Waste molasses is one of the most important feedstock for ethanol production in Brazil as well as in many Southeast Asian countries, including China. Sulfuric acid pretreatment is employed in most ethanol distilleries in China to control bacterial contamination, which results in difficulties in the treatment of wastewater containing high levels of sulfate ions. In this study, a high efficiency, non-sterilized, continuous ethanol fermentation process without sulfuric acid pretreatment was developed using the flocculating yeast strain KF-7 and the widely utilized, traditional, stirred tank reactors. An alternative molasses medium feeding method, which differs from traditional methods, is proposed that effectively controls bacterial contamination. Separate feeding of 1.2-fold diluted molasses and tap water into the reactor proved to be effective against bacterial contamination during long-term continuous fermentation. By feeding yeast cells with high metabolic activity to the second reactor, a two-stage continuous fermentation process that yielded a high ethanol concentration of 80 g/l as well as high ethanol productivity of 6.6 g/l/h was successfully operated for more than one month. This fermentation process can be applied to ethanol distilleries in which traditional tank reactors are used. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.