Ethanol fermentation characteristics of the 100% respiratory-deficient nuclear petite amylolytic Saccharomyces cerevisiae NPB-G strain was investigated in both shake-flask and controlled bioreactor cultivation conditions, and comparison with the earlier reported results revealed 54.6% increase in ethanol yield. Efforts to improve the starch utilization rate by increasing the selection pressure or supplying the fermentation medium with glucose did not prevent the observed decrease in time-dependent amylolytic activity. Response surface methodology (RSM) was then used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both the respiratory-deficient NPB-G and the parental respiratory-sufficient WTPB-G strains, with concomitant improvement in their amylolytic potentials. High ethanol yields on substrate values of the bioreactor cultures, which were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient NPB-G strain was effective in the direct fermentation of starch into ethanol. Copyright (c) 2006 John Wiley & Sons, Ltd.