Refermentation and maturation of lambic beer in bottles: a necessary step for gueuze production.

The production of gueuze beers through refermentation and maturation of blends of lambic beer in bottles is a way for lambic brewers to cope with the variability among different lambic beer batches. The resulting gueuze beers are more carbonated than lambic beers and are supposed to possess a unique flavor profile that varies over time. To map this refermentation and maturation process for gueuze production, a blend of lambic beers was made and bottled, whereby one of them was produced with the old wheat landrace Zeeuwse Witte. Through the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high-throughput sequencing of bacterial and fungal amplicons, in combination with metabolite target analysis, new insights into gueuze production were obtained. During the initial stages of refermentation, the conditions in the bottles were similar to those encountered during the maturation phase of lambic beer productions in wooden barrels, which was also reflected microbiologically (presence of Brettanomyces species, Pediococcus damnosus, and Acetobacter lambici) and biochemically (ethanol, higher alcohols, lactic acid, acetic acid, volatile phenolic compounds, and ethyl esters). However, after a few weeks of maturation, a switch from a favorable environment to one with nutrient and dissolved oxygen depletion resulted in several changes. Concerning the microbiology, a sequential prevalence of three lactic acid bacterial species occurred, namely, P. damnosus, Lentilactobacillus buchneri, and Lactobacillus acetotolerans, while the diversity of the yeasts decreased. Concerning the metabolites produced, mainly those of the Brettanomyces yeasts determined the metabolic profiles encountered during later stages of the gueuze production.IMPORTANCEGueuze beers are the result of a refermentation and maturation process of a blend of lambic beers carried out in bottles. These gueuze beers are known to have a long shelf life, and their quality typically varies over time. However, knowledge about gueuze production in bottles is scarce. The present study provided more insights into the varying microbial and metabolite composition of gueuze beers during the first 2 years of this refermentation and maturation process. This will allow gueuze producers to gain more information about the influence of the refermentation and maturation time on their beers. These insights can also be used by gueuze producers to better inform their customers about the quality of young and old gueuze beers.

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.

Effect of overexpression of partial TDH1 and TDH2/3 gene sequences in a starter strain of industrial bioethanol fermentation on the Brettanomyces bruxellensis contaminant growth.

Selected Saccharomyces cerevisiae strains, such as the commercial Ethanol-Red (ER) strain, are used as starters in the bioethanol industry. Yet, bioethanol fermentations are prone to microbial contaminations, mainly by Brettanomyces bruxellensis and lactic acid bacteria. Chemicals, such as sulphuric acid and antibiotics, are commonly used to combat those contaminations, but they have negative environmental impacts. Recently, ER strain was found to secrete antimicrobial peptides (AMPs) active against B. bruxellensis. Therefore, the partial TDH1 and TDH2/3 genes sequences that codify those AMPs were inserted into the pSR41k plasmid and cloned in ER strains. The relative expression levels (plasmidic/genomic) of those sequences in the respective modified ER strains were quantified by real-time quantitative polimerase chain reaction (RT-qPCR), confirming their overexpression. The effect of the modified strains on B. bruxellensis (Bb) growth was then evaluated during synthetic must (SM) and carob syrup (CS) fermentations, co-inoculated with 105 cells ml-1 of ER and Bb in SM and with 106 of ER and 5 × 103 cells ml-1 of Bb in CS. Results showed that modified ER strains exerted a much higher inhibitory effect against B. bruxellensis (72-fold in SM and 10-fold in CS) than the non-modified ER strain. In those fermentations, 90-100 g l-1 of ethanol was produced in 3-6 days.

Addition of Saccharomyces eubayanus to SCOBY fermentations modulates the chemical and volatile compound profiles in kombucha.

Kombucha is a fermented beverage derived from a sweetened tea fermentation inoculated with a bacteria-yeast consortium referred to as Symbiotic Culture of Bacteria and Yeast (SCOBY). Different SCOBY cultures can impact the beverage's quality and make the whole process highly variable. Adding Saccharomyces yeast cultures to the fermentation process can avoid stalled fermentations, providing a reproducible beverage. Here, we explored using different Saccharomyces eubayanus strains together with SCOBY in the context of kombucha fermentation. Our results show that yeast x SCOBY co-cultures exhibited a robust fermentation profile, providing ethanol and acetic acid levels ranging from 0,18-1,81 %v/v and 0,35-1,15 g/L, respectively. The kombucha volatile compound profile of co-cultures was unique, where compounds such as Isopentyl acetate where only found in yeast x SCOBY fermentations. Metabarcoding revealed that the SCOBY composition was also dependent on the S. eubayanus genotype, where besides Saccharomyces, amplicon sequence variants belonging to Brettanomyces and Starmerella were detected. These differences concomitated global changes in transcript levels in S. eubayanus related to the metabolism of organic molecules used in kombucha fermentation. This study highlights the potential for exploring different S. eubayanus strains for kombucha fermentation, and the significant yeast genotype effect in the profile differentiation in this process.

