Liquorilactobacillus: A Context of the Evolutionary History and Metabolic Adaptation of a Bacterial Genus from Fermentation Liquid Environments.

In the present work, we carried out a comparative genomic analysis to trace the evolutionary trajectory of the bacterial species that make up the Liquorilactobacillus genus, from the identification of genes and speciation/adaptation mechanisms in their unique characteristics to the identification of the pattern grouping these species. We present phylogenetic relationships between Liquorilactobacillus and related taxa such as Bacillus, basal lactobacilli and Ligilactobacillus, highlighting evolutionary divergences and lifestyle transitions across different taxa. The species of this genus share a core genome of 1023 genes, distributed in all COGs, which made it possible to characterize it as Liquorilactobacillus sensu lato: few amino acid auxotrophy, low genes number for resistance to antibiotics and general and specific cellular reprogramming mechanisms for environmental responses. These species were divided into four clades, with diversity being enhanced mainly by the diversity of genes involved in sugar metabolism. Clade 1 presented lower (< 70%) average amino acid identity with the other clades, with exclusive or absent genes, and greater distance in the genome compared to clades 2, 3 and 4. The data pointed to an ancestor of clades 2, 3 and 4 as being the origin of the genus Ligilactobacillus, while the species of clade 1 being closer to the ancestral Bacillus. All these traits indicated that the species of clade 1 could be soon separated in a distinct genus.

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.

Lawsone, a 2-hydroxy-1,4-naphthoquinone from Lawsonia inermis (henna), produces mitochondrial dysfunctions and triggers mitophagy in Saccharomyces cerevisiae.

Lawsone is a natural naphthoquinone present in the henna leaf extract with several cytotoxic activities and used as precursor for synthesis of various pharmaceutical compounds. Its biological activities are thought to be the result of oxidative stress generated, although the hydroxy group at position C-2 in its structure tends to reduce its electrophilic potential. In view of lack of knowledge on its activity, the present work aimed to elucidate the biological effect of lawsone using the yeast Saccharomyces cerevisiae. In the model strain BY4741 it was defined 229 mmol/L as the minimal inhibitory concentration (MIC). Using 172 mmol/L as sub-MIC value it was observed that yap1 deletion mutant was sensitive to lawsone independent the presence of oxygen. Lawsone affected yeast growth in glycerol, indicating interference in the respiratory metabolism. Intracellular content of thiol groups did not indicate intensive oxidative stress and the presence of the anti-oxidant N-acetylcysteine (NAC) exacerbated lawsone toxicity. By analysing the sensitivity of atg mutant strains and the localization of GFP-Atg8 fusion protein, it was concluded that lawsone primarily produces mitochondrial malfunctioning, leading to indirect oxidative stress. It triggers the autophagic response that ultimately induces mitophagy.

The effects of the Ncw2 protein of Saccharomyces cerevisiae on the positioning of chitin in response to cell wall damage.

The recently described NCW2 gene encodes a protein that is assumed to be located in the cell wall (CW). This protein was proposed to participate in the repair of CW damages induced by polyhexamethylene biguanide (PHMB). However, much of the information on the biological function(s) of Ncw2p still remains unclear. In view of this, this study seeks to extend the analysis of this gene in light of the way its protein functions in the Cell Wall Integrity (CWI) mechanism. Deletion of the NCW2 gene led to constitutive overexpression of some key CWI genes and increased chitin deposition in the walls of cells exposed to PHMB. This means the lack of Ncw2p might activate a compensatory mechanism that upregulates glucan CWI genes for cell protection by stiffening the CW. This condition seems to alleviate the response through the HOG pathway and makes cells sensitive to osmotic stress. However, Ncw2p may not have been directly involved in tolerance to osmotic stress itself. The results obtained definitely place the NCW2 gene in the list of CWI genes of S. cerevisiae and indicate that its protein has an auxiliary function in the maintenance of the glucan/chitin balance and ensuring the correct structure of the yeast cell wall.

Biological diversity of carbon assimilation among isolates of the yeast Dekkera bruxellensis from wine and fuel-ethanol industrial processes.

