# Biotechnological Approaches to Lowering the Ethanol Yield during Wine Fermentation
## 4 Aerobic Fermentation
### S. cerevisiae Strains Improved for Aerobic Fermentation  
As mentioned above, one major drawback for the use of _S. cerevisiae_ under the aerobic growth conditions required for respiration was that strains of this species produced unacceptable acetic acid levels under these conditions. In order to overcome this problem, the researchers turned to other wine yeast species. Although this strategy is yielding some promising results (see the previous section), and though the use of non-_Saccharomyces_ species could bring some other benefits to the wine, it also hinders the fermentation management in the winery. The use of a single yeast starter that will at the same time be able to reduce alcohol levels, and drive fermentation to the end, while not increasing volatile acidity, would be an optimal alternative.  
A wine strain of _S. cerevisiae_ improved for lower acetic acid production was expected to be able to meet all of these requirements. Indeed, recombinant wine yeast strains partly defective in carbon catabolite repression (CCR) due to _REG1_ gene knockout were shown to produce reduced levels of acetic acid under anaerobic conditions [75]. Interestingly, _reg1_ mutant strains can grow on non-preferred carbon sources (glycerol or galactose, for example) in the presence of the non-metabolizable glucose analogue 2-deoxyglucose (2 DG). In contrast, strains with an intact CCR will be inhibited under these growth conditions. Although this link between CCR and acetic acid production under aerobic conditions is not yet clarified, it is possible to take a practical advantage of it to design experimental evolution strategies to develop _S. cerevisiae_ wine yeast strains which are better suited for alcohol level reduction. This was recently performed by [PERSON] et al. [76] using galactose as a carbon source in the presence of growing amounts of 2 DG for around 100 generations. Several evolved strains from different wine yeast genetic backgrounds were indeed derepressed for glucose, and had been indirectly selected for lower acetic acid production under aerobic conditions. As a trend, the glycerol yields were also increased for the selected evolved strains. However, some strains had to be discarded due to impaired fermentation kinetics. Indeed, there is always a risk of unwanted change during adaptive laboratory evolution. This must be managed by the careful design of experimental conditions and a thorough characterization of the derived strains before they can by promoted to industrial applications.  
On the other side, in the framework of the European project CoolWine (ERA CoBioTech), researchers are working on the development of rational strategies for adaptive laboratory evolution aiming at alcohol level reduction. For _S. cerevisiae_, the aim is to develop new strategies to reduce the above-mentioned problem of excess acetic acid production in the presence of oxygen, while for non-_Saccharomyces_ strains, the aim is to improve their survival and competitiveness in the context of winemaking.  
Although the general trend for _S. cerevisiae_ strains is the production of high volatile acidity under aerated wine fermentation conditions, we recently found rare isolates showing acceptable acetic acid yields under aerobic conditions [77]. These strains show promise to be used for alcohol level reduction by sugar respiration, without resourcing to non-_Saccharomyces_ yeast or the requirement for genetic improvement. However, genetic improvement will still be necessary to ensure enough genetic diversity for industrial applications (a single strain cannot fulfil all industry requirements), and to take advantage of the features of commercial strains that have previously shown optimal features for winemaking.