Brazil is currently responsible for about 33 % of the bioethanol produced worldwide and can play an important role in satisfying future increase in bioethanol demand (Goldman and Buckeridge 2011; Leite et al. 2009). Nowadays, there are more than 400 plants in operation crushing 625 million tons of sugarcane per year, approximately one-half being used for sugar and the other half for bioethanol production (Conab 2011). In 2010, approximately 27.7 billion liters of bioethanol were produced using 8.0 million hectares of land (Conab 2011). The efficiency of sugarcane-to-ethanol production can still be increased through improvements in the agricultural and industrial phases of the production process (Goldemberg 2007; Goldemberg 2008; Leite et al. 2009). Sugarcane bagasse contains one-third of the energy in the sugarcane, and is the current source of all of the energy needed in the bioethanol mills. The other two-thirds are split between sucrose and the tops and leaves (Goldemberg 2008). Presently the production of bioethanol in Brazil relies exclusively on first generation technologies that are based on the utilization of the sucrose content of sugarcane that is efficiently converted directly to bioethanol by the yeast Saccharomyces cerevisiae. This fermentation process works with cultures of very high yeast cell densities in a semicontinuous fed-batch mode to ferment broths (cane juice and/or diluted molasses) containing up to 150-200 g/L of total sugar, producing high ethanol concentrations (9-12 % v/v) with high yield (90-92 % of the theoretical maximum) and productivity (each fermentation cycle lasts 6-10 h, allowing two to three fermentations per day). By the end of each fermentation cycle, the cells are harvested by centrifugation, washed in diluted sulfuric acid, and then reused for another fermentation cycle (Basso et al. 2008; Wheals et al. 1999). This routine happens during the whole crop season for about 6 to 9 months of the year. The bioethanol fermentation process performed in Brazil is quite unusual because yeast cells are intensively recycled (Basso et al. 2008). Since high cell densities are used, it is possible to obtain the desired product in a very short fermentation time.
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The industrial S. cerevisiae strains responsible for the efficient production of bioethanol were selected based on the pioneering studies of the dynamics of microbial populations into industrial fermenters (Basso et al. 2008, Silva-Filho et al. 2005). These studies showed the existence of a succession of yeast strains during bioethanol fermentations; the original "starter'' yeast (most often commercial baker's yeast strains) is completely replaced by other strains in a few weeks. The dominant S. cerevisiae strains were able to replace the inoculated populations and take control of the fermentation process during the entire production season. These strains were isolated and some of them, such as CAT-1 and PE-2, became the most used commercial strains for bioethanol production available in Brazil. Around 60 % of all Brazilian distilleries start the fermentation process with selected yeast strains and 80 % of these industries have been using S. cerevisiae PE-2 and CAT-1 strains for bioethanol production. Although they are extensively used in the bioethanol production system, very little is known about the biological properties that make them superior strains for industrial bioethanol production.
As above mentioned, Brazilian processes of alcoholic fermentation have particular characteristics. For each fermentative cycle, we can identify distinct phases such as sugarcane must feedingÂ and fermentation, centrifugation of yeast cells at the end of fermentation and treatment of yeast cells with diluted sulfuricÂ acid (Basso et al., 2008). After acid treatment the yeast cells return to fermenters to start a new fermentation cycle. During the must feeding yeast cells sense the sugar in the medium and drive their metabolism to ethanol production. After the end of must feeding (3 hours), the content of sugars drops, the pH decreases and the density of fermentation medium changes due to the increasing concentrations of ethanol. The ethanol concentration reaches the maximum around 9 hours. After the end of fermentation, the yeast cells are centrifuged to separate the cells from the wine.Â While the wine goes to the distillation step, the concentrated yeast cells go to acid treatment. These cells are submitted to a new environment without sugar and very low pH before returning to the fermentation tanks. Then, there are at least three distinct environments: (i) low sugar and low ethanol during the acid treatment step, (ii) high sugar and low ethanol (during the feeding step, 3 hours) and finally, (iii) low sugar and high ethanolÂ at the end of fermentationÂ (9 hours). Our major interest is to understand how the yeast cells respond to must feeding and at the end of fermentation when compared to acid treatment step. This will surely provide information about which biological properties at population levels keep these cells productive during the whole fermentation season. Thus, as a preliminary step to understand it, we performed microarray hybridization analyses for the two most commonly used S. cerevisiae strains for bioethanol production in Brazil, PE-2 and CAT-1. The goal was to understand which genes are modulated during bioethanol fermentation on sugarcane substrates. We mimicked at small scale the same conditions traditionally used for bioethanol fermentation in the Brazilian fermentation units and performed microarray hybridization analysis for these strains. We observed that S. cerevisiae PE-2 and CAT-1 strains have increased mRNA accumulation of genes involved in oxidative stress responses and cell wall integrity pathway. Accordingly, these industrial strains have increased tolerance to several stressing conditions that either increase the reactive oxygen species (ROS) into the cell or affect the cell wall integrity (CWI) pathway. In addition, we showed increased activation of the unfolded protein response (UPR) in these strains.
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