Development of a molecular diagnostic test for the specific detection of Brettanomyces bruxellensis in red wine.

Brettanomyces bruxellensis is considered the main source of spoilage in red wine. This yeast, by producing volatile phenols, is responsible for the development of unpleasant aromas affecting the quality of final products and resulting in substantial economic losses for wine producers. This work therefore describes the development of an easy to-use colorimetric molecular diagnostic test for the rapid and specific detection of B. bruxellensis in wine. Detection was achieved using a sandwich hybridization format in which the target RNA was recognized by an immobilized DNA capture probe and a labelled DNA signal probe. The proposed device was highly specific to B. bruxellensis and showed a linear relationship between measured signal and target RNA concentration in the range 0.1-5 ng µL-1, with a limit of detection value of 0.1 ng µL-1 of total RNA. The colorimetric assay was validated on red wine samples, with a detection limit of 102 CFU mL-1. This study suggests that the reported method could be used for early detection of spoilage yeasts in wine and other alcoholic beverages.

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.

Comparative Zymocidial Effect of Three Different Killer Toxins against Brettanomyces bruxellensis Spoilage Yeasts.

Three killer toxins that were previously investigated, one excreted by Kluyveromyces wickerhamii and two by different strains of Wickerhamomyces anomalus, were produced at the pilot scale, lyophilized and characterized, and the formulates were assessed for their zymocidial effect against Brettanomyces bruxellensis spoilage yeast. A comparative analysis allowed the evaluation of the minimum inhibitory concentration (MIC) against a sensitive strain. Fungicidal and fungistatic concentrations were used to evaluate the cytocidal effect using a cytofluorimetric approach that confirmed the lethal effect of all lyophilized formulates against B. bruxellensis spoilage yeasts. Moreover, the potential killer toxins' cytotoxicity against human intestinal cells (Caco-2) were evaluated to exclude any possible negative effect on the consumers. Finally, the effective lethal effect of all three lyophilized killer toxins toward B. bruxellensis sensitive strain were tested. The results indicated that all of them acted without dangerous effects on the human epithelial cells, opening the way for their possible commercial application. In particular, D15 showed the lowest MIC and the highest activity, was evaluated also in wine, revealing a strong reduction of Brettamonyces yeast growth and, at the same time, a control of ethyl phenols production.

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.

Evaluating the influence of operational parameters of pulsed light on wine related yeasts: focus on inter- and intra-specific variability sensitivity.

In oenology, there is a growing demand by consumers for wines produced with less inputs (such as sulphite, frequently used for microbial control). Emerging control methods for managing microorganisms in wine are widely studied. In this study, the efficiency of pulsed light (PL) treatment was investigated. A drop-platted system was used to evaluate the impact of three PL operational parameters: the fluence per flash, the total fluence and the flash frequency. Fluence per flash appeared to be a key parameter prior to total fluence, thus demonstrating the importance of the effect of peak voltage during PL treatments. The efficiency of PL treatment was assessed on 198 strains distributed amongst fourteen yeast species related to wine environment, and an important variability in PL response was observed. Brettanomyces bruxellensis strains were strongly sensitive to PL, with intraspecific variation. PL was then applied to red wines inoculated with 9 strains of B. bruxellensis, Saccharomyces cerevisiae and Lachancea thermotolerans. Results confirmed interspecific response variability and a higher sensitivity of B. bruxellensis species to PL. Wine treatments with a total fluence of 22.8 J cm-2 resulted in more than 6 log reduction for different B. bruxellensis strains. These results highlight the potential of PL for wine microbial stabilization.

Rapid Detection of Brettanomyces bruxellensis in Wine by Polychromatic Flow Cytometry.

