RESUMO
AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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). SIGNIFICANCE AND IMPACT OF STUDY: 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.
Assuntos
Dekkera , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Peróxido de Hidrogênio/metabolismo , Nitratos/metabolismo , Antioxidantes/metabolismo , Dekkera/genética , Dekkera/metabolismo , Fermentação , Nitrogênio/metabolismoRESUMO
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.
Assuntos
Dekkera , Saccharomyces cerevisiae , Acetatos/metabolismo , Brettanomyces , Dekkera/genética , Dekkera/metabolismo , Etanol/metabolismo , Fermentação , Glucose/metabolismo , Microbiologia Industrial , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sacarose/metabolismoRESUMO
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.
Assuntos
Dekkera , Brettanomyces , Fermentação , Nitratos , ProteômicaRESUMO
Dekkera bruxellensis is one of the most important contaminant yeasts of alcoholic fermentation. The use of propolis, which can selectively target contaminating yeasts without affecting the starter one, Saccharomyces cerevisiae, could be a useful nonconventional strategy for controlling the growth of contaminant yeasts. The objective of this research was to evaluate four samples of propolis produced by Apis mellifera honeybees from different regions of Argentina as antimicrobial agents against the growth of D. bruxellensis and S. cerevisiae. Hydroalcoholic extracts of propolis were prepared with ethanol:water (70:30 v/v), and the specific absorbance and final concentration of the samples were evaluated. A qualitative in vitro assay in solid medium was performed with different propolis concentrations, and the evaluation of yeast growth was based on a qualitative scale. A quantitative in vitro assay in liquid medium was also performed to assess the yeast cell number, using two different propolis concentrations. The cell number of D. bruxellensis decreased 1.52 and 1.85 log cycles with the two propolis extracts utilised at a concentration of 4.5 mg mL-1 while the cell number of S. cerevisiae decreased 0.48 and 0.76 log cycles with the same samples of propolis. The results from both assays demonstrated the selectivity of propolis use on the yeast species, leading to a higher inhibition of D. bruxellensis growth. This indicates a good potential for using propolis at the concentration of 4.5 mg mL-1, as a nonconventional strategy to control the growth of D. bruxellensis without significantly affecting S. cerevisiae, the yeast starter of ethanol fermentation.(AU)
Dekkera bruxellensis é uma das mais importantes leveduras contaminantes da fermentação alcoólica. O uso de própolis, que pode seletivamente afetar a levedura contaminante mas não a levedura do processo, Saccharomyces cerevisiae, pode ser uma estratégia não convencional útil para o controle de leveduras contaminantes. O objetivo deste trabalho foi avaliar quatro amostras de própolis produzidas por Apis mellifera de diferentes regiões da Argentina como agentes antimicrobianos no controle do crescimento de D. bruxellensis e S. cerevisiae. Foram preparados extratos hidroalcoólicos de própolis com etanol: água (70:30 v/v) e avaliadas a absorbância específica e concentração final das amostras. Um ensaio qualitativo in vitro foi realizado em meio sólido com diferentes concentrações de própolis e a avaliação do crescimento da levedura foi baseada em uma escala qualitativa. Um ensaio quantitativo in vitro em meio líquido foi realizado com duas concentrações de própolis, avaliando-se o número de leveduras. O número de células de D. bruxellensis diminuiu 1,52 e 1,85 ciclos log com dois extratos de própolis na concentração de 4,5 mg mL-1 enquanto para S. cerevisiae, a diminuição no número de células foi de 0,48 e 0,76 ciclos log com as mesmas amostras de própolis. Os resultados de ambos os ensaios demonstraram claramente a seletividade do efeito do emprego de própolis nas leveduras estudadas, resultando em maior inibição no crescimento da levedura D. bruxellensis. Isso indica a boa perspectiva do uso de própolis, na concentração de 4,5 mg mL-1, como uma estratégia não convencional para controlar o crescimento de D. bruxellensis sem afetar substancialmente S. cerevisiae, a levedura agente da fermentação etanólica.(AU)
Assuntos
Própole , Anti-Infecciosos/análise , Dekkera/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , 26016 , Leveduras/efeitos dos fármacos , Fermentação/efeitos dos fármacosRESUMO
