Effect of abiotic and biotic factors on Brettanomyces bruxellensis bioadhesion properties.

Biofilms are central to microbial life because of the advantage that this mode of life provides, whereas the planktonic form is considered to be transient in the environment. During the winemaking process, grape must and wines host a wide diversity of microorganisms able to grow in biofilm. This is the case of Brettanomyces bruxellensis considered the most harmful spoilage yeast, due to its negative sensory effect on wine and its ability to colonise stressful environments. In this study, the effect of different biotic and abiotic factors on the bioadhesion and biofilm formation capacities of B. bruxellensis was analyzed. Ethanol concentration and pH had negligible effect on yeast surface properties, pseudohyphal cell formation or bioadhesion, while the strain and genetic group factors strongly modulated the phenotypes studied. From a biotic point of view, the presence of two different strains of B. bruxellensis did not lead to a synergistic effect. A competition between the strains was rather observed during biofilm formation which seemed to be driven by the strain with the highest bioadhesion capacity. Finally, the presence of wine bacteria reduced the bioadhesion of B. bruxellensis. Due to biofilm formation, O. oeni cells were observed attached to B. bruxellensis as well as extracellular matrix on the surface of the cells.

Impact of the acquired subgenome on the transcriptional landscape in Brettanomyces bruxellensis allopolyploids.

Gene expression variation can provide an overview of the changes in regulatory networks that underlie phenotypic diversity. Certain evolutionary trajectories such as polyploidization events can have an impact on the transcriptional landscape. Interestingly, the evolution of the yeast species Brettanomyces bruxellensis has been punctuated by diverse allopolyploidization events leading to the coexistence of a primary diploid genome associated with various haploid acquired genomes. To assess the impact of these events on gene expression, we generated and compared the transcriptomes of a set of 87 B. bruxellensis isolates, selected as being representative of the genomic diversity of this species. Our analysis revealed that acquired subgenomes strongly impact the transcriptional patterns and allow discrimination of allopolyploid populations. In addition, clear transcriptional signatures related to specific populations have been revealed. The transcriptional variations observed are related to some specific biological processes such as transmembrane transport and amino acids metabolism. Moreover, we also found that the acquired subgenome causes the overexpression of some genes involved in the production of flavor-impacting secondary metabolites, especially in isolates of the beer population.

High intraspecific variation of the cell surface physico-chemical and bioadhesion properties in Brettanomyces bruxellensis.

Brettanomyces bruxellensis is the most damaging spoilage yeast in the wine industry because of its negative impact on the wine organoleptic qualities. The strain persistence in cellars over several years associated with recurrent wine contamination suggest specific properties to persist and survive in the environment through bioadhesion phenomena. In this work, the physico-chemical surface properties, morphology and ability to adhere to stainless steel were studied both on synthetic medium and on wine. More than 50 strains representative of the genetic diversity of the species were considered. Microscopy techniques made it possible to highlight a high morphological diversity of the cells with the presence of pseudohyphae forms for some genetic groups. Analysis of the physico-chemical properties of the cell surface reveals contrasting behaviors: most of the strains display a negative surface charge and hydrophilic behavior while the Beer 1 genetic group has a hydrophobic behavior. All strains showed bioadhesion abilities on stainless steel after only 3 h with differences in the concentration of bioadhered cells ranging from 2.2 × 102 cell/cm2 to 7.6 × 106 cell/cm2. Finally, our results show high variability of the bioadhesion properties, the first step in the biofilm formation, according to the genetic group with the most marked bioadhesion capacity for the beer group.

Brettanomyces bruxellensis: Overview of the genetic and phenotypic diversity of an anthropized yeast.

