Bruxellensis Research Articles

Potential Utilization of Sugarcane Bagasse as Raw Material for Bioethanol Production

Bagasse has the potential to generate alternative energy resources such as bioethanol owing to its lignocellulose structure. This research aims for utilizing bagasse as the raw material for producing bioethanol. The process for producing bioethanol consists of delignification, hydrolysis, fermentation and distillation using biocatalyst namely Fungi Dekkera bruxellensis and yeast Saccharomyces cerevisiae. Delignification was conducted at the substrate variation of 5, 10, 15 grams and 3, 5, 7 days contact time. The hydrolysis process used H2SO4 2% with 30, 45, 60 minutes contact time. Fermentation used concentration ratio for fungi and yeast at 1:1 with 3, 5, 7 days contact time. The distillation process went through the temperature of 78-80°C to obtain ethanol. The gravimetric analysis shows the optimum removal efficiency at 10.90% using 5 gram substrates with 3 days contact time. The hydrolysis process shows the highest glucose concentration at 16.64 g/L for each contact time using 15 gram substrates and H2SO4 2%. The highest amount of ethanol was 3.1% with the ratio of substrates 1:1 and 3 days contact time. This research shows that bagasse can produce bioethanol by the utilization of Fungi Dekkera bruxellensis and yeast Saccharomyces cerevisiae.

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.

Which microorganisms contribute to mousy off-flavour in our wines?

In recent years, the frequency of occurrence of mousy off-flavours in wines has increased. This could be caused by the significant decrease in sulphur dioxide addition during processing, the increase in pH or even the trend for spontaneous fermentation in wine. This off-flavour was associated with Brettanomyces bruxellensis or lactic acid bacteria metabolisms. Three N-heterocyclic compounds (APY, ETHP, ATHP) were described as involved in mousiness perception. Thus far, no study addressed the variability in that N-heterocycles production according to microorganism strains from different species. Twenty-five wines presenting mousy off-flavour were analysed. In total, 252 bacteria with 90.5 % of Oenococcus oeni and 101 yeast strains with 53.5 % of Saccharomyces cerevisiae were isolated and identified. Their capacity to produce mousy compounds was investigated using Stir Bar Sorptive Extraction-Gas Chromatography-Mass Spectrometry (SBSE-GC-MS) and a standardised N-heterocycle assay medium. While four and three species of yeast and bacteria, respectively, were isolated from mousy wines, only three species of microorganisms were associated with N-heterocycles production: B. bruxellensis, Lentilactobacillus hilgardii and Oenococcus oeni. The screening was then extended to collection strains for these three species to improve their genetic representativity. Our results show that the levels and the ratios of the three N-heterocycles present huge variations according to the species. In addition, it has been shown that in most mousy wines, B. bruxellensis was not found. Finally, an interesting correlation between ATHP and ETHP was identified.

Influence of mineral ions on the growth and fermentation performance of Dekkera bruxellensis GDB248.

The excess of minerals in the industrial substrates is detrimental for Saccharomyces cerevisiae ethanol fermentation performance. In this work, we sought to understand the effect of some of those minerals on the physiology of Dekkera bruxellensis. Three groups of minerals were classified on the basis of the aerobic growth profiles on glucose: neutrals (K+, Mg2+, P5+ and Zn2+), inducers (Mn2+ and Ca2+) and inhibitors (Al3+, Cu2+ and Fe2+). Cu2+ showed the highest mineral toxicity, and its effect was dependent of the level of medium aeration. On the other hand, copper stimulated respiration by increasing growth on respiratory carbon sources. Most growth inhibitors also hampered glucose fermentation, with changes in carbon distribution to metabolic routes dedicated to anabolic reactions and for alternative reduced co-factors oxidations to maintain cellular homeostasis. The negative effect of Cu2+ on yeast fermentation was partially alleviated by Mg2+ and Mn2+, similar to magnesium antagonism observed for S. cerevisiae. All these results might contribute to understand the action of these minerals in sugarcane substrates on the physiology of D. bruxellensis cells. Therefore, it represents one more step for the consolidation of the industrial use of this yeast in the production of fuel-ethanol as well as other biotechnological goods.

