Novel Non-Cerevisiae Saccharomyces Yeast Species Used in Beer and Alcoholic Beverage Fermentations

: A great deal of research in the alcoholic beverage industry was done on non- Saccharomyces yeast strains in recent years. The increase in research interest could be attributed to the changing of consumer tastes and the search for new beer sensory experiences, as well as the rise in popularity of mixed-fermentation beers. The search for unique ﬂavors and aromas, such as the higher alcohols and esters, polyfunctional thiols, lactones and furanones, and terpenoids that produce fruity and ﬂoral notes led to the use of non- cerevisiae Saccharomyces species in the fermentation process. Additionally, a desire to invoke new technologies and techniques for making alcoholic beverages also led to the use of new and novel yeast species. Among them, one of the most widely used non- cerevisiae strains is S. pastorianus , which was used in the production of lager beer for centuries. The goal of this review is to focus on some of the more distinct species, such as those species of Saccharomyces sensu stricto yeasts: S. , S. paradoxus , S. mikatae , S. uvarum , and S. . the factors professional brewers might consider when selecting a strain of yeast for fermentation, are reviewed herein. The factors include the metabolism and fermentation potential of carbon sources, attenuation, ﬂavor proﬁle of fermented beverage, ﬂocculation, optimal temperature range of fermentation, and commercial availability of each species. While there is a great deal of research regarding the use of some of these species on a laboratory scale wine fermentation, much work remains for their commercial use and e ﬃ cacy for the production of beer.