Human-associated microorganisms are ideal models to study the impact of environmental changes on species evolution and adaptation because of their small genome, short generation time, and their colonization of contrasting and ever-changing ecological niches. The yeast Brettanomyces bruxellensis is a good example of organism facing anthropogenic-driven selective pressures. It is associated with fermentation processes in which it can be considered either as a spoiler (e.g., winemaking, bioethanol production) or as a beneficial microorganism (e.g., production of specific beers, kombucha). In addition to its industrial interests, noteworthy parallels and dichotomies with Saccharomyces cerevisiae propelled B. bruxellensis as a valuable complementary yeast model. In this review, we emphasize that the broad genetic and phenotypic diversity of this species is only beginning to be uncovered. Population genomic studies have revealed the coexistence of auto- and allotriploidization events with different evolutionary outcomes. The different diploid, autotriploid and allotriploid subpopulations are associated with specific fermented processes, suggesting independent adaptation events to anthropized environments. Phenotypically, B. bruxellensis is renowned for its ability to metabolize a wide variety of carbon and nitrogen sources, which may explain its ability to colonize already fermented environments showing low-nutrient contents. Several traits of interest could be related to adaptation to human activities (e.g., nitrate metabolization in bioethanol production, resistance to sulphite treatments in winemaking). However, phenotypic traits are insufficiently studied in view of the great genomic diversity of the species. Future work will have to take into account strains of varied substrates, geographical origins as well as displaying different ploidy levels to improve our understanding of an anthropized yeast's phenotypic landscape.

Molecular approaches improving our understanding of Brettanomyces physiology.

Brettanomyces species, and particularly B. bruxellensis as the most studied representative, are strongly linked to industrial fermentation processes. This association is considered either positive or undesirable depending on the industry. While in some brewing applications and in kombucha production Brettanomyces yeasts contribute to the flavour and aroma profile of these beverages, in winemaking and bioethanol production Brettanomyces is considered a spoilage or contaminant microorganism. Nevertheless, understanding Brettanomyces biology and metabolism in detail will benefit all industries. This review discusses recent molecular biology tools including genomics, transcriptomics, and genetic engineering techniques that can improve our understanding of Brettanomyces physiology and how these approaches can be used to make the industrial potential of this species a reality.

Non-Saccharomyces as Biotools to Control the Production of Off-Flavors in Wines.

Off-flavors produced by undesirable microbial spoilage are a major concern in wineries, as they affect wine quality. This situation is worse in warm areas affected by global warming because of the resulting higher pHs in wines. Natural biotechnologies can aid in effectively controlling these processes, while reducing the use of chemical preservatives such as SO2. Bioacidification reduces the development of spoilage yeasts and bacteria, but also increases the amount of molecular SO2, which allows for lower total levels. The use of non-Saccharomyces yeasts, such as Lachancea thermotolerans, results in effective acidification through the production of lactic acid from sugars. Furthermore, high lactic acid contents (>4 g/L) inhibit lactic acid bacteria and have some effect on Brettanomyces. Additionally, the use of yeasts with hydroxycinnamate decarboxylase (HCDC) activity can be useful to promote the fermentative formation of stable vinylphenolic pyranoanthocyanins, reducing the amount of ethylphenol precursors. This biotechnology increases the amount of stable pigments and simultaneously prevents the formation of high contents of ethylphenols, even when the wine is contaminated by Brettanomyces.

Survival and metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines.

Volatile phenols in wines are responsible for unpleasant aromas, which negatively affect the quality of the wine. These compounds are produced from the metabolism of hydroxycinnamic acids, mainly by the yeasts Brettanomyces/Dekkera. Relevant data, potentially useful to support decisions on how to manage the risk of contamination of wines by Brettanomyces/Dekkera, according to the grape varieties used in the vinification, is important to the wine industry. Therefore, the aim of this work was to evaluate the survival and the metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines. Yeast growth and survival were monitored in fifteen wines, five from each of the grape varieties Touriga Nacional, Cabernet Sauvignon and Syrah, inoculated with a strain of D. bruxellensis. Yeast culturable populations of 107 CFU mL-1 were reduced to undetectable numbers in 24 h in all wines. Plate counts of 104-106 CFU mL-1 were, however, detected after 48 h in most of Touriga Nacional and Cabernet Sauvignon wines and later in Syrah. Viability measurement by flow cytometry showed that a significant part of the populations was in a viable but non-culturable state (VBNC). The time required for the recovery of the culturable state was dependent on the wine, being longer on Syrah wines. Besides the production of ethylphenols, the metabolism of hydroxycinnamic acids by VBNC cells led to the accumulation of vinylphenols at relatively high levels, independently of the grape variety. The flow cytometry methodology showed a higher survival capacity of D. bruxellensis in Touriga Nacional wines, which corroborates with the higher amounts of volatile phenols found on this variety.

Carbohydrate composition of red wines during early aging and incidence on spoilage by Brettanomyces bruxellensis.

