Saccharomyces cerevisiae and its industrial applications (2024)

2.1. Application of S. cerevisiae in the wine industry

The relationship between wine and man dates back thousands of years: detection of calcium salt of tartaric acid and terebinth resin in a pottery jar constitute the first experimental evidence for the presence of wine in Iran, around 5400–5000 BC [24]. The relationship between wine and S. cerevisiae is equally long-lasting as it was proven by the presence of ribosomal DNA from S. cerevisiae in a wine jar from Egypt dated back to 3150 BC [25]. However, this relationship was revealed not earlier than 1860 when Louis Pasteur established for the first time the ‘hidden’ world of yeast activity during the wine fermentation [26] and eventually in 1890, when Müller-Thurgau proposed the process of controlled wine fermentations with starter cultures [27]. This innovative practice, which found broad application after almost a century, in the 1970s, revolutionized the wine industry and has resulted in the improvement of wine quality by offering a better control and consequently better repeatability and reliability of the fermentations [27].

From the very beginning, the vinification environment exposes microorganisms present in grape must to many different types of stress applying selective pressure on them [27]–[30]: Natural grape must is hostile due to its low pH and high sugar concentrations; in the majority of industrial fermentations high concentrations of the antioxidant and antimicrobial preservative sulphur dioxide are also added intensifying harsh conditions, whereas, as the fermentation proceeds, stress is multiplied for a plethora of reasons including anaerobic conditions, depletion of nutrient reserves (nitrogen, lipids and vitamins), increased acid concentrations, ethanol toxicity and temperature variations. S. cerevisiae although is found in very low populations in vineyards or grapes, prevails fermentation by dominating over the other yeast species abundant in natural must as it is able to overcome all fermentation stresses [28],[31]. This is the reason it has gained itself the title of ‘the wine yeast’ being the main workhorse of the wine industry worldwide [31]. Another set of criteria for a successful choice of a starter culture in industry is the production levels of several metabolites that, form the ‘fermentation bouquet’ and determine the complex sensorial and organoleptic character of the produced wine [29]. As it has been reported the compounds that have the greatest impact on ‘fermentation bouquet’ include higher alcohols, esters, aldehydes and terpenes [32]. S. cerevisiae is primarily responsible for the formation of the first three categories as it is not a sufficient terpenes producer [33].

Higher alcohols (fusel alcohols), from a quantitative point of view, is the most important group of compounds that S. cerevisiae produces during fermentation. Their biosynthesis is conducted via amino acid catabolism, through a route known as the Ehrlich pathway [34]. As it is reviewed by Hazelwood et al., 2008 [35] this pathway consists of three steps: Initially transaminases, encoded by the genes ARO8, ARO9, BAT1 and BAT2, deaminate amino acids to the corresponding α-ketoacids. Secondly, α-ketoacids are converted to their corresponding aldehydes by one of the five decarboxylases (Pdc1p, Pdc5p, Pdc6p, Aro10p and Thi3p) present in S. cerevisiae genome. Finally, the alcohol dehydrogenases, Adh1p to Adh6p and Sfa1p, catalyze the reduction of aldehydes to their corresponding higher alcohols. Styger et al., 2011, 2013 [36],[37] exploited the yeast deletion library EUROSCARF in order to access the genes that have the most important contribution on higher alcohol production. Their results highlight BAT2 as the dominant gene of the pathway suggesting that the initial transamination step is rate-limiting. Typical representatives of higher alcohols found in wine are 1-propanol (stupefying), 1-butanol (fusel odor), isobutanol (alcoholic flavour), 2 phenylethanol (floral, rose notes) and isoamyl alcohol (marzipan flavours) [38]. Because if their total concentration exceeds 400 mg/lt, they contribute negatively on the wine bouquet [38], industry demands for strains with a relatively low fusel alcohol production [29]). The correlation between the concentration of amino acids and the amount of higher alcohols produced follows a pattern according to which at YAN (Yeast Assimilable Nitrogen) levels below 200 mg/L, the production of higher alcohols increases along with YAN concentrations whereas above 200 mg/L the relationship turns inversible [39],[40], a trend that should be taken into account to modulate higher alcohols formation in industry. The concentrations that are reached however are strongly related to the strain used [41].

