Acidity in wine can be defined by two main parameters: the pH and the Titratable Acidity (TA) (Ribereau-Gayon et al., 2006b). Broadly, pH is defined by the expression: pH = log 1/[H⁺] = - log[H⁺]; the pH of a wine is the measure of free protons concentration in the solution, calculated as pH = - log[H⁺], while TA refers to the concentration of titratable H₃O⁺ ions in wine. In hydroalcoholic solutions like wine, weak organic acids are partially dissociated, and their dissociation degree is represented by their pKa. The lower the pKa, the stronger the dissociation and in turn the concentration of H₃O⁺ ions in the solution. Typical values for white wine are a pH of 3.0–3.4 and a TA of 6–9 g/L as tartaric acid, and for red wine a pH of 3.3–3.7 and a TA of 5–8 g/L as tartaric acid (Waterhouse et al., 2016). The TA is a good proxy for the perceived sourness in wine, while pH is weakly correlated with sourness perception (Plane et al., 1980). In practice, the goals of achieving low wine pH and of avoiding excessively high TA (and thus sourness) often compete with each other. In addition to pH and TA, another parameter of oenological importance is the buffer capacity. The buffer capacity can be defined as the ability of a solution to maintain a stable pH upon addition of a strong acid or base. This property is directly correlated with the concentration of weak acids and their conjugate bases. Consequently, wines with higher TA have a higher buffering capacity.

The main organic acids that contribute to wine TA are presented in Table 1. Two of them, tartaric acid and malic acid, contribute to up to 90 % of the titratable acidity of grape juices and wines. Tartaric acid is present in some fruits (Jantwal et al., 2022) and notably in grapes, in which it represents quantitatively the main organic acid of grape juice and wine (Ribereau-Gayon et al., 2006b). The isomer of tartaric acid found in the grape is the L (+) form. Its concentration varies between 2 and 10 g/L (Chidi et al., 2018). Malic acid takes its name from the apple (malus in Latin), in which it is present in high concentrations. Mature grapes contain between 2 and 6.5 g/L of L-malic acid (Chidi et al., 2018). This C4-dicarboxylic organic acid takes three acid-base forms: malic acid (H2M), hydrogen malate (HM) or malate (M). In grape juice and wine, the protonated forms are predominant (pK_a1 = 3.40) while malate is mostly found in cytosolic conditions (pK_a2 = 5.11). Malic acid has two stereoisomeric forms (L and D), but only the L-isomer exists naturally.

The concentrations of organic acids in grapes are influenced by many factors, including grape variety, ripening stage, climatic conditions, soil potassium levels, plant nutrition, and canopy management, as reviewed elsewhere (Gerós et al., 2012; Volschenk et al., 2006). While both tartaric and malic acids can be found in grapes early in the growing season, their behaviour during ripening and winemaking differs. Tartaric acid is synthesised during initial berry cell division and remains stable more or less throughout the ripening process of healthy berries. It is not metabolised during winemaking but can be lost through physiochemical mechanisms like precipitation. While malic acid is present at very high concentrations prior to véraison, it is actively metabolised during berry ripening and is significantly impacted by microbial activity, as described in the following sections of this review. Other organic acids that modulate wine acidity, such as succinic, lactic, citric and acetic acids, can be synthetised or metabolised by yeasts and bacteria during winemaking. Finally, gluconic acid, naturally present in trace amounts in healthy grapes, is found in a larger concentration in wines produced from rotten grapes; in fact, Botrytis cinerea and acetic bacteria are able to produce gluconic acid by glucose oxidation (Ribereau-Gayon et al., 2006a).

Table 1. Main organic acids present in healthy grape must and wine, sourced from Waterhouse et al. (2016).

*Polyprotic acids have one pK_a for each -COOH group. The pK_a values in water are slightly different to those in wines, as they are affected by ethanol concentration, ionic strength, and temperature. As a rule of thumb, the first pK_a of an organic acid is 0.10-0.15 units higher in wine than water and 0.10-0.15 units lower for second pK_a.

** G: grape, Y: yeast, LAB: lactic acid bacteria, AAB: acetic acid bacteria.

In recent decades, several attempts have been made to modulate acidity by using genetically modified (GM) yeasts, mostly focusing on the modulation of lactic and malic acids. Some of them have been applied at industrial scale.

