The present study uses a mathematical-empirical approach to estimate the cardinal growth temperature parameters (T min , the temperature below which growth is no longer observed; T opt , the temperature at which the max equals its optimal value; opt , the optimal value of max ; and T max , the temperature above which no growth occurs) of 27 yeast strains belonging to different Saccharomyces and non-Saccharomyces species. S. cerevisiae was the yeast best adapted to grow at high temperatures within the Saccharomyces genus, with the highest optimum (32.3°C) and maximum (45.4°C) growth temperatures. On the other hand, S. kudriavzevii and S. bayanus var. uvarum showed the lowest optimum (23.6 and 26.2°C) and maximum (36.8 and 38.4°C) growth temperatures, respectively, confirming that both species are more psychrophilic than S. cerevisiae. The remaining Saccharomyces species (S. paradoxus, S. mikatae, S. arboricolus, and S. cariocanus) showed intermediate responses. With respect to the minimum temperature which supported growth, this parameter ranged from 1.3 (S. cariocanus) to 4.3°C (S. kudriavzevii). We also tested whether these physiological traits were correlated with the phylogeny, which was accomplished by means of a statistical orthogram method. The analysis suggested that the most important shift in the adaptation to grow at higher temperatures occurred in the Saccharomyces genus after the divergence of the S. arboricolus, S. mikatae, S. cariocanus, S. paradoxus, and S. cerevisiae lineages from the S. kudriavzevii and S. bayanus var. uvarum lineages. Finally, our mathematical models suggest that temperature may also play an important role in the imposition of S. cerevisiae versus non-Saccharomyces species during wine fermentation.The estimation of the temperature range in which microorganisms are able to grow is very important for the food industry, to guarantee food safety or optimize fermentative conditions, for example, but also in ecological and taxonomic studies to classify and identify the different species of microorganisms. In this way, several works have shown the marked importance of temperature for the growth of industrial yeasts (1,4,22,24), as well as the influence of this environmental factor in determining the natural distribution of wild species (12,21,23,25). Specifically, there is an increasing interest in determining the influence of temperature in the adaptation of Saccharomyces species to both wild and fermentative environments (8,21,25).The Saccharomyces genus includes several species associated only with natural habitats (S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus, and S. arboricolus) and others that are present in both fermentative and wild habitats (S. cerevisiae and S. bayanus). The ability of the latter to ferment a broad range of beverages (cider, beer, and wines) and foods (bread, vegetables, etc.) (19) has unconsciously favored their selection by humans for thousands of years. It is thought that temperature could play an important role in the imposition and presence ...

In this work, we apply statistical modelling techniques to study the influence of increasing concentrations of ethanol on the overall growth of 29 yeast strains belonging to different Saccharomyces and non-Saccharomyces species. A modified Gompertz equation for decay was used to objectively estimate the noninhibitory concentration (NIC) and minimum inhibitory concentration (MIC) for the assayed strains to ethanol, which are related to the susceptibility and resistance of yeasts to this compound, respectively. A first ANOVA analysis, grouping strains as a function of their respective Saccharomyces species, revealed that S. cerevisiae was the yeast with the highest, and statistically significant, ethanol resistance value. Then, a second factorial ANOVA analysis, using the origin of strains (wild or fermentative) and their taxonomic classification (S. cerevisiae, S. paradoxus or S. bayanus var. uvarum) as categorical predictor variables, showed that no significant differences for the NIC and MIC parameters were found between both ecological niches within the same species, indicative that these physiological characteristics were presumably not modified throughout the adaptation to human-manipulated fermentative environments. Finally, differences among selected strains with respect to ethanol tolerance were correlated to the initial contents of unsaturated fatty acids, mainly oleic acid.

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The inoculation of active dry wine yeast (ADWY) is one of the most common practices in winemaking. This inoculation exposes the yeast cells to strong osmotic, acidic and thermal stresses, and adaptation to the new medium is crucial for successful fermentation. We have analysed the changes that occur in the ADWY protein profile in the first hours after inoculation under enological-like conditions at a low temperature. Protein changes mainly included enzymes of the nitrogen and carbon metabolism and proteins related to the cellular stress response. Most of the enzymes of the lower part of the glycolysis showed an increase in their concentration 4 and 24 h after inoculation, indicating an increase in glycolytic flux and in ATP production. However, the shift from respiration to fermentation was not immediate in the inoculation because some mitochondrial proteins involved in oxidative metabolism were induced in the first hours after inoculation. Inoculation in this fresh medium also reduced the cellular concentration of stress proteins produced during industrial production of the ADWY. The only exception was Cys3p, which might be involved in glutathione synthesis as a response to oxidative stress. A better understanding of the yeast stress response to rehydration and inoculation will lead to improvements in the handling efficiency of ADWY in winemaking and presumably to better control of fermentation startup.