Dekkera bruxellensis is one of the most important contaminant yeasts of alcoholic fermentation. The use of propolis, which can selectively target contaminating yeasts without affecting the starter one, Saccharomyces cerevisiae, could be a useful nonconventional strategy for controlling the growth of contaminant yeasts. The objective of this research was to evaluate four samples of propolis produced by Apis mellifera honeybees from different regions of Argentina as antimicrobial agents against the growth of D. bruxellensis and S. cerevisiae. Hydroalcoholic extracts of propolis were prepared with ethanol:water (70:30 v/v), and the specific absorbance and final concentration of the samples were evaluated. A qualitative in vitro assay in solid medium was performed with different propolis concentrations, and the evaluation of yeast growth was based on a qualitative scale. A quantitative in vitro assay in liquid medium was also performed to assess the yeast cell number, using two different propolis concentrations. The cell number of D. bruxellensis decreased 1.52 and 1.85 log cycles with the two propolis extracts utilised at a concentration of 4.5 mg mL-1 while the cell number of S. cerevisiae decreased 0.48 and 0.76 log cycles with the same samples of propolis. The results from both assays demonstrated the selectivity of propolis use on the yeast species, leading to a higher inhibition of D. bruxellensis growth. This indicates a good potential for using propolis at the concentration of 4.5 mg mL-1, as a nonconventional strategy to control the growth of D. bruxellensis without significantly affecting S. cerevisiae, the yeast starter of ethanol fermentation.
Dekkera bruxellensis é uma das mais importantes leveduras contaminantes da fermentação alcoólica. O uso de própolis, que pode seletivamente afetar a levedura contaminante mas não a levedura do processo, Saccharomyces cerevisiae, pode ser uma estratégia não convencional útil para o controle de leveduras contaminantes. O objetivo deste trabalho foi avaliar quatro amostras de própolis produzidas por Apis mellifera de diferentes regiões da Argentina como agentes antimicrobianos no controle do crescimento de D. bruxellensis e S. cerevisiae. Foram preparados extratos hidroalcoólicos de própolis com etanol: água (70:30 v/v) e avaliadas a absorbância específica e concentração final das amostras. Um ensaio qualitativo in vitro foi realizado em meio sólido com diferentes concentrações de própolis e a avaliação do crescimento da levedura foi baseada em uma escala qualitativa. Um ensaio quantitativo in vitro em meio líquido foi realizado com duas concentrações de própolis, avaliando-se o número de leveduras. O número de células de D. bruxellensis diminuiu 1,52 e 1,85 ciclos log com dois extratos de própolis na concentração de 4,5 mg mL-1 enquanto para S. cerevisiae, a diminuição no número de células foi de 0,48 e 0,76 ciclos log com as mesmas amostras de própolis. Os resultados de ambos os ensaios demonstraram claramente a seletividade do efeito do emprego de própolis nas leveduras estudadas, resultando em maior inibição no crescimento da levedura D. bruxellensis. Isso indica a boa perspectiva do uso de própolis, na concentração de 4,5 mg mL-1, como uma estratégia não convencional para controlar o crescimento de D. bruxellensis sem afetar substancialmente S. cerevisiae, a levedura agente da fermentação etanólica.
Assuntos
Anti-Infecciosos/análise , Dekkera/efeitos dos fármacos , Própole , Saccharomyces cerevisiae/efeitos dos fármacos , 26016 , Fermentação/efeitos dos fármacos , Leveduras/efeitos dos fármacosRESUMO
Dekkera bruxellensis is an industrial yeast mainly regarded as a contaminant species in fermentation processes. In winemaking, it is associated with off-flavours that cause wine spoilage, while in bioethanol production this yeast is linked to a reduction of industrial productivity by competing with Saccharomyces cerevisiae for the substrate. In spite of that, this point of view is gradually changing, mostly because D. bruxellensis is also able to produce important metabolites, such as ethanol, acetate, fusel alcohols, esters and others. This dual role is likely due to the fact that this yeast presents a set of metabolic traits that might be either industrially attractive or detrimental, depending on how they are faced and explored. Therefore, a proper industrial application for D. bruxellensis depends on the correct assembly of its central metabolic puzzle. In this sense, researchers have addressed issues regarding the physiological and genetic aspects of D. bruxellensis, which have brought to light much of our current knowledge on this yeast. In this review, we shall outline what is presently understood about the main metabolic features of D. bruxellensis and how they might be managed to improve its current or future industrial applications (except for winemaking, in which it is solely regarded as a contaminant). Moreover, we will discuss the advantages and challenges that must be overcome in order to take advantage of the full biotechnological potential of this yeast.
