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.

Production of ethanol fuel from enzyme-treated sugarcane bagasse hydrolysate using d-xylose-fermenting wild yeast isolated from Brazilian biomes.

In this study, we evaluated the potential of yeasts isolated from Amazon to produce second-generation ethanol from sugarcane bagasse delignified with alkaline hydrogen peroxide and hydrolysed with commercial enzyme preparation. The best efficiency savings in glucose and release of xylose were determined by considering the solids and enzyme loads. Furthermore, we selected Spathaspora passalidarum UFMG-CM-Y473 strain with the best fermentative parameters. Fermentations used bagasse hydrolysate without any nutritional supplementation, a significant difference from previous studies, which is closer to industrial conditions. Ethanol yield of 0.32 g/g and ethanol productivity of 0.34 g/L h were achieved after the consumption of 78% of the sugar. This hydrolysis/fermentation technology package could represent the input of an additional 3180 L of ethanol per hectare in areas of average sugarcane productivity such as 60 ton/ha. Thus, we concluded that Sp. passalidarum UFMG-CM-Y473 has a clear potential for the production of second-generation ethanol from delignified and enzyme-hydrolysed bagasse.

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.

Glutamine: a major player in nitrogen catabolite repression in the yeast Dekkera bruxellensis.

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.

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.

Oxygen-limited cellobiose fermentation and the characterization of the cellobiase of an industrial Dekkera/Brettanomyces bruxellensis strain.

The discovery of a novel yeast with a natural capacity to produce ethanol from lignocellulosic substrates (second-generation ethanol) is of great significance for bioethanol technology. While there are some yeast strains capable of assimilating cellobiose in aerobic laboratory conditions, the predominant sugar in the treatment of lignocellulosic material, little is known about this ability in real industrial conditions. Fermentations designed to simulate industrial conditions were conducted in synthetic medium with glucose, sucrose, cellobiose and hydrolyzed pre-treated cane bagasse as a different carbon source, with the aim of further characterizing the fermentation capacity of a promising Dekkera bruxellensis yeast strain, isolated from the bioethanol process in Brazil. As a result, it was found (for the first time in oxygen-limiting conditions) that the strain Dekkera bruxellensis GDB 248 could produce ethanol from cellobiose. Moreover, it was corroborated that the cellobiase activity characterizes the enzyme candidate in semi-purified extracts (ß-glucosidase). In addition, it was demonstrated that GDB 248 strain had the capacity to produce a higher acetic acid concentration than ethanol and glycerol, which confirms the absence of the Custer effect with this strain in oxygen-limiting conditions. Moreover, it is also being suggested that D. bruxellensis could benefit Saccharomyces cerevisiae and outcompete it in the industrial environment. In this way, it was confirmed that D. bruxellensis GDB 248 has the potential to produce ethanol from cellobiose, and is a promising strain for the fermentation of lignocellulosic substrates.

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.

5-Hydroxymethylfurfural induces ADH7 and ARI1 expression in tolerant industrial Saccharomyces cerevisiae strain P6H9 during bioethanol production.

The aims of this work were to obtain, by evolutionary engineering, an industrial strain of Saccharomyces cerevisiae tolerant to high concentrations of HMF and to determine the expression levels of genes previously described as responsible for this tolerance. Cells were grown under anaerobic and oxygen limited conditions, in the presence of glucose or sucrose as carbon sources. P6H9 strain presented high expression levels for genes ADH7 and ARI1 in presence of HMF. This tolerant strain also showed higher ethanol productivity, biomass formation and alcohol dehydrogenase activity comparing to sensitive strains. Results suggest that S. cerevisiae P6H9 strain presents potential to be used for second-generation ethanol production.

Construction of integrative plasmids suitable for genetic modification of industrial strains of Saccharomyces cerevisiae.

The development of efficient tools for genetic modification of industrial yeast strains is one of the challenges that face the use of recombinant cells in industrial processes. In this study, we examine how the construction of two complementary integrative vectors can fulfill the major requirements of industrial recombinant yeast strains: the use of lactose assimilation genes as a food-grade yeast selection marker, and a system of integration that does not leave hazardous genes in the host genome and involves minimal interference in the yeast physiology. The pFB plasmid set was constructed to co-integrate both LAC4-based and LAC12-based cassettes into the ribosomal DNA (rDNA) locus to allow yeast cells to be selected in lactose medium. This phenotype can also be used to trace the recombinant cells in the environment by simply being plated on X-gal medium. The excisable trait of the LAC12 marker allows the introduction of many different heterologous genes, and makes it possible to introduce a complete heterologous metabolic pathway. The cloned heterologous genes can be highly expressed under the strong and constitutive TPI1 gene promoter, which can be exchanged for easy digestion of enzymes if necessary. This platform was introduced into Saccharomyces cerevisiae JP1 industrial strain where a recombinant with high stability of markers was produced without any change in the yeast physiology. Thus, it proved to be an efficient tool for the genetic modification of industrial strains.

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.

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 ability to use nitrate confers advantage to Dekkera bruxellensis over S. cerevisiae and can explain its adaptation to industrial fermentation processes.

The yeast Dekkera bruxellensis has been regarded as a contamination problem in industrial ethanol production because it can replace the originally inoculated Saccharomyces cerevisiae strains. The present study deals with the influence of nitrate on the relative competitiveness of D. bruxellensis and S. cerevisiae in sugar cane ethanol fermentations. The industrial strain D. bruxellensis GDB 248 showed higher growth rates than S. cerevisiae JP1 strain in mixed ammonia/nitrate media, and nitrate assimilation genes were only slightly repressed by ammonia. These characteristics rendered D. bruxellensis cells with an ability to overcome S. cerevisiae populations in both synthetic medium and in sugar cane juice. The results were corroborated by data from industrial fermentations that showed a correlation between high nitrate concentrations and high D. bruxellensis cell counts. Moreover, the presence of nitrate increased fermentation efficiency of D. bruxellensis cells in anaerobic conditions, which may explain the maintenance of ethanol production in the presence of D. bruxellensis in industrial processes. The presence of high levels of nitrate in sugar cane juice may be due to its inefficient conversion by plant metabolism in certain soil types and could explain the periodical episodes of D. bruxellensis colonization of Brazilian ethanol plants.