351 Research Article Received: 27 May 2011 Revised: 19 July 2011 Accepted: 22 July 2011 Published online in Wiley Online Library: 14 September 2011 (wileyonlinelibrary.com) DOI 10.1002/jctb.2725 Potential use of wine yeasts immobilized on Penicillium chrysogenum for ethanol production Teresa Garc´ ıa-Mart´ ınez, a Anna Puig-Pujol, b Rafael A. Peinado, c Juan Moreno c and Juan C. Mauricio a* Abstract BACKGROUND: Six different wine yeast strains (G1, X4, X5, P29, QA23, Uvaferm BC) were co-immobilized in a natural, spontaneous way with Penicillium chrysogenum under special conditions without the need for an external support or chemical binder and provided six different ‘yeast biocapsules’. The purpose was to characterize and evaluate the biocapsules obtained in terms of yeast cell viability, ethanol production and reusability to assess their suitability for ethanol production and the development of industrially competitive alternative wine and beer production methods. RESULTS: Biocapsule size was found to decrease and quantity to increase with increasing shaking rate during the immobilization process. The fermentations were realized in YPD medium containing 18% (w/v) glucose with repeated fermentations reaching 10% (v/v) ethanol. X4 and Uvaferm BC biocapsules afforded at least seven uses with no significant decrease in ethanol production; P29 and QA23 biocapsules five times; and G1 and X5 three times each. Seemingly, ethanol production was directly related to the viability of yeast cells in the immobilizate under defined assay conditions. CONCLUSIONS: X4 and Uvaferm BC may be the most suitable yeast strains for autoimmobilization on P. chrysogenum with a view to their use in alcoholic fermentation processes. c  2011 Society of Chemical Industry Keywords: immobilized cells; S. cerevisiae; P. chrysogenum; ethanol fermentation INTRODUCTION The energy crisis caused by the dependence on fossil fuels, and the environmental effects of their use, has raised the demand for biofuels. Bioethanol fuel has been deemed the great alternative to fossil fuels; also, it is currently considered a profitable commodity, and is being increasingly used as a renewable energy source and car fuel. 1 The increasing demand for ethanol has raised the need for more cost-effective technologies for its production. The yeast Saccharomyces cerevisiae is the microorganism most widely used in alcoholic fermentation; however, the bacterium Zymomonas mobilis, which is currently being used mostly in sugar fermentation processes, might be an advantageous alternative. 2,3 Immobilization procedures allow cells to be confined in a well-defined spatial region in order to preserve their catalytic properties and make them reusable. 4,5 Ethanol production by immobilized yeasts has been the subject of extensive research during the last few decades as an alternative procedure, with technical and economic advantages over traditional systems based on free cells. Using immobilized cells for fermentation avoids the inhibitory effects of high concentrations of substrate and product, thereby enhancing ethanol productivity and yield. 6–10 Cells can be immobilized by natural or artificial means. Artificial immobilization is the more common and can be accomplished by binding to a support, cross-linking through binding to bifunctional compounds, trapping in a semi-permeable membrane or a polymer, 5,8,11,12 or a combination of the three methods. Because the cells used in artificial immobilization methods are not in their natural form, they can experience strong changes in metabolism and viability. However, some microorganisms can be spontaneously immobilized naturally under special conditions by aggregation into small pellets, flocs, microspheres, mycelia or biofilms. No immobilization treatment is required, so no metabolic alteration in the immobilized cells is to be expected, which can open the door to advantageous uses relative to artificially immobilized cells. 5,13 – 20 In addition, the natural adhesion method has been paid more and more attention, because maximum cell viability and biochemical activity were obtained due to the formation of biofilms. 21 ∗ Correspondence to: Juan C. Mauricio, Departamento de Microbiolog´ ıa, Fac- ultad de Ciencias, Edificio Severo Ochoa, Campus Universitario de Rabanales. Universidad de C´ ordoba. 14071-C´ ordoba, Spain. E-mail: mi1gamaj@uco.es a Departamento de Microbiolog´ ıa, Facultad de Ciencias, Universidad de C´ ordoba, Spain b INCAVI-IRTA, Estaci´ o de Viticultura i Enologia. Secci´ o d’Investigaci´ o Enol` ogica, Vilafranca del Pened` es (Barcelona), Spain c Departamento de Qu´ ımica Agr´ ıcola y Edafolog´ ıa, Facultad de Ciencias, Universidad de C´ ordoba, Spain J Chem Technol Biotechnol 2012; 87: 351–359 www.soci.org c  2011 Society of Chemical Industry