Effect of overexpression of partial TDH1 and TDH2/3 gene sequences in a starter strain of industrial bioethanol fermentation on the Brettanomyces bruxellensis contaminant growth.

Selected Saccharomyces cerevisiae strains, such as the commercial Ethanol-Red (ER) strain, are used as starters in the bioethanol industry. Yet, bioethanol fermentations are prone to microbial contaminations, mainly by Brettanomyces bruxellensis and lactic acid bacteria. Chemicals, such as sulphuric acid and antibiotics, are commonly used to combat those contaminations, but they have negative environmental impacts. Recently, ER strain was found to secrete antimicrobial peptides (AMPs) active against B. bruxellensis. Therefore, the partial TDH1 and TDH2/3 genes sequences that codify those AMPs were inserted into the pSR41k plasmid and cloned in ER strains. The relative expression levels (plasmidic/genomic) of those sequences in the respective modified ER strains were quantified by real-time quantitative polimerase chain reaction (RT-qPCR), confirming their overexpression. The effect of the modified strains on B. bruxellensis (Bb) growth was then evaluated during synthetic must (SM) and carob syrup (CS) fermentations, co-inoculated with 105 cells ml-1 of ER and Bb in SM and with 106 of ER and 5 × 103 cells ml-1 of Bb in CS. Results showed that modified ER strains exerted a much higher inhibitory effect against B. bruxellensis (72-fold in SM and 10-fold in CS) than the non-modified ER strain. In those fermentations, 90-100 g l-1 of ethanol was produced in 3-6 days.

Wine Spoilage Control: Impact of Saccharomycin on Brettanomyces bruxellensis and Its Conjugated Effect with Sulfur Dioxide.

The yeast Brettanomyces bruxellensis is one of the most dangerous wine contaminants due to the production of phenolic off-flavors such as 4-ethylphenol. This microbial hazard is regularly tackled by addition of sulfur dioxide (SO2). Nevertheless, B. bruxellensis is frequently found at low levels (ca 103 cells/mL) in finished wines. Besides, consumers health concerns regarding the use of sulfur dioxide encouraged the search for alternative biocontrol measures. Recently, we found that Saccharomyces cerevisiae secretes a natural biocide (saccharomycin) that inhibits the growth of different B. bruxellensis strains during alcoholic fermentation. Here we investigated the ability of S. cerevisiae CCMI 885 to prevent B. bruxellensis ISA 2211 growth and 4-ethylphenol production in synthetic and true grape must fermentations. Results showed that B. bruxellensis growth and 4-ethylphenol production was significantly inhibited in both media, although the effect was more pronounced in synthetic grape must. The natural biocide was added to a simulated wine inoculated with 5 × 102 cells/mL of B. bruxellensis, which led to loss of culturability and viability (100% dead cells at day-12). The conjugated effect of saccharomycin with SO2 was evaluated in simulated wines at 10, 12, 13 and 14% (v/v) ethanol. Results showed that B. bruxellensis proliferation in wines at 13 and 14% (v/v) ethanol was completely prevented by addition of 1.0 mg/mL of saccharomycin with 25 mg/L of SO2, thus allowing to significantly reduce the SO2 levels commonly used in wines (150-200 mg/L).

Proteomics insights into the responses of Saccharomyces cerevisiae during mixed-culture alcoholic fermentation with Lachancea thermotolerans.

The response of Saccharomyces cerevisiae to cocultivation with Lachancea thermotolerans during alcoholic fermentations has been investigated using tandem mass tag (TMT)-based proteomics. At two key time-points, S. cerevisiae was sorted from single S. cerevisiae fermentations and from mixed fermentations using flow cytometry sorting. Results showed that the purity of sorted S. cerevisiae was above 96% throughout the whole mixed-culture fermentation, thereby validating our sorting methodology. By comparing protein expression of S. cerevisiae with and without L. thermotolerans, 26 proteins were identified as significantly regulated proteins at the early death phase (T1), and 32 significantly regulated proteins were identified at the late death phase (T2) of L. thermotolerans in mixed cultures. At T1, proteins involved in endocytosis, increasing nutrient availability, cell rescue and resistance to stresses were upregulated, and proteins involved in proline synthesis and apoptosis were downregulated. At T2, proteins involved in protein synthesis and stress responses were up- and downregulated, respectively. These data indicate that S. cerevisiae was stressed by the presence of L. thermotolerans at T1, using both defensive and fighting strategies to keep itself in a dominant position, and that it at T2 was relieved from stress, perhaps increasing its enzymatic machinery to ensure better survival.

