# Biotechnological Approaches to Lowering the Ethanol Yield during Wine Fermentation
## 4 Aerobic Fermentation  
### Non-Saccharomyces Species  
Glycerol has been the target in many attempts to reduce ethanol yields by genetic improvement (see above). However, reducing the alcohol content by 1% (\(v/v\)), by diverting all excess carbon to glycerol, can result in more than 15 g/L extra glycerol in the wine. This sets a technological limit to this strategy, as a further increase in the glycerol yield would lead to values well above the commercially acceptable levels. However, almost every alternative carbon sink can have a detrimental sensory impact if produced in excess, even for compounds with positive sensory attributes. In this context, some authors, including our research group [69; 70], proposed CO\({}_{2}\) generated by the aerobic respiration of yeast as a neutral carbon sink. By respiration, all of the carbon present in sugar is converted to CO\({}_{2}\), in a process that requires molecular oxygen. However, _S. cerevisiae_ is a Crabtree-positive yeast. This implies that, except under very small concentrations of glucose, it will mostly ferment sugars and produce ethanol, even in the presence of oxygen. Therefore, the proposal was made to perform a multi-starter fermentation with _S. cerevisiae_ and alternative yeasts with higher respiration capacities [70]. The appropriate yeast would be inoculated into the must, and during the first hours, oxygen would be provided to the fermentation tank. After some time, the forced aeration would be stopped, and the traditional process would follow. _S. cerevisiae_ could be inoculated from the beginning (co-inoculation), or when the aeration is stopped (sequential inoculation). The more sugar is respired during the aeration process, the lower the ethanol yield of the overall process will be. The availability of suitable yeast strains was a prerequisite to reach this goal. To be useful, these strains must be able to respire in the stressful conditions of grape must, with no negative impact on the wine sensory properties. In a screening of 65 strains from 28 yeast species, [PERSON] et al. (2014) [71] found an interesting result for some _S. cerevisiae_ strains. Despite the Crabtree effect, the residual respiration activity of some of them was enough to allow for a substantial reduction in the ethanol yield according to the measured respiration quotient (RQ) values. However, _S. cerevisiae_ strains showed a boost in their acetic acid production when provided with air. In contrast, several non-_Saccharomyces_ strains showed a combination of metabolic features suggesting that they might be suited for use in the reduction of alcohol levels. Namely, some strains showed a moderate-to-high sugar consumption rate, very low acetic acid production, and a low RQ (which means a low expected ethanol yield). Indeed, one strain of _M. pulcherrima_ was used in a co-inoculation with _S. cerevisiae_ in laboratory-scale bioreactors for the fermentation of natural grape must (260 g/L sugar content). Several inoculation and aeration rates were assayed. The alcohol level reduction reached 3.6% (\(\varepsilon/\varepsilon\)) with a somehow too-high volatile acidity, or 2.2% (\(\varepsilon/v\)) with acceptable volatile acidity levels [43]. Some environmental factors that can be controlled during wine fermentation--such as nutrients, temperature, and dissolved oxygen levels--will certainly affect both the ethanol and acetic acid yield by different yeast strains. Interestingly, strains of _M. pulcherrima_ and _Candida sake_ show low acetic acid production almost independently of the fermentation conditions [72]. Fermentation conditions leading to lower ethanol yields based on the use of non-_Saccharomyces_ strains under aerobic conditions have been studied by these and other authors, using _T. delbrueckii_, _M. pulcherrima_, _Zyososacharoumyes bailii_, _C. zemplinina_, _M. pulcherrima_, _Pichia guililermundii_, _Pichia kluyveri_, _Candida oleophila_, _Starmerella bombicola_, or _Hanseni'spora vineae_[44; 45; 48; 51; 57; 73; 74].