Wine composition is the product of complex interactions among yeast and bacteria that take place in vineyards and wineries, although one yeast species, Saccharomyces cerevisiae, is generally the main microorganism responsible for winemaking process (Pretorius, 2000). Its vigorous fermentative capacity, even in the presence of oxygen (Crabtree effect), makes S. cerevisiae a very efficient ethanol producer, strategy that allows its imposition over the rest of the microbiota during fermentation due to the toxicity of this compound (Thomson et al., 2005; Piškur et al., 2006).

However, this high ethanol production capability may be disadvantageous taking into account the challenges currently faced by the wine industry. In the first place, global warming provokes a gap during grape ripening between phenolic maturity and sugar content. If grapes are harvested when the sugar content is appropriate but the phenolic maturity has not been reached, wines can show altered aroma, flavor, mouth feel, and astringency. On the contrary, if grapes are harvested when their phenolic maturity is the appropriate, their sugar contents are higher, giving rise to wines with increasing ethanol concentrations (Jones et al., 2005). This higher ethanol content is undesirable according to consumers' new demands, because affects flavor complexity sensing (Goldner et al., 2009), and its excessive consumption is harmful for health and road safety.

A variety of measures are taken at the different winemaking stages to overcome the problem of the higher ethanol levels in wines. These include new agronomical methods for grape cultivation (Intrigliolo and Castel, 2009), the use of mixed musts from grapes at different ripening stages (Kontoudakis et al., 2011), the use of engineered yeasts producing lower ethanol yields (Varela et al., 2012), or the partial dealcoholisation of wines by chemical or physical procedures (Gómez-Plaza et al., 1999; Pilipovik and Riverol, 2005; Diban et al., 2008; Hernández et al., 2010; Offeman et al., 2010; Belisario-Sánchez et al., 2012). However, some of these approaches have little impact on ethanol contents, negatively affect the quality of wine, are highly expensive industrial processes, or contravene the current regulations about the use of GMO.

In addition, a wide range of different biological strategies have been proposed to reduce alcohol contents in wines (Kutyna et al., 2010). The use of non-conventional yeast strains in winemaking stands out for its potential. Several non-Saccharomyces yeasts, usually in combination with S. cerevisiae, have been tested to reduce ethanol yields during wine fermentation (Comitini et al., 2011; Sadoudi et al., 2012; Contreras et al., 2014, 2015; Quirós et al., 2014; Ciani et al., 2016). Different strategies have been carried out to improve the fermentation performance of these non-Saccharomyces yeasts, such as, sequential inoculation or co-inoculation at increased proportions with S. cerevisiae, to provide new characteristics to the final wines (Andorrà et al., 2012; Gobbi et al., 2013; Izquierdo Cañas et al., 2014; Jolly et al., 2014; Loira et al., 2014; Canonico et al., 2016). Another approach to reduce alcohol content in wines is the supply of oxygen to the fermenters, under a controlled flowrate, to promote the respiratory consumption of sugars by these non-Saccharomyces yeasts (Gonzalez et al., 2013; Rodrigues et al., 2016). However, temperature under industrial winemaking conditions is generally close to 25°C, which does not allow for any of these alternative yeasts to survive the first hours of the process (Nissen and Arneborg, 2003; Torija, 2003; Pérez-Nevado et al., 2006; Williams et al., 2015).

Alternative Saccharomyces yeasts, such as, Saccharomyces kudriavzevii or S. uvarum, can help to solve some of the new challenges of the wine industry. These species exhibit physiological properties that are especially relevant during the winemaking process, such as, their good fermentative capabilities at low temperatures, resulting in wines with lower alcohol and higher glycerol amounts (Varela et al., 2016; Pérez-Torrado et al., 2017a). In the case of S. kudriavzevii, this species displays a different metabolic regulation concerning ethanol and glycerol syntheses (Arroyo-López et al., 2010; Oliveira et al., 2014; Pérez-Torrado et al., 2016). Moreover, it recently showed an ethanol reducing capability in mixed fermentation with S. cerevisiae at low temperatures (Alonso-del-Real et al., 2017). Again, temperature appears as the most important factor to determine the preponderance of S. cerevisiae during wine fermentation (Nissen and Arneborg, 2003; Torija, 2003; Pérez-Nevado et al., 2006; Arroyo-López et al., 2011; Salvado et al., 2011; Williams et al., 2015; Alonso-del-Real et al., 2017).