Biocontrol of Brettanomyces/Dekkera bruxellensis in alcoholic fermentations using saccharomycin-overproducing Saccharomyces cerevisiae strains.

Microbial contamination of alcoholic fermentation processes (e.g. winemaking and fuel-ethanol production) is a serious problem for the industry since it may render the product unacceptable and/or reduce its productivity, leading to large economic losses. Brettanomyces/Dekkera bruxellensis is one of the most dangerous microbial contaminant of ethanol industrial fermentations. In the case of wine, this yeast species can produce phenolic compounds that confer off-flavours to the final product. In fuel-ethanol fermentations, D. bruxellensis is a persistent contaminant that affects ethanol yields and productivities. We recently found that Saccharomyces cerevisiae secretes a biocide, which we named saccharomycin, composed of antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Saccharomycin is active against several wine-related yeast species, namely D. bruxellensis. However, the levels of saccharomycin naturally secreted by S. cerevisiae during alcoholic fermentation are not sufficient to ensure the complete death of D. bruxellensis. Therefore, the aim of the present work was to construct genetically modified S. cerevisiae strains to overproduce these GAPDH-derived AMPs. The expression levels of the nucleotides sequences encoding the AMPs were evaluated in the modified S. cerevisiae strains by RT-qPCR, confirming the success of the recombinant approach. Furthermore, we confirmed by immunological tests that the modified S. cerevisiae strains secreted higher amounts of the AMPs by comparison with the non-modified strain, inducing total death of D. bruxellensis during alcoholic fermentations.

Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux rates.

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.

Saccharomyces cerevisiae accumulates GAPDH-derived peptides on its cell surface that induce death of non-Saccharomyces yeasts by cell-to-cell contact.

During wine fermentations, Saccharomyces cerevisiae starts to excrete antimicrobial peptides (AMPs) into the growth medium that induce death of non-Saccharomyces yeasts at the end of exponential growth phase (24-48 h). Those AMPs were found to derive from the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). On the other hand, the early death of non-Saccharomyces yeasts during wine fermentations was also found to be mediated by a cell-to-cell contact mechanism. Since GAPDH is a cell-wall-associated protein in S. cerevisiae, we put forward the hypothesis that the GAPDH-derived AMPs could accumulate on the cell surface of S. cerevisiae, thus inducing death of non-Saccharomyces yeasts by cell-to-cell contact. Here we show that 48-h grown (stationary phase) cells of S. cerevisiae induce death of Hanseniaspora guilliermondii and Lachancea thermotolerans by direct cell-to-cell contact, while 12-h grown cells (mid-exponential phase) do not. Immunological tests performed with a specific polyclonal antibody against the GAPDH-derived AMPs revealed their presence in the cell wall of S. cerevisiae cells grown for 48 h, but not for 12 h. Taken together, our data show that accumulation of GAPDH-derived AMPs on the cell surface of S. cerevisiae is one of the factors underlying death of non-Saccharomyces yeasts by cell-to-cell contact.

Antimicrobial properties and death-inducing mechanisms of saccharomycin, a biocide secreted by Saccharomyces cerevisiae.

We recently found that Saccharomyces cerevisiae (strain CCMI 885) secretes antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that are active against various wine-related yeast and bacteria. Here, we show that several other S. cerevisiae strains also secrete natural biocide fractions during alcoholic fermentation, although at different levels, which correlates with the antagonistic effect exerted against non-Saccharomyces yeasts. We, therefore, term this biocide saccharomycin. The native AMPs were purified by gel-filtration chromatography and its antimicrobial activity was compared to that exhibited by chemically synthesized analogues (AMP1 and AMP2/3). Results show that the antimicrobial activity of the native AMPs is significantly higher than that of the synthetic analogues (AMP1 and AMP2/3), but a conjugated action of the two synthetic peptides is observed. Moreover, while the natural AMPs are active at pH 3.5, the synthetic peptides are not, since they are anionic and cannot dissolve at this acidic pH. These findings suggest that the molecular structure of the native biocide probably involves the formation of aggregates of several peptides that render them soluble under acidic conditions. The death mechanisms induced by the AMPs were also evaluated by means of epifluorescence microscopy-based methods. Sensitive yeast cells treated with the synthetic AMPs show cell membrane disruption, apoptotic molecular markers, and internalization of the AMPs. In conclusion, our work shows that saccharomycin is a natural biocide secreted by S. cerevisiae whose activity depends on the conjugated action of GAPDH-derived peptides. This study also reveals that S. cerevisiae secretes GAPDH-derived peptides as a strategy to combat other microbial species during alcoholic fermentations.

Dominance of Saccharomyces cerevisiae in alcoholic fermentation processes: role of physiological fitness and microbial interactions.

Winemaking, brewing and baking are some of the oldest biotechnological processes. In all of them, alcoholic fermentation is the main biotransformation and Saccharomyces cerevisiae the primary microorganism. Although a wide variety of microbial species may participate in alcoholic fermentation and contribute to the sensory properties of end-products, the yeast S. cerevisiae invariably dominates the final stages of fermentation. The ability of S. cerevisiae to outcompete other microbial species during alcoholic fermentation processes, such as winemaking, has traditionally been ascribed to its high fermentative power and capacity to withstand the harsh environmental conditions, i.e. high levels of ethanol and organic acids, low pH values, scarce oxygen availability and depletion of certain nutrients. However, in recent years, several studies have raised evidence that S. cerevisiae, beyond its remarkable fitness for alcoholic fermentation, also uses defensive strategies mediated by different mechanisms, such as cell-to-cell contact and secretion of antimicrobial peptides, to combat other microorganisms. In this paper, we review the main physiological features underlying the special aptitude of S. cerevisiae for alcoholic fermentation and discuss the role of microbial interactions in its dominance during alcoholic fermentation, as well as its relevance for winemaking.

Cell-to-cell contact and antimicrobial peptides play a combined role in the death of Lachanchea thermotolerans during mixed-culture alcoholic fermentation with Saccharomyces cerevisiae.

The roles of cell-to-cell contact and antimicrobial peptides in the early death of Lachanchea thermotolerans CBS2803 during anaerobic, mixed-culture fermentations with Saccharomyces cerevisiae S101 were investigated using a commercially available, double-compartment fermentation system separated by cellulose membranes with different pore sizes, i.e. 1000 kDa for mixed- and single-culture fermentations, and 1000 and 3.5-5 kDa for compartmentalized-culture fermentations. SDS-PAGE and gel filtration chromatography were used to determine an antimicrobial peptidic fraction in the fermentations. Our results showed comparable amounts of the antimicrobial peptidic fraction in the inner compartments of the mixed-culture and 1000 kDa compartmentalized-culture fermentations containing L. thermotolerans after 4 days of fermentation, but a lower death rate of L. thermotolerans in the 1000 kDa compartmentalized-culture fermentation than in the mixed-culture fermentation. Furthermore, L. thermotolerans died off even more slowly in the 3.5-5 kDa than in the 1000 kDa compartmentalized-culture fermentation, which coincided with the presence of less of the antimicrobial peptidic fraction in the inner compartment of that fermentation than of the 1000 kDa compartmentalized-culture fermentation. Taken together, these results indicate that the death of L. thermotolerans in mixed cultures with S. cerevisiae is caused by a combination of cell-to-cell contact and antimicrobial peptides.

Antimicrobial peptides (AMPs) produced by Saccharomyces cerevisiae induce alterations in the intracellular pH, membrane permeability and culturability of Hanseniaspora guilliermondii cells.

Saccharomyces cerevisiae produces antimicrobial peptides (AMPs) during alcoholic fermentation that are active against several wine-related yeasts (e.g. Hanseniaspora guilliermondii) and bacteria (e.g. Oenococcus oeni). In the present study, the physiological changes induced by those AMPs on sensitive H. guilliermondii cells were evaluated in terms of intracellular pH (pHi), membrane permeability and culturability. Membrane permeability was evaluated by staining cells with propidium iodide (PI), pHi was determined by a fluorescence ratio imaging microscopy (FRIM) technique and culturability by a classical plating method. Results showed that the average pHi of H. guilliermondii cells dropped from 6.5 (healthy cells) to 5.4 (damaged cells) after 20 min of exposure to inhibitory concentrations of AMPs, and after 24 h 77.0% of the cells completely lost their pH gradient (∆pH=pHi-pHext). After 24h of exposure to AMPs, PI-stained (dead) cells increased from 0% to 77.7% and the number of viable cells fell from 1×10(5) to 10 CFU/ml. This means that virtually all cells (99.99%) became unculturable but that a sub-population of 22.3% of the cells remained viable (as determined by PI staining). Besides, pHi results showed that after 24h, 23% of the AMP-treated cells were sub-lethally injured (with 0<∆pH<3). Taken together, these results indicated that this subpopulation was under a viable but non-culturable (VBNC) state, which was further confirmed by recuperation assays. In summary, our study reveals that these AMPs compromise the plasma membrane integrity (and possibly also the vacuole membrane) of H. guilliermondii cells, disturbing the pHi homeostasis and inducing a loss of culturability.

Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactions.

Saccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.

Survival rate of wine-related yeasts during alcoholic fermentation assessed by direct live/dead staining combined with fluorescence in situ hybridization.

Real-time detection of microorganisms involved in complex microbial process, such as wine fermentations, and evaluation of their physiological state is crucial to predict whether or not those microbial species will be able to impact the final product. In the present work we used a direct live/dead staining (LDS) procedure combined with fluorescence in situ hybridization (FISH) to simultaneously assess the identity and viability of Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) during fermentations performed with single and mixed cultures. The population evolution of both yeasts was determined by plating and by LDS combined with species-specific FISH-probes labeled with Fluorescein. Since the FISH method involves the permeabilization of the cell membrane prior to hybridization and that it may influence the free diffusion of PI in and out of the cells, we optimized the concentration of this dye (0.5 µg of PI per 10(6) cells) for minimal diffusion (less than 2%). Fluorescent cells were enumerated by hemocytometry and flow cytometry. Results showed that the survival rate of Sc during mixed cultures was high throughout the entire process (60% of viable cells at the 9th day), while Hg began to die off at the 2nd day, exhibited 98% of dead cells at the 3rd day (45 g/l of ethanol) and became completely unculturable at the 4th day. However, under single culture fermentation the survival rate and culturability of Hg decreased at a much slower pace, exhibiting at the 7th day (67 g/l of ethanol) 8.7×10(4) CFU/ml and 85% of dead cells. Thus, our work demonstrated that the LDS-FISH method is able to simultaneously assess the viability and identity of these wine-related yeast species during alcoholic fermentation in a fast and reliable way. In order to validate PI-staining as a viability marker during alcoholic fermentation, we evaluated the effect of ethanol on the membrane permeability of Sc and Hg cells, as well as their capacity to recover membrane integrity after being exposed to different levels of ethanol (1%, 6%, 10%, 12% v/v). Results showed that while Sc cells were able to recover membrane integrity after ethanol exposure, Hg cells were not. However, under alcoholic fermentation Sc cells didn't recover membrane integrity after the mid-term (4-5 days) of alcoholic fermentation.

Analysis and direct quantification of Saccharomyces cerevisiae and Hanseniaspora guilliermondii populations during alcoholic fermentation by fluorescence in situ hybridization, flow cytometry and quantitative PCR.

Traditionally, it was assumed that non-Saccharomyces (NS) yeasts could only survive in the early stages of alcoholic fermentations. However, recent studies applying culture-independent methods have shown that NS populations persist throughout the fermentation process. The aim of the present work was to analyze and quantify Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) populations during alcoholic fermentations by plating and culture-independent methods, such as fluorescence in situ hybridization (FISH) and quantitative PCR (QPCR). Species-specific FISH probes labeled with fluorescein (FITC) were used to directly hybridize Sc and Hg cells from single and mixed cultures that were enumerated by epifluorescence microscopy and flow cytometry. Static and agitated fermentations were performed in synthetic grape juice and cell density as well as sugar consumption and ethanol production were determined throughout fermentations. Cell density values obtained by FISH and QPCR revealed the presence of high populations (107-108 cells/ml) of Sc and Hg throughout fermentations. Plate counts of both species did not show significant differences with culture-independent results in pure cultures. However, during mixed fermentations Hg lost its culturability after 4-6 days, while Sc remained culturable (about 108 cells/ml) throughout the entire fermentation (up to 10 days). The rRNA content of cells during mixed fermentations was also analyzed by flow cytometry in combination with FISH probes. The fluorescence intensity conferred by the species-specific FISH probes was considerably lower for Hg than for Sc. Moreover, the rRNA content of Hg cells, conversely to Sc cells, remained almost unchanged after boiling, which showed that rRNA stability is species-dependent.

Use of interdelta polymorphisms of Saccharomyces cerevisiae strains to monitor population evolution during wine fermentation.

The industrial use of starter cultures containing a consortium of different strains from the same species is nowadays seen as a possible strategy to enhance the organoleptic complexity of wines. To assess the relative contribution of each strain to the final product it is essential to quantify population evolution during the wine fermentation process, which requires strain-specific methods to identify and differentiate each strain. In the present study, a molecular method based on analysis of the polymorphisms exhibited by the PCR-amplification of the delta regions of three Saccharomyces cerevisiae strains was developed. A set of three pairs of primers (delta1-delta2, delta12-delta2, delta12-delta21) was used for each strain, and analysis of the resulting polymorphism patterns showed that the delta12-delta2 primer pair exhibited the highest resolution and discriminatory power. Thus, this pair of primers was selected to monitor the population evolution of a laboratory-scale wine fermentation performed in synthetic grape juice that was inoculated with similar amounts of each strain. The results showed that all strains grew together during the exponential growth phase (2-3 days) and maintained high cell density values (10(6)-10(7) cfu ml(-1)) throughout the stationary growth phase without significantly changing their relative population proportion, thus indicating that each strain can influence the chemical composition and final flavor of wine, albeit at different levels. This study also showed that PCR-amplification of DNA delta sequences of S. cerevisiae strains is a reproducible, strain-specific and simple method that can be used successfully to monitor yeast strain population dynamics during wine fermentations.

Saccharomyces cerevisiae CCMI 885 secretes peptides that inhibit the growth of some non-Saccharomyces wine-related strains.

The nature of the toxic compounds produced by Saccharomyces cerevisiae CCMI 885 that induce the early death of Hanseniaspora guilliermondii during mixed fermentations, as well as their ability to inhibit the growth of other non-Saccharomyces wine-related strains, was investigated. The killing effect of mixed supernatants towards H. guilliermondii was inactivated by protease treatments, thus revealing the proteinaceous nature of the toxic compounds. Analysis of the protein pattern of mixed supernatants on Tricine SDS-PAGE showed that this S. cerevisiae strain secretes peptides (<10 kDa), which were detected only when death of H. guilliermondii was already established. Death-inducing supernatants were ultrafiltrated by 10 and 2 kDa membranes, respectively, and the inhibitory effect of those permeates were tested in H. guilliermondii cultures. Results indicated that the (2-10) kDa protein fraction of those supernatants seemed to contain antimicrobial peptides active against H. guilliermondii. Thus, the (2-10) kDa protein fraction was concentrated and its inhibitory effect tested against strains of Kluyveromyces marxianus, Kluyveromyces thermotolerans, Torulaspora delbrueckii and H. guilliermondii. Under the growth conditions used for these tests, the (2-10) kDa protein fraction of S. cerevisiae CCMI 885 supernatants exhibited a fungistatic effect against all the strains and a fungicidal effect against K. marxianus.

Physiological behaviour of Hanseniaspora guilliermondii in aerobic glucose-limited continuous cultures.

The physiology of Hanseniaspora guilliermondii was studied under aerobic glucose-limited conditions using the accelerostat procedure (continuous acceleration of dilution rate) and classical chemostat cultures. By both cultivation techniques this yeast was found to be Crabtree-positive. Up to a dilution rate of 0.25 h(-1), glucose was completely metabolised into biomass, glycerol and carbon dioxide. Above this value, an increase in the dilution rate was accompanied by the production of other metabolites like ethanol, acetic and malic acids. Biomass yield during the purely oxidative growth was 0.49 g g(-1) and decreased to 0.26 g g(-1) for D=0.34 h(-1). A maximal specific ethanol production rate of 1.36 mmol g(-1) h(-1) and a maximal ethanol yield of 0.05 g g(-1) were achieved at D=0.34 h(-1).