Recent studies have focused on elucidating the natural variation in the production of organic acids by fermenting S. cerevisiae strains in an oenological context using quantitative genetics approaches. Several QTLs were linked to the variation of succinate production located on the chromosome IV, VI, XI, XV, XIII, and XIV (Ambroset et al., 2011; Eder et al., 2018; Salinas et al., 2012). For succinic acid production, the impact of two genes FLX1 (Chr IX QTL) and MDH2 (Chr XV QTL) were experimentally validated. FLX1 encodes a transporter of flavin adenine dinucleotide (FAD) across the mitochondrial membrane that can modulate the activity of the succinate dehydrogenase. MDH2 encodes the cytosolic malate dehydrogenase involved in malate/oxalacetate interconversion that play a role in the glyoxylate cycle. More recently, the genetic determinism of malic acid has also been investigated in a multi-environmental QTL-mapping program (Peltier et al., 2021). The percentage of malic acid consumed by a wide population of yeast strains was calculated (MAC %) and eleven QTLs linked to malic acid consumption were identified (Peltier et al., 2021; Vion et al., 2021). Six genes affecting the variation of MAC % among progeny were validated by functional genetics experiments. The genes MAE1, PYC2, and SDH2 are directly related to malic acid, pyruvic acid and oxaloacetate metabolism and their position on the metabolic map are shown in Figure 2. MAE1 encodes the mitochondrial malic enzyme, PYC2 encodes an isoform of pyruvate kinase, and SDH2 the catalytic subunit of the succinate dehydrogenase complex. Interestingly, the gene MAE1 carries a single nonsynonymous allelic variation MAE^I605V that has been previously described to modify the production of branched ethyl esters, with are directly connected to malic acid catabolism (Eder et al., 2018). In addition, two other genes, PMA1 and PNC1, have a role in proton and NAD⁺/NADH, H⁺ homeostasis. Finally, the gene YBL036c encodes for a putative alanine racemase with a connection to the mitochondrial pyruvate pool. Interestingly, most of the allelic forms of QTLs involved in malic acid consumption were derived from the same parental strain. Phylogenomic analyses demonstrated that those alleles were derived from the flor yeast genome (Peltier et al., 2021), which constitutes a specific genetic group of wine yeasts (Coi et al., 2017). Flor yeasts are adapted to surviving in harsh environments that are depleted of sugar and rich in ethanol. Recently, we demonstrated that, compared to other S. cerevisiae strains, the flor yeast population can consume a large fraction of malic acid present in grape juice (Vion et al., 2023b).

The expression and contribution of the different QTLs mapped for malic acid consumption have been investigated in breeding programmes aiming to control the malic acid level at the end of the AF. First, a marker-assisted selection of malic-consuming strains was achieved demonstrating that individuals carrying a high proportion of enhancer alleles statistically consumed more malic acid than those carrying a proportion of preserver alleles. Although each allele had a low impact on the final MAC % value, their cumulative effect strongly impacted the MAC % (Vion et al., 2021). Second, malic producer strains were obtained by crossing together strains consuming low amounts of malic acid. After two cycles of segregation and selection, individuals producing up to 3.5 g/L of malic acid at the end of alcoholic fermentation were obtained. These extreme strains were significantly enriched in preserver alleles (Vion et al., 2023c).

Figure 2. Metabolomic map of S. cerevisiae. Genes impacting Malic acid consumption (MAC %) are shown in blue. Figure inspired from Peltier et al. (2021).

Several studies have investigated the ability of S. cerevisiae strains to consume malic acid during alcoholic fermentation. Some strains have been reported to consume up to 45 % of malic acid, while the role of other strains is to conserve acidity and consume little or no malic acid (Delcourt et al., 1995; Peltier et al., 2018; Redzepovic et al., 2003). The natural variability of Saccharomyces strains regarding the consumption of malic acid in different grape juices has been recently reevaluated for genetically distinct populations (Vion et al., 2023b). The Flor yeast population consumed significantly more malic acid than wine and fruit populations. This higher consumption might be regarded as a sign of the adaptation of these yeasts to growing in harsh media with depleted sugars and high ethanol concentrations. This property might be due to complex genetic regulation and adaptation, as indicated by the recent findings discussed in the previous section. Indeed, flor yeasts have been reported to shift to oxidative metabolism when sugar is depleted (David-Vaizant & Alexandre, 2018). They have also shown higher intracellular metabolites load than wine yeast (Vion et al., 2023a). Hence, flor yeasts might be able to consume more malic acid at the end of fermentation than wine yeasts. By using genetic selection strategies, strains able to consume around 70 % of malic acid have been successfully obtained (Vion et al., 2021), enabling efficient wine acidity management. Such strains have proven to facilitate malolactic fermentation by reducing wine malic acid concentration and increasing its pH (Vion et al., 2021). To our knowledge, no study has shown consumption higher than 80 % of initial malic acid or less than 0.5 g/L of malic acid remaining after fermentation by a strain of S. cerevisiae, regardless of the initial medium.