The antioxidant defence of Dekkera bruxellensis against hydrogen peroxide and its relationship to nitrate metabolism.

The yeast Dekkera bruxellensis is a Crabtree-positive yeast that tends towards the oxidative/respiratory metabolism in aerobiosis. However, it is more sensitive to H2O2 than Saccharomyces cerevisiae. In order to investigate this metabolic paradox, the present work aimed to uncover the biological defence mechanism used by this yeast to tolerate the presence of exogenous H2O2. Growth curves and spot tests were performed to establish the values of minimal inhibitory concentration and minimal biocidal concentration of H2O2 for different combinations of carbon and nitrogen sources. Cells in exponential growth phase in different culture conditions were used to measure superoxide and thiols [protein (PT) and non-PT], enzyme activities and gene expression. The combination of glutathione peroxidase (Gpx) and sulfhydryl-containing PT formed the preferred defence mechanism against H2O2, which was more efficiently active under respiratory metabolism. However, the action of this mechanism was suppressed when the cells were metabolizing nitrate (NO3). These results were relevant to figure out the fitness of D. bruxellensis to metabolize industrial substrates containing oxidant molecules, such as molasses and plant hydrolysates, in the presence of a cheaper nitrogen source such as NO3.

Differences in Formation Mechanisms of Phenolic Off-Flavor Compounds among Yeast Species

Phenolic off-flavor compounds such as 4-vinylguaiacol (4-VG) affect the quality of fermented foods. In Saccharomyces cerevisiae, 4-VG production requires prenylated FMN (flavin mononucleotide) produced by the mitochondrial protein Pad1. To examine this mechanism further, we expressed Escherichia coli ubiX, whose gene product prenylates FMN in E. coli, in an S. cerevisiae Δpad1 strain, which recovered the ability to produce 4-VG. Because E. coli UbiX is expected to localize to the cytoplasm in the transformed yeast, next, we expressed a truncated Pad1 (tPad1) variant lacking the mitochondrial translocation signal sequence in S. cerevisiae Δpad1. The transformed strain also recovered the ability to produce 4-VG; furthermore, it produced a larger amount of 4-VG than wild-type S. cerevisiae, indicating that 4-VG production in S. cerevisiae may be mediated by the partial mislocalization of Pad1 to the cytoplasm. We also expressed the gene encoding Dekkera (Brettanomyces) bruxellensis phenolic acid decarboxylase (DbPAD) in S. cerevisiae Δpad1. The transformed yeast recovered the ability to produce 4-VG, indicating that the D. bruxellensis phenolic acid decarboxylation reaction does not require prenylated FMN. Sequencing of the cloned DbPAD gene showed that the translation start codon differs from previous reports; therefore, we recloned the DbPAD gene from the position predicted to be the translation initiation point (designated DbPADs). Transformation of S. cerevisiae Δpad1 with DbPADs further increased the amount of 4-VG produced. Collectively, these findings provide insight into the mechanism of off-flavor production by different yeast species.

Evaluation of the application of <i>Wickerhamomyces anomalus</i> supernatant for the control of relevant spoilage yeasts in wines

SO2 is traditionally used to limit or nullify the proliferation of spoilage yeasts in must/wine, but its use has become controversial due to its negative effects on human health. An alternative strategy for the control of spoilage yeasts is the use of biocontrol yeasts. Wickerhamomyces anomalus is an outstanding biocontroller yeast that has been examined by our group and that is a good biocontrol agent against Brettanomyces bruxellensis and Zygosaccharomyces rouxii. The challenge regarding the application of the results of microbial biocontrol studies lies in exploring technologically simple and economical ways to make use of microbial biocontrol activity. The introduction of foreign microbiota adds complexity to the fermentation medium and can cause technological difficulties and have oenological consequences inherent to the implementation of mixed cultures. Therefore, the objective of this study was to characterise the concentrated culture supernatant of W. anomalus and evaluate its biocontrol action in liquid medium on two main wine spoilage yeasts. W. anomalus BWa156 supernatant demonstrated inhibitory killer protein characteristics. The production of the inhibitory supernatant by the biocontroller yeast was independent of the presence or absence of the spoilage yeast. Supernatant can be produced faster under aerobic conditions than in traditional fermentation (i.e., with around 24 h), thus increasing its potential for technological development. The treatment with the supernatant of BWa156 was effective against the two spoilage populations Zygosaccharomyces rouxii and Brettanomyces bruxellensis, which are considered problematic for the wine industry. The supernatant of the biocontrol yeast can be considered to be a relevant additional stress factor for the spoilage population, which together with other factors such as ethanol, competition for nutrients, oxygen and pH, contributes to the elimination of the polluting population. This technology would allow a simple future application, through its production in a bioreactor parallel to the fermentation and subsequent inoculation in the must/wine.

