AimTo evaluate the effect of temperature, pH and SO2 on growth and glycerol production improvement by Saccharomyces cerevisiae mc(2), Kloeckera apiculata mF and Oenococcus oeni X2L using the response surface method (RSM). Methods and ResultsMultifactorial design of cultures with physicochemical factors variations was performed. The micro-organisms grew in all cultures conditions. Overall, after 6days yeasts prevailed, especially S.cerevisiae (10(9)CFUml(-1)), while O.oeni reached 10(7)CFUml(-1). At initial fixed pH 55, metabolic behaviour of cultures showed a temperature-dependent response. Total malate consumption occurred at 26 degrees C, 50mgl(-1) SO2. Glucose and pentoses utilization was highly modified when varying SO2. Ethanol showed negative interaction with temperature-SO2 relationship. At low SO2, glycerol and acetate production increased when temperature enhanced. Predictive results of RSM indicate that 26 degrees C, 6024mgl(-1) SO2 and pH 55 were the optimal conditions for glycerol and organic acids synthesis compatible with wine quality. ConclusionsWe propose a predictive condition to improve the performance of mixed cultures for must fermentations. Significance and Impact of the StudyTo optimize the culture conditions to design mixed starters containing autochthonous yeasts and O.oeni strains for winemaking and to obtain products with high glycerol content, low acidity and maintenance of regional characteristics.