Dekkera bruxellensis is considered a spoilage yeast in winemaking, brewing and fuel-ethanol production. However, there is growing evidence in the literature of its biotechnological potential. In this work, we surveyed 29 D. bruxellensis isolates from three countries and two different industrial origins (winemaking and fuel-ethanol production) for the metabolization of industrially relevant sugars. The isolates were characterized by the determination of their maximum specific growth rates, and by testing their ability to grow in the presence of 2-deoxy-d-glucose and antimycin A. Great diversity was observed among the isolates, with fuel-ethanol isolates showing overall higher specific growth rates than wine isolates. Preferences for galactose (three wine isolates) and for cellobiose or lactose (some fuel-ethanol isolates) were observed. Fuel-ethanol isolates were less sensitive than wine isolates to glucose catabolite repression (GCR) induction by 2-deoxy-d-glucose. In strictly anaerobic conditions, isolates selected for having high aerobic growth rates were able to ferment glucose, sucrose and cellobiose at fairly high rates without supplementation of casamino acids or yeast extract in the culture medium. The phenotypic diversity found among wine and fuel-ethanol isolates suggests adaptation to these environments. A possible application of some of the GCR-insensitive, fast-growing isolates in industrial processes requiring co-assimilation of different sugars is considered.

Respiratory deficiency in yeast mevalonate kinase deficient may explain MKD-associate metabolic disorder in humans.

Mevalonate kinase deficiency (MKD) an orphan drug rare disease affecting humans with different clinical presentations, is still lacking information about its pathogenesis; no animal or cell model mimicking the genetic defect, mutations at MVK gene, and its consequences on the mevalonate pathway is available. Trying to clarify the effects of MVK gene impairment on the mevalonate pathway we used a yeast model, the erg12-d mutant strain Saccharomyces cerevisiae (orthologous of MKV) retaining only 10% of mevalonate kinase (MK) activity, to describe the effects of reduced MK activity on the mevalonate pathway. Since shortage of isoprenoids has been described in MKD, we checked this observation using a physiologic approach: while normally growing on glucose, erg12-d showed growth deficiency in glycerol, a respirable carbon source, that was not rescued by supplementation with non-sterol isoprenoids, such as farnesol, geraniol nor geranylgeraniol, produced by the mevalonate pathway. Erg12-d whole genome expression analysis revealed specific downregulation of RSF2 gene encoding general transcription factor for respiratory genes, explaining the absence of growth on glycerol. Moreover, we observed the upregulation of genes involved in sulphur amino acids biosynthesis that coincided with the increasing in the amount of proteins containing sulfhydryl groups; upregulation of ubiquinone biosynthesis genes was also detected. Our findings demonstrated that the shortage of isoprenoids is not the main mechanism involved in the respiratory deficit and mitochondrial malfunctioning of MK-defective cells, while the scarcity of ubiquinone plays an important role, as already observed in MKD patients.

On the catabolism of amino acids in the yeast Dekkera bruxellensis and the implications for industrial fermentation processes.

In the last years several reports have reported the capacity of the yeast Dekkera (Brettanomyces) bruxellensis to survive and adapt to the industrial process of alcoholic fermentation. Much of this feature seems to relate to the ability to assimilate limiting sources of nutrients, or somehow some that are inaccessible to Saccharomyces cerevisiae, in particular the sources of nitrogen. Among them, amino acids (AA) are relevant in terms of beverage musts, and could also be important for bioethanol. In view of the limited knowledge on the control of AA, the present work combines physiological and genetic studies to understand how it operates in D. bruxellensis in response to oxygen availibility. The results allowed separation of the AA in three groups of preferentiality and showed that glutamine is the preferred AA irrespective of the presence of oxygen. Glutamate and aspartate were also preferred AA in anaerobiosis, as indicated by the physiological data. Gene expression experiments showed that, apart from the conventional nitrogen catabolic repression mechanism that is operating in aerobiosis, there seems to be an oxygen-independent mechanism acting to overexpress key genes like GAP1, GDH1, GDH2 and GLT1 to ensure adequate anaerobic growth even in the presence of non-preferential nitrogen source. This could be of major importance for the industrial fitness of this yeast species.