Esters are the most desirable group of compounds contributing fruity and floral aromas to the wine bouquet [42]. S. cerevisiae synthesizes two major groups of esters during fermentation, namely the acetate esters of higher alcohols and the ethyl esters of medium-chain fatty acids [MCFA]. Such esters include ethyl acetate (varnish, nail polish, fruity), isoamyl acetate (banana, pear), isobutyl acetate (banana) and phenylethyl acetate (rose, honey, fruity, flowery), ethyl hexanoate (apple, banana, violets), ethyl octanoate (pineapple, pear) and ethyl decanoate (floral) [43]. Recently, Ruiz et al. 2019 [44] reviewed extensively the correlation of the most important volatile compounds in wines with their corresponding odor in many different vine varieties. At this point it should be noted that ethyl acetate is desirable at concentrations below 150 mg/L, otherwise it confers spoilage character to wine [43]. Because the concentration of most esters is low and therefore very close to the human's smell nose detection limit, we understand that minimal variations in their concentration can to be of great importance for the quality of the final product of fermentation (wine) [30],[45]. The biosynthesis of acetate esters takes place intracellularly through an enzymatic reaction between acetyl-coenzyme A and an alcohol catalyzed by an alcohol acetyltransferase (AATase). In S. cerevisiae, two such enzymes have been extensively studied: AATase I and AATase II encoded by genes ATF1 and ATF2, respectively [42]–[48], whereas recently an ethanol acetyltransferase, encoded by EAT1, was also identified [49] and reported to have the potential to produce acetate and propanoate esters [50]. Eht1p and Eeb1p are the enzymes that catalyze the reaction between a medium chain fatty acid [MCFA]-CoA with ethanol synthesizing the MCFA-ethyl esters, possessing also as well as an esterase activity [51]. With regard to acetate ester hydrolysis, the only enzyme identified in the S. cerevisiae proteome is isoamyl acetate-hydrolyzing esterase [Iah1p] [48],[52],[53]. Experiments by Kruis et al., 2018 [50] revealed that even when all known ester synthases were deleted in S. cerevisiae, ester biosynthesis was not completely abolished, suggesting the existence of other ester synthases in the S. cerevisiae proteome, which have not been discovered yet. In addition, the determination of esterase activity in partially purified protein fragments of S. cerevisiae cells leads to the conclusion that other unidentified enzymes with esterase activity are present in the S. cerevisiae proteome [42]. Based on the needs of modern wine making, successful starters should possess moderate esterase activity [29].

The major aldehyde synthesized by S. cerevisiae during wine fermentation is acetaldehyde constituting over 90% of the total aldehyde content of wine [54],[55]. Acetaldehyde, is the last precursor in the anaerobic pathway before ethanol. The pyruvate, end product of glycolysis, is converted to acetaldehyde by the pyruvate decarboxylase enzymes, encoded by genes PDC1, PDC5, and PDC6 with the first two pdc enzymes, being the major contributors to the decarboxylation activity controlling directly levels of acetaldehyde [56]. Acetaldehyde is further converted to ethanol, by the ADH1-encoded dehydrogenase, whereas ADH2-encoded enzyme [Adh2p] catalyzes the reverse reaction. Acetaldehyde when present in low concentrations confers a fruity pleasant aroma but when in excess produces green and grassy off-flavours [54]. Worth of notice though is that the concentrations of acetaldehyde vary significantly between different types of wine with average values of approximately 80 mg/L for white wine, 30 mg/L for red wine, and 300 mg/L for sherries [54]. In addition to the direct effect of acetaldehyde on the aromatic profile of wine, it is equally important, if not more, its indirect effect due to its high reactivity with other compounds [36],[54]. Of special interest for the wine industry it is acetaldehyde's binding activity with SO₂, the basic antimicrobial and antioxidant agent, forming a complex compound which offers limited protection to the produced wine [54]. Acetaldehyde also mediates condensation reactions of grape-derived anthocyanins with tannins into stable red wine pigments during winemaking [57]. As different strains of S. cerevisiae synthesize considerably different amounts of acetaldehyde, it is critical to choose a strain suitable depending on the type of wine produced [54],[58],[59].

