol was efficiently produced with
the combination of sweet sorghum variety FS902 and S. cerevisiae
strain NBRC 0216 or Hitachi, next it was attempted to establish a
repeated-batch fermentation procedure with the reuse of S. cerevisiae
cells. A repeated-batch fermentation, which was composed of the 1st
cycle involving the growth of inoculated S. cerevisiae cells and the
following cycles reusing the cells recovered from the preceding
fermentation broth, was designed. First of all, the glycerol stock
solutions of S. cerevisiae NBRC 0216 and Hitachi were prepared by
adding an equal volume of sterilized 50% (v/v) glycerol into the culture
broth of YMmediumfor 2 days at 30°C and kept at−80°C. To determine
the inoculum size of cycle 1, the prepared glycerol stock solution was
transferred into the YM medium with 0.1% (v/v)-inoculation as a preculture,
and after cultivation at 30°C for 2 days, the cells of the preculture
were added into the juice of FS902 (10 ml) with 0.01% (v/v)-,
0.05% (v/v)-, and 0.1% (v/v)-inoculation.After inoculation, fermentation
was conducted at 30°C for 5 days with standing and then the ethanol
production, residual sugar, and yeast growth, were analyzed every day
(Fig. 1). In the 0.01% (v/v)-inoculation, neither strain could grow well,
and the insufficient sugar consumption resulted in a less than 20%
ethanol yield even after 5 days of cultivation (Fig. 1Aand D).