Assuntos
Dekkera/genética , Dekkera/metabolismo , Microbiologia Industrial , Ácido Acético/metabolismo , Etanol/metabolismo , Fermentação , Saccharomyces cerevisiae/metabolismo , Vinho/microbiologiaRESUMO
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.
Assuntos
Biodiversidade , Biocombustíveis/microbiologia , Carbono/metabolismo , Dekkera/metabolismo , Vinho/microbiologia , Anaerobiose , Dekkera/classificação , Etanol , Fermentação , Microbiologia IndustrialRESUMO
In the past few years, the yeast Dekkera bruxellensis has gained much of attention among the so-called non-conventional yeasts for its potential in the biotechnological scenario, especially in fermentative processes. This yeast has been regarded as an important competitor to Saccharomyces cerevisiae in bioethanol production plants in Brazil and several studies have reported its capacity to produce ethanol. However, our current knowledge concerning D. bruxellensis is restricted to its aerobic metabolism, most likely because wine and beer strains cannot grow in full anaerobiosis. Hence, the present work aimed to fulfil a gap regarding the lack of information on the physiology of Dekkera bruxellensis growing in the complete absence of oxygen and the relationship with assimilation of nitrate as nitrogen source. The ethanol strain GDB 248 was fully capable of growing anaerobically and produces ethanol at the same level of S. cerevisiae. The presence of nitrate in the medium increased this capacity. Moreover, nitrate is consumed faster than ammonium and this increased rate coincided with a higher speed of glucose consumption. The profile of gene expression helped us to figure out that even in anaerobiosis, the presence of nitrate drives the yeast cells to an oxidative metabolism that ultimately incremented both biomass and ethanol production. These results finally provide the clues to explain most of the success of this yeast in industrial processes of ethanol production.
Assuntos
Ácido Acético/metabolismo , Dekkera/efeitos dos fármacos , Etanol/metabolismo , Nitratos/metabolismo , Compostos de Amônio/metabolismo , Anaerobiose , Cerveja/microbiologia , Biomassa , Brasil , Dekkera/metabolismo , Fermentação , Manipulação de Alimentos , Microbiologia de Alimentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Desidrogenase de Glutamato (NADP+)/genética , Desidrogenase de Glutamato (NADP+)/metabolismo , Nitrogênio/metabolismo , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vinho/microbiologiaRESUMO
Dekkera bruxellensis is a spoilage yeast in wine and fuel ethanol fermentations able to produce volatile phenols from hydroxycinnamic acids by the action of the enzymes cinnamate decarboxylase (CD) and vinyphenol reductase (VR) in wine. However, there is no information about this ability in the bioethanol industry. This work evaluated CD and VR activities and 4-ethylphenol production from p-coumaric acid by three strains of D. bruxellensis and PE-2, an industrial Saccharomyces cerevisiae strain. Single and multiple-cycle batch fermentations in molasses and sugarcane juice were carried out. Dekkera bruxellensis strains showed similar CD activity but differences in VR activity. No production of 4-ethylphenol by S. cerevisiae in any fermentation system or media was observed. The concentrations of 4-ethylphenol peaked during active growth of D. bruxellensis in single-cycle fermentation but they were lower than in multiple-cycle fermentation. Higher concentrations were observed in molasses with molar conversion (p-coumaric acid to 4-ethylphenol) ranging from 45% to 85%. As the first report on 4-ethylphenol production in sugarcane musts by D. bruxellensis in industry-like conditions, it opens up a new avenue to investigate its effect on the viability and fermentative capacity of S. cerevisiae as well as to understand the interaction between the yeasts in the bioethanol industry.