However, none of the techniques used to favor the growth of non-Saccharomyces yeasts, such as, co-inoculation, sequential inoculation, or microoxigenation, have been applied to S. kudriavzevii species to favor their presence during wine fermentation. In this work, we first analyzed the presence of S. kudriavzevii during co-fermentation with a S. cerevisiae wine strain under different aeration conditions to select the most suitable one. Next, we studied the effect of S. kudriavzevii enrichment in the inoculum with and without external oxygen supply, and finally the effect of sequential inoculation of the strains.

In the last century, alcohol abuse became considered as one of the most important health problems in the world, and promoted new behavioral strategies against alcohol consumption. In addition, because of global warming, in wine-growing regions with a Mediterranean climate there is excessive ripening of the grape, which produces musts with a higher concentration of sugars (Jones et al., 2005), and hence, higher alcohol yields, implying a higher tax burden, which makes wines less competitive, and a rejection by the consumer for health reasons, road safety, etc.

Therefore, wine industry must respond to these challenges posed both by new consumer demands and by changes in the composition and properties of the grape must due to climate change. These demands have a significant impact on the quality and acceptance of the final wines and require improvements in the enological practices, among which the development of new yeast starters exhibiting lower ethanol yields during wine fermentation is of chief importance.

Different approaches in the use of yeast starters have been proposed to reduce alcohol contents in wines (Schmidtke et al., 2012; Varela et al., 2015). They include controlled aeration, starter strain proportion adjustment, or inoculation of dominant yeast species after a non-Saccharomyces yeast of interest (Comitini et al., 2011; Sadoudi et al., 2012; Contreras et al., 2014, 2015; Quirós et al., 2014; Ciani et al., 2016). In the present study, we adapted these strategies to foster a Saccharomyces non-cerevisiae strain (S. kudriavzevii CR85) presence in synthetic must fermentation. This yeast had been proved to foster decreased ethanol content, and also to increase fermentation kinetics and glycerol concentration in a 1:1 inoculum proportion with S. cerevisiae under low temperatures conditions. In contrast, this effect was not found under regular red winemaking temperatures (Alonso-del-Real et al., 2017), probably due to some of the already proposed competition mechanisms, such as, antimicrobial GAPDH-derived peptides produced by S. cerevisiae (Branco et al., 2016), lower sulfite tolerance and efflux capacity (Pérez-Torrado et al., 2017b), or early nutrient depletion by S. cerevisiae (Fleet, 2003). However, the results reported in the present work show that S. kudriavzevii presence during an important period of the fermentation was achieved at regular industrial temperatures.

Although S. kudriavzevii and S. cerevisiae show long-term Crabtree effect, the carbon flux ratio between respiration and fermentation under aerobic conditions seem to be slightly higher in S. kudriavzevii CR85 compared to S. cerevisiae T73 (our unpublished data). Thus, an external oxygen supply to a fermentation co-inoculated with these two yeast species may benefit S. kudriavzevii growth. Nevertheless, high oxygen levels can deteriorate important compounds of must, originating undesired metabolites correlated to respiration such as acetic acid (Salmon, 2006). Therefore, a fine tuning of the amount of oxygen introduced into the system seems to be critical for the final wine quality. A wide range of airflow rates, from 2.4 to 60 VVH have been used at laboratory scale (Vilanova et al., 2007; Shekhawat et al., 2016). Nevertheless, an air flow rate of 20 VVH has been showed to be on the top limit for acetic acid production when applied to S. cerevisiae microvinification (Morales et al., 2015), therefore the screening for the most suitable condition was performed always below this value.

S. kudriavzevii performance under air supply conditions was observed to improve its competitive fitness against S. cerevisiae (Arroyo-López et al., 2011; Alonso-del-Real et al., 2017). Our results suggest, though, that despite maintaining an air supply during the whole fermentation, after 48 h, S. kudriavzevii was outcompeted by S. cerevisiae. This, together with the fact that an aerobic environment produces a higher acetic acid accumulation up to 70%, led us to reduce aeration just for the first 48 h of fermentation for the successive experiments. Nevertheless, it is noteworthy that, as observed by Moruno et al. (1993) and later confirmed by Beltrán et al. (2008), synthetic and natural musts have different impact on the final product composition, acetic acid levels are much higher for synthetic must, as can also be observed for our aerated conditions. Thus, due to laboratory experimental conditions, acetic acid values obtained in the present work are high even for non-aerated synthetic must fermentations performed with the S. cerevisiae wine strain, compared to natural must fermentation under industrial conditions (0.35 g/L). Therefore, acetic acid levels produced during fermentations with air supply could still be under the limits of regulation (~1 g/L) and consumers' acceptance when tested at industrial scale.