The process of producing the fermentation bouquet is complex. It includes a significant number of biosynthetic pathways and genes and is affected by various parameters including the composition of the fermentation medium, the fermentation conditions and the inoculum used [32]. Although genetic manipulation of S. cerevisiae strains has already been proposed since the beginning of the century in order to generate strains with an ideal combination of desired oenological traits [60], however, as practice proves, it is not an alternative that finds ground in industry that prefers natural strains to cover consumers' demands. Many studies have shown that the use of starter cultures consisting of indigenous to the winemaking environment strains enhance wine flavour and confer to wine a special distinctive character reflecting the area of origin (terroir) [61]–[65]. However, in industry spontaneous fermentations involving indigenous microflora are not carried out due to their very serious disadvantages including inconsistent results from vintage to vintage and prevailing of undesired microorganisms resulting in the production of off-flavours spoilage of wine [66]. Instead, winemaking in industry worldwide is usually carried out with the use of a limited number (<150) of commercially available S. cerevisiae strains [60]. Although this practice ensures reliability and reproducibility, however, it leads to the production of ‘industrialized’ wines in which the positive influence of indigenous flora has been minimized [62]. Given the fact that the microflora of the vineyards is characteristic, demonstrating the existence of a nonrandom microbial-terroir, depending on regional, varietal and climatic factors, it is easily concluded that there is a rich source of indigenous S. cerevisiae strains to be exploited by the wine industry [67]. To this end, this practice can guarantee the preserving of the microbial biodiversity and the improvement of product quality leading to better consumer acceptance and consequently higher economic profit of the wine industries.

2.2. Alternative vinifications employing mixed yeast inocula of S. cerevisiae and non-Saccharomyces species as starters

In recent years, several researchers and many wine industries have turned their attention to vinification processes involving fermentations with mixed yeast inocula. There are two main reasons for this approach: i) the attempt to improve the organoleptic characteristics of the resulting wines. ii) The need for production of wines with lower alcohol content.

The mixed starter cultures usually consist of one commercial, or laboratory S.cerevisiae strain and one non-Saccharomyces strain, usually isolated from grapes. As non-Saccharomyces inocula have served member species of several genera, among them Candida, Debaryomyces Hanseniaspora (and its anamorph Kloekera), Issatchenkia, Kluyveromyces/Lanchancea, Metschnikowia, Pichia, Torulaspora, Wickerhamomyces (Table 1). The mixed starters are applied in a co-, or sequential fashion and often in varying ratios of cell numbers.

2.3. Applications of S. cerevisiae in the bread industry

2.4. The application of S. cerevisiae in the chocolate industry

The beans of the tropical plant Theobroma cacao are the basic raw material for the production of chocolate [108]–[110]. However, raw cocoa beans are inedible, being bitter and astringent, while their aroma and flavours are not those of chocolate; thus, are subjected to fermentation to reduce the levels of polyphenols and alcaloids, causing the bitterness and astringency, and to develop flavours determining the fine organoleptics of cocoa and chocolate [108]–[110]. To this end, after the cocoa rods are opened, cocoa beans covered by the acidic [high concentration of citric acid] and sugar-rich (10–15% sugars) cocoa pulp are exposed to the naturally existing wild microflora and left to undergo a spontaneous fermentation [108]–[111].

The micro-ecosystem of the cocoa fermentation is complex and dynamic including mainly yeasts followed by lactic acid bacteria (LAB) and acetic acid bacteria [AAB] [109],[112],[113]. At the beginning of fermentation, yeast under anaerobic and low pH (3–4) conditions start to ferment the pulp-sugars producing ethanol as well as numerous flavour metabolites that will determine the quality of the final products [108]–[111],[114]. In addition, through the action of pectinolytic enzymes, they degrade gradually the highly viscous cocoa pulp (containing 1,5% pectin) allowing the air to penetrate into the pulp [108]–[111] while they also metabolise citric acid causing a pH increase [108],[110],[111] conditions that favor the growth of LAB and AAB [108]–[111]. LAB increase pH further as they metabolize citric acid and AAB oxidize ethanol to acetic acid [[108]–[111]. As both ethanol and acetic acid productions are exothermic reactions the temperature grows up to 50 °C [108]–[110].