Wine is generally considered as hostile medium in which spoilage microbes have to manage with many abiotic factors among which low nutrient content. Wines elaborated in 8 wineries were sampled during the first summer of aging over two consecutive vintages, and analysed for carbohydrate composition. This revealed the systematic presence of many carbohydrates including those useful for the spoilage yeast Brettanomyces bruxellensis. However, during the first summer of aging, the changes in wine carbohydrate composition were low and it was difficult to assess how much carbohydrate composition contributed to wine spoilage by B. bruxellensis. Subsequent laboratory experiments in inoculated wines showed that the sugars preferentially consumed in wine by the spoilage yeast are d-glucose, d-fructose, and trehalose, whatever the yeast strain considered. The addition of these sugars to red wines accelerates the yeast growth and the volatile phenols formation. Although probably not the only promoting factor, the presence of high amounts of metabolisable sugars thus really increases the risk of "brett" spoilage.

Fermentation profiles of the yeast Brettanomyces bruxellensis in d-xylose and l-arabinose aiming its application as a second-generation ethanol producer.

The yeast Brettanomyces bruxellensis is able to ferment the main sugars used in first-generation ethanol production. However, its employment in this industry is prohibitive because the ethanol productivity reached is significantly lower than the observed for Saccharomyces cerevisiae. On the other hand, a possible application of B. bruxellensis in the second-generation ethanol production has been suggested because this yeast is also able to use d-xylose and l-arabinose, the major pentoses released from lignocellulosic material. Although the latter application seems to be reasonable, it has been poorly explored. Therefore, we aimed to evaluate whether or not different industrial strains of B. bruxellensis are able to ferment d-xylose and l-arabinose, both in aerobiosis and oxygen-limited conditions. Three out of nine tested strains were able to assimilate those sugars. When in aerobiosis, B. bruxellensis cells exclusively used them to support biomass formation, and no ethanol was produced. Moreover, whereas l-arabinose was not consumed under oxygen limitation, d-xylose was only slightly used, which resulted in low ethanol yield and productivity. In conclusion, our results showed that d-xylose and l-arabinose are not efficiently converted to ethanol by B. bruxellensis, most likely due to a redox imbalance in the assimilatory pathways of these sugars. Therefore, despite presenting other industrially relevant traits, the employment of B. bruxellensis in second-generation ethanol production depends on the development of genetic engineering strategies to overcome this metabolic bottleneck.

Biotransformation of Major Ginsenoside Rb1 to Rd by Dekkera anomala YAE-1 from Mongolian Fermented Milk (Airag).

Dekkera anomala YAE-1 strain separated from "airag" (Mongolian fermented mare's milk) produces ß-glucosidase, which can convert ginsenoside Rb1 from Panax ginseng. Ginseng-derived bioactive components such as ginsenoside Rb1 have various immunological and anticancer activities. Airag was collected from five different mare milk farms located near Ulaanbaatar, Mongolia. YAE-1 strains were isolated from airag to examine the hydrolytic activities of ß-glucosidase on Korean Panax ginseng using an API ZYM kit. Supernatants of selected cultures having ß-glucosidase activity were examined for hydrolysis of the major ginsenoside Rb1 at 40°C, pH 5.0. The YAE-1 strain was found to be nearly identical at 99.9% homology with Dekkera anomala DB-7B, and was thus named Dekkera anomala YAE-1. This strain exerted higher ß-glucosidase activity than other enzymes. Reaction mixtures from Dekkera anomala YAE-1 showed great capacity for converting ginsenoside Rb1 to ginsenoside Rd. The ß-glucosidase produced by Dekkera anomala YAE-1 was able to hydrolyze ginsenoside Rb1 and convert it to Rd during fermentation of the ginseng. The amount of ginsenoside Rd was highly increased from 0 to 1.404 mg/ml in fermented 20% ginseng root at 7 days.

Targeted gene deletion in Brettanomyces bruxellensis with an expression-free CRISPR-Cas9 system.