Despite the acetic acid increase, ethanol reduction is notable for the aerated fermentations, in concordance with previous studies (Morales et al., 2015; Shekhawat et al., 2016), and similar to ethanol reductions obtained in other works in which similar co-inoculation strategies with non-Saccharomyces yeasts have been followed (Contreras et al., 2015; Ciani et al., 2016; Englezos et al., 2016). However, this is the first study in which S. kudriavzevii was used to reduce ethanol yields, which, together with a recent study on the sequential inoculation of S. uvarum and S. cerevisiae (Varela et al., 2016), opens new approaches to the use of other Saccharomyces species. These species, in addition to their ethanol metabolic characteristics, also provide richer aroma profiles to wine (Stribny et al., 2015).

The analysis of the non-aerated fermentations also showed a slight ethanol yield reductions clearly correlated with the S. kudriavzevii proportions during the fermentation process under the different assayed conditions. Moreover, there also is a clear direct correlation between S. kudriavzevii proportions and glycerol production, another desirable enological characteristic of importance for wine quality because it contributes to wine body and astringency masking (Jolly et al., 2014). Glycerol and ethanol metabolism has been proven to differ in S. kudriavzevii with respect to S. cerevisiae (Arroyo-López et al., 2010; Pérez-Torrado et al., 2016). In fact, cryotolerant Saccharomyces species, such as, S. kudriavzevii and S. uvarum, have been proven to produce wines and ciders with higher glycerol contents than S. cerevisiae (Bertolini et al., 1996; Masneuf-Pomarède et al., 2010; Peris et al., 2016; González Flores et al., 2017), so their use could be of great interest for wine industry.

Among the strategies followed to favor S. kudriavzevii growth against S. cerevisiae, the co-inoculation with a proportion of S. cerevisiae lower than 10% and the sequential inoculation showed the more promising results. Air supply showed a synergistic effect in proportion S. cerevisiae/S. kudriavzevii 1:9, whereas it did not have a significant impact on the rest of the assayed inoculum proportions. These results agree with the fact that S. cerevisiae is better adapted to anaerobic conditions such as, wine fermentation, and air supply produces an imbalance in this environment, which promotes S. kudriavzevii survival. According to our results, it also seems feasible that a certain threshold in S. cerevisiae cell density is necessary to trigger S. kudriavzevii lack of viability. This also agrees with the previous observations indicating that the viability of a competitor strain is affected by its interaction with S. cerevisiae due to cell-to-cell contacts (Nissen et al., 2003; Arneborg et al., 2005; Branco et al., 2016; Pérez-Torrado et al., 2017b), or by microenvironment modifications produced by S. cerevisiae (Goddard, 2008). A rise in temperature due to the higher fermentative rate of S. cerevisiae (Goddard, 2008) can affect S. kudriavzevii viability (Arroyo-López et al., 2011).

In summary, the most promising results were obtained from the combination of different strategies for promoting S. kudriavzevii prevalence during wine fermentation, such as, co-inoculation with a low proportion of S. cerevisiae (<10%) or sequential inoculation together with limited aeration, resulting in an ethanol yield reduction as well as a higher glycerol production. Aeration requires costly additional technology, but it is already implemented in the wine industry (Vivas and Glories, 1996; Vidal and Aagaard, 2008) to improve wine quality by accelerating the transformations of phenols reducing the astringency.

Finally, these results have to be confirmed in real grape must to evaluate not only the effect of aeration on yeast physiology but also a potential effect on sensory profile. In addition, lower aeration rates can also be tested at industrial scale, particularly for S. cerevisiae/S. kudriavzevii proportions lower than 1:9. In addition a deeper understanding of the interactions among Saccharomyces yeasts, are also needed in order to finely tune the optimal use of these tools to reduce ethanol contents in wine.

JA, GC, EB, and AQ conceived and designed the experiments. JA, AC, and GC performed the experiments. JA, EB, and AQ analyzed the data and wrote the paper.