**1. Introduction**

After carbon dioxide (CO2), ethanol and glycerol are the most abundant compounds produced during alcoholic fermentation. The levels of ethanol and glycerol in wine depend upon many factors, such as seasonal events affecting the concentration of grape sugar, and winemaking decisions, including fermentation conditions and fermenting yeasts [1].

At a commercial scale, inoculations with *Saccharomyces cerevisiae* strains are often preferred over those with non-*Saccharomyces* or *S.* non-*cerevisiae* yeasts, because the latter is considered responsible for incomplete fermentations (and consequently high levels of residual sugar in wine), and they produce high concentrations of acetic acid and ethyl acetate [2,3]. Nevertheless, non-*Saccharomyces* or *S.* non-*cerevisiae* yeasts are important to winemakers, particularly to those who target wines with unique sensory characters that are popularly recognised as typical of their geographical origin or variety [4–7]. These yeasts are also popular among winemakers who choose to produce less alcoholic wines [8].

Although non-*Saccharomyces* and *S.* non-*cerevisiae* yeasts are sought after for their specific oenological characteristics, it is a challenge for some of these yeasts to conduct a complete fermentation

to a desired level of dryness. This is very important to winemakers, in part because finished wines with higher levels of residual sugars above 0.5 g/<sup>L</sup> require high doses of sulfur dioxide (SO2) to ensure their microbial stability to prevent wine spoilage. Therefore, inoculations of non-*Saccharomyces*/*S.* non-*cerevisiae* in mixed cultures with *S. cerevisiae* strains, which have higher fermentation rates, have been studied to ensure complete fermentation.

The strategy of using selected mixed cultures for alcoholic fermentation is believed to be the key to produce wines with desirable characteristics that meet changing market demands with less ethanol but still with flavours comparable to standard wines [9]. This strategy is carried out by two different methods of inoculation: (1) co-inoculation, which involves concurrent inoculations of non-*Saccharomyces*/*S.* non-*cerevisiae* yeasts at high cell concentration (e.g., 10<sup>7</sup> cell/mL) with *S. cerevisiae*; and/or (2) sequential inoculation, which involves inoculating non-*Saccharomyces*/*S.* non-*cerevisiae* yeasts to start the fermentation and continue for a determined amount of time alone, and inoculating *S. cerevisiae* to take over and complete the fermentation [9,10]. The time period before carrying out the sequential *S. cerevisiae* inoculation and the *Saccharomyces*/non-*Saccharomyces* or *Saccharomyces* non-*cerevisiae* inoculum ratio are both important parameters that affect the fermentation kinetics and oenological outcomes, and the former generally varies between 1 and 3 days [11–14].

Many reviews have studied different perspectives of non-*Saccharomyces*/*S.* non-*cerevisiae* yeasts for modern winemaking practices, including their influence on different wine quality parameters with an emphasis on traits such as the primary (or varietal) and secondary (or fermentative) aromas of wines, acidity, freshness, as well as specific styles of wines (such as traditional method sparkling wines and red table wines) [3,9,15–21].

The aim of this review is to highlight those studies that have shown a direct link between the use of non-*Saccharomyces* or *S.* non-*cerevisiae* and the concentration of ethanol and glycerol in wine or synthetic media. The first part of this review provides an overview of ethanol and glycerol as contributors to wine sensory characteristics, and a general overview of non-*S. cerevisiae* or *S.* non-*cerevisiae* yeasts. The second part of this review provides more specific details of individual non-*S. cerevisiae* or *S.* non-*cerevisiae* species that are relevant to the wine industry. We conclude this review by suggesting what additional research might help winemakers have greater control over wine quality outcomes.

## **2. Ethanol Reduction**

Ethanol is produced by yeas<sup>t</sup> during the alcoholic fermentation and is generally found in the range of 11.5–15% *v*/*v* in wines. It is an important wine component that directly effects organoleptic properties, aging, and wine stability [22]. The impact of ethanol on the sensory profile of wines and other alcoholic beverages has been recently reviewed [23]. Ethanol influences taste and mouthfeel sensations, alters the sensation of sweetness, increases bitterness, decreases sourness, and contributes to the hotness sensation and body of the wine [24–27]. Ethanol can also decrease the volatility of aroma compounds by increasing their solubility in the wine [28], making small compounds such as fruity-driven ethyl esters and acetates less recognisable by human senses.

According to the International Organisation of Vine and Wine (OIV), the alcohol strength of wines must be a minimum of 8.5% *<sup>v</sup>*/*<sup>v</sup>*, although in cool climate wine regions, this value can be lowered to 7% *v*/*v* [29]. Over the past two decades, ethanol content in wines has been noticeably increasing in some regions by 0.1–1% per year [30,31]. Apart from hotter climates leading to higher sugar berry levels at harvest and therefore, higher alcohol contents in wine [32], one of the main reasons behind this progressive increase is consumer demand for specific wine styles, which are described as rich, well-structured, with a flavour profile dominated by dark, ripe fruits [33]. This style requires optimal grape maturity and higher sugar content of 240 g/<sup>L</sup> or more [34].