NCW2, a Gene Involved in the Tolerance to Polyhexamethylene Biguanide (PHMB), May Help in the Organisation of ß-1,3-Glucan Structure of Saccharomyces cerevisiae Cell Wall.

In the present work, we provide biological evidences supporting the participation of NCW2 gene in the mechanism responsible for cell tolerance to polyhexamethylene biguanide (PHMB), an antifungal agent. The growth rate of yeast cells exposed to this agent was significantly reduced in ∆ncw2 strain and the mRNA levels of NCW2 gene in the presence of PHMB showed a 7-fold up-regulation. Moreover, lack of NCW2 gene turns yeast cell more resistant to zymolyase treatment, indicating that alterations in the ß-glucan network do occur when Ncw2p is absent. Computational analysis of the translated protein indicated neither catalytic nor transmembrane sites and reinforced the hypothesis of secretion and anchoring to cell surface. Altogether, these results indicated that NCW2 gene codes for a protein which participates in the cell wall biogenesis in yeasts and that Ncw2p might play a role in the organisation of the ß-glucan assembly.

Biosynthesis of higher alcohol flavour compounds by the yeast Saccharomyces cerevisiae: impact of oxygen availability and responses to glucose pulse in minimal growth medium with leucine as sole nitrogen source.

Higher alcohol formation by yeast is of great interest in the field of fermented beverages. Among them, medium-chain alcohols impact greatly the final flavour profile of alcoholic beverages, even at low concentrations. It is widely accepted that amino acid metabolism in yeasts directly influences higher alcohol formation, especially the catabolism of aromatic and branched-chain amino acids. However, it is not clear how the availability of oxygen and glucose metabolism influence the final higher alcohol levels in fermented beverages. Here, using an industrial Brazilian cachaça strain of Saccharomyces cerevisiae, we investigated the effect of oxygen limitation and glucose pulse on the accumulation of higher alcohol compounds in batch cultures, with glucose (20 g/l) and leucine (9.8 g/l) as the carbon and nitrogen sources, respectively. Fermentative metabolites and CO2 /O2 balance were analysed in order to correlate the results with physiological data. Our results show that the accumulation of isoamyl alcohol by yeast is independent of oxygen availability in the medium, depending mainly on leucine, α-keto-acids and/or NADH pools. High-availability leucine experiments showed a novel and unexpected accumulation of isobutanol, active amyl alcohol and 2-phenylethanol, which could be attributed to de novo biosynthesis of valine, isoleucine and phenylalanine and subsequent outflow of these pathways. In carbon-exhausted conditions, our results also describe, for the first time, the metabolization of isoamyl alcohol, isobutanol, active amyl alcohol but not of 2-phenylethanol, by yeast strains in stationary phase, suggesting a role for these higher alcohols as carbon source for cell maintenance and/or redox homeostasis during this physiological phase.

Production of sensory compounds by means of the yeast Dekkera bruxellensis in different nitrogen sources with the prospect of producing cachaça.

The distilled spirit made from sugar cane juice, also known as cachaça, is a traditional Brazilian beverage that in recent years has increased its market share among international distilled beverages. Several volatile compounds produced by yeast cells during the fermentation process are responsible for the unique taste and aroma of this drink. The yeast Dekkera bruxellensis has acquired increasing importance in the fermented beverage production, as the different metabolites produced by this yeast may be either beneficial or harmful to the end-product. Since D. bruxellensis is often found in the fermentation processes carried out in ethanol fuel distillation in Brazil, we employed this yeast to analyse the physiological profile and production of aromatic compounds and to examine whether it is feasible to regard it as a cachaça-producing microorganism. The assays were performed on a small scale and simulated the conditions for the production of handmade cachaça. The results showed that the presence of aromatic and branched-chain amino acids in the medium has a strong influence on the metabolism and production of flavours by D. bruxellensis. The assimilation of these alternative nitrogen sources led to different fermentation yields and the production of flavouring compounds. The influence of the nitrogen source on the metabolism of fusel alcohols and esters in D. bruxellensis highlights the need for further studies of the nitrogen requirements to obtain the desired level of sensory compounds in the fermentation. Our results suggest that D. bruxellensis has the potential to play a role in the production of cachaça.