The ability to genetically manipulate microorganisms has been essential for understanding their biology and metabolism. Targeted genome editing relies on highly efficient homologous recombination, and while this is readily observed in the yeast Saccharomyces cerevisiae, most non-conventional yeast species do not display this trait and remain recalcitrant to targeted editing methods. CRISPR-based editing can bypass the requirement for high levels of native homologous recombination, enabling targeted modification to be more broadly implemented. While genetic transformation has been reported previously in Brettanomyces bruxellensis, a yeast with broad biotechnological potential and responsible for significant economic losses during the production of fermented beverages, targeted editing approaches have not been reported. Here, we describe the use of an expression-free CRISPR-Cas9 system, in combination with gene transformation cassettes tailored for B. bruxellensis, to provide the means for targeted gene deletion in this species. Deletion efficiency was shown to be dependent on homologous flanking DNA length, with higher targeting efficiencies observed with cassettes containing longer flanking regions. In a diploid strain, it was not possible to delete multiple alleles in one step, with heterozygous deletants only obtained when using DNA cassettes with long flanking regions. However, stepwise transformations (using two different marker genes) were successfully used to delete both wild-type alleles. Thus, the approach reported here will be crucial to understand the complex physiology of B. bruxellensis. Key points • The use of CRISPR-Cas9 enables targeted gene deletion in Brettanomyces bruxellensis. • Homozygous diploid deletions are possible with step-wise transformations. • Deletion of SSU1 confirmed the role of this gene in sulphite tolerance.

Transcriptomics unravels the adaptive molecular mechanisms of Brettanomyces bruxellensis under SO2 stress in wine condition.

Sulfur dioxide is generally used as an antimicrobial in wine to counteract the activity of spoilage yeasts, including Brettanomyces bruxellensis. However, this chemical does not exert the same effectiveness on different B. bruxellensis yeasts since some strains can proliferate in the final product leading to a negative sensory profile due to 4-ethylguaiacol and 4-ethylphenol. Thus, the capability of deciphering the general molecular mechanisms characterizing this yeast species' response in presence of SO2 stress could be considered strategic for a better management of SO2 in winemaking. A RNA-Seq approach was used to investigate the gene expression of two strains of B. bruxellensis, AWRI 1499 and CBS 2499 having different genetic backgrounds, when exposed to a SO2 pulse. Results revealed that sulphites affected yeast culturability and metabolism, but not volatile phenol production suggesting that a phenotypical heterogeneity could be involved for the SO2 cell adaptation. The transcriptomics variation in response to SO2 stress confirmed the strain-related response in B. bruxellensis and the GO analysis of common differentially expressed genes showed that the detoxification process carried out by SSU1 gene can be considered as the principal specific adaptive response to counteract the SO2 presence. However, nonspecific mechanisms can be exploited by cells to assist the SO2 tolerance; namely, the metabolisms related to sugar alcohol (polyols) and oxidative stress, and structural compounds.

Brettanomyces bruxellensis phenotypic diversity, tolerance to wine stress and wine spoilage ability.

Brettanomyces bruxellensis is a yeast species found in many fermented matrices. A high level of genetic diversity prevails in this species and was recently connected with tolerance to sulfur dioxide, the main preservative used in wine. We therefore examine other phenotypes that may modulate the ability of the species to spoil wine, in a selection of representative strains. The species shows a fairly high homogeneity with respect to the carbohydrates that can support growth, but more diverse behaviors regarding tolerance to low pH or ethanol. Thought no clear link can be drawn with genotype, some strains appear more tolerant than the others, mainly in the AWRI1499 like genetic group. Volatile phenol production is ubiquitous within the species, independent from yeast growth profile and not affected by the nature of the growth substrate. The specific production. n rate of volatile phenol production raises in case of increased aeration. It is little affected by pH decrease until 3.0 or by ethanol concentration increase up to 12% vol, but it decreased in case of increased constraint (pH < 3.0, Ethanol ≥14% vol) or combination of constraints. All the strain studied have thus the ability to spoil wine but some outstanding dangerous strains can even spoil the wine with high level of constrainst.

Identification and characterisation of two high-affinity glucose transporters from the spoilage yeast Brettanomyces bruxellensis.

