BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Optimization of temperature, sugar concentration, and inoculum size to maximize ethanol production without significant decrease in yeast cell viability Cecilia Laluce & João Olimpio Tognolli & Karen Fernanda de Oliveira & Crisla Serra Souza & Meline Rezende Morais Received: 27 October 2008 / Revised: 16 January 2009 / Accepted: 19 January 2009 / Published online: 21 February 2009 # Springer-Verlag 2009 Abstract Aiming to obtain rapid fermentations with high ethanol yields and a retention of high final viabilities (responses), a 2 3 full-factorial central composite design combined with response surface methodology was employed using inoculum size, sucrose concentration, and temperature as independent variables. From this statistical treatment, two well-fitted regression equations having coefficients significant at the 5% level were obtained to predict the viability and ethanol production responses. Three-dimensional response surfaces showed that increas- ing temperatures had greater negative effects on viability than on ethanol production. Increasing sucrose concentra- tions improved both ethanol production and viability. The interactions between the inoculum size and the sucrose concentrations had no significant effect on viability. Thus, the lowering of the process temperature is recommended in order to minimize cell mortality and maintain high levels of ethanol production when the temperature is on the increase in the industrial reactor. Optimized conditions (200 g/l initial sucrose, 40 g/l of dry cell mass, 30 °C) were experimentally confirmed and the optimal responses are 80.8±2.0 g/l of maximal ethanol plus a viability retention of 99.0±3.0% for a 4-h fermentation period. During consecutive fermenta- tions with cell reuse, the yeast cell viability has to be kept at a high level in order to prevent the collapse of the process. Keywords RSM . Viability . Ethanol production . Temperature . Sugar concentration . Inoculum size Introduction High ethanol yields in a short fermentation time are an economically relevant factor in industrial ethanol produc- tion. However, this is dependent on the yeast strain, type of process (batch or fed-batch), cell density, temperature, and sugar concentration and enrichment of the medium with the proper nutrients, along with other factors that influence the microbial activity. Studies related to ethanol production have been carried out in complex and synthetic media. Although these have not yet being implemented on an industrial scale due to economical reasons, a synthetic medium exhibits favorable characteristics over the tradi- tional complex or natural media since it is composed of pure chemicals in precisely known proportions (Zhang and Greasham 1999). Appl Microbiol Biotechnol (2009) 83:627–637 DOI 10.1007/s00253-009-1885-z C. Laluce (*) : M. R. Morais Department of Biochemistry and Biotechnological Chemistry, Instituto de Química de Araraquara-UNESP, Caixa Postal 355, 14801-970 Araraquara, Sao Paulo, Brazil e-mail: claluce@iq.unesp.br M. R. Morais e-mail: spg@iq.unesp.br J. O. Tognolli Department Analytical Chemistry, Instituto de Química de Araraquara-UNESP, Caixa Postal 355, 14801-970 Araraquara, Sao Paulo, Brazil e-mail: tognolli@iq.unesp.br K. F. de Oliveira : C. S. Souza Programa de Pós-Graduação Interunidades em Biotecnologia, Institute of Biomedical Sciences, Avenida Prof. Lineu Prestes, 1730-Edifício ICB-IV, Sala 03-Cidade Universitária, CEP: 05508-900 Sao Paulo, Sao Paulo, Brazil K. F. de Oliveira e-mail: biotec@icb.usp.br C. S. Souza e-mail: biotec@icb.usp.br