Despite this huge variability, S. cerevisiae is considered a relatively poor metaboliser of extracellular malate compared to other species. This is due to the weak malate dehydrogenase (Mdh2p) affinity for malate (Pines et al., 1996), the mitochondrial location of the malic enzyme (Mae1p) and its low affinity for malate (Km = 50 mM) (Boles et al., 1998). In addition, malic acid has been reported to enter the cell in its undissociated form (H2M) by simple diffusion due to the lack of active transport of malate through the membrane (Salmon, 1987). Malic acid has two pKa (pK_a1 = 3.40 and pK_a2 = 5.11), while the pH of grape juice ranges between 3.2 and 4.0. Extracellular malic acid can be found mostly in its undissociated (H2M) and mono-dissociated (HM) forms. Once it enters the cell, it acquires its deprotonated form (M). A proton pump ensures the exit of H⁺ and helps maintain an intracellular pH of around 5-6. When entering the cell by diffusion, malic acid is in its undissociated form, which represents about 50 % of the total malic acid available in grape juice at a pH of 3.5. As low pH values enhance the H2M/HM ratio, more di-protonated form is consumed, triggering the deacidification of the medium. This explains why more malic acid is consumed in grape juice at higher acidity levels. For all these reasons, S. cerevisiae consumes less malic acid than other yeasts, such as Z. bailii or S. pombe.

Malic acid consumption by S. cerevisiae depends on environmental factors, such as grape juice pH, and the concentration of assimilable nitrogen (Delcourt et al., 1995; Vilanova et al., 2007). Several studies have indicated that a high initial malic acid concentration will lead to its greater consumption (Delcourt et al., 1995; Vion et al., 2021) with malic acid production repressed in what would normally be malic acid-producing yeasts (Farris et al., 1989; Yéramian et al., 2007). However, Redzepovic et al. did not report any differences in malic acid consumption between two media with 3 g/L and 8 g/L of initial malic acid (Redzepovic et al., 2003). Low biotin content also favours malic acid degradation (Salmon et al., 1987; Schwartz & Radler, 1988), as does an elevated glucose concentration (Delcourt et al., 1995). Finally, low pH promotes the consumption of malate (Delcourt et al., 1995; Ramon-Portugal et al., 1999), since malic acid enters the cell in its undissociated form by simple diffusion. Finally, the addition of thiamine also facilitates malic acid consumption by S. cerevisiae (Carre et al., 1983).

The genus Schizosaccharomyces encompasses four related species (S. japonicus, S. octosporus, S. cryophilus, and S. pombe) (Hironori, 2014), the latter being particularly efficient for malic acid consumption. S. pombe is mostly isolated from tropical regions and from high sugar habitats (Jeffares, 2018) but is rarely detected in winemaking conditions, because it is out-competed by S. cerevisiae (Yokotsuka et al., 1993). It is characterised by its ability to completely metabolise the malic acid from grapes. This specific feature is due to the action of a constitutive active malic acid transporter encoded by the mae2 gene (Grobler et al., 1995). The incorporated malic acid is decarboxylated to pyruvic acid by the malic enzyme (in presence of NAD⁺ and one of the divalent cations Mn²⁺ or Mg²⁺) (Osothsilp & Subden, 1986). The high affinity of the malic enzyme for its substrate (Km 3.2 mM) and its cytosolic location contribute to the stronger efficiency of malo-ethanolic fermentation with respect to S. cerevisiae. The resulting pyruvate follows the alcoholic fermentation pathway, producing ethanol and CO₂. In this pathway, known as malo-ethanolic fermentation, one molecule of malic acid is fermented to produce one molecule of ethanol and two molecules of CO₂ in anaerobic conditions (Volschenk et al., 2003). In S. Pombe, both malic acid transporter and malic enzyme activities are induced by the presence of malic acid in the medium (Osothsilp & Subden, 1986).

