Plasmid-mediate transfer of FLO1 into industrial Saccharomyces cerevisiae PE-2 strain creates a strain useful for repeat-batch fermentations involving flocculation–sedimentation Daniel G. Gomes, Pedro M.R. Guimarães, Francisco B. Pereira, José A. Teixeira, Lucília Domingues ⇑ IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal article info Article history: Received 19 July 2011 Received in revised form 4 November 2011 Accepted 16 December 2011 Available online 24 December 2011 Keywords: Yeast flocculation VHG bio-ethanol production Repeat-batch operation Industrial Saccharomyces cerevisiae strains abstract The flocculation gene FLO1 was transferred into the robust industrial strain Saccharomyces cerevisiae PE-2 by the lithium acetate method. The recombinant strain showed a fermentation performance similar to that of the parental strain. In 10 repeat-batch cultivations in VHG medium with 345 g glucose/L and cell recycling by flocculation–sedimentation, an average final ethanol concentration of 142 g/L and an ethanol productivity of 2.86 g/L/h were achieved. Due to the flocculent nature of the recombinant strain it is pos- sible to reduce the ethanol production cost because of lower centrifugation and distillation costs. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Bio-ethanol fermentation can be improved by media and pro- cess optimization and selection of robust microorganisms (Pereira et al., 2010a,b). One of the process strategies currently applied in the fermentation industry is very high gravity (VHG) technology. Compared to conventional fermentations, this approach decreases process water requirements and increases overall plant productiv- ity and final ethanol concentrations (usually above 15%, v/v), and thus allows considerable savings in energy for distillation (Li et al., 2009). However, high osmotic stress and ethanol inhibition affect the microorganism and result in incomplete fermentations. Saccharomyces cerevisiae strains are the most frequently used microorganisms for large-scale industrial bio-ethanol fermenta- tions due to their ability to ferment a wide range of sugars and to deal with the harmful VHG stresses (Bai et al., 2008). S. cerevisiae CA1185 and PE-2 are industrial strains exhibiting high fermenta- tion performance and stress tolerance under VHG conditions (Pereira et al., 2011). PE-2, isolated from a Brazilian sugar cane-to-ethanol distillery and now intensively used for bio-ethanol production in Brazil, consumed 330 g glucose/L achieving 19.2% (v/ v) ethanol, using a VHG optimized medium (Pereira et al., 2010a). The application of a repeat-batch system with yeast recycling achieves high ethanol productivities (Choi et al., 2009; Li et al., 2009; Ma et al., 2009); however, the need for separating the fer- mented medium from cells at the end of each batch by filtration or centrifugation (Bai et al., 2008; Sakurai et al., 2000) makes the process expensive. A yeast flocculation–sedimentation process would appear to be able to reduce the cost of the repeat-batch pro- cess (Li et al., 2009). Flocculation is already utilized in a variety of applications such as winemaking (Govender et al., 2010), brewing (Van Mulders et al., 2010) or wastewater treatment (Liu et al., 2009). Wang et al. (2008) transferred a vector harboring the FLO1 gene into S. cerevisiae and Verstrepen et al. (2001) and Zhao et al. (in press) integrated the gene into the yeast’s genome to introduce a floccu- lation phenotype. FLO1 is the most studied gene related to floccu- lation (Russell et al., 1980). It encodes a large flocculation protein rich in Ser/Thr amino acids (Watari et al., 1994) and when the pro- tein is incorporated into the cell wall, its N-terminal part has the capacity to selectively bind mannose-sugars present on the wall of other cells (Bidard et al., 1995). The binding efficiency depends on several factors such as pH, temperature or the amount of spe- cific components (e.g. cations, sugars, oxygen, ethanol) (Soares, 2011). In the present work, FLO1-mediate flocculation capacity was introduced into the robust industrial strain, S. cerevisiae PE-2, and the recombinant strain was exploited in a VHG repeat-batch system. 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.12.089 ⇑ Corresponding author. Tel.: +351 253 604402; fax: +351 253 678986. E-mail addresses: dgg.ebiol@gmail.com (D.G. Gomes), pmrguimaraes@gmail. com (P.M.R. Guimarães), franciscopereira@deb.uminho.pt (F.B. Pereira), jateixeir a@deb.uminho.pt (J.A. Teixeira), luciliad@deb.uminho.pt (L. Domingues). Bioresource Technology 108 (2012) 162–168 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech