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.

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.

Comparative proteomic analyses reveal the metabolic aspects and biotechnological potential of nitrate assimilation in the yeast Dekkera bruxellensis.

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.

Incomplete mitophagy in the mevalonate kinase-deficient Saccharomyces cerevisiae and its relation to the MKD-related autoinflammatory disease in humans.

Mevalonate kinase deficiency (MKD) is an autosomal recessive disorder in humans that causes systemic autoinflammatory problems to children. Previously, we used a yeast model to show that MKD results in mitochondrial malfunctioning that may finally induce mitophagy. Here, we proved that MKD indeed induced general autophagy as well as mitophagy in yeast, but these mechanisms did not go to completion. Therefore, the limitation of mevalonate kinase activity produces dysfunctional mitochondria that might not be recycled, causing metabolic dysfunctions in the cells. Understanding this mechanism may provide a piece in solving the nonspecific autoinflammatory response puzzle observed in MKD patients.

Second-generation ethanol production by Wickerhamomyces anomalus strain adapted to furfural, 5-hydroxymethylfurfural (HMF), and high osmotic pressure.

The aims of this work were to improve cell tolerance towards high concentrations of furfural and 5-hydroxymethylfurfural (HMF) of an osmotolerant strain of Wickerhamomyces anomalus by means of evolutionary engineering, and to determine its ethanol production under stress conditions. Cells were grown in the presence of furfural, HMF, either isolated or in combination, and under high osmotic pressure conditions. The most toxic condition for the parental strain was the combination of both furans, under which it was unable to grow and to produce ethanol. However, the tolerant adapted strain achieved a yield of ethanol of 0.43 g g-1glucose in the presence of furfural and HMF, showing an alcohol dehydrogenase activity of 0.68 mU mg protein-1. For this strain, osmotic pressure, did not affect its growth rate. These results suggest that W. anomalus WA-HF5.5strain shows potential to be used in second-generation ethanol production systems.

Extreme Low Cytosolic pH Is a Signal for Cell Survival in Acid Stressed Yeast.

Yeast biomass is recycled in the process of bioethanol production using treatment with dilute sulphuric acid to control the bacterial population. This treatment can lead to loss of cell viability, with consequences on the fermentation yield. Thus, the aim of this study was to define the functional cellular responses to inorganic acid stress. Saccharomyces cerevisiae strains with mutation in several signalling pathways, as well as cells expressing pH-sensitive GFP derivative ratiometric pHluorin, were tested for cell survival and cytosolic pH (pHc) variation during exposure to low external pH (pHex). Mutants in calcium signalling and proton extrusion were transiently sensitive to low pHex, while the CWI slt2Δ mutant lost viability. Rescue of this mutant was observed when cells were exposed to extreme low pHex or glucose starvation and was dependent on the induced reduction of pHc. Therefore, a lowered pHc leads to a complete growth arrest, which protects the cells from lethal stress and keeps cells alive. Cytosolic pH is thus a signal that directs the growth stress-tolerance trade-off in yeast. A regulatory model was proposed to explain this mechanism, indicating the impairment of glucan synthesis as the primary cause of low pHex sensitivity.

Biodiversity among Brettanomyces bruxellensis Strains Isolated from Different Wine Regions of Chile: Key Factors Revealed about its Tolerance to Sulphite.

Brettanomyces bruxellensis is regarded as the main spoilage microorganism in the wine industry, owing to its production of off-flavours. It is difficult to eradicate owing to its high tolerance of adverse environmental conditions, such as low nutrient availability, low pH, and high levels of ethanol and SO2. In this study, the production of volatile phenols and the growth kinetics of isolates from various regions of Chile were evaluated under stressful conditions. Through randomly amplified polymorphic DNA (RAPD) analysis, 15 strains were identified. These were grown in the presence of p-coumaric acid, a natural antimicrobial and the main precursor of off-flavours, and molecular sulfur dioxide (mSO2), an antimicrobial synthetic used in the wine industry. When both compounds were used simultaneously, there were clear signs of an improvement in the fitness of most of the isolates, which showed an antagonistic interaction in which p-coumaric acid mitigates the effects of SO2. Fourteen strains were able to produce 4-vinylphenol, which showed signs of phenylacrylic acid decarboxylase activity, and most of them produced 4-ethylphenol as a result of active vinylphenol reductase. These results demonstrate for the first time the serious implications of using p-coumaric acid, not only for the production of off-flavours, but also for its protective action against the toxic effects of SO2.

