The effect of ethanol on the inactivation of Saccharomyces pastorianus by a two-stage system with low-pressure carbon dioxide microbubbles (two-stage MBCO2) was investigated. Zero and >5 log reductions of S. pastorianus populations suspended in physiological saline (PS) containing 0% and 10% ethanol, respectively, occurred by the two-stage MBCO2 at a mixing vessel pressure of 1 MPa and a heating coil temperature of 40 degrees C. Conversely, the detected number of surviving S. pastorianus cells in PS containing 5% ethanol was higher in yeast and mold agar (YMA, an optimum agar) than YMA with 2.5% sodium chloride, followed by yeast nitrogen base agar (YNBA, a minimum agar). The fluorescence polarization of S. pastorianus in PS containing 5% and 10% ethanol increased similarly with exposure time in the heating coil of two-stage MBCO2 and was correlated with the surviving cell number measured in YNBA. The intracellular pH (pH(in)) of S. pastorianus in PS containing 5% ethanol decreased linearly with exposure time in the heating coil of two-stage MBCO2. Also, the pH(in)-lowering of S. pastorianus in PS containing 10% ethanol was drastically caused by two-stage MBCO2 at 1 min exposure time in the heating coil but then stayed constant until 5 min, agreeing with the inactivation efficiency. Therefore, ethanol in S. pastorianus suspension was suggested to accelerate the cell membrane injury caused by two-stage MBCO2. (c) 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:282-286, 2018