Adaptation of Dekkera bruxellensis to lignocellulose-based substrate.

Adaptation of Dekkera bruxellensis to lignocellulose hydrolysate was investigated. Cells of D. bruxellensis were grown for 72 and 192 H in batch and continuous culture, respectively (adapted cells). Cultivations in semisynthetic medium were run as controls (nonadapted cells). To test the adaptation, cells from these cultures were reinoculated in the lignocellulose medium, and growth and ethanol production characteristics were monitored. Cells adapted to lignocellulose hydrolysate had a shorter lag phase, grew faster, and produced a higher ethanol concentration as compared with nonadapted cells. A stability test showed that after cultivation in rich medium, cells partially lost the adapted phenotype but still showed faster growth and higher ethanol production as compared with nonadapted cells. Because alcohol dehydrogenase genes have been described to be involved in the adaptation to furfural in Saccharomyces cerevisiae, an analogous mechanism of adaptation to lignocelluloses hydrolysate of D. bruxellensis was hypothesized. However, gene expression analysis showed that genes homologous to S. cerevisiae ADH1 were not involved in the adaptation to lignocelluloses hydrolysate in D. bruxellensis.

The fermentation of sugarcane molasses by Dekkera bruxellensis and the mobilization of reserve carbohydrates.

The yeast Dekkera bruxellensis is considered to be very well adapted to industrial environments, in Brazil, USA, Canada and European Countries, when different substrates are used in alcoholic fermentations. Our previous study described its fermentative profile with a sugarcane juice substrate. In this study, we have extended its physiological evaluation to fermentation situations by using sugarcane molasses as a substrate to replicate industrial working conditions. The results have confirmed the previous reports of the low capacity of D. bruxellensis cells to assimilate sucrose, which seems to be the main factor that can cause a bottleneck in its use as fermentative yeast. Furthermore, the cells of D. bruxellensis showed a tendency to deviate most of sugar available for biomass and organic acids (lactic and acetic) compared with Saccharomyces cerevisiae, when calculated on the basis of their respective yields. As well as this, the acetate production from molasses medium by both yeasts was in marked contrast with the previous data on sugarcane juice. Glycerol and ethanol production by D. bruxellensis cells achieved levels of 33 and 53 % of the S. cerevisiae, respectively. However, the ethanol yield was similar for both yeasts. It is worth noting that this yeast did not accumulate trehalose when the intracellular glycogen content was 30 % lower than in S. cerevisiae. The lack of trehalose did not affect yeast viability under fermentation conditions. Thus, the adaptive success of D. bruxellensis under industrial fermentation conditions seems to be unrelated to the production of these reserve carbohydrates.

Oenococcus alcoholitolerans sp. nov., a lactic acid bacteria isolated from cachaça and ethanol fermentation processes.

Four strains of lactic acid bacteria isolated from cachaça and alcohol fermentation vats in Brazil were characterised in order to determine their taxonomic position. Phylogenetic analysis revealed that they belong to the genus Oenococcus and should be distinguished from their closest neighbours. The 16S rRNA gene sequence similarity against the type strains of the other two species of the genus was below 94.76 % (Oenococcus kitaharae) and 94.62 % (Oenococcus oeni). The phylogeny based on pheS gene sequences also confirmed the position of the new taxon. DNA-DNA hybridizations based on in silico genome-to-genome comparison, Average Amino Acid Identity, Average Nucleotide Identity and Karlin genomic signature confirmed the novelty of the taxon. Distinctive phenotypic characteristics are the ability to metabolise sucrose but not trehalose. The name Oenococcus alcoholitolerans sp. nov. is proposed for this taxon, with the type strain UFRJ-M7.2.18(T) ( = CBAS474(T) = LMG27599(T)). In addition, we have determined a draft genome sequence of the type strain.

The influence of nitrate on the physiology of the yeast Dekkera bruxellensis grown under oxygen limitation.