Assuntos
Biocombustíveis , Dekkera/metabolismo , Etanol/metabolismo , Microbiologia Industrial , Fenóis/metabolismo , Brasil , Carboxiliases/análise , Cinamatos/metabolismo , Ácidos Cumáricos , Fermentação , Propionatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharum/metabolismoRESUMO
Dekkera bruxellensis is continuously changing its status in fermentation processes, ranging from a contaminant or spoiling yeast to a microorganism with potential to produce metabolites of biotechnological interest. In spite of that, several major aspects of its physiology are still poorly understood. As an acetogenic yeast, minimal oxygen concentrations are able to drive glucose assimilation to oxidative metabolism, in order to produce biomass and acetate, with consequent low yield in ethanol. In the present study, we used disulfiram to inhibit acetaldehyde dehydrogenase activity to evaluate the influence of cytosolic acetate on cell metabolism. D. bruxellensis was more tolerant to disulfiram than Saccharomyces cerevisiae and the use of different carbon sources revealed that the former yeast might be able to export acetate (or acetyl-CoA) from mitochondria to cytoplasm. Fermentation assays showed that acetaldehyde dehydrogenase inhibition re-oriented yeast central metabolism to increase ethanol production and decrease biomass formation. However, glucose uptake was reduced, which ultimately represents economical loss to the fermentation process. This might be the major challenge for future metabolic engineering enterprises on this yeast.
Assuntos
Acetatos/metabolismo , Dekkera/metabolismo , Etanol/metabolismo , Fermentação , Acetatos/análise , Aldeído Oxirredutases/antagonistas & inibidores , Carbono/metabolismo , Meios de Cultura , Dekkera/efeitos dos fármacos , Dissulfiram/farmacologia , Glucose/metabolismo , Microbiologia Industrial , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismoRESUMO
The alcoholic fermentation for fuel ethanol production in Brazil occurs in the presence of several microorganisms present with the starter strain of Saccharomyces cerevisiae in sugarcane musts. It is expected that a multitude of microbial interactions may exist and impact on the fermentation yield. The yeast Dekkera bruxellensis and the bacterium Lactobacillus fermentum are important and frequent contaminants of industrial processes, although reports on the effects of both microorganisms simultaneously in ethanolic fermentation are scarce. The aim of this work was to determine the effects and interactions of both contaminants on the ethanolic fermentation carried out by the industrial yeast S. cerevisiae PE-2 in two different feedstocks (sugarcane juice and molasses) by running multiple batch fermentations with the starter yeast in pure or co-cultures with D. bruxellensis and/or L. fermentum. The fermentations contaminated with D. bruxellensis or L. fermentum or both together resulted in a lower average yield of ethanol, but it was higher in molasses than that of sugarcane juice. The decrease in the CFU number of S. cerevisiae was verified only in co-cultures with both D. bruxellensis and L. fermentum concomitant with higher residual sucrose concentration, lower glycerol and organic acid production in spite of a high reduction in the medium pH in both feedstocks. The growth of D. bruxellensis was stimulated in the presence of L. fermentum resulting in a more pronounced effect on the fermentation parameters than the effects of contamination by each microorganism individually.
Assuntos
Biocombustíveis/microbiologia , Dekkera/metabolismo , Etanol , Fermentação , Microbiologia Industrial , Limosilactobacillus fermentum/metabolismo , Saccharomyces cerevisiae/metabolismo , Ácido Acético , Brasil , Contagem de Células , Técnicas de Cocultura , Dekkera/crescimento & desenvolvimento , Glicerol , Concentração de Íons de Hidrogênio , Limosilactobacillus fermentum/crescimento & desenvolvimento , Interações Microbianas , Melaço , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharum/metabolismo , Saccharum/microbiologia , SacaroseRESUMO
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.
Assuntos
Aminoácidos/metabolismo , Dekkera/metabolismo , Dekkera/enzimologia , Fermentação , Indústria Alimentícia , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão GênicaRESUMO
In the present work we studied the expression of genes from nitrogen central metabolism in the yeast Dekkera bruxellensis and under regulation by the Nitrogen Catabolite Repression mechanism (NCR). These analyses could shed some light on the biological mechanisms involved in the adaptation and survival of this yeast in the sugarcane fermentation process for ethanol production. Nitrogen sources (N-sources) in the form of ammonium, nitrate, glutamate or glutamine were investigated with or without the addition of methionine sulfoximine, which inhibits the activity of the enzyme glutamine synthetase and releases cells from NCR. The results showed that glutamine might act as an intracellular sensor for nitrogen availability in D. bruxellensis, by activating NCR. Gene expression analyses indicated the existence of two different GATA-dependent NCR pathways, identified as glutamine-dependent and glutamine-independent mechanisms. Moreover, nitrate is sensed as a non-preferential N-source and releases NCR to its higher level. After grouping genes according to their regulation pattern, we showed that genes for ammonium assimilation represent a regulon with almost constitutive expression, while permease encoding genes are mostly affected by the nitrogen sensor mechanism. On the other hand, nitrate assimilation genes constitute a regulon that is primarily subjected to induction by nitrate and, to a lesser extent, to a repressive mechanism by preferential N-sources. This observation explains our previous reports showing that nitrate is co-consumed with ammonium, a trait that enables D. bruxellensis cells to scavenge limiting N-sources in the industrial substrate and, therefore, to compete with Saccharomyces cerevisiae in this environment.