New insights into the role of key microorganisms and wooden barrels during lambic beer fermentation and maturation

Belgian lambic beers are still produced through traditional craftsmanship. They rely on a spontaneous fermentation and maturation process that is entirely carried out in wooden barrels. The latter are used repetitively and may introduce some batch-to-batch variability. The present systematic and multiphasic study dealt with two parallel lambic beer productions carried out in nearly identical wooden barrels making use of the same cooled wort. It encompassed a microbiological and metabolomic approach. Further, a taxonomic classification and metagenome-assembled genome (MAG) investigation was based on shotgun metagenomics. These investigations provided new insights into the role of these wooden barrels and key microorganisms for this process. Indeed, besides their role in traditionality, the wooden barrels likely helped in establishing the stable microbial ecosystem of lambic beer fermentation and maturation by acting as an inoculation source of the necessary microorganisms, thereby minimizing batch-to-batch variations. They further provided a microaerobic environment, which aided in achieving the desirable succession of the different microbial communities for a successful lambic beer production process. Moreover, these conditions prevented excessive growth of acetic acid bacteria and, therefore, uncontrolled production of acetic acid and acetoin, which may lead to flavor deviations in lambic beer. Concerning the role of less studied key microorganisms for lambic beer production, it was shown that the Acetobacter lambici MAG contained several acid tolerance mechanisms toward the harsh environment of maturing lambic beer, whereas genes related to sucrose and maltose/maltooligosaccharide consumption and the glyoxylate shunt were absent. Further, a Pediococcus damnosus MAG possessed a gene encoding ferulic acid decarboxylase, possibly contributing to 4-vinyl compound production, as well as several genes, likely plasmid-based, related to hop resistance and biogenic amine production. Finally, contigs related to Dekkera bruxellensis and Brettanomyces custersianus did not possess genes involved in glycerol production, emphasizing the need for alternative external electron acceptors for redox balancing.

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.

Cell morphology observations for discriminating between <i>Brettanomyces bruxellensis</i> strains among genetic groups

It is essential to discriminate between B. bruxellensis isolates at the strain level, because stress resistance capacities are strain dependent and also related to the genetic groups (GG). In this work, we investigated further the correlation between genetic groups and cell polymorphism by analysing optical microscopy images via deep learning. A Convolutional Neural Network (CNN) was trained to discriminate between 74 different B. bruxellensis isolates belonging to 4 of the 6 genetic groups described. Compared to the microsatellite analysis, the CNN enabled the prediction of the genetic groups of B. bruxellensis isolates with 96.6 % accuracy in a faster and cheaper way and with the same genetic group affiliations. Based on these very promising results, further research is needed to validate this technique for all genetic groups.

Use of red grape pulp, marc and must in the production of beer

The aim of the paper was to determine the potential of using grape pulp, marc and must in the beer production process. Samples were fermented using non-Saccharomyces yeasts (Dekkera bruxellensis 3429, Metschnikowia pulcherrima MG970690), while Saccharomyces cerevisiae Safale US-05 was used as a control. Grape marc was obtained by pressing grape must. The grape marc, must and pulp were pasteurized and, together with wort, volumetrically introduced into fermentation flasks for fermentation. Mass changes taking place during the process were analyzed. Real extract, alcohol content, free amino nitrogen (FAN) content, titratable acidity, pH, color, organic acid profile and content of sugars were determined in obtained beers. The addition of grape marc, must and pulp increased the value of most of the tested parameters. It did not adversely affect the fermentation process. This offers the possibility of using grape marc, must and pulp in the brewing industry, even with the use of non-Saccharomyces yeast monocultures.