The yeast Brettanomyces bruxellensis (syn. Dekkera bruxellensis) is an emerging and undesirable contaminant in industrial low-sugar ethanol fermentations that employ the yeast Saccharomyces cerevisiae. High-affinity glucose import in B. bruxellensis has been proposed to be the mechanism by which this yeast can outcompete S. cerevisiae. The present study describes the characterization of two B. bruxellensis genes (BHT1 and BHT3) believed to encode putative high-affinity glucose transporters. In vitro-generated transcripts of both genes as well as the S. cerevisiae HXT7 high-affinity glucose transporter were injected into Xenopus laevis oocytes and subsequent glucose uptake rates were assayed using 14C-labelled glucose. At 0.1 mM glucose, Bht1p was shown to transport glucose five times faster than Hxt7p. pH affected the rate of glucose transport by Bht1p and Bht3p, indicating an active glucose transport mechanism that involves proton symport. These results suggest a possible role for BHT1 and BHT3 in the competitive ability of B. bruxellensis.

Chromosomal genome assembly of the ethanol production strain CBS 11270 indicates a highly dynamic genome structure in the yeast species Brettanomyces bruxellensis.

Here, we present the genome of the industrial ethanol production strain Brettanomyces bruxellensis CBS 11270. The nuclear genome was found to be diploid, containing four chromosomes with sizes of ranging from 2.2 to 4.0 Mbp. A 75 Kbp mitochondrial genome was also identified. Comparing the homologous chromosomes, we detected that 0.32% of nucleotides were polymorphic, i.e. formed single nucleotide polymorphisms (SNPs), 40.6% of them were found in coding regions (i.e. 0.13% of all nucleotides formed SNPs and were in coding regions). In addition, 8,538 indels were found. The total number of protein coding genes was 4897, of them, 4,284 were annotated on chromosomes; and the mitochondrial genome contained 18 protein coding genes. Additionally, 595 genes, which were annotated, were on contigs not associated with chromosomes. A number of genes was duplicated, most of them as tandem repeats, including a six-gene cluster located on chromosome 3. There were also examples of interchromosomal gene duplications, including a duplication of a six-gene cluster, which was found on both chromosomes 1 and 4. Gene copy number analysis suggested loss of heterozygosity for 372 genes. This may reflect adaptation to relatively harsh but constant conditions of continuous fermentation. Analysis of gene topology showed that most of these losses occurred in clusters of more than one gene, the largest cluster comprising 33 genes. Comparative analysis against the wine isolate CBS 2499 revealed 88,534 SNPs and 8,133 indels. Moreover, when the scaffolds of the CBS 2499 genome assembly were aligned against the chromosomes of CBS 11270, many of them aligned completely, some have chunks aligned to different chromosomes, and some were in fact rearranged. Our findings indicate a highly dynamic genome within the species B. bruxellensis and a tendency towards reduction of gene number in long-term continuous cultivation.

Competition experiments between Brettanomyces bruxellensis strains reveal specific adaptation to sulfur dioxide and complex interactions at intraspecies level.

Recent studies have suggested a strong niche adaptation for Brettanomyces bruxellensis strains according to human-related fermentation environments, including beer, wine and bioethanol. This is further supported by a correlation between B. bruxellensis genetic grouping and tolerance to SO2, the main antimicrobial used in wine. The allotriploid AWRI1499-like cluster, in particular, shows high SO2 tolerance suggesting that the genetic configuration observed for these strains may confer a selective advantage in winemaking conditions. To test this hypothesis, we evaluated the relative selective advantage of representatives of the three main B. bruxellensis genetic groups in presence of SO2. As a proof-of-concept and using recently developed transformation cassettes, we compared strains under different SO2 concentrations using pairwise competitive fitness experiments. Our results showed that AWRI1499 is specifically adapted to environments with high SO2 concentrations compared to other B. bruxellensis wine strains, indicating a potential correlation between allotriploidisation origin and environmental adaptation in this species. Additionally, our findings suggest different types of competition between strains, such as coexistence and exclusion, revealing new insights on B. bruxellensis interactions at intraspecies level.

Influence of wine composition on consumer perception and acceptance of Brettanomyces metabolites using temporal check-all-that-apply methodology.