Brettanomyces bruxellensis is found in several fermented matrices and produces relevant alterations to the wine quality. The methods usually used to identify B. bruxellensis contamination are based on conventional microbiological techniques that require long procedures (15 days), causing the yeast to spread in the meantime. Recently, a flow cytometry kit for the rapid detection (1-2 h) of B. bruxellensis in wine has been developed. The feasibility of the method was assessed in a synthetic medium as well as in wine samples by detecting B. bruxellensis in the presence of other yeast species (Saccharomyces cerevisiae and Pichia spp.) and at the concentrations that produce natural contaminations (up to 105 cells/mL), as well as at lower concentrations (103-102 cells/mL). Wine samples naturally contaminated by B. bruxellensis or inoculated with four different strains of B. bruxellensis species together with Saccharomyces cerevisiae and Pichia spp., were analyzed by flow cytometry. Plate counts were carried out in parallel to flow cytometry. We provide evidence that flow cytometry allows the rapid detection of B. bruxellensis in simple and complex mixtures. Therefore, this technique has great potential for the detection of B. bruxellensis and could allow preventive actions to reduce wine spoilage.

Metabolic and biotechnological insights on the analysis of the Pdh bypass and acetate production in the yeast Dekkera bruxellensis.

The advancement of knowledge about the physiology of Dekkera bruxellensis has shown its potential for the production of fuel ethanol very close to the conventional fermenting yeast S. cerevisiae. However, some aspects of its metabolism remain uncovered. In the present study, the respiro-fermentative parameters of D. bruxellensis GDB 248 were evaluated under different cultivation conditions. The results showed that sucrose was more efficiently converted to ethanol than glucose, regardless the nitrogen source, which points out for the industrial efficiency of this yeast in sucrose-based substrate. The blockage of the cytosolic acetate production incremented the yeast fermentative efficiency by 27% (in glucose) and 14% (in sucrose). On the other hand, the presence of nitrate as inducer of acetate production reducing the production of ethanol. Altogether, these results settled the hypothesis that acetate metabolism is the main constraint for ethanol production. Besides, this acetate-generating pathway seems to exert some regulatory action on the flux and distribution of the carbon flowing through the central metabolism. These physiological aspects were corroborated by the relative expression analysis of key genes in the crossroad to ethanol, acetate and biomass formation. All the results were discussed in the light of the industrial potential of this yeast.

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.

Off-Flavor Removal from Sheep Placenta via Fermentation with Novel Yeast Strain Brettanomyces deamine kh3 Isolated from Traditional Apple Vinegar.

Animal placentae can be used as health-promoting food ingredients with various therapeutic efficacies, but their use is limited by their unpleasant odor and taste. This study aimed to investigate the possibility of deodorization of sheep placenta via yeast fermentation. A yeast strain was successfully isolated and identified as a novel Brettanomyces strain (Brettanomyces deamine kh3). The deodorizing efficacy of fermentation of the sheep placenta with B. deamine kh3 was evaluated by 42 panels, based on evaluation of preference, ranking, and aroma profiles, and compared with normal placenta and placenta fermented with B. bruxellensis. The results of the sensory evaluation indicated that fermentation of the sheep placenta with B. deamine kh3 may improve its palatability by increasing flavors such as that of grass (tree), rubber, and burnt, and by decreasing the odor and soy sauce flavor. Solid-phase microextraction-gas chromatography (SPME-GC) showed that major off-flavors in sheep placenta, such as ammonia, dimethyl disulfide, and 1,3-dioxolane, were completely diminished in the sheep placenta fermented with B. deamine kh3. This study presents those major volatile compounds, including 2-isobutyl\-4,4-dimethyl-1,3-dioxane, and 3-methyl-1-butanol, could be crucial in improving the palatability of the sheep placentae fermented with B. deamine kh3. This study provides a good starting point for the industrial application of a new deodorization method.

Application of Micropore Device for Accurate, Easy, and Rapid Discrimination of Saccharomyces pastorianus from Dekkera spp.

Traceability analysis, such as identification and discrimination of yeasts used for fermentation, is important for ensuring manufacturing efficiency and product safety during brewing. However, conventional methods based on morphological and physiological properties have disadvantages such as time consumption and low sensitivity. In this study, the resistive pulse method (RPM) was employed to discriminate between Saccharomyces pastorianus and Dekkera anomala and S. pastorianus and D. bruxellensis by measuring the ionic current response of cells flowing through a microsized pore. The height and shape of the pulse signal were used for the simultaneous measurement of the size, shape, and surface charge of individual cells. Accurate discrimination of S. pastorianus from Dekkera spp. was observed with a recall rate of 96.3 ± 0.8%. Furthermore, budding S. pastorianus was quantitatively detected by evaluating the shape of the waveform of the current ionic blockade. We showed a proof-of-concept demonstration of RPM for the detection of contamination of Dekkera spp. in S. pastorianus and for monitoring the fermentation of S. pastorianus through the quantitative detection of budding cells.