Nonetheless, an increasing trend for reduced alcohol in beverages (broadly defined as containing 9% to 13% *v*/*v* ethanol), and low-alcohol (0.5–2% *v*/*v*) wines by consumers has been recently observed [35,36]. Increasing health and safety consciousness and global initiatives towards moderating alcohol consumption are reasons for producing lower alcohol wines that appeal to wine drinkers [37]. Wines may also be subjected to higher taxation depending on their alcohol content, which increases the final cost of wines to the consumer [38]. Since ethanol is the main source of caloric content in wine, there is also a risk of a negative impact on wine export to countries where health labeling of foods and beverages served at restaurants is voluntary or mandatory [39].

During winemaking, high sugar and therefore ethanol can cause sluggish and stuck alcoholic fermentations and can be challenging for successful malolactic fermentations [40,41]. As a means to address these issues, methods have been studied that include lowering the final ethanol content of wine using a wide selection of interventions. These can be grouped into (1) pre- (e.g., viticultural, juice dilution, and fermentation of early harvest fruit); (2) concurrent (e.g., non-*Saccharomyces* yeasts, modified yeasts, and arrested fermentation) [42,43]; and (3) post-fermentation (e.g., non-membrane and membrane ethanol removal) [43–45]. The use of microbiological approaches such as inoculation with non-*Saccharomyces* yeasts for producing wines with less ethanol is a promising alternative to the removal of ethanol by membrane based-approaches [11,46–48]. The advantages associated with the use of low-ethanol/high-glycerol yielding yeasts include their relatively easy application and lower costs when compared to more expensive and less eco-friendly approaches, such as membrane contactors, nanofiltration, or the spinning cone column [49,50]. However, it is important to acknowledge that low-ethanol/high-glycerol yielding yeasts are much less e ffective than the membrane-based processes in terms of ethanol reductions [44], and they are perhaps only suitable when winemakers want to achieve a reduction in ethanol content by up to 3.0% *v*/*v* [49].

Inoculations with non-*Saccharomyces* yeasts can be used as a strategy to produce lower alcohol wines due to the yeasts di fferent sugar utilisation pathways, including respiration, alcoholic fermentation, and glycerol–pyruvic metabolisms, and di fferent regulatory mechanisms, in comparison to *S. cerevisiae* [51]. While the theoretical sugar-to-ethanol yield for a complete fermentation by *S. cerevisiae* generally ranges from 90% to 95%, the residual sugar is consumed via alternative metabolic pathways and biomass biosynthesis [52]. On the other hand, ethanol yield and the by-products formed vary immensely amongs<sup>t</sup> non-*Saccharomyces* yeasts [23]. For example, due to the Crabtree e ffect, *S. cerevisiae* prefers fermentation metabolism rather than respiration when the sugar amount exceeds 10 g/<sup>L</sup> [40]. In contrast, among non-*Saccharomyces* yeasts, there are strains and species that can consume sugar with aerobic respiration regardless of sugar concentration [53,54] without contributing significantly to the final ethanol level of the wine. Therefore, non-*Saccharomyces* yeasts have been studied under partial and controlled aeration strategies during fermentation to achieve lower ethanol by allowing part of the sugar to be consumed via respiration rather than alcoholic fermentation [47,55]. However, an increase in undesirable volatile compounds, such as acetic acid and ethyl acetate, are the main limiting factors of the aeration strategies that require the application of a proper aeration regime [55–57].

As a response to interest in reduced alcohol levels in wines, researchers studied non-*S. cerevisiae* and *S.* non-*cerevisiae* yeas<sup>t</sup> species [49,56,58–60] (Table 1). Several non-*Saccharomyces* yeas<sup>t</sup> strains were identified, including *Metschnikowia pulcherrima,* and two species of *Kluyveromyces,* which have the capacity to decrease ethanol yields by respiration [59]. *M. pulcherrima* AWRI 1149 was identified as a potential yeas<sup>t</sup> to produce wine with a reduced ethanol concentration, having been identified following the evaluation of 50 non-*Saccharomyces* isolates under limited aeration conditions, and in sequential inoculations with *S. cerevisiae* [49]. A similar study with 48 non-*Saccharomyces* yeas<sup>t</sup> isolates identified *Torulaspora delbrueckii* AWRI 1152 and *Zygosaccharomyces bailii* AWRI 1578 yeasts as suitable for reducing ethanol [56]. More recently, the respiratory, fermentative, and physiological characteristics of 114 non-*Saccharomyces* yeasts were evaluated [60]. Taking into account their ability to reduce ethanol content *Hanseniaspora uvarum* BHu9 and BHu11, *Hanseniaspora osmophila* BHo51, *Starmerella bacillaris* (synonym. *Candida zemplinina*) BSb55, and *Candida membranaefaciens* BCm71 were selected as candidates for co-fermentations.