Assuntos
Repressão Catabólica/fisiologia , Dekkera/metabolismo , Regulação Fúngica da Expressão Gênica , Glutamina/metabolismo , Nitrogênio/metabolismo , Compostos de Amônio/metabolismo , Repressão Catabólica/genética , Dekkera/genética , Dekkera/crescimento & desenvolvimento , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/biossíntese , Microbiologia Industrial , Metionina Sulfoximina/metabolismo , Metionina Sulfoximina/toxicidade , Nitratos/metabolismo , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real , RegulonRESUMO
A comprehensive understanding of the presence and role of yeasts in bottled wines helps to know and control the organoleptic quality of the final product. The South Region of Brazil is an important wine producer, and the state of "Rio Grande do Sul" (RS) accounts for 90% of Brazilian wines. The state of "Santa Catarina" (SC) started the production in 1975, and is currently the fifth Brazilian producer. As there is little information about yeasts present in Brazilian wines, our main objective was to assess the composition of culturable yeasts associated to bottled wines produced in RS and SC, South of Brazil. We sampled 20 RS and 29 SC bottled wines produced between 2003 and 2011, and we isolated culturable yeasts in non-selective agar plates. We identified all isolates by sequencing of the D1/D2 domain of LSU rDNA or ITS1-5.8 S-ITS2 region, and comparison with type strain sequences deposited in GenBank database. Six yeast species were shared in the final product in both regions. We obtained two spoilage yeast profiles: RS with Zygosaccharomyces bailii and Pichia membranifaciens (Dekkera bruxellensis was found only in specific table wines); and SC with Dekkera bruxellensis and Pichia manshurica. Knowledge concerning the different spoilage profiles is important for winemaking practices in both regions.
Assuntos
Análise de Sequência de DNA/métodos , Vinho/microbiologia , Leveduras/classificação , Leveduras/isolamento & purificação , Brasil , DNA Fúngico/análise , Dekkera/classificação , Dekkera/genética , Dekkera/isolamento & purificação , Microbiologia de Alimentos , Pichia/classificação , Pichia/genética , Pichia/isolamento & purificação , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificação , Leveduras/genética , Zygosaccharomyces/classificação , Zygosaccharomyces/genética , Zygosaccharomyces/isolamento & purificaçãoRESUMO
UNLABELLED: Dekkera bruxellensis hit the spotlight in the past decade mostly due to its rather high ability to adapt to several different fermentation processes. This yeast relies on different genetic and physiological aspects to achieve and preserve its high industrial fitness and some of these traits are shared with Saccharomyces cerevisiae. We have previously described that D. bruxellensis is unable to make use of accumulating trehalose as a strategy for cell adaptation and survival in the industrial scenario, as opposed to S. cerevisiae. Since trehalose is often involved in mechanisms related to cell protection, we aimed to investigate both cause and effect of the absence of this metabolite in the cell adaptive capacity in the industrial environment. Our results indicate that the major cause for the nonaccumulation of trehalose is the high constitutive activity of neutral trehalase. Therefore, the rate of trehalose degradation could be higher than its rate of synthesis, preventing accumulation. Altogether, our data elucidate the mechanisms involved in the lack of trehalose accumulation in D. bruxellensis as well as evaluates the implications of this feature. SIGNIFICANCE AND IMPACT OF THE STUDY: Dekkera bruxellensis can successfully take advantage of its peculiar physiological and genetic traits in order to adapt and survive in fermentation processes. So far, tolerance to stress has been credited to trehalose synthesis. The data presented in this work provided information on the underlying mechanism that prevents trehalose accumulation and corroborated the recent information that trehalose itself is not implicated in yeast stress tolerance. Second, it showed that D. bruxellensis responds differently to Saccharomyces cerevisiae to excess of sugar, which may explain its preference for respiration (oxidative metabolism) over fermentation (reductive metabolism) even at limited oxygen supply. These findings help to understand the drop on ethanol production in processes overtaken by this yeast.