Several authors have proposed adding S. pombe in grape juices for either partial or complete consumption of malic acid as an alternative to MLF (Benito et al., 2012; Ciani et al., 2009; Redzepovic et al., 2003). The proposed itineraries involve pure culture fermentations of S. pombe, and their co-cultures with S. cerevisiae or, as described more recently, with L. thermotolerans (Benito et al., 2015). To date, only one strain of S. pombe is commercially available in an immobilised form (Suárez-Lepe et al., 2012) for uses in a controlled biological deacidification process; in this process, the immobilized S. pombe cells use malic acid (Ciani et al., 2009), whereas S. cerevisiae achieves fermentation using almost all the available sugar. Despite the advantages of deacidifying wines with S. pombe, its industrial use in winemaking is limited due to the production of off-flavours including acetic acid (Benito et al., 2012) and a loss in typicity and fruitiness (Carre et al., 1983; Redzepovic et al., 2003).

Zygosaccharomyces bailii is a fructophilic yeast which can degrade high concentrations of malic acid during alcoholic fermentation (Baranowski & Radler, 1984). This species is considered a spoilage organism in the food industry because of its strong resistance to weak organic acids, chemical preservatives (sulfites, sorbic acid), ethanol, and high sugar concentrations (Martorell et al., 2007; Radler et al., 1993; Sousa et al., 1996). Different studies have reported the use of this species in wineries for mixed fermentation with S. cerevisiae (Escribano et al., 2018; Escribano-Viana et al., 2019; Garavaglia et al., 2015). Z. bailii preferably degrades fructose, followed by glucose; malic acid is only degraded during the glucose degradation step. Most of malate is oxidatively decarboxylated to pyruvate by the malic enzyme, while a small fraction is reduced by fumarase and fumarate reductase (Kuczynski & Radler, 1982). The malic enzyme of Z. bailii, has a notable affinity for malate (K_m = 10 mM) and is constitutively expressed (Baranowski & Radler, 1984). The same authors reported that this species has a L-malate transporter which is induced by glucose and inactivated by fructose. These properties allow Z. bailii to metabolise large amount of malic acid or acetic acid (Rodrigues et al., n.d.) in the presence of glucose. Although Z. bailii cannot be used alone as a starter for winemaking, the use of multi-starters that comprise a strain of S. cerevisiae and a non-Saccharomyces yeast for fermentation are being increasingly studied for different purposes, such as biological deacidification, bio-protection, and confering aroma complexity to wines. In this light of this, active dried yeasts of Z. bailli have become available (Ciani et al., 2009).

The main purposes of acidifying wines using organic acids are to increase TA and decrease pH, which can be necessary to maintain the freshness of a wine. The indirect aims are to enhance and stabilise the colour and the tannin structure of the wine, and to prevent microbial spoilage. The appropriate acidity levels help preserve wine over time, and leads to a reduction in sulfur dioxide content and microbiological stabilisation.

Acid-producing yeasts are generally less common than non-acid-producing yeasts (Kuczynski & Radler, 1982) because of their slightly lower rate of multiplication and growth. For this reason, acid-producing strains are rarely dominant in natural yeast populations of grape must. Nevertheless, if a sufficiently large population of acid-producing strains is inoculated in the must, they can become dominant and increase the acidity of the resulting wine.

The acidity of wine is a key component of its overall quality. With climate change posing a significant challenge to the winemaking industry, the emergence of yeast strains for wine acidity management has become an essential tool for winemakers. Current trends mainly focus on acidification to improve the analytical and sensory profiles of wines in the context of climate change. Deacidification can nonetheless be of interest to reduce acidity in cooler regions as well as to shorten malolactic fermentation by lowering malic acid content post-AF. Besides the use of specific Saccharomyces cerevisiae strains, novel oenological practices also combine the use of different non-Saccharomyces yeasts to either increase or decrease wine acidity. A diverse range of yeast starters could therefore be used in specific vinification strategies tailored to the climate, terroir, and desired wine style.