Meyerozyma caribbica Isolated from Vinasse-Irrigated Sugarcane Plantation Soil: A Promising Yeast for Ethanol and Xylitol Production in Biorefineries.

The production of fuels and other industrial products from renewable sources has intensified the search for new substrates or for the expansion of the use of substrates already in use, as well as the search for microorganisms with different metabolic capacities. In the present work, we isolated and tested a yeast from the soil of sugarcane irrigated with vinasse, that is, with high mineral content and acidic pH. The strain of Meyerozyma caribbica URM 8365 was able to ferment glucose, but the use of xylose occurred when some oxygenation was provided. However, some fermentation of xylose to ethanol in oxygen limitation also occurs if glucose was present. This strain was able to produce ethanol from molasses substrate with 76% efficiency, showing its tolerance to possible inhibitors. High ethanol production efficiencies were also observed in acidic hydrolysates of each bagasse, sorghum, and cactus pear biomass. Mixtures of these substrates were tested and the best composition was found for the use of excess plant biomass in supplementation of primary substrates. It was also possible to verify the production of xylitol from xylose when the acetic acid concentration is reduced. Finally, the proposed metabolic model allowed calculating how much of the xylose carbon can be directed to the production of ethanol and/or xylitol in the presence of glucose. With this, it is possible to design an industrial plant that combines the production of ethanol and/or xylitol using combinations of primary substrates with hydrolysates of their biomass.

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.

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

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

The Saccharomyces cerevisiae Ncw2 protein works on the chitin/ß-glucan organisation of the cell wall.

The NCW2 gene was recently described as encoding a GPI-bounded protein that assists in the re-modelling of the Saccharomyces cerevisiae cell wall (CW) and in the repair of damage caused by the polyhexamethylene biguanide (PHMB) polymer to the cell wall. Its absence produces a re-organization of the CW structure that result in resistance to lysis by glucanase. Hence, the present study aimed to extend the analysis of the Ncw2 protein (Ncw2p) to determine its physiological role in the yeast cell surface. The results showed that Ncw2p is transported to the cell surface upon O-mannosylation mediated by the Pmt1p-Pmt2p enzyme complex. It co-localises with the yeast bud scars, a region in cell surface formed by chitin deposition. Once there, Ncw2p enables correct chitin/ß-glucan structuring during the exponential growth. The increase in molecular mass by hyper-mannosylation coincides with the increasing in chitin deposition, and leads to glucanase resistance. Treatment of the yeast cells with PHMB produced the same biological effects observed for the passage from exponential to stationary growth phase. This might be a possible mechanism of yeast protection against cationic biocides. In conclusion, we propose that Ncw2p takes part in the mechanism involved in the control of cell surface rigidity by aiding on the linkage between chitin and glucan layers in the modelling of the cell wall during cell growth.

Fermentation profiles of the yeast Brettanomyces bruxellensis in d-xylose and l-arabinose aiming its application as a second-generation ethanol producer.

The yeast Brettanomyces bruxellensis is able to ferment the main sugars used in first-generation ethanol production. However, its employment in this industry is prohibitive because the ethanol productivity reached is significantly lower than the observed for Saccharomyces cerevisiae. On the other hand, a possible application of B. bruxellensis in the second-generation ethanol production has been suggested because this yeast is also able to use d-xylose and l-arabinose, the major pentoses released from lignocellulosic material. Although the latter application seems to be reasonable, it has been poorly explored. Therefore, we aimed to evaluate whether or not different industrial strains of B. bruxellensis are able to ferment d-xylose and l-arabinose, both in aerobiosis and oxygen-limited conditions. Three out of nine tested strains were able to assimilate those sugars. When in aerobiosis, B. bruxellensis cells exclusively used them to support biomass formation, and no ethanol was produced. Moreover, whereas l-arabinose was not consumed under oxygen limitation, d-xylose was only slightly used, which resulted in low ethanol yield and productivity. In conclusion, our results showed that d-xylose and l-arabinose are not efficiently converted to ethanol by B. bruxellensis, most likely due to a redox imbalance in the assimilatory pathways of these sugars. Therefore, despite presenting other industrially relevant traits, the employment of B. bruxellensis in second-generation ethanol production depends on the development of genetic engineering strategies to overcome this metabolic bottleneck.

Gene regulation of the Lactobacillus vini in response to industrial stress in the fuel ethanol production.