Introduction
Fermented beverages have played an important and special role over the course of human history due to their economic and cultural importance, perhaps even lending to the beginning of modern civilizations [1,2]. Archaeological evidence places the oldest fermented beverage in the fertile crescent, as far back as 11,000 BCE [3,4], and based on the agricultural evidence of the time and region, that beverage was likely beer. While beer originally could have been an accidental beverage, it progressed into one of the most artfully crafted beverages known to man. No longer thought of as just an art, the science of beer led to several very important landmarks in scientific history ( Table 1). As scientific discoveries keep developing, there are some amazing innovations that led to advances in the quality and stability of beer, over the past 40 years [5]. However, minimal advancement was made when considering the raw ingredients used in the brewing process.
side to the cold side, wort is subsequently chilled for fermentation ( Figure 1a); yeast is added, metabolizing 50 to 80 percent of the sugar and nutrients to fermentation products, leaving behind non metabolized proteins, oligosaccharides, and other compounds [12][13][14]. The resultant sugar profile of brews can vary, based on the malting and mashing conditions, but typical mashes might contain 60.0% maltose, 20.0% glucose, 10.0% maltotriose, and 5.0% of both sucrose and fructose [15].  [14]. (b). Schematic diagram of the winemaking process as outlined and described by Water house et al. 2016 [16].  [14]. (b). Schematic diagram of the winemaking process as outlined and described by Water house et al. 2016 [16].
In Stage 1 of alcoholic fermentation, free glucose is assimilated first, followed by the hydrolyzation of maltose or other disaccharides into two glucose, by the enzyme alpha glucosidase (a.k.a maltase, EC 3.2. 1.20). Several other enzymatic destabilization and phosphorylation reactions then happen in Stage 1, which turns the substrate into glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). Stage 2 oxidizes G3P and DHAP, as well as the ADP generated previously, to create ATP as energy for the cell and pyruvate. Stage 3 enzymatically decarboxylates pyruvate to acetaldehyde and CO 2 that leaves the cell, before the alcohol dehydrogenase converts the acetaldehyde to ethanol in Stage 4 ( Figure 2). In brewing, yeast is typically reused (repitched) for ten generations or more [9], while in wine, the yeast is generally used far lesser times, due to the prominence of other microorganisms and the higher mortality from more stressful conditions of osmotic pressure and higher ethanol concentrations [28]. In most cases, serial repitching can cause genetic mutation within the cells and the desired flavor profile might no longer be attainable [29][30][31][32].
solution [9,12,23]. S. cerevisiae, when used at the proper pitching rate, takes the maltose and other sugars produced from the hot side of the brewing process [15], and anaerobically converts the disaccharides into carbon dioxide (CO2) and ethanol. More than 600 flavor active compounds can also be produced during the alcoholic fermentation process, depending on type of beverage produced ( Figure 2) [24][25][26]. Yeast works via an anaerobic pathway of glycolysis; if oxygen is present it performs respiration and cell reproduction [27]. The metabolic role of Saccharomyces yeast in the development of flavor for fermented alcoholic beverages. The sole products of yeast fermentation are not just ethanol and CO2, this schematic representation shows the derivation and synthesis of flavor active compounds from sugar, amino acids, and sulfur metabolism, delineated by the arrows on the diagram. Alcoholic fermentation of beer by Saccharomyces is the substrate level phosphorylation anaerobic pathway of glycolysis, which converts maltose sugar into ethanol and carbon dioxide.
In Stage 1 of alcoholic fermentation, free glucose is assimilated first, followed by the hydrolyzation of maltose or other disaccharides into two glucose, by the enzyme alpha glucosidase (a.k.a maltase, EC 3.2. 1.20). Several other enzymatic destabilization and phosphorylation reactions then happen in Stage 1, which turns the substrate into glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). Stage 2 oxidizes G3P and DHAP, as well as the ADP generated previously, to create ATP as energy for the cell and pyruvate. Stage 3 enzymatically decarboxylates pyruvate to acetaldehyde and CO2 that leaves the cell, before the alcohol Interest in brewing beer with novel yeast strains and applying Saccharomyces cerevisiae in new methods outside of traditional beer fermentation [33,34] has increased in recent years, due to the growing consumer tastes of sour and wild mixed-fermentation beers, as well as using some of these novel species for low or no alcohol beer production [35,36]. A great deal of research in the brewing industry was done on non-Saccharomyces yeast strains, such as Brettanomyces, Pichia, Hanseniaspora, Metschnikowia, and Torulaspora [37][38][39]. Furthermore, the search for unique flavors and aromas, and a desire to invoke new technologies and techniques for making alcoholic beverages led to the use of non-cerevisiae Saccharomyces spp. in the alcoholic fermentation process [40,41].
While the most widely used non-cerevisiae species is S. pastorianus, traditionally used in the production of lager beer around the world [42][43][44], this review focuses on some of the more distinct species. The focus is on five species of Saccharomyces sensu stricto (Sss) yeasts, S. kudriavzevii, S. paradoxus, S. mikatae, S. uvarum, and S. bayanus, as well as other novel species not currently in the Sss, such as S. abulensis and S. florentinus. When selecting yeast strains for fermentation, brewers consider its attenuation (the amount of sugar consumed by the yeast), flocculation (the yeast's ability to clump together and fall out of solution), fermentation temperature range, effects on flavor profile, capacity for reuse, and supply chain availability [45]. These facets, as well as a yeast's ability to ferment various carbon sources, morphological characteristics, and genetic hybridization can all assist brewers, when adopting a new strain.

Saccharomyces Species Diversity
Since Louis Pasteur's groundbreaking and historic report that fermentation was caused by a microorganism instead of a spontaneous mystery [8], the Saccharomyces genome was continuously studied, with several distinct species identified [46]. This diversity was termed the Saccharomyces sensu stricto (Sss) complex and is currently composed of ten genetically distinct species, all of which are capable of metabolizing glucose to produce ethanol ( Figure 3). Each of these species was perceptibly delineated from other Saccharomyces species, through studies of reproductive isolation and application of the biological species concept [31,[47][48][49]. All Sss species were isolated from unique sources in nature, including tree bark, flowers, fruit, and insects, demonstrating their lineage from wild type to the cultured stock of Saccharomyces spp. While all members of the Sss were proven to produce energy with fermentation, and many of these species are novel, some were used and studied for their potential use in commercial alcohol production for human consumption. The distribution of S. cerevisiae and S. pastorianus were long linked to alcoholic beverage production, along with minor mentions of other species in the Sss complex. Cultured species, specific to beer production, were shown to have evolved from European wine and Asian sake fermentations [21,50], therefore, its relation to wine production proliferates much of the research.