The effects of the Ncw2 protein of Saccharomyces cerevisiae on the positioning of chitin in response to cell wall damage.

The recently described NCW2 gene encodes a protein that is assumed to be located in the cell wall (CW). This protein was proposed to participate in the repair of CW damages induced by polyhexamethylene biguanide (PHMB). However, much of the information on the biological function(s) of Ncw2p still remains unclear. In view of this, this study seeks to extend the analysis of this gene in light of the way its protein functions in the Cell Wall Integrity (CWI) mechanism. Deletion of the NCW2 gene led to constitutive overexpression of some key CWI genes and increased chitin deposition in the walls of cells exposed to PHMB. This means the lack of Ncw2p might activate a compensatory mechanism that upregulates glucan CWI genes for cell protection by stiffening the CW. This condition seems to alleviate the response through the HOG pathway and makes cells sensitive to osmotic stress. However, Ncw2p may not have been directly involved in tolerance to osmotic stress itself. The results obtained definitely place the NCW2 gene in the list of CWI genes of S. cerevisiae and indicate that its protein has an auxiliary function in the maintenance of the glucan/chitin balance and ensuring the correct structure of the yeast cell wall.

Lawsone, a 2-hydroxy-1,4-naphthoquinone from Lawsonia inermis (henna), produces mitochondrial dysfunctions and triggers mitophagy in Saccharomyces cerevisiae.

Lawsone is a natural naphthoquinone present in the henna leaf extract with several cytotoxic activities and used as precursor for synthesis of various pharmaceutical compounds. Its biological activities are thought to be the result of oxidative stress generated, although the hydroxy group at position C-2 in its structure tends to reduce its electrophilic potential. In view of lack of knowledge on its activity, the present work aimed to elucidate the biological effect of lawsone using the yeast Saccharomyces cerevisiae. In the model strain BY4741 it was defined 229 mmol/L as the minimal inhibitory concentration (MIC). Using 172 mmol/L as sub-MIC value it was observed that yap1 deletion mutant was sensitive to lawsone independent the presence of oxygen. Lawsone affected yeast growth in glycerol, indicating interference in the respiratory metabolism. Intracellular content of thiol groups did not indicate intensive oxidative stress and the presence of the anti-oxidant N-acetylcysteine (NAC) exacerbated lawsone toxicity. By analysing the sensitivity of atg mutant strains and the localization of GFP-Atg8 fusion protein, it was concluded that lawsone primarily produces mitochondrial malfunctioning, leading to indirect oxidative stress. It triggers the autophagic response that ultimately induces mitophagy.

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.

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.

Selection of Saccharomyces cerevisiae isolates for ethanol production in the presence of inhibitors.

Eight yeast isolates identified as Saccharomyces cerevisiae were recovered from molasses-using Cuban distilleries and discriminated by nucleotide sequence analysis of ITS locus. The isolates L/25-7-81 and L/25-7-86 showed the highest ethanol yield from sugarcane juice, while L/25-7-12 and L/25-7-79 showed high ethanol yield from sugarcane molasses. The isolate L/25-7-86 also displayed high fermentation capacity when molasses was diluted with vinasse. In addition, stress tolerance was evaluated on the basis of growth in the presence of inhibitors (acetic acid, lactic acid, 5-hydroxymethylfurfural and sulfuric acid) and the results indicated that L/25-7-77 and L/25-7-79 congregated the highest score for cross-tolerance and fermentation capacity. Hence, these isolates, especially L/25-7-77, could serve as potential biological platform for the arduous task of fermenting complex substrates that contain inhibitors. The use of these yeasts was discussed in the context of second-generation ethanol and the environmental and economic implications of the use of vinasse, saving the use of water for substrate dilution.

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.

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.

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.

Enzymatic hydrolysis of cactus pear varieties with high solids loading for bioethanol production.