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.

UV-C treatment: A non-thermal inactivation method for microbiological stabilisation of must and wine

UV-C treatment is discussed as an effective and efficient method to inactivate harmful microorganisms in wine and other viticultural products. In comparison to other stabilisation techniques, the application of UV-C is thought to be beneficial to reduce energy costs and to minimize SO2 addition. The object of this work was to determine the lethal UV-C dose for harmful microorganisms such as Brettanomyces bruxellensis and Acetobacter aceti. The concept of 5-log inactivation was applied and the Weibull model was used to compare different microbial and wine parameters. Microbial relevant UV-C doses and 2-fold overdose treatments and how they affected chemical and sensory changes of wine were investigated. Riesling and Pinot noir wine, which have different absorbance at 254 nm, were individually inoculated with microorganisms at different inoculation numbers. The results showed that the Weibull model is appropriate to predict the lethal UV-C dose. Already at microbially relevant doses, UV-C treatment can lead to significant changes in the colour and concentration of aroma compounds in white wine. Higher concentrations of 2-aminoacetophenone were found with increasing UV-C doses. Hence, UV-C overdosing can cause the “atypical ageing” off-flavour in wine. However, microbially relevant UV-C doses change the sensory properties of wine more towards a typical ageing character.

Evaluation of plant phenolic extracts as an alternative to sulfur dioxide for the control of Oenococcus oeni and Brettanomyces bruxellensis

In the current work, the potential of plant phenolic extracts to replace the ability of SO2 to inhibit spoilage microorganisms was evaluated. The study included the application of different doses of plant phenolic concentrates to test their antimicrobial activity against O. oeni, and Brettanomyces yeasts. For the evaluation of O. oeni, Chardonnay and Riesling wines were inoculated with the lactic acid bacteria, and antimicrobial agents were added: SO2 or phenolic concentrates. Then within seven weeks and six months, the levels of malic and lactic acids in the control and treated samples were monitored. In the tests to verify the control of Brettanomyces, oak cubes were contaminated with the spoilage yeasts and later treated with water (control) or two aqueous sanitizing solutions (conventional one: water with citric acid and SO2; experimental one: containing phenolic concentrates). Later, the corresponding solutions and water were analyzed for the presence of Brettanomyces yeasts by observation and plate numbering. Results confirm the inhibitory capacity of the phenolic concentrates tested regarding these two microorganisms, showing promising results.

Grape stalks: From wastes to source of antioxidants and nutraceuticals

Wine production is one of the most significant agricultural activities worldwide. The winemaking process generates large amounts of by-products: grape marc, stalks, and exhausted grape marc. Until now, many studies have been focused on marc valorization, very few on stalks. The aim of this research was to deeply explore the potential of residual stalks in the wine industry from a circular economy perspective. Polyphenols were extracted from stalks with new sustainable technologies in the frame of “green chemistry” without the use of hazardous solvents. Extracts were analyzed for total phenolic compound content (TPC) and their antioxidant activity was determined. As polyphenols can have antimicrobial activity, the effect of the extracted polyphenols against wine-spoiling yeast Brettanomyces bruxellensis was determined. The percent reduction of the culture optical density, in the presence or absence of polyphenols, was compared to assess the antimicrobial activity of the samples. The results obtained underline the importance of winemaking by-products (stalks) and their eco-friendly valorization to obtain molecules for food, nutraceutical and cosmetic industries.

