The growth of the fission yeast Schizosaccharomyces pombe on glucose and glycerol was monitored on-line in shake flasks and microtiter plates. The Edinburgh Minimal Medium 2 was improved by doubling its concentrations, improving its buffer and increasing its sulphur and iron concentrations additionally. By growing S. pombe on mixed carbon sources, it was shown that glycerol and glucose complement one another. Several tests were performed to establish the cultivation of S. pombe with non-fermentable glycerol as the main carbon source in minimal medium. Interestingly, a synergistic effect of glycerol and acetate was discovered which can significantly improve the growth of the fission yeast on glycerol. S. pombe showed optimal respiration activity, growth, and product formation by co-utilizing 20g/L glycerol and 2.5g/L sodium acetate.