The industrial ethanol fermentation imposes several stresses to microorganisms. However, some bacterial species are well adapted and manage to endure these harmful conditions. Lactobacillus vini is one of the most found bacteria in these environments, indicating the existence of efficient tolerance mechanisms. In view of this premise, the present study aimed to describe the tolerance of L. vini to several stressing agents encounter in industrial environments and the genetic components of the stress response. In general, L. vini showed significant tolerance to stressors commonly found in fuel-ethanol fermentations, and only doses higher than normally reached in processes restrained its growth. The lag phase and the growth rate were the most responsive kinetic parameter affected. Gene expression analysis revealed that uspII gene positively responded to all conditions tested, a typical profile of a general stress response gene. In addition, the results also revealed aspects of regulatory modules of co-expressed genes responding to different stresses, and also the similarities of response mechanism with basis in common cellular damages. Altogether, these data contribute to uncover the factors that could favour L. vini in the industrial fermentation which could be shared with other well adapted species and reports the first stress response genes in this bacterium.

The biotechnological potential of the yeast Dekkera bruxellensis.

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.

Lactobacillus vini: mechanistic response to stress by medium acidification.

This work describes the response of Lactobacillusvini, a bacterium found as a contaminant in winemaking and fuel ethanol fermentation processes, to acid stress caused by inorganic or weak organic acids. First, we observed for the first time that bacterial cells become resistant to lysis by lysozyme when submitted to acidic stress. Then, the predicted intracellular acidification can be reversed by the presence of arginine, histidine and glutamine. However, these molecules were not able to reverse the effect of resistance to lysis, indicating the independence of these mechanisms. In general, a reduction in the expression of the main genes involved in the synthesis and deposition of material in the cell wall was observed, whereas the genes involved in the reabsorption of this structure showed increased expression. These data suggested that L. vini responds to the acidification of the medium through early entry into the stationary phase, firing two signals for cell wall remodelling and maintenance of intracellular pHin a coordinated way, most probably by alkalization and the proton extrusion process. If this picture is conserved among lactobacilli, it may not only have an impact on research associated with fermentation processes, but also on that associated with probiotic improvement.

Nitrate boosts anaerobic ethanol production in an acetate-dependent manner in the yeast Dekkera bruxellensis.

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.

First aspects on acetate metabolism in the yeast Dekkera bruxellensis: a few keys for improving ethanol fermentation.

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.

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.

Magnesium ions in yeast: setting free the metabolism from glucose catabolite repression.

In a recent work we showed that magnesium (MgII) plays an important role in industrial ethanol production, overcoming the negative effect of the excess of minerals, particularly copper, present in sugarcane juice, with a consequent increase in ethanol yield. This cation has been reported to be involved in several steps of yeast metabolism, acting mainly as a co-factor of several enzymes of fermentation metabolism and protecting yeast cells from stressful conditions. However, despite many physiological investigations, its effect in the molecular mechanisms that control such metabolic activities remains unclear and to date no information concerning its influence on gene expression has been provided. The present work took advantage of the DNA microarray technology to analyse the global gene expression in yeast cells upon fermentation in MgII-supplemented medium. The results of the fermentation parameters confirmed the previous report on the increase in ethanol yield by MgII. Moreover, the gene expression data revealed an unexpected set of up-regulated genes currently assigned as being negatively-regulated by glucose, which belong to respiratory and energy metabolism, the stress response and the glyoxalate cycle. On the other hand, genes involved in ribosome biogenesis were down-regulated. Computational analysis provided evidence for a regulatory network commanded by key transcriptional factors that may be responsible for the biological action of MgII in yeast cells. In this scenario, MgII seems to act by reprogramming the yeast metabolism by releasing many genes from glucose catabolite repression with positive consequences for ethanol production and maintenance of cell viability.

Occurrence and analysis of irp2 virulence gene in isolates of Klebsiella pneumoniae and Enterobacter spp. from microbiota and hospital and community-acquired infections.

Eighty-five isolates of Klebsiella pneumoniae and Enterobacter spp., originating from hospital- and community-acquired infections and from oropharyngeal and faecal microbiota from patients in Recife-PE, Brazil, were analyzed regarding the presence of irp2 gene. This is a Yersinia typical gene involved in the synthesis of siderophore yersiniabactin. DNA sequencing confirmed the identity of irp2 gene in five K. pneumoniae, five Enterobacter aerogenes and one Enterobacter amnigenus isolates. To our knowledge in the current literature, this is the first report of the irp2 gene in E. amnigenus, a species considered an unusual human pathogen, and in K. pneumoniae and E. aerogenes isolates from the normal microbiota and from community infections, respectively. Additionally, the analyses of nucleotide and amino acid sequences suggest the irp2 genes derived from isolates used in this study are more closely related to that of Yersinia pestis P.CE882 than to that of Yersinia enterocolitica 8081. These data demonstrated that K. pneumoniae and Enterobacter spp. from normal microbiota and from community- and hospital-acquired infections possess virulence factors important for the establishment of extra-intestinal infections.

