Factors affecting simultaneous hydrogen and ethanol production from waste glycerol by a newly isolated bacterium Enterobacter aerogenes KKU-S1 were investigated employing response surface methodology (RSM) with central composite design (CCD). The Plackett-Burman design was first used to screen the factors influencing simultaneous hydrogen and ethanol production, i.e., initial pH, temperature, amount of vitamin solution, yeast extract (YE) concentration and glycerol concentration. Results indicated that initial pH, temperature, YE concentration, and glycerol concentration had a statistically significant effect ( p <= 0.05) on hydrogen production rate (HPR) and ethanol production. The significant factors were further optimized using CCD. Optimum conditions for simultaneously maximizing HPR and ethanol production were YE concentration of 1.00 g/L, glycerol concentration of 37 g/L, initial pH of 8.14, and temperature of 37 degrees C in which a maximum HPR and ethanol production of 0.24 mmol H-2/L h and 120 mmol/L were achieved. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.