Effect of Brown Algae and Lichen Extracts on the SCOBY Microbiome and Kombucha Properties

Kombucha tea was made by the fermentation of SCOBY culture of green tea broth with the addition of Fucus vesiculosus algae extract, Cetraria islandica lichen extract and their mixture. Kombucha was also made without the herbal supplements as a control. After 11 days of fermentation, in addition to the yeast Brettanomyces bruxellensis and the bacteria Komagataeibacter rhaeticus and Komagataeibacter hansenii contained in all of the samples, the yeast Zygosaccharomyces bailii and bacteria Komagataeibacter cocois were detected in the samples with the herbal extracts. In all of the kombucha with herbal additives, the total fraction of yeast was decreased as compared to the control. The total content of polyphenols and the antioxidant activity of the beverages with and without the addition of herbal extracts were comparable. The kombucha made with the algae extract showed an increased content of sucrose and organic acids, while the fructose and glucose content in the samples with algae and the mixture of extracts were lower than in the other samples. The samples with the algae extract had the highest organoleptic indicators "aroma", "clarity" and "acidity", while the control samples had slightly higher indicators of "taste" and "aftertaste". The results of this study indicate the potential of algae and lichens as functional supplements for obtaining non-alcoholic fermented beverages with additional nutraceutical value.

Physicochemical characteristics of beer with grape must addition produced using non-Saccharomyces yeasts

The aim of the paper was to determine the potential of using grape must and unconventional yeasts in the beer production process. Samples were fermented using non-Saccharomyces yeasts (Dekkera bruxellensis 3429, Wickerhamomyces anomalus MG971261, Kluyveromyces lactis MG971263), and Saccharomyces cerevisiae Safale US-05 was used as a control. Grape must was pasteurized and, together with wort, volumetrically introduced into fermentation flasks for fermentation. Mass changes taking place during the process were analyzed. Real extract, alcohol content, free amino nitrogen (FAN) content, titratable acidity, color, and sugar profile were determined in obtained beers. The obtained results have proven that the tested non-Saccharomyces yeasts can adapt to fermentation conditions, just like S. cerevisiae Safale US-05, and can be used to produce beer-wine hybrid alcoholic beverages. The yeast K. lactis MG971263 produced a greater amount of alcohol in beers compared to other yeast strains. There were no significant differences in the color of the obtained beers and in the degree of FAN utilization during the fermentation process. The unconventional yeasts used are capable of producing beers without and with the addition of grape must with parameters similar to beers obtained using S. cerevisiae Safale US-05.

Flow cytometry, a sustainable method for the identification and quantification of microorganisms in enology - Part 1/2 Review of the usual methods applied in wine microbiology and the principle of flow cytometry

Flow cytometry (FC) is a powerful technique for the detection, characterization and quantification of microbial populations in enology. Depending on the fluorescent markers and specific probes used, FC can provide information on the physiological state of the cell and allows the quantification of a microorganism of interest, or spoilage microorganisms such as Brettanomyces bruxellensis, within a mixed population.

Identification of spoilage microflora in draught beer using culture-dependent methods.

To determine whether the culture-dependent spoilage microflora found in draught beer are influenced by beer style. Four beer styles-lager, ale, stout and cask ale - were sampled twice from five different public houses (accounts) in four different locations. The microbiological quality of the dispensed beers was determined by a culture-dependent method ('forcing'), measuring the increase in turbidity after incubation at 30°C. The quality of draught beer varied from 'excellent' to 'poor' with cask beer samples having a higher Quality Index (90%) with keg ale the lowest (67.5%). With PCR amplified DNA (ITS1, ITS4, 16S rRNA primers) and blast identification of microflora, 386 colonies from agar plates were identified with 28 different micro-organisms from five genera of yeast and six of bacteria. Seven micro-organisms were found in all beer styles with Brettanomyces bruxellensis, B. anomalus and Acetobacter fabarum representing 53% of the identified micro-organisms. A subsequent, limited study using PALL multiplex PCR GeneDisc technology on forced samples (without selection on plates) suggests that draught beer microflora is qualitatively broader. It is noteworthy that the microflora of spoilt draught beer resembles that involved in the production of Belgian Lambic sour beers. Draught beer was of variable quality. Culture-dependent analysis suggests that species of Brettanomyces and Acetobacter are core microflora with some micro-organisms being associated with beer style. The microbiological quality of draught beer is important both commercially and to the consumer. Here, we report the core and diverse microflora found in different styles of draught beer using culture-dependent methods.

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.