According to experimental results, yeast activity is of paramount importance for the production of high-quality chocolate. Specifically, in pilot-scale cocoa fermentations carried out in the presence or absence of yeasts Ho et al., 2014 [115] observed that without yeasts there is a reduced production of ethanol, higher alcohols and esters throughout the fermentation while the chocolate produced was of inferior quality compared to the one produced when yeasts were present in fermentation. On the contrary, as the same research team reports LAB and AAB, were not proven necessary for the completion of cocoa fermentation, while their absence did not affect the organoleptic characteristics of the chocolate produced [116],[117].

A great variety of yeasts have been isolated and characterized from cocoa beans fermentations, with S. cerevisiae being among the most prevalent in several studies [112],[118],[119],[120]–[126], This fact is attributed to the specific properties of S. cerevisiae including its pectinolytic activity, rapid growth at a slightly increased pH and better adaptation to stress conditions of high ethanol concentrations and high temperatures [108],[109]. As unlike the other industrial fermentation, cocoa fermentation is spontaneous and consequently poorly controlled; inoculation with selected starter cultures could ensure successful fermentations with guaranteed reproducibility [108]. To this end, S. cerevisae has gained a lot of interest and several studies have exploited strains of the species as starters in mixed or mono-cultures fermentation schemes [127],[128]. However, the dynamics and contribution of S. cerevisiae to cocoa fermentation are clearly depicted in the experimental proof of the studies in which S. cerevisiae served as the only starter culture or in comparative studies in which its presence or absence was the only parameter of differentiation. In specific, the pectinolytic activity of S. cerevisiae in inoculated cocoa fermentations has been reported to improve pulp draining up to 127% [129]. In addition, Meersman et. al., 2017 [130] in order to evaluate the role of endo-polygalacturonase [EPG] conducted cocoa pulp fermentations inoculated either with a wild type S. cerevisiae or with a deletion strain in which PGU1 gene, encoding EPG, was knocked out. As a control served the fermentation with non-inoculated pulp. As Meersman et al., 2017 [130] reported the viscosity of the pulp inoculated with the deletion strain wasn't significantly different from that of the corresponding non-fermented pulp whereas it was also by a 23.5% higher when compared to that of its counterpart fermented with the wild type strain [130]. These results render EPG the major pectinolytic enzyme of S. cerevisiae for pulp degradation.

In addition, Lefeber et al., 2012 [131] and Ramos et al., 2014 [122], have reported that the inoculation with S. cerevisiae strains leads to a quicker consumption of pulp-sugars and to a higher production of ethanol, accelerating the fermentation process. Several studies have also focused on the impact of S. cerevisiae inoculation on the organoleptic features of the chocolates produced. Lefeber et al., 2012 [131] reported that the chocolates produced in presence of S. cerevisiae in a mixed starter culture along with LAB and AAB were characterized as fruity and were the most preferred by a trained panel compared to their counterparts produced in its absence in the starter or through spontaneous cocoa bean fermentation. To the same conclusion resulted Visintin et al., 2017 [132] that observed more fruity odors in chocolates produced in presence of S. cerevisiae and T. delbrueckii than in their counterparts fermented in presence of only T. delbrueckii. This positive influence of S. cerevisiae on the sensory characteristics can be attributed to its ability to produce desirable flavour compounds such as esters, alcohols and aldehydes conferring fruity, flowery and candy, fruity and cocoa notes, respectively [133],[134]. More specifically S. cerevisiae has been related to key flavour compounds in cocoa beans such as ethyl octanoate, 2-phenylethyl acetate, ethyl acetate, 2-methyl-butanal, 3-methyl-butanol, 2-phenylethanol, and 2-heptanol [124],[127].