Saccharomyces Kudriavzevii
S. kudriavzevii was first isolated from a decaying leaf and has since been isolated repeatedly from the bark of oak trees in Portugal and France [52,53]. The yeast is a multipolar budding species, with a size of 5-10 µm, and an oval to slightly elongated shape [47]. It was shown to ferment glucose, sucrose, and maltose, but it did not ferment lactose, melibiose, or starch, which are common characteristics of Sss yeast (Table 2). S. kudriavzevii is a naturally occurring S. cerevisiae hybrid that might constitute 23-100% of the genome for some yeast [54,55], including Belgian trappist ale strains, such as Chimay, Westmalle, and Orval, and wass also genetically isolated in draft beer from the United Kingdom, Germany, and New Zealand [56]. This implies that the attenuation, flocculation, and flavor profiles of S. kudriavzevii might be similar to that of most Belgian strains. This meant low flocculation, high attenuation, and phenolic off-flavor positive (POF+) [45,57], though there is research in the wine industry that suggests S. kudriavzevii ferments slowly and produces less ethanol when used on grape juice [58]. Other research suggests it has high flocculation, as in overnight liquid culture, it grew into spherical 2-3 mm pellets [31].

Saccharomyces kudriavzevii
S. kudriavzevii was first isolated from a decaying leaf and has since been isolated repeatedly from the bark of oak trees in Portugal and France [52,53]. The yeast is a multipolar budding species, with a size of 5-10 µm, and an oval to slightly elongated shape [47]. It was shown to ferment glucose, sucrose, and maltose, but it did not ferment lactose, melibiose, or starch, which are common characteristics of Sss yeast (Table 2). S. kudriavzevii is a naturally occurring S. cerevisiae hybrid that might constitute 23-100% of the genome for some yeast [54,55], including Belgian trappist ale strains, such as Chimay, Westmalle, and Orval, and wass also genetically isolated in draft beer from the United Kingdom, Germany, and New Zealand [56]. This implies that the attenuation, flocculation, and flavor profiles of S. kudriavzevii might be similar to that of most Belgian strains. This meant low flocculation, high attenuation, and phenolic off-flavor positive (POF+) [45,57], though there is research in the wine industry that suggests S. kudriavzevii ferments slowly and produces less ethanol when used on grape juice [58]. Other research suggests it has high flocculation, as in overnight liquid culture, it grew into spherical 2-3 mm pellets [31]. Table 2. Physiological characteristics that distinguish each species of the Saccharomyces sensu stricto complex are discussed. Growth ability scored as positive (+), negative (−), evidence of both positive and negative (−,+), and unknown (u). Ethanol tolerance is defined as being able to grow in the presence of 2.5% v/v EtOH, the low-end strength of standard beer. Attenuation and flocculation scored on a relative basis scale, ranging from low, to moderate, to high. Type strain as defined in MycoBank (mycobank.org), origin, isolation, and commercial availability, as defined in the cited literature. It is advised to ferment S. kudriavzevii in tandem with a traditional Saccharomyces [59] and it was shown to form a triple hybrid complex with S. cerevisiae and S. uvarum, as it was isolated as such from farmhouse ciders made in France [60,61]. S. kudriavzevii is a cryophilic strain in the Sss that prefers fermentation temperatures in the 10-15 • C range [52,62,63], and is currently used to ferment lower temperature pinot noir and lager beer in Europe [54]. The only current commercially available example is Anchor Oenology's Vin7 strain, developed in Stellenbosch, South Africa, for enhancing thiol aromas in white wine [64,65], but it stands to reason that it can be isolated from previously noted commercial beer examples. Due to its cryophilic tendencies and aromatic potential, S. kudriavzevii has potential for use in the production of hoppy lager beers in the brewing industry. Further research remains to be done on this species, considering it is POF+ and it is likely also diastaticus (STA1) positive, meaning it could ferment residual maltodextrins. Additionally, minimal commercial production was done with the direct intention of using S. kudriavzevii, as most fermentations did not take place with the intention of the use of this species.