Assuntos
Dekkera/enzimologia , Dekkera/metabolismo , Saccharomyces cerevisiae/metabolismo , Trealase/metabolismo , Trealose/metabolismo , Metabolismo dos Carboidratos , Carboidratos , Dekkera/genética , Etanol/metabolismo , Fermentação/genética , Microbiologia Industrial/métodos , Fosforilação Oxidativa , Oxigênio/metabolismoRESUMO
UNLABELLED: We investigated the presence of the yeast Dekkera bruxellensis in samples collected at three points surrounding the industrial alcoholic fermentation plants of two distilleries where there are often cases of contamination caused by this yeast: this involved sugar cane wash water, feeding sugar cane juice and vinasse from the treatment pond. Total yeast was isolated in WLN medium with bromocresol green and cycloheximide and further selected on the basis of its ability to grow in synthetic medium containing nitrate. Following this, colonies were selected from the distribution on nitrate plates and identified by amplification with species-specific primers and DNA sequencing of the 26S-D1/D2 locus. The results showed that D. bruxellensis is introduced through the feeding substrate, which suggests that its cells originated with the harvested cane. Subsequently, its population circulates as a result of the reuse of water for washing the cane, in a continuous re-inoculation of the plant with yeasts. Furthermore, the yeast population is formed in the vinasse by the addition of wash water into the treatment ponds and then reintroduced to the culture fields by fertigation, so that the process can be renewed in the following season. It is now possible to adopt sanitation procedures that can prevent the entry of the contamination to the fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of the yeast Dekkera bruxellensis is sometimes attributed to a decline in the industrial productivity of ethanol since it has a more limited fermentation capacity than Saccharomyces cerevisiae. Although its adaptability to the industrial environment has been noted, so far, there has been no evidence to determine the source of this contamination. In this study, we provide evidence to show that D. bruxellensis comes from the fields together with the harvested cane and is then accumulated and recirculated. It might be possible to prevent the accumulation of this yeast by carrying out sanitation controls during the harvesting season.
Assuntos
Reatores Biológicos/microbiologia , Dekkera/crescimento & desenvolvimento , Dekkera/metabolismo , Etanol/metabolismo , Saccharum/microbiologia , Dekkera/genética , Fermentação/fisiologia , Microbiologia Industrial/métodos , Nitratos , Saccharomyces cerevisiae/metabolismo , Microbiologia da ÁguaRESUMO
Dekkera/Brettanomyces bruxellensis is considered a major cause of wine spoilage, and 4-ethylphenol and 4-ethylguaiacol are the most abundant off-aromas produced by this species. They are produced by decarboxylation of the corresponding hydroxycinnamic acids (HCAs), followed by a reduction of the intermediate 4-vinylphenols. The aim of the present study was to examine coumarate decarboxylase (CD) and vinylphenol reductase (VR) enzyme activities in 5 native D. bruxellensis strains and determine their relation with the production of ethylphenols under 'wine-like' conditions. In addition, biomass, cell culturability, carbon source utilization and organic acids were monitored during 60 days. All strains assayed turned out to have both enzyme activities. No significant differences were found in CD activity, whilst VR activity was variable among the strains. Growth of D. bruxellensis under 'wine-like' conditions showed two growth phases. Sugars were completely consumed during the first growth phase. Transformation of HCAs into ethylphenols also occurred during active growth of the yeast. No statistical differences were observed in volatile phenol levels produced by the strains growing under 'wine-like' conditions, independently of the enzyme activity previously recorded. Furthermore, our results demonstrate a relationship between the physiological state of D. bruxellensis and its ability to produce ethylphenols. Inhibition of growth of D. bruxellensis in wine seems to be the most efficient way to avoid ethylphenol production and the consequent loss of wine quality.