Assi-Clair et al., 2019 [135] conducted a comparative study on the performance of two S. cerevisiae strains when used as starters in cocoa fermentations. According to their results the two strains presented different profiles in regard to the production of flavour metabolites, depicted also on the organoleptic attributes of the produced chocolates, indicating that the selection of a successful starter is rather strain than species depending. Another parameter that must be taken into account is the cocoa variety used as the it was evidenced by the experimental results of Ramos et al., 2014 [122] and Menezes et al., 2016 [134] that inoculated different cocoa varieties with the same S. cerevisiae CA11 leading to chocolates with different sensory profiles, concluding that a starter culture can be appropriate for a certain cocoa variety but not for all.

One step further, tailoring of S. cerevisiae strains has been proposed in order to obtain superior strains with a combination of desirable features [113],[114]. First, in 2015 the Meersamn et al. [114] team mated selected strains and developed a hybrid that exhibited increased thermotolerance and fermentation capacity, compared to parental strains, whereas it also produced chocolate of superior quality. Following other mating experiments in 2016, the same team reported even improved hybrids that apart from being temperature tolerant, and robust fermentors, could also produce high concentrations of desirable esters modulating the flavour of chocolate produced [113].

Taken together, these studies underline the importance of S. cerevisiae in cocoa fermentations and point out to its exploitation as the starter culture to improve the efficiency and consistency of fermentations and thereafter the quality of commercial chocolate production.

3.1. General aspects of the bioethanol production

Bioethanol can be used alone or mixed with gasoline and exhibits several advantages over petroleum fuel such as higher octane number (108), broader flammability limits, higher flame speeds and increased heats of vaporization [138], while on the other hand is less toxic, readily biodegradable and produces lesser air-borne pollutants [139].

Bioethanol is produced from the fermentation of sugars originating by a variety of sources, since its synthetic production is prohibitive due to its high cost. Primarily, the substrates used for sugar fermentation involve plants rich in sucrose from food crops such as sugarcane, sugar beet and a variety of fruits, and starch (corn, rice, wheat, etc): the bioethanol produced from fermentation of food crops is called ‘first generation’ biofuel. However, since these feed stocks fulfill the needs of animal and human nutrition, there is a controversy concerning their use as fermentation substrates for ethanol production. Therefore, a strategy of ‘second-generation’ biofuel has been developed, in which non-food substrates belonging to the lignocellulosic biomass (wood, straw, crop and food wastes, etc.) are exploited. Subsequently, a ‘third-generation’ bioethanol has been derived from algal biomass including microalgae and macroalgae [137],[140],[141].

United States of America use corn as the dominant feedstock for ethanol production, while Canada uses corn and wheat, Brazil sugar cane, China corn, wheat, and cassava, and European countries use primarily wheat and sugar beet to produce bioethanol [137]. Up today, USA is the largest ethanol producer worldwide for fuel utilization [142]. Ethanol production averages over a million barrels (159 million liters) per day with an annualized rate of 16 billion gallons (60 billion liters) in 2017, as reported by H. T. Kennedy in the February 17 issue of Biofuels Digest. USA and Brazil are the dominant countries in ethanol production manufacturing over 85% of the world's fuel alcohol [143].

3.2. Fermentation microorganisms

Alcoholic fermentation is pretty much synonymous to Saccharomyces cerevisiae, the protagonist in the industrial ethanol production among various other yeasts that synthesize ethanol by sugar fermentation. Under anaerobic conditions, S. cerevisiae uses glycolysis to catabolize sugars reaching the step of pyruvic acid formation. In follows, the latter is converted by pyruvate decarboxylase to acetaldehyde and carbon dioxide, which in turn is reduced to ethanol by alcohol dehydrogenase and releasing NAD⁺ at the same time. The terminal step reactions that lead to ethanol are therefore very important and constitute the basis for major fermentation industries [144].