Saccharomyces paradoxus
S. paradoxus is one of the first isolates of the Sss [66], a wild-type strain commonly isolated from the bark of deciduous trees and occasionally from fruit and insects in North America and Eastern Europe [67][68][69]. Even though genetically S. cerevisiae and S. paradoxus were proven to be distinct species [70], phylogenetically the two were the closest relatives in the Sss (Figure 3) and were 90% genetically similar [55]. They share the same morphological and phenotypic characteristics, such as being spherical or ellipsoid in shape, with a diameter of 1-5 µm [71]. Previous research indicates mixed results of the fermentative capacity of S. paradoxus, but it has the ability to convert glucose into ethanol and a relatively high alcohol tolerance [47,72,73]. It is a positive fermenter for glucose, sucrose, and maltose, but it does not ferment lactose, melibiose, or starch ( Table 2). Little evidence exists for the domestication and commercial use of S. paradoxus in alcohol fermentation, but it was found to be naturally co-fermenting with S. cerevisiae in Eastern European wine fermentations [73,74], as well as with S. cerevisiae, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, and S. pastorianus in indigenous African sorghum beer [75].
In laboratory fermentations, the optimal growth temperature for S. paradoxus falls 7 • C lower than S. cerevisiae, and is likely cryophilic, due to the climates in which it is found, but S. paradoxus is yet to be trialed extensively in a production environment [76,77]. Unfortunately, no information  [73,74]. There is a commercially available hybrid of S. paradoxus and S. cerevisiae produced by Anchor Oenology, which when used for Syrah and Merlot wine fermentations, shows increased aromas of cherries, strawberries, cocoa, and floral notes, and the wine is described as full-bodied, well-balanced, complex and intense [78]. Much work remains to be done on the versatility of this species for the brewing industry, but it might have potential for unique and novel flavor characteristics if a pure culture from a genetic bank is obtained for further experimentation.

Saccharomyces mikatae
S. mikatae is a natural genetic hybrid that results from introgression events with S. cerevisiae and S. paradoxus [55,79], and its current hybrids are described for use in industrial wine fermentation. This hybrid was deliberate, created in a lab with the intent of creating greater complexity in resultant wines, akin to those that are spontaneously fermented, but more easily controlled due to the inclusion of typical S. cerevisiae yeast [80,81] S. mikatae was first isolated from decaying leaves and soil in Japan [47,60]. It is ovoid in shape and approximately 5-9 µm in diameter; it reproduces by multipolar budding, and generally appears in pairs or short chains. S. mikatae was also shown to form a pellicle after 25 days at 20 • C, similar to Brettanomyces and other wild-type yeasts [47]. The inclusion of S. mikatae in the Sss means it is capable of alcoholic fermentation and assimilation of glucose, it is also capable of fermenting maltose, sucrose, and melibiose, but not lactose or starch (Table 2). However, S. mikatae might have a lower attenuation, due to genetic diversion from S. cerevisiae, while still exhibiting similar levels of flocculence [31].
S. mikatae readily creates hybrids with S. cerevisiae, and these hybrids were shown to produce higher concentrations of multiple compounds that yield fruity, banana, floral, and sweet perfume aromas in the fermentation of white wine [80,81]. Although no information on beer fermentation with either the type strain or any hybrids exist, the additional amounts of certain volatile compounds in the research by Bellon et al. (2013Bellon et al. ( , 2019 might show signs of this yeast's production of phenolic off flavors. S. mikatae grows readily in temperatures from 4-30 • C, with expected slower growth in the range limits and no growth outside the range, making it a cryotolerant fermenter [47,63]. Commercial availability is limited, but yeast manufacturer AB Biotek commenced exploratory production of an S. mikatae and S. cerevisiae hybrid, AWRI 2526; brewers and winemakers can expect the hybrid as an active dried yeast product that is expected to be available for trials, by the fall of 2020 [81].