The optimization of enzymatic hydrolysis, with high solids loading, of two species of cactus pear for bioethanol production was tested evaluating the influence of surfactant Tween 80 and pretreatment with H2O and H2SO4 (1% v/v) (50 °C, 150 rpm, 3 h). XRD and FTIR analyzes were performed. Afterwards, the influence of the factors cellulase (FPU g-1), pectinase (U g-1) and solids load (% w/v), on the hydrolysis of varieties (50 °C, 150 rpm, 48 h), and the fermentation of the optimal point (33 °C, 8 h) were evaluated. The pretreatments and the Tween 80 did not increase the hydrolysis yields and Rotacional Central Compound Design indicated that the pectinase factor was not significant. The best cellulase and solids load conditions were 10 FPU g-1 of biomass and 30% w/v for both species. The fermentation efficiency of hydrolysates for Nopalea cochenillifera and Opuntia ficus-indica were 76.3% and 82.8%, respectively, showing their potential for bioethanol production.

Recycling the liquid fraction of alkaline hydrogen peroxide in the pretreatment of corn stover.

The aim of this study was to evaluate the influence of recycling the liquid fraction of pretreatment with alkaline hydrogen peroxide (AHP) on the hydrolysis of corn stover. Corn stover was pretreated in the traditional condition with 7.5% v/v H2O2. After pretreatment, the solids were separated from the liquid fraction and five successive reuse cycles of the liquid fraction were tested. The solid fraction from pretreatment in each recycle was submitted to enzymatic hydrolysis. The number of recycles had a linear negative effect (R2=0.98) on biomass delignification efficiency and also affected negatively the enzymatic conversion efficiency. Despite the decrease in efficiency after each recycling step, reuse of the liquid fraction leads to reduction in water, H2O2 and NaOH consumption of up to 57.6%, 59.6% and 57.6%, respectively. These findings point to an efficient recycling technology, which may reduce costs and save water.

NCW2, a Gene Involved in the Tolerance to Polyhexamethylene Biguanide (PHMB), May Help in the Organisation of ß-1,3-Glucan Structure of Saccharomyces cerevisiae Cell Wall.

In the present work, we provide biological evidences supporting the participation of NCW2 gene in the mechanism responsible for cell tolerance to polyhexamethylene biguanide (PHMB), an antifungal agent. The growth rate of yeast cells exposed to this agent was significantly reduced in ∆ncw2 strain and the mRNA levels of NCW2 gene in the presence of PHMB showed a 7-fold up-regulation. Moreover, lack of NCW2 gene turns yeast cell more resistant to zymolyase treatment, indicating that alterations in the ß-glucan network do occur when Ncw2p is absent. Computational analysis of the translated protein indicated neither catalytic nor transmembrane sites and reinforced the hypothesis of secretion and anchoring to cell surface. Altogether, these results indicated that NCW2 gene codes for a protein which participates in the cell wall biogenesis in yeasts and that Ncw2p might play a role in the organisation of the ß-glucan assembly.

First identification of Tn916-like element in industrial strains of Lactobacillus vini that spread the tet-M resistance gene.

The open process used to ferment sugar cane juice or molasses to produce ethanol fuel is prone to contamination by bacterial cells of different species, in particular Lactobacilli. The situation can be exacerbated by the emergence of resistant cells to industrial antibiotics that are normally used to combat this contamination. In this work, two Lactobacillus vini isolates from ethanol distilleries were identified and found to be resistant to doxycycline, a tetracycline derivative, although sensitive to other antibiotics tested. The identification of these isolates was confirmed by sequencing the pheS gene and their clonal origin was shown by PCR-fingerprinting analysis. Moreover, the isolates were shown to carry the transposable element Tn916 that harboured the tet-M gene. Furthermore, conjugation experiments showed that both isolates were capable of transferring this element, and as a result, the tet-M gene, to Enterococcus faecalis reference strain. Finally, the identification of tetracycline resistance in the same distilleries in other Lactobacilli, suggested that inter-species transfer of antibiotic resistance may be occurring in the industrial environment, and thus impairing the efficiency of the antibiotic treatment and causing serious health concerns.