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The isolation and selection of yeast strains to improve the quality of the cachaça-Brazilian Spirit-have been studied in our research group. Our strategy considers Saccharomyces cerevisiae as the predominant species involved in sugarcane juice fermentation and the presence of different stressors (osmolarity, temperature, ethanol content, and competition with other microorganisms). It also considers producing balanced concentrations of volatile compounds (higher alcohols and acetate and/or ethyl esters), flocculation capacity, and ethanol production. Since the genetic bases behind these traits of interest are not fully established, the whole genome sequencing of 11 different Saccharomyces cerevisiae strains isolated and selected from different places was analyzed to identify the presence of a specific genetic variation common to cachaça yeast strains. We have identified 20,128 single-nucleotide variants shared by all genomes. Of these shared variants, 37 were new variants (being six missenses), and 4,451 were identified as missenses. We performed a detailed functional annotation (using enrichment analysis, protein-protein interaction network analysis, and database and in-depth literature searches) of these new and missense variants. Many genes carrying these variations were involved in the phenotypes of flocculation, tolerance to fermentative stresses, and production of volatile compounds and ethanol. These results demonstrate the existence of a genetic profile shared by the 11 strains under study that could be associated with the applied selective strategy. Thus, this study points out genes and variants that may be used as molecular markers for selecting strains well suited to the fermentation process, including genetic improvement by genome editing, ultimately producing high-quality beverages and adding value.IMPORTANCEThis work demonstrates the existence of new genetic markers related to different phenotypes used to select yeast strains and mutations in genes directly involved in producing flavoring compounds and ethanol, and others related to flocculation and stress resistance.

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The fractionation of sugarcane bagasse (SB) by hydrothermal pretreatment (HP, autohydrolysis) followed by alkaline extraction (AE) and advanced oxidative pretreatment (AOP) for production of second-generation ethanol and biogas was investigated. The AOP of SB was optimized using a Doehlert design, varying the applied H2O2 load, liquid-to-solid ratio (LSR), and time. The responses evaluated were yield (Y), residual cellulose (RC), delignification (DE), and enzymatic conversion (EC). The AE of SB pretreated by HP led to 61.8% DE (using 0.2 mol L-1 NaOH). This high lignin removal enabled substantial savings of H2O2 in the AOP. The optimized AOP conditions led to 78% Y, 82.2% RC, 42.7% DE, and 88.9% EC (overall glucose yield of 60.9%). Fermentation of the enzymatic hydrolysate with Saccharomyces cerevisiae yielded 190.8 Lethanol tonSB-1. Biogas production by anaerobic digestion of residual liquid streams of the pretreatment steps yielded 27.46 NLCH4 kgSB-1. An energy balance was estimated for the SB fractionation.

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Wickerhamomyces anomalus strain LBCM1105 was originally isolated from the wort of cachaça (the Brazilian fermented sugarcane juice-derived Brazilian spirit) and has been shown to grow exceptionally well at high amounts of glycerol. This paramount residue from the biodiesel industry is a promising cheap carbon source for yeast biotechnology. The assessment of the physiological traits underlying the W. anomalus glycerol consumption ability in opposition to Saccharomyces cerevisiae is presented. A new WaStl1 concentrative glycerol-H+ symporter with twice the affinity of S. cerevisiae was identified. As in this yeast, WaSTL1 is repressed by glucose and derepressed/induced by glycerol but much more highly expressed. Moreover, LBCM1105 aerobically growing on glycerol was found to produce ethanol, providing a redox escape to compensate the redox imbalance at the level of cyanide-resistant respiration (CRR) and glycerol 3P shuttle. This work is critical for understanding the utilization of glycerol by non-Saccharomyces yeasts being indispensable to consider their industrial application feeding on biodiesel residue.

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This work was performed to verify the potential of yeast strains isolated from cachaça distilleries for two specific biotechnological applications: beer and bioethanol production. In the beer production, the strains were tested for characteristics required in brewery practices, such as: capacity to ferment maltose and maltotriose, ability to grow at lowest temperatures, low H2S production, and flocculation profile. Among the strains tested, two of them showed appropriate characteristics to produce two different beer styles: lager and ale. Moreover, both strains were tested for cachaça production and the results confirmed the capacity of these strains to improve the quality of cachaça. In the bioethanol production, the fermentation process was performed similarly to that used by bioethanol industries: recycling of yeast biomass in the fermentative process with sulfuric acid washings (pH 2.0). The production of ethanol, glycerol, organic acids, dry cell weight, carbohydrate consumption, and cellular viability were analyzed. One strain presented fermentative parameters similar to PE2, industrial/commercial strain, with equivalent ethanol yields and cellular viability during all fermentative cycles. This work demonstrates that cachaça distilleries seem to be an interesting environment to select new yeast strains to be used in biotechnology applications as beer and bioethanol production.

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In yeast, as in other eukaryotes, calcium plays an essential role in signaling transduction to regulate different processes. Many pieces of evidence suggest that glucose-induced activation of plasma membrane H+-ATPase, essential for yeast physiology, is related to calcium signaling. Until now, no protein that could be regulated by calcium in this context has been identified. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, by using different approaches, we obtained many pieces of evidence suggesting that the requirement of calcium signaling for activation of the plasma membrane H+-ATPase is due to its requirement for activation of Lpx1p. According to the current model, activation of Lpx1p would cause hydrolysis of an acetylated tubulin that maintains the plasma membrane H+-ATPase in an inactive state. Therefore, after its activation, Lpx1p would hydrolyze the acetylated tubulin making the plasma membrane H+-ATPase accessible for phosphorylation by at least one protein kinase.