Saccharomyces uvarum
S. uvarum is a fairly well-known member of the Sss, originally believed to be identical to S. bayanus and often referred to as S. bayanus var. uvarum, it was shown to be a genetically distinct Saccharomyces species [82][83][84]. S. uvarum is also similar in size and shape to S. bayanus, being spherical or ellipsoid in shape, with a diameter of 1-5 µm, and reproducing by multipolar budding. S. uvarum was isolated in natural European wine and cider fermentations [85][86][87], as well as in South American chicha fermentations [88,89], but was first isolated in 1894 and described in 1898 by M.W. Beijerinck, from spontaneous wine fermentation [90]. S. uvarum is known to hybridize with S. cerevisiae, S. bayanus, and S. pastorianus [85,91,92], and thus can show signs of being POF+ and possibly might have the STA1 gene for diastaticus [91,93]. S. uvarum showed the capacity to ferment glucose, sucrose, melibiose, and maltose, but it does not ferment lactose. S. uvarum is a known bottom-fermenting yeast, meaning it acts similar to a S. bayanus or S. pastorianus when not in hybrid form, offering cryotolerance [94], moderate attenuation, and high flocculation ( Table 2).
Research with wine showed that S. uvarum produces comparatively higher amounts of volatile aromatics when fermented cold [95], implying potential use as a lager strain. In Chardonnay winemaking trials, wines were described as showing apricot, cooked orange peel, citrus, lime, honey, and nutty aromas with some tasters, and estery, pineapple, peach, melon, and floral aromas with others [80,96]. While S. uvarum continues to predominate in spontaneous European wine fermentations [86,87,97] and is a known species in some Norwegian kveik hybrid strains [91], its commercial availability is limited to the Australian Wine Research Institute at this time [98]. Cryotolerance and increased aromatic potential means S. uvarum could be used in the production of some complex and eccentric lagers in the brewing industry, but currently, it has only been isolated as part of a hybrid culture in the aforementioned kveik beer. Further research remains to be done on the brewing potential of S. uvarum, but brewers should be aware of the increased aromatic character that might come from the POF+ genes.

Saccharomyces bayanus
S. bayanus is a well-studied species in the Sss and is often used as a model organism for comparative functional genomics studies of yeast, based on introgression and interspecific hybridization [99]. It is genetically similar to S. cerevisiae, but evolved to be a distinct member of the Saccharomyces sensu stricto complex [55,100], and is now referred to as S. bayanus var. bayanus, in order to delineate it from S. eubayanus and S. uvarum [43,101]. S. bayanus was previously thought to be the parent of the lager strain, S. pastorianus [43,60,85], but the hybridization event that produced lager brewing yeast is now proven to have occurred between S. cerevisiae and S. eubayanus [21,50,102,103]. While S. bayanus might not be the true hybrid parent of the most used brewing yeast in the world, it still forms natural hybrids with other members of the Sss and was identified in these complexes in the fermentation of wine [62,85,104]. While it was first isolated from turbid beer in 1927 [105], S. bayanus was also isolated from beer, wine, fruit, and even soda [47]. S. bayanus is ellipsoid to elongate in shape, with a diameter of 1-5 µm, and reproduces by multipolar budding. Research showed it to be a positive fermenter for glucose, sucrose, and maltose. Other studies reported S. bayanus to show both positive and negative fermentation of melibiose, but it does not ferment lactose or starch ( Table 2).
S. bayanus is well-known as a fermenter of beer and cider [43,105,106], but is most commonly used in wine [96,103,107]. It can be purchased from several commercial suppliers, but unfortunately several commercially available strains were genetically identified as S. cerevisiae, including the famous Lalvin EC-1118 strain that was originally typed S. bayanus [108,109]. S. bayanus ferments best in the upper end of the lager strain temperature range of 10 to 21 • C [77,101], is moderately flocculant [31], and has a fairly standard attenuation [110], as expected, given its genetic similarity to S. pastorianus lager yeast. The commercially available hybrid of S. bayanus and S. cerevisiae available from Lallemand, Lalvin S6U, is known to increase the varietal characteristics in white wine, and might produce elevated levels of POF [111,112]. More research needs to be carried out with regards to the flavor profile of beers made with S. bayanus, but the research on wine and its history as a potential lager strain means, it is capable of fermenting remarkable lager style beers.