Although Saccharomycescerevisiae is the dominant sugar fermenter, other yeast species are capable of producing bioethanol from sugar fermentation as well [143]. Kluyveromycesmarxianus has been investigated (among other applications) for the production of bioethanol from polyfructan substrates [145]. Dekkera bruxellensis has been used for bioethanol production from hexoses as products of starch hydrolysis [146], while Scheffersomyces (Pichia) stipitis utilizes lignocelluloses substrates [147] or algal biomass [148].

3.3. Contribution of S. cerevisiae to the synthesis of other types of biofuel

Apart from bioethanol, higher alcohols such as propanol and butanol are synthesized by genetically modified or metabolically engineered S. cerevisiae strains [149]. Propanol is suitable for engine fuel usage due to its high octane numbers. Since, however, its synthesis is very expensive, microbial strains have been tested for its production by fermentation of sugar substrates. Production of propanol in wine by yeast strains has been reported [150]. Based on this reaction, a genetically modified S. cerevisiae strain with 2-Keto acid decarboxylase (KDC) and alcohol/aldehyde dehydrogenase (ADH) activity synthesized increased amounts of propanol via 2-ketobutyrate (2KB) and could therefore be a potential for this application [151].

Butanol, as propanol, possesses a range of physical properties such as less hygroscopy, less corrosiveness, higher energy density and octane value compared with ethanol. Therefore, it can be blended with gasoline in much higher proportions than ethanol [152]. Butanol production from yeast is genetically monitored by introducing a synthetic acetone-butanol-ethanol (ABE) pathway using adh1 mutants of S. cerevisiae A267T/E568K, which significantly improves the n-butanol yield [153].

The isobutanol biosynthesis includes ketoisovalerate synthesis (an intermediate of valine biosynthesis) in the mitochondria and catabolism of this ketoacid into isobutanol in the cytosol [35].

A number of heterologous bacterial genes encoding appropriate enzymes which lead to the increased production of isobutanol have been introduced into S. cereviasiae strains. The molecular mechanisms and genetic engineering of S. cereviasiae strains have been analytically described and reviewed by Buijs et al., 2013 [152]. This synthesis has been applied by Butalco, Butamax and Gevoare companies that have developed commercial production of isobutanol. Butamax applied the mitochondrial pathway [154], while Butalco [155] and Gevo [156] based their production on a cytosolic pathway.

3.4. Characteristics of S. cerevisiae influencing the fermentation process

The benefits of S. cerevisiae as GRAS ethanologen have been extensively reviewed and include high rates of ethanol tolerance and production, stress tolerance, flexibility in genetic improvement and effective adaptation for large scale fermentations. On the other hand, S. cerevisiae cannot ferment certain sugars such as pentoses. Another obstacle is the accumulation of trehalose to boost the membrane as a stress response. Also, in response to osmotic stress, S. cerevisiae synthesizes the compatible solute glycerol (especially in the presence of high sugar concentrations) to protect the cells from water loss; this response, however, results in reduction of the proportion of alcohol production, which is undesirable in bioethanol fermentations. Subsequently, flocculation (the aggregation of yeast cells into clumps) is a phenomenon that complicates the procedure in fuel alcohol plants, because flocculent yeasts do not remain in suspension to be in contact with the fermentable sugars for the duration of the fermentation [143].

Since bioethanol is produced in fuel plants (often called biorefineries to be discriminated from the petrochemical industry), the fermentation takes place in a bioreactor, where yeast should possess tolerance in high temperature and alcohol concentration, pH alteration, and, most importantly, the achievement of the highest level of alcohol production in the shortest possible time. Therefore, because yeast is the fermentation biocatalyst, understanding yeast physiology is a key to optimizing industrial alcohol production. Various types of yeast strains have been used in fermentation for ethanol production, including wild type and recombinants obtained as hybrids, by genetic engineering, immobilization, or yeast synthetic biology approaches [141],[143].

3.5. The process of bioethanol production

Nutrients supporting the fermentation of certain substrates are key factors as they influence the yeast growth, stress tolerance and ethanol production along with undesired by-products. For optimal alcohol production, yeast fermentation should be supplied with appropriate nutrients which include fermentable carbohydrates, sufficient nitrogen, vitamins, as well as oxygen at the beginning of the fermentation so that yeast synthesizes compounds as e.g. sterols to strengthen its membrane [157]. Minerals are also very important, as the decarboxylation of pyruvate by yeast is catalyzed by pyruvate decarboxylase and alcohol dehydrogenase, which in turn require magnesium and zinc, respectively [144].