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The development of hybrids has been an effective approach to generate novel yeast strains with optimal technological profile for use in beer production. This study describes the generation of a new yeast strain for lager beer production by direct mating between two Saccharomyces cerevisiae strains isolated from cachaça distilleries: one that was strongly flocculent, and the other with higher production of acetate esters. The first step in this procedure was to analyze the sporulation ability and reproductive cycle of strains belonging to a specific collection of yeasts isolated from cachaça fermentation vats. Most strains showed high rates of sporulation, spore viability, and homothallic behavior. In order to obtain new yeast strains with desirable properties useful for lager beer production, we compare haploid-to-haploid and diploid-to-diploid mating procedures. Moreover, an assessment of parental phenotype traits showed that the segregant diploid C2-1d generated from a diploid-to-diploid mating experiment showed good fermentation performance at low temperature, high flocculation capacity, and desirable production of acetate esters that was significantly better than that of one type lager strain. Therefore, strain C2-1d might be an important candidate for the production of lager beer, with distinct fruit traces and originating using a non-genetically modified organism (GMO) approach.IMPORTANCE Recent work has suggested the utilization of hybridization techniques for the generation of novel non-genetically modified brewing yeast strains with combined properties not commonly found in a unique yeast strain. We have observed remarkable traits, especially low temperature tolerance, maltotriose utilization, flocculation ability, and production of volatile aroma compounds, among a collection of Saccharomyces cerevisiae strains isolated from cachaça distilleries, which allow their utilization in the production of beer. The significance of our research is in the use of breeding/hybridization techniques to generate yeast strains that would be appropriate for producing new lager beers by exploring the capacity of cachaça yeast strains to flocculate and to ferment maltose at low temperature, with the concomitant production of flavoring compounds.

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This study identified phenotypic traits appropriate for biotechnological applications of 118 yeasts isolated from cachaça distilleries. Different properties were verified: capacity to use alternative carbon sources; ability to tolerate high concentrations of sucrose, ethanol, methanol, aluminum and zinc as well as different pH values and foam production. Pichia guilliermondii and Pichia anomala strains were identified as the most promising ones for application in the second-generation biofuel industry, showing ability to grow on high glycerol concentrations. Other isolates, identified as Saccharomyces cerevisiae, produced bioethanol comparable to the industrial strains, and were therefore ideal for use in the first-generation ethanol industry. Some of these strains also showed high resistance to aluminum, as observed in sugarcane juice, and to inter-cycle washings with diluted sulphuric acid, as performed in the industrial bioethanol production process. In summary, yeast isolates from cachaça distilleries displayed robustness and phenotypic plasticity, which makes them interesting for biotechnological applications.

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In cachaça production, the use of yeast cells as starters with predictable flocculation behavior facilitates the cell recovery at the end of each fermentation cycle. Therefore, the aim of this work was to explain the behavior of cachaça yeast strains in fermentation vats containing sugarcane through the determination of biochemical and molecular parameters associated with flocculation phenotypes. By analyzing thirteen cachaça yeast strains isolated from different distilleries, our results demonstrated that neither classic biochemical measurements (e.g., percentage of flocculation, EDTA sensitivity, cell surface hydrophobicity, and sugar residues on the cell wall) nor modern molecular approaches, such as polymerase chain reaction (PCR) and real-time PCR (q-PCR), were sufficient to distinctly classify the cachaça yeast strains according to their flocculation behavior. It seems that flocculation is indeed a strain-specific phenomenon that is difficult to explain and/or categorize by the available methodologies.

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In this work, we have used classical genetics techniques to find improved starter strains to produce cachaça with superior sensorial quality. Our strategy included the selection of yeast strains resistant to 5,5',5″-trifluor-D: ,L: -leucine (TLF) and cerulenin, since these strains produce higher levels of higher alcohols and esters than parental strains. However, no clear relationship was observed when levels of flavoring compounds were compared with the levels expression of the genes (BAT1, BAT2, ATF2, EEB1 genes) involved with the biosynthesis of flavoring compounds. Furthermore, we determined the stability of phenotypes considered as the best indicators of the quality of the cachaça for a parental strain and its segregants. By applying the principal component analysis, a cluster of segregants, showing a high number of characteristics similar to the parental strain, was recognized. One segregant, that was resistant to TLF and cerulenin, also showed growth stability after six consecutive replications on plates containing high concentrations of sugar and ethanol. "Cachaça" produced at laboratory scale using a parental strain and this segregant showed a higher level of flavoring compounds. Both strains predominated in an open fermentative process through seven cycles, as was shown by mitochondrial restriction fragment length polymorphisms analysis. Based on the physical chemical composition of the obtained products, the results demonstrate the usefulness of the developed strategies for the selection of yeast strains to be used as starters in "cachaça" production.

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Saccharomyces cerevisiae strains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachaça, the Brazilian sugar cane spirit. The methodology established took into account the screening for biochemical traits desirable in a yeast cachaça producer, such as no H2S production, high tolerance to ethanol and high temperatures, high fermentative capacity, and the abilities to flocculate and to produce mycocins. Furthermore, the yeasts were exposed to drugs such as 5,5',5"-trifluor-D,L-leucine and cerulenin to isolate those that potentially overproduce higher alcohols and esters. The utilization of a random amplified polymorphic DNA-PCR method with primers based on intron splicing sites flanking regions of the COX1 gene, as well as microsatellite analysis, was not sufficient to achieve good differentiation among selected strains. In contrast, karyotype analysis allowed a clear distinction among all strains. Two selected strains were experimentally evaluated as cachaça producers. The results suggest that the selection of strains as fermentation starters requires the combined use of biochemical and molecular criteria to ensure the isolation and identification of strains with potential characteristics to produce cachaça with a higher quality standard.

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