A previous study showed that the use of nitrate by Dekkera bruxellensis might be an advantageous trait when ammonium is limited in sugarcane substrate for ethanol fermentation. The aim of the present work was to evaluate the influence of nitrate on the yeast physiology during cell growth in different carbon sources under oxygen limitation. If nitrate was the sole source of nitrogen, D. bruxellensis cells presented slower growth, diminished sugar consumption and growth-associated ethanol production, when compared to ammonium. These results were corroborated by the increased expression of genes involved in the pentose phosphate (PP) pathway, the tricarboxylic acid (TCA) cycle and ATP synthesis. The presence of ammonium in the mixed medium restored most parameters to the standard conditions. This work may open up a line of investigation to establish the connection between nitrate assimilation and energetic metabolism in D. bruxellensis and their influence on its fermentative capacity in oxygen-limited or oxygen-depleted conditions.

Quantitative aerobic physiology of the yeast Dekkera bruxellensis, a major contaminant in bioethanol production plants.

Dekkera bruxellensis has been described as the major contaminant yeast of industrial ethanol production, although little is known about its physiology. The aim of this study was to investigate the growth of this yeast in diverse carbon sources and involved conducting shake-flask and glucose- or sucrose-limited chemostats experiments, and from the chemostat data, the stoichiometry of biomass formation during aerobic growth was established. As a result of the shake-flask experiments with hexoses or disaccharides, the specific growth rates were calculated, and a different behavior in rich and mineral medium was observed concerning to profile of acetate and ethanol production. In C-limited chemostats conditions, the metabolism of this yeast was completely respiratory, and the biomass yields reached values of 0.62 gDW gS(-1) . In addition, glucose pulses were applied to the glucose- or sucrose-limited chemostats. These results showed that D. bruxellensis has a short-term Crabtree effect. While the glucose pulse was at the sucrose-limited chemostat, sucrose accumulated at the reactor, indicating the presence of a glucose repression mechanism in D. bruxellensis.

Physiology and gene expression profiles of Dekkera bruxellensis in response to carbon and nitrogen availability.

The assimilation of nitrate, a nitrogenous compound, was previously described as an important factor favoring Dekkera bruxellensis in the competition with Saccharomyces cerevisiae for the industrial sugarcane substrate. In this substrate, nitrogen sources are limited and diverse, and a recent report showed that amino acids enable D. bruxellensis to grow anaerobically. Thus, understanding the regulation of nitrogen metabolism is one fundamental aspect to comprehend the competiveness of D. bruxellensis in the fermentation environment. In the present study, we evaluated the physiological and transcriptional profiles of D. bruxellensis in response to different carbon and nitrogen supplies to determine their influence on growth, sugar consumption, and ethanol production. Besides, the expression of genes coding for nitrogen permeases and enzymes involved in the biosynthesis of glutamate and energetic metabolism were investigated under these conditions. Our data revealed that genes related to nitrogen uptake in D. bruxellensis are under the control of nitrogen catabolite repression. Moreover, we provide indications that glutamate dehydrogenase and glutamate synthase may switch roles as the major pathway for glutamate biosynthesis in D. bruxellensis. Finally, our data showed that in nonoptimal growth conditions, D. bruxellensis leans toward the respiratory metabolism. The results presented herein show that D. bruxellensis and S. cerevisiae share similar regulation of GDH­GOGAT pathway, while D. bruxellensis converts less glucose to ethanol than S. cerevisiae do when nitrogen is limited. The consequence of this particularity to the industrial process is discussed.

The Metabolism of Respiring Carbon Sources by Dekkera bruxellensis and Its Relation with the Production of Acetate.

Dekkera bruxellensis has been studied for several aspects of its metabolism over the past years, which has expanded our comprehension on its importance to industrial fermentation processes and uncovered its industrial relevance. Acetate is a metabolite often found in D. bruxellensis aerobic cultivations, whereas its production is linked to decreased ethanol yields. In a previous work, we aimed to understand how acetate metabolism affected the fermentation capacity of D. bruxellensis. In the present work, we evaluated the role of acetate metabolism in respiring cells using ammonium or nitrate as nitrogen sources. Our results showed that galactose is a strictly respiratory sugar and that a relevant part of its carbon is lost, while the remaining is metabolised through the Pdh bypass pathway before being assimilated into biomass. When this pathway was blocked, yeast growth was reduced while more carbon was assimilated to the biomass. In nitrate, more acetate was produced as expected, which increased carbon assimilation, although less galactose was uptaken from the medium. This scenario was not affected by the Pdh bypass inhibition. The confirmation that acetate production was crucial for carbon assimilation was brought by cultivations in pyruvate. All physiological data were connected to the expression patterns of PFK1, PDC1, ADH1, ALD3, ALD5 and ATP1 genes. Other respiring carbon sources could only be properly used by the cells when some external acetate was supplied. Therefore, the results reported herein helped in providing valuable contributions to the understanding of the oxidative metabolism in this potential industrial yeast.