Assuntos
Carboxiliases/metabolismo , Dekkera/enzimologia , Microbiologia de Alimentos , Oxirredutases/metabolismo , Fermentação , Fenóis/metabolismo , Saccharomyces cerevisiae/metabolismo , Vinho/microbiologiaRESUMO
UNLABELLED: Dekkera bruxellensis is an important contaminant yeast of fuel ethanol fermentations in Brazil, whose system applies cell repitching between the fermentative cycles. This work evaluated the addition of potassium metabisulphite (PMB) on yeast growth and fermentative yields in pure and co-cultures of Saccharomyces cerevisiae and D. bruxellensis in two situations: addition to the acidic solution in which the cells are treated between the fermentative cycles or to the fermentation medium. In the range of 200-400 mg l(-1) , PMB was effective to control the growth of D. bruxellensis depending on the culture medium and strain. When added to the acidic solution (250 mg l(-1) ), a significant effect was observed in mixed cultures, because the inactivation of SO2 by S. cerevisiae most likely protected D. bruxellensis from being damaged by PMB. The physiological response of S. cerevisiae to the presence of PMB may explain the significant decrease in alcohol production. When added to the fermentation medium, PMB resulted in the control but not the death of D. bruxellensis, with less intensive effect on the fermentative efficiency. In co-culture with the addition of PMB, the fermentative efficiency was significantly lower than in the absence of PMB. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to evaluate the action of potassium metabisulphite to control the growth of Dekkera bruxellensis in the fermentation process for fuel alcohol production. As near as possible of industrial conditions, the study simulates the addition of that substance in different points in the fermentation process, verifying in which situation the effects over the starter yeast and alcohol yield are minimal and over D. bruxellensis are maximal. Co-culture fermentations were carried out in cell-recycled batch system. The feasibility of using this substance for this specific fermentation is discussed in light of the possible biological and chemical interactions.
Assuntos
Biocombustíveis , Dekkera/efeitos dos fármacos , Desinfetantes/farmacologia , Fermentação , Saccharomyces cerevisiae/metabolismo , Sulfitos/farmacologia , Brasil , Técnicas de Cocultura , Meios de Cultura/metabolismo , Etanol/metabolismo , Microbiologia Industrial , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Bebidas Alcoólicas/microbiologia , Dekkera/metabolismo , Aromatizantes/metabolismo , Nitrogênio/metabolismo , Saccharum/microbiologia , Brasil , Meios de Cultura/metabolismo , Fermentação , Saccharum/metabolismoRESUMO
Dekkera bruxellensis is an industrially relevant yeast, especially in bioethanol production. The capacity of D. bruxellensis to assimilate nitrate can confer advantages of this yeast over Saccharomyces cerevisiae at industrial conditions. In the present work we present the consequences of nitrate assimilation, using ammonium as reference, to the proteomics of D. bruxellensis. Thirty-four protein spots were overproduced in nitrate medium and were identified by MS-TOF/TOF analysis and were putatively identified by using local Mascot software. Apart from the overexpression of genes of nitrate metabolism, ATP synthesis and PPP and TCA pathways previously reported, cultivation on nitrate induced overproduction of glycolytic enzymes, which corroborate the high energy demand and NADH availability for nitrate assimilation. Overproduction of alcohol dehydrogenase (Adh) protein was also observed. Proteomic profile of D. bruxellensis cultivated in nitrate and described in the present work agrees with the hypothesis of metabolic flux regulation, making available the energy in the form of NADH to support nitrate assimilation. This work contributes with an initial picture of proteins presenting differential accumulation in industrial contaminant yeast, in strict association with possible metabolic responses to nitrate as sole nitrogen source in cultivation medium. BIOLOGICAL SIGNIFICANCE: The present study investigated the gene expression at translational level of yeast D. bruxellensis for nitrate assimilation. This study corroborated with biological models that consider the ability to assimilate this nitrogen source confers advantages on this yeast during the fermentation process industry. However, larger studies are needed in this way as our group is investigating new proteins under LC-MS/MS approach. Together, these studies will help in understanding the operation of networks and cellular regulation of the process of assimilation of nitrogen sources for the D. bruxellensis, unravelling new aspects of the physiology of this yeast by proteomic analysis. This article is part of a Special Issue entitled: Environmental and structural proteomics.