The effect of the main environmental factors governing wine fermentation on the fitness of industrial yeast strains has barely received attention. In this study, we used the concept of fitness advantage to measure how increasing nitrogen concentrations (0 to 200 mg N/liter), ethanol (0 to 20%), and temperature (4 to 45°C) affects competition among four commercial wine yeast strains (PDM, ARM, RVA, and TTA). We used a mathematical approach to model the hypothetical time needed for the control strain (PDM) to out-compete the other three strains in a theoretical mixed population. The theoretical values obtained were subsequently verified by competitive mixed fermentations in both synthetic and natural musts, which showed a good fit between the theoretical and experimental data. Specifically, the data show that the increase in nitrogen concentration and temperature values improved the fitness advantage of the PDM strain, whereas the presence of ethanol significantly reduced its competitiveness. However, the RVA strain proved to be the most competitive yeast for the three enological parameters assayed. The study of the fitness of these industrial strains is of paramount interest for the wine industry, which uses them as starters of their fermentations. Here, we propose a very simple method to model the fitness advantage, which allows the prediction of the competitiveness of one strain with respect to different abiotic factors. Wine is the product of complex interactions among fungi, yeasts, and bacteria that commence in the vineyard and continue throughout the fermentation process until packaging. Alcoholic fermentation is the sugar transformation of must (glucose and fructose) into ethanol and CO 2 . This process is carried out exclusively by yeasts. During the first days of fermentation, the predominant genera are often Hanseniaspora (anamorph, Kloeckera) and Candida and, to a lesser extent, non-Saccharomyces species of the genera Torulaspora, Kluyveromyces, Hansenula, Pichia, Brettanomyces, Rhodotorula, and Metschnikowia, which are also isolated from freshly extracted grape must (1, 2, 3). During the consecutive days of fermentation, the populations of these lowfermentative-power species progressively decrease, and they are replaced by Saccharomyces cerevisiae, which is the species with the greatest fermentative capacity (3, 4). The ecological advantage of S. cerevisiae over its non-Saccharomyces competitors has been traditionally explained by this species' high tolerance to ethanol (5). Recently, Goddard (6) proposed that temperature may also play an important role in the ecological advantage of S. cerevisiae during wine fermentation. Fermentation temperature as a key factor in the competitive advantage of S. cerevisiae was later confirmed by Salvadó et al. (7), who individually studied the effects of temperature and ethanol on the growth of a S. cerevisiae strain and of various non-Saccharomyces yeasts isolated from wine fermentations.The wine fermentation process has been traditionally carried out spontaneously...

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This work was designed to identify yeast cellular functions specifically affected by the stress factors predominating during the early stages of wine fermentation, and genes required for optimal growth under these conditions. The main experimental method was quantitative fitness analysis by means of competition experiments in continuous culture of whole genome barcoded yeast knockout collections. This methodology allowed the identification of haploinsufficient genes, and homozygous deletions resulting in growth impairment in synthetic must. However, genes identified as haploproficient, or homozygous deletions resulting in fitness advantage, were of little predictive power concerning optimal growth in this medium. The relevance of these functions for enological performance of yeast was assessed in batch cultures with single strains. Previous studies addressing yeast adaptation to winemaking conditions by quantitative fitness analysis were not specifically focused on the proliferative stages. In some instances our results highlight the importance of genes not previously linked to winemaking. In other cases they are complementary to those reported in previous studies concerning, for example, the relevance of some genes involved in vacuolar, peroxisomal, or ribosomal functions. Our results indicate that adaptation to the quickly changing growth conditions during grape must fermentation require the function of different gene sets in different moments of the process. Transport processes and glucose signaling seem to be negatively affected by the stress factors encountered by yeast in synthetic must. Vacuolar activity is important for continued growth during the transition to stationary phase. Finally, reduced biogenesis of peroxisomes also seems to be advantageous. However, in contrast to what was described for later stages, reduced protein synthesis is not advantageous for the early (proliferative) stages of the fermentation process. Finally, we found adenine and lysine to be in short supply for yeast growth in some natural grape musts.

Fermentations carried out at low temperatures, that is, 10–15 °C, not only enhance the production and retention of flavor volatiles, but also increase the chances of slowing or arresting the process. In this study, we determined the transcriptional activity of 10 genes that were previously reported as induced by low temperatures and involved in cold adaptation, during fermentation with the commercial wine yeast strain QA23. Mutant and overexpressing strains of these genes were constructed in a haploid derivative of this strain to determine the importance of these genes in growth and fermentation at low temperature. In general, the deletion and overexpression of these genes did affect fermentation performance at low temperature. Most of the mutants were unable to complete fermentation, while overexpression of CSF1, HSP104, and TIR2 decreased the lag phase, increased the fermentation rate, and reached higher populations than that of the control strain. Another set of overexpressing strains were constructed by integrating copies of these genes in the delta regions of the commercial wine strain QA23. These new stable overexpressing strains again showed improved fermentation performance at low temperature, especially during the lag and exponential phases. Our results demonstrate the convenience of carrying out functional analysis in commercial strains and in an experimental set‐up close to industrial conditions.