In the present work, the optimal design of pre-fermentation and fermentation operations for ethanol production is obtained developing a superstructure mathematical model. Different configurations of both operations are simultaneously considered in an overall model which also includes detailed kinetics equations. The zero wait is the transfer policy selected for these stages for ensuring the quality of these operations, given the nature and characteristics of microbiological sugary substrates. From the overall proposed model, the optimal configuration of the stages, the number of duplicated units in each stage, the size of each process unit, the process variables as concentrations and flows, and the total investment and production cost are obtained. This model is formulated as a non-linear programming problem, which is solved by the Professional Software, General Algebraic Modeling System (GAMS) with the application of CONOPT solver. The optimal design and operation of pre-fermentation and fermentation stages are obtained and the attained results are compared with the structures in conventional distillery. (C) 2014 Elsevier Ltd. All rights reserved.