The physiological characteristics of the yeast Dekkera bruxellensis in fully fermentative conditions with cell recycling and in mixed cultures with Saccharomyces cerevisiae.

The yeast Dekkera bruxellensis plays an important role in industrial fermentation processes, either as a contaminant or as a fermenting yeast. In this study, an analysis has been conducted of the fermentation characteristics of several industrial D. bruxellensis strains collected from distilleries from the Southeast and Northeast of Brazil, compared with Saccharomyces cerevisiae. It was found that all the strains of D. bruxellensis showed a lower fermentative capacity as a result of inefficient sugar assimilation, especially sucrose, under anaerobiosis, which is called the Custer effect. In addition, most of the sugar consumed by D. bruxellensis seemed to be used for biomass production, as was observed by the increase of its cell population during the fermentation recycles. In mixed populations, the surplus of D. bruxellensis over S. cerevisiae population could not be attributed to organic acid production by the first yeast, as previously suggested. Moreover, both yeast species showed similar sensitivity to lactic and acetic acids and were equally resistant to ethanol, when added exogenously to the fermentation medium. Thus, the effects that lead to the employment of D. bruxellensis in an industrial process and its effects on the production of ethanol are multivariate. The difficulty of using this yeast for ethanol production is that it requires the elimination of the Custer effect to allow an increase in the assimilation of sugar under anaerobic conditions.

A new set of reference genes for RT-qPCR assays in the yeast Dekkera bruxellensis.

The yeast Dekkera bruxellensis has been recently regarded as an important microorganism for bioethanol production owing to its ability to convert glucose, sucrose, and cellobiose to ethanol. The aim of this work was to validate a new set of reference genes for gene expression analysis by quantitative real-time PCR in D. bruxellensis and compare the influence of the method of choice for quantification of mRNA levels with the reliability of our data. Three candidate reference genes, DbEFA1, DbEFB1, and DbYNA1, were used in a quantitative analysis of 4 genes of interest, DbYNR1, DbTPS1, DbADH7, and DbUBA4, based on an approach for calculating the normalization factors by means of the geNorm applet. Each reference gene was also individually used for a 2(-ΔΔC(q)) (comparative C(q) method) calculation of the relative expression of genes of interest. Our results showed that the 3 reference genes provided enough stability and were complementary to the normalization factors method in different culture conditions. This work was able to confirm the usefulness of a previously reported reference gene, EFA1/TEF1, and increased the set of possible reference genes in D. bruxellensis to 4. Moreover, this can improve the reliability of the analysis of the regulation of gene expression in the industrial yeast D. bruxellensis.

The consequences of Lactobacillus vini and Dekkera bruxellensis as contaminants of the sugarcane-based ethanol fermentation.

This work describes the effects of the presence of the yeast Dekkera bruxellensis and the bacterium Lactobacillus vini on the industrial production of ethanol from sugarcane fermentation. Both contaminants were quantified in industrial samples, and their presence was correlated to a decrease in ethanol concentration and accumulation of sugar. Then, laboratory mixed-cell fermentations were carried out to evaluate the effects of these presumed contaminants on the viability of Saccharomyces cerevisiae and the overall ethanol yield. The results showed that high residual sugar seemed the most significant factor arising from the presence of D. bruxellensis in the industrial process when compared to pure S. cerevisiae cultures. Moreover, when L. vini was added to S. cerevisiae cultures it did not appear to affect the yeast cells by any kind of antagonistic effect under stable fermentations. In addition, when L. vini was added to D. bruxellensis cultures, it showed signs of being able to stimulate the fermentative activity of the yeast cells in a way that led to an increase in the ethanol yield.