Other Saccharomyces spp. Used in Alcoholic Fermentation
Several other novel species of Saccharomyces used in alcoholic fermentation were determined to be genetically distinct by current research but might not yet be included in the Saccharomyces sensu stricto complex. S. abulensis is a novel species dubbed the "Santa Maria strain" and was isolated from yeast originating from breweries in Madrid and Sevilla, Spain [92]. S. florentinus, formerly known as S. pyriformis, is a species of yeast isolated from the scoby of traditionally fermented ginger beer, known as "bees wine," but is yet to be used in commercial production of beer [113,114]. Three other strains included in the phylogenetic tree of the Sss (Figure 3) exist-S. arboricola, S. jurei, and S. cariocanus-but research is limited on their fermentation capacity. S. arboricola is a wild-type hybrid of S. bayanus and S. kudriavzevii, which was isolated from oak and beechwood bark in China [115,116], and is currently being used in sake production [117]. S. jurei is closely related to S. mikatae and S. paradoxus and was isolated from a high altitude tree bark in France; little is known of its fermentative capacity [118]. S. cariocanus, isolated from insects in South America [119], is a wild-type hybrid of S. paradoxus, which is capable of fermenting sucrose and shows ethanol tolerance [120]. These yeasts are not members of the Sss, but is likely to be included, as the complex underwent many changes over the years, in accordance with the system employed in classifying yeast cultures. Very little information exists on these yeasts' ability to ferment beer or their use in a commercial setting, but by contacting genetic banks and yeast culture collections directly, the strain type could be obtained for further experimentation. There also exists multiple variants of S. cerevisiae, such as var. boulardii, which is known to produce higher levels of polyphenols, and can thus be used in functional probiotic beer [121][122][123][124][125][126]. Another variant, var. diastaticus, can cause over-attenuation [127][128][129], which was discussed earlier as having the STA1 gene.

Conclusions
The non-cerevisiae Saccharomyces species discussed in this review, have the ability to ferment glucose and maltose into ethanol, anaerobically. Most are members of the Saccharomyces sensu stricto complex, while some are yet to be defined within the species complex classification. S. kudriavzevii is available commercially as a hybrid with S. cerevisiae and can increase thiol aromatic qualities during fermentation, at lower temperatures, making it ideal for distinctive lagers. S. paradoxus is also available commercially as a hybrid with S. cerevisiae and showed increased fruity and floral esters in wine, and also lends unique characteristics to African umqombothi beer. Hybrids of S. mikatae and S. cerevisiae are not yet commercially available, but they were known to produce increased fruity and perfume aromas even when fermented at low temperatures, marking its potential for remarkable lager beer production. Due to debate on the classification and isolation process of S. uvarum in genetic research, there is no commercially available version of this species, but it shows potential as a cryotolerant lager yeast, with more character than S. pastorianus. After recent research, many commercially available S. bayanus strains were reclassified as variants of S. cerevisiae, but the true hybrids of S. bayanus and S. cerevisiae showed increased ethyl esters and spicy notes that could add a complexity to beer production. While much of the research regarding flavor and aroma that is presented in this review might be focused on wine, these species all have potential for novel fermentations and new sensory experiences, if used in beer.