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.

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.

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.

Journey of the Probiotic Bacteria: Survival of the Fittest.

This review aims to bring a more general view of the technological and biological challenges regarding production and use of probiotic bacteria in promoting human health. After a brief description of the current concepts, the challenges for the production at an industrial level are presented from the physiology of the central metabolism to the ability to face the main forms of stress in the industrial process. Once produced, these cells are processed to be commercialized in suspension or dried forms or added to food matrices. At this stage, the maintenance of cell viability and vitality is of paramount for the quality of the product. Powder products requires the development of strategies that ensure the integrity of components and cellular functions that allow complete recovery of cells at the time of consumption. Finally, once consumed, probiotic cells must face a very powerful set of physicochemical mechanisms within the body, which include enzymes, antibacterial molecules and sudden changes in pH. Understanding the action of these agents and the induction of cellular tolerance mechanisms is fundamental for the selection of increasingly efficient strains in order to survive from production to colonization of the intestinal tract and to promote the desired health benefits.

Production of 1,3-propanediol by Lactobacillus diolivorans from agro-industrial residues and cactus cladode acid hydrolyzate.

This study evaluated the bioproduction of 1,3-propanediol by Lactobacillus diolivorans in the medium based on agro-industrial residues and vegetal biomass substituting the MRS medium components. It was performed on a set of acid treatments and batch fermentations assays with crude glycerol (TCG) from biodiesel production, corn steep liquor (CSL), and cactus cladode hydrolyzate (CCH). Firstly, it was carried out on batch fermentation with different pure glycerol concentrations in MRS medium which was carried out, and the best condition achieved 4.66 g/L and 0.61 g/g of 1,3-PDO production and yield, respectively. Then, the TCG was evaluated, and a discrete increase of 1,3-PDO was observed. The replacement of the MRS medium nutrients by CLS was assessed, at different concentrations, for bacteria growth, and 5% of CLS reproduced the same biomass formation compared to the bacteria growth in MRS medium. It was also added cactus cladode hydrolyzate as the only sugar source, which showed a 1,3-PDO production close to the medium with pure glucose. Finally, a B-complex vitamin was added to the batch fermentation medium composed of TCG, CLS, and CCH, replacing all the costly MRS components. In this medium, the production of 1,3-propanediol was 6.57 g/L with a yield of 0.75 g/g. It means an increment of 29% and 19%, respectively, compared to MRS medium. Therefore, the combination of treated crude glycerol, corn steep liquor, and cactus cladode hydrolyzate has excellent potential for 1,3-PDO production by L. diolivorans.

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.

Nutritional requirements for Lactobacillus vini growth in sugarcane derivative substrate of ethanol fermentation.

Lactobacillus vini is a bacterial contaminant found in industrial environments of winemaking and fuel-ethanol fermentation. However, there has been no standard analysis of its physiology that can pinpoint its adaptive traits to these kinds of environments. In view of this lack of information, the aim of this study is to determine the nutritional factors that lead to the growth of L. vini in the industrial plants of fuel-ethanol. First of all, the limited growth of this bacterium was studied in the industrial substrate, which was improved by nutritional supplementation with amino acids, and its homofermentative status was confirmed. Metabolite analysis showed that citrate is a growth factor of paramount importance for this bacterium in industrial processes through pyruvate metabolization, and increases ATP production and biomass formation. Furthermore,e acetate uptake, either from the medium or generated from citrate metabolism, was assimilated for biomass production. Hence, a metabolic model was designed to describe the role of citrate and acetate in the growth of L. vini that could be tested on other lactobacilli.

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.

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

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

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.

Mineral composition of the sugarcane juice and its influence on the ethanol fermentation.

In the present work, we evaluated the mineral composition of three sugarcane varieties from different areas in northeast Brazil and their influence on the fermentation performance of Saccharomyces cerevisiae. The mineral composition was homogeneous in the different areas investigated. However, large variation coefficients were observed for concentrations of copper, magnesium, zinc and phosphorus. Regarding the fermentation performances, the sugarcane juices with the highest magnesium concentration showed the highest ethanol yield. Synthetic media supplemented with magnesium also showed the highest yield (0.45 g g(-1)) while the excess of copper led to the lowest yield (0.35 g g(-1)). According to our results, the magnesium is the principal responsible for the increase on the ethanol yield, and it also seems to be able to disguise the inhibitory effects of the toxic minerals present in the sugarcane juice.

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

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