Brettanomyces spoilage in wine is due to the production of metabolites, which together create the distinctive 'Bretty' aroma and flavor profile associated with wine. The objective of this study was to assess the influence of three wine flavor matrices on consumer acceptance and the temporal sensory properties of wines containing high and low concentrations of Brettanomyces-metabolites. A commercial Shiraz red wine was altered through additions of whiskey lactone (oaky) and 2-isobutyl-3-methoxypyrazine (green). The Shiraz wines (unaltered, oak and green) were spiked with either low or high concentrations of 4-ethylphenol (4-EP), 4-ethylguaiacol (4-EG), and isovaleric acid (IA). All wines were evaluated by consumers (n = 105) using check-all-that-apply (CATA) for wine aroma. In-mouth flavor and mouthfeel perceptions were evaluated with temporal check-all-that-apply (TCATA) and a ranking evaluation where the top three most prominent attributes were reported. Lastly, consumers evaluated each sample on overall liking. Consumers were classified as having low, medium, or high wine knowledge level, in addition to wine industry experience. Differences in flavor and aroma attribute citation across all wine samples were described by consumers. In comparing oak and green treatments, the presence of whiskey lactone in the oak wine more strongly masked Brettanomyces associated aromas than did a 2-isobutyl-3-methoxypyrazine in the green wine. Brettanomyces metabolite-associated flavor terms commonly increased in citations by consumers when concentrations of 4-EP, 4-EG, and IA were increased from the low to high Brett levels (p < .05). At the high Brett treatments, citations of Band-Aid®, smoky, and leather flavor attributes were all significantly lower when oak was present. Consumers identified as having wine industry experience had lower liking ratings for the wine samples as compared to those without experience (p < .05). Results demonstrated the influence of wine composition on the perception of Brettanomyces metabolites, and provided valuable information to the wine industry as to how composition, or further wine style may influence the perception of wine spoilage aroma and flavors.

4-Ethylphenol, 4-ethylguaiacol and 4-ethylcatechol in red wines: Microbial formation, prevention, remediation and overview of analytical approaches.

The presence of 4-ethylphenol, 4-ethylguaiacol and 4-ethylcatechol in red wines affect negatively their aroma conferring horsy, barnyard, smoky and medicinal aromatic notes. These volatile phenols formed from free hydroxycinnamic acids and their ethyl esters by Dekkera/Brettanomyces yeasts, can contaminate wines. Their formation can cause serious negative economic impact to the wine industry worldwide as consumers tend to reject these wines. For these reasons various preventive and remedial treatments have been studied. This review summarises the wine microbial volatile phenols formation, preventive measures during winemaking and remedial treatments in finished wines along with their advantages and limitations for dealing with this sensory defect and impact on wine quality. Also it is important to control the levels of volatile phenols in wines using fast and convenient analytical methods namely with a detection limit below their olfactory perception threshold. The analytical methods available for quality control and performance characteristics as well their advantages and disadvantages when dealing with a complex matrix like wine are discussed in detail.

Novel antimicrobial peptides produced by Candida intermedia LAMAP1790 active against the wine-spoilage yeast Brettanomyces bruxellensis.

Brettanomyces bruxellensis negatively impacts on the sensorial quality of wine by producing phenolic compounds associated with unpleasant odors. Thus, the control of this spoilage yeast is a critical factor during the winemaking process. A recent approach used to biocontrol undesired microorganisms is the use of yeast released antimicrobial peptides (AMPs), but this strategy has been poorly applied to wine-related microorganisms. The aim of this study was to evaluate the antifungal capacity of Candida intermedia LAMAP1790 against wine-spoilage strains of B. bruxellensis and fermentative strains of Saccharomyces cerevisiae, and also to determine the chemical nature of the compound. The exposure of strains to the supernatant of C. intermedia saturated cultures showed antifungal activity against B. bruxellensis, without affecting the growth of S. cerevisiae. By fractionation and concentration of C. intermedia supernatants, it was determined that the antifungal activity was related to the presence of heat-labile peptides with molecular masses under 5 kDa. To our knowledge, this is the first report of AMPs secreted by C. intermedia that control B. bruxellensis. This could lead to the development of new biocontrol strategies against this wine-spoilage yeast.

The raise of Brettanomyces yeast species for beer production.

The adequate application of Brettanomyces species could raise a potential opportunity for the beer industry, generating new products and optimizing production processes. Several valuable properties like high ethanol yield, tolerance to low pH and production of unique flavors have brought this yeast species into the spotlight. Aroma and flavor production of Brettanomyces in beer is currently under discussion, and it can be adjusted if the mechanism insights are understood. This review summarizes the recent findings in physiological, genetic and biochemical traits related to the application of Brettanomyces species for brewing.