Regardless of the kind of feed stocks used, these should firstly undergo a pretreatment in order to reduce in size and facilitate the next steps. Usually, steam explosion is the most efficient procedure combining low cost, non-environmental hazard and complete sugar recovery [158]. The substrates should then be converted to fermentable sugars, a process carried out by enzymatic treatment which provides high selectivity for each substrate, gentle treatment and low energy cost [159]. Usually, starch is converted to hexoses by alpha amylases, while hemicelluloses are converted to pentoses by glucoamylases. Lignocellulosic material is often converted to sugars by acidic hydrolysis. Fermentation is then carried out by yeasts and ethanol is further isolated by distillation and dehydration [137],[141].

The above processes are performed by three possible mechanisms: separate hydrolysis and fermentation (SHF) followed mainly for lignocellulosic materials, simultaneous saccharification and fermentation (SSF) and simultaneous saccharification and co-fermentation (SSCF). In SSF and SSCF, enzymatic hydrolysis and fermentation process occur simultaneously and therefore are preferred for their lower cost, higher ethanol yield and shorter processing time [160],[161].

Bioethanol is produced in the bioreactor by in fed-batch, repeated batch, continuous and semicontinuous conditions [141]. In batch fermentation, where everything is added in a closed system at the beginning, the reaction manipulation is simple [162], but the high sugar concentration can act as an inhibition factor for yeast growth and therefore ethanol production [163]. In continuous fermentation, on the contrary, a bioreactor containing the fermenting yeast is constantly supplied with substrates and supporting nutrients, while the products are steadily separated from the reaction mixture [164]. The benefits include smaller volumes which lead to increased product yield and lower cost [161]. On the other hand, the long cultivation time as well as contamination risks are disadvantages for this method [165]. In fed-batch fermentation, procedures of both batch and continuous conditions are combined in order to minimize the inhibitor effects on yeast activity caused by the substrate during the batch mode by keeping it in small amounts [165]. This process exhibits many benefits and has been reported to be productive in combination with non-uniform SSF system [166].

3.6. Manipulation of S. cerevisiae to confront stress conditions in the fermentor

In a bioreactor, factors like temperature increase, pH alteration, osmotic stress and ethanol concentration can cause S. cerevisiae multiple stress phenomena which, in turn, will affect ethanol production. Α variation between 20–35 °C is acceptable for S. cerevisiae with optimum temperature growth of 30 °C, while an increase to approx. 40 °C would induce the biosynthesis of stress-response factors as heat-shock proteins and trehalose among them. The upregulation of trehalose metabolizing genes induce, in turn, a number of other genes, (for instance, those involved in ergosterol biosynthesis [167]. Subsequently, enzymes produced by S. cerevisiae or added externally are temperature-sensitive ant thus inactivated [168], hence temperature is monitored during the whole process. Ethanol causes toxic effects in yeast membrane, which are confronted by the addition of protective nutrients [169],[170]. On the other hand, osmotic stress can occur from the overproduction of glycerol by yeast, mainly observed in biofuel production plants that utilize starch and sugar feedstocks. This causes toxic effects on cells and decreased alcohol fermentation and could be avoided by simultaneous saccharification and fermentation [143]. SSF can be applied in fermentations of lignocelluloses feed stocks for second-generation bioethanol production as well, because during this procedure, acetic acid, which inhibits yeast growth, is reduced [171].

Non-Saccharomyces strains and some bacteria have been observed as contaminants in bioethanol production and compete against Saccharomyces starters by synthesizing inhibitory products which inhibit yeast growth and ethanol productivity. The majority of the reports come from plant industries in Brazil using sugarcane, since Brazil is the largest producer of bioethanol from this feed stock. Dekkera, Schizosaccharomyces, and Candida spp. are often isolated, due to their tolerance to stress conditions [172]. On the other hand, the dominant bacteria belong to the genus Lactobacillus and exhibit high ethanol tolerance [173]. To overcome this obstacle, the cleaning and sterilization regulation should be strictly followed in plant industries [143].