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.

Tailor-made microbial consortium for Kombucha fermentation: Microbiota-induced biochemical changes and biofilm formation.

Kombucha is a very distinct naturally fermented sweetened tea that has been produced for thousands of years. Fermentation relies on metabolic activities of the complex autochthonous symbiotic microbiota embedded in a floating biofilm and used as a backslop for successive fermentations. Here, we designed a tailor-made microbial consortium representative of the core Kombucha microbiota to drive this fermentation. Microbial (counts, metagenetics), physico-chemical (pH, density) and biochemical (organic acids, volatile compounds) parameters were monitored as well as biofilm formation by confocal laser scanning microscopy and scanning electron microscopy. While nine species were co-inoculated, four (Dekkera bruxellensis, Hanseniaspora uvarum, Acetobacter okinawensis and Liquorilactobacillus nagelii) largely dominated. Microbial activities led to acetic, lactic, succinic and oxalic acids being produced right from the start of fermentation while gluconic and glucuronic acids progressively increased. A distinct shift in volatile profile was also observed with mainly aldehydes identified early on, then high abundances of fatty acids, ketones and esters at the end. Correlation analyses, combining metabolomic and microbial data also showed a shift in species abundances during fermentation. We also determined distinct bacteria-yeast co-occurence patterns in biofilms by microscopy. Our study provides clear evidence that a tailor-made consortium can be successfully used to drive Kombucha fermentations.

Adaptive evolution of sulfite tolerance in Brettanomyces bruxellensis.

Brettanomyces bruxellensis is considered one of the most problematic microbes associated with wine production. Sulfur dioxide is commonly used to inhibit the growth of B. bruxellensis and limit the potential wine spoilage. Brettanomyces bruxellensis wine isolates can grow at higher concentrations of this preservative than isolates from other sources. Thus, it has been suggested that the use of sulfite may have selected for B. bruxellensis strains better adapted to survive in the winemaking environment. We utilized laboratory adaptive evolution to determine the potential for this to occur. Three B. bruxellensis strains, representative of known genetic variation within the species, were subjected to increasing sublethal sulfur dioxide concentrations. Individual clones isolated from evolved populations displayed enhanced sulfite tolerance, ranging from 1.6 to 2.5 times higher than the corresponding parental strains. Whole-genome sequencing of sulfite-tolerant clones derived from two of the parental strains revealed structural variations affecting 270 genes. The region containing the sulfite efflux pump encoding gene, SSU1, showed clear copy number variants in all sequenced clones. Regardless of parental strain genetic background, SSU1 copy number changes were reproducibly associated with one SSU1 haplotype. This work clearly demonstrates adaptive evolution of B. bruxellensis when exposed to sublethal sulfites and suggests that, similar to Saccharomyces cerevisiae wine yeast, the mechanism responsible involves the gene SSU1.

Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae.

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.

Comparative proteomic analyses reveal the metabolic aspects and biotechnological potential of nitrate assimilation in the yeast Dekkera bruxellensis.

The yeast Dekkera bruxellensis is well-known for its adaptation to industrial ethanol fermentation processes, which can be further improved if nitrate is present in the substrate. To date, the assimilation of nitrate has been considered inefficient because of the apparent energy cost imposed on cell metabolism. Recent research, however, has shown that nitrate promotes growth rate and ethanol yield when oxygen is absent from the environment. Given this, the present work aimed to identify the biological mechanisms behind this physiological behaviour. Proteomic analyses comparing four contrasting growth conditions gave some clues on how nitrate could be used as primary nitrogen source by D. bruxellensis GDB 248 (URM 8346) cells in anaerobiosis. The superior anaerobic growth in nitrate seems to be a consequence of increased cell metabolism (glycolytic pathway, production of ATP and NADPH and anaplerotic reactions providing metabolic intermediates) regulated by balanced activation of TORC1 and NCR de-repression mechanisms. On the other hand, the poor growth observed in aerobiosis is likely due to an oxidative stress triggered by nitrate when oxygen is present. These results represent a milestone regarding the knowledge about nitrate metabolism and might be explored for future use of D. bruxellensis as an industrial yeast. KEY POINTS: • Nitrate can be regarded as preferential nitrogen source for D. bruxellensis. • Oxidative stress limits the growth of D. bruxellensis in nitrate in aerobiosis. • Nitrate is a nutrient for novel industrial bioprocesses using D. bruxellensis.