Transcriptomic response of Saccharomyces cerevisiae for its adaptation to sulphuric acid-induced stress.

In bioethanol production plants, yeast cells are generally recycled between fermentation batches by using a treatment with sulphuric acid at a pH ranging from 2.0 to 2.5. We have previously shown that Saccharomyces cerevisiae cells exposed to sulphuric acid treatment induce the general stress response pathway, fail to activate the protein kinase A signalling cascade and requires the mechanisms of cell wall integrity and high osmolarity glycerol pathways in order to survive in this stressful condition. In the present work, we used transcriptome-wide analysis as well as physiological assays to identify the transient metabolic responses of S. cerevisiae under sulphuric acid treatment. The results presented herein indicate that survival depends on a metabolic reprogramming of the yeast cells in order to assure the yeast cell viability by preventing cell growth under this harmful condition. It involves the differential expression of a subset of genes related to cell wall composition and integrity, oxidation-reduction processes, carbohydrate metabolism, ATP synthesis and iron uptake. These results open prospects for application of this knowledge in the improvement of industrial processes based on metabolic engineering to select yeasts resistant to acid treatment.

Emergence of extensively drug-resistant OXA-72-producing Acinetobacter baumannii in Recife, Brazil: risk of clonal dissemination?

Two new examples of OXA-72-producing Acinetobacter baumannii isolate resistant to a broad spectrum of antimicrobials, but not polymyxin B, have been identified in Recife, Brazil. Molecular typing indicated a close genetic link with the OXA-72-producing A. baumannii previously isolated in São Paulo, suggesting the possibility of clonal dissemination within the country.

Genome sequences of the ethanol-tolerant Lactobacillus vini strains LMG 23202T and JP7.8.9.

We report on the genome sequences of Lactobacillus vini type strain LMG 23202(T) (DSM 20605) (isolated from fermenting grape musts in Spain) and the industrial strain L. vini JP7.8.9 (isolated from a bioethanol plant in northeast Brazil). All contigs were assembled using gsAssembler, and genes were predicted and annotated using Rapid Annotation using Subsystem Technology (RAST). The identified genome sequence of LMG 23202(T) had 2.201.333 bp, 37.6% G+C, and 1,833 genes, whereas the identified genome sequence of JP7.8.9 had 2.301.037 bp, 37.8% G+C, and 1,739 genes. The gene repertoire of the species L. vini offers promising opportunities for biotechnological applications.

Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification.

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90.6% of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.

Genotoxic activity of 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid and its peptidyl derivatives determined by Ames and SOS response tests.

Compounds derived from 1,2,4-oxadiazole have being reported for their anti-inflammatory activity. However, those compounds should be devoid of any genotoxic side effect. In this work, the genotoxic activity of peptidomimetic moiety-containing 1,2,4-oxadiazoles derivatives was tested based on the Ames and SOS Chromotest. The results showed no mutagenic activity on the Ames test for 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid (POPA) parental drug, but a weak SOS response induction on Chromotest. The chemical modifications reduced that response to a non-significative level, with l-phenylalanine peptidomimetic derivative being showing the lowest induction response. The results pointed out for the effectiveness of promoting chemical modifications of biological active compounds to increase its mode of action, showed in previous work, without increasing and even decreasing its DNA damage effect.

Molecular typing of Klebsiella pneumoniae isolates from public hospitals in Recife, Brazil.

Thirty nosocomial isolates of Klebsiella pneumoniae, collected from three public hospitals in Recife, Brazil, between 1999 and 2000, were analysed in order to determine their epidemiological relatedness and genetic characteristics. The isolates were characterised by biotyping, antibiotyping, protein analysis, plasmid profile and random amplified polymorphic DNA (RAPD). The majority of the clinical isolates were resistant to multiple antibiotics, in particular to beta-lactams, and 30% were found to be ESBLs producers. RAPD proved to be the most effective technique in discriminating unrelated K. pneumoniae isolates. It was confirmed by the highly genetic similarity found among related isolates from an hospital outbreak. We conclude that K. pneumoniae infections in Recife has been caused by a variety of bacterial genotypes. This is the first report that revealed the subtypes of K. pneumoniae in Brazil by plasmid analysis and RAPD.