3.7. Strain improvement and manipulation

There is a tremendous number of S. cerevisiae strains used in bioethanol industry described in detail (e. g. [143],[174]). The strain requirements for an application in plant industry should include, among other parameters, stress tolerance and ethanol productivity. It has been shown that wild-type S. cerevisiae strains exhibit a high potential in fermenting sugars to ethanol compared to the commercial ones. This is the case of Brazilian fuel ethanol plants where wild-type Saccharomyces strains have been isolated from sugarcane molasses [175], or wild-type S. cerevisiae KL17 which reached an exceptionally high ethanol concentration of 96.9 g/L with a productivity of 3.46 g/L/h after simultaneous fermentation of glucose and galactose [176].

On the other hand, improved strains can be obtained by evolutionary adaptation, in which a strain is subjected to a particular selective stress by serial inoculations in order to obtain spontaneous mutants that responded to the above conditions [177]. By this method a series of strains possessing important properties for ethanol production have been achieved, such as xylose utilization by yeast strains used in lignocellulose fermentation [178]. Moreover, by exhibiting yeast strains xylose plus acetic acid selective pressure, Wright et al., 2011 [179] isolated an improved strain fermenting xylose and resistant to acetic acid and proceeded thus furthermore in strain improvement to be used in second-generation ethanol fermentation.

In addition, enhanced strains for certain properties can be developed by application of classical genetics, such as hybridization by crossing or protoplast fusion, as well as mutagenesis. These methods have been extensively studied by Steensels et al., 2014 [174]. For instance, hybrid strains developed by protoplast fusion between S. cerevisiae and non-Saccharomyces strains which ferment xylose have been used for fermentation of Ipomea carnea biomass containing both hexoses and pentoses [180]. By hybrid strategies, appropriate strains have been developed (especially in ethanol tolerance) with natural method and therefore are not regarded as genetically modified (GM). On the other hand, many industrial strains are polyploid or aneuploid and therefore cannot be improved by mating procedures. By the crossing methods, many other characteristics apart from the desired are altered and could affect the fermentation procedure.

Cell immobilization is a common technology followed in fermentation procedures. It exhibits a series of advantages over free cell fermentation such as high density of active cells, higher rates of conversion, while the reaction time is shortened and the product isolation is facilitated. The immobilization types include adsorption, crosslinking, encapsulation and entrapment. The most popular immobilization method for yeast is adsorption, while calcium alginate has been the most suitable carrier. A number of immobilization procedures concerning strains, carriers and ethanol productivity have been reviewed by Azhar et al., 2017 [141]. Among them, a high ethanol production of 98.48 g/L has been achieved by fermentation of sweet sorghum juice from S. cerevisiae NP 01 immobilized by adsorption on sorghum stalk [181].

There is a remarkable variety of genetic modified yeast strains used in alcohol fuel plants, in which heterologous enzymes providing products of high added value have been introduced. Recombinant DNA technology is applied depending of the desired properties that should be provided by the recombinant strains. The reduction of glycerol production has been a challenge achieved for ethanol-producing yeasts [182]. For the fermentation of starch obtained from corn mashes (mainly in USA), commercial strains modified to express glucoamylase genes have been constructed. This way, starch is breaking down to glucose, which, in turn, is fermented in a SSF system, where glucose is slowly released and the osmotic stress is diminished [183]. Another obstacle confronted by GM yeasts is the release of pentoses after the pretreatment of lignocellulosic substrate, not fermented by wild S. cerevisiae strains. The introduction of heterologous xylose isomerase genes in wild type strains allows the complete utilization of lignocellulosic hydrolysates [184]. Thus, the development of robust yeast strains with improved metabolic pathways will continue to be critical for the fruitful operation of large-scale ethanol biorefineries.

Conflict of interest: All authors declare no conflicts of interest in this paper.

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