Stuck Fermentations Research Articles

A Future Place for Saccharomyces Mixtures and Hybrids in Wine Making

Each year, winemakers can face sluggish or stuck fermentations during wine making, especially when a spontaneous fermentation is performed, even if strains of the classical wine yeast Saccharomyces cerevisiae are applied. Problems are inevitable when low ammonium concentrations (<160 mg L−1 grape must) or an excess of fructose compared to glucose are observed during grape must fermentation. S. cerevisiae strains cannot use all kinds of amino acids as the sole nitrogen source but usually need free ammonium (optimal concentration: 600 mg L−1 grape must). It preferably consumes glucose, leading often to an excess of fructose in the fermenting must, which contains glucose and fructose in an equal ratio at the beginning of fermentation. Yeast hybrids have been isolated from wines several times and different strains are already commercially available. The united properties of the parent strains can provide advantages under sophisticated fermentation conditions. However, the involvement of a hybrid yeast for the rectification of fermentation disorders in spontaneous fermentations has only been described recently in the literature. Recent investigations have provided convincing evidence that fermentation problems can be overcome when must fermentations are successively performed with Saccharomyces bayanus strain HL 77 and the triple hybrid S. cerevisiae × Saccharomyces kudriavzevii × S. bayanus strain HL 78. The triple hybrid strain HL 78 uses amino acids as a nitrogen source in the absence of ammonium and it also exhibits a fructophilic character with an enhanced uptake of fructose in comparison to glucose. The application of genetically modified yeast strains is not allowed for starter cultures in wine making, but the usage of yeast mixtures and hybrid strains could be a promising tool for winemakers to solve fermentation problems during spontaneous fermentation or for the creation of novel wine types with desired sensory characteristics under more challenging conditions, especially when the composition of the must components is not optimal because of, e.g., critical climatic or soil conditions.

Resolving bacterial contamination of fuel ethanol fermentations with beneficial bacteria – An alternative to antibiotic treatment

Fuel ethanol fermentations are not performed under aseptic conditions and microbial contamination reduces yields and can lead to costly “stuck fermentations”. Antibiotics are commonly used to combat contaminants, but these may persist in the distillers grains co-product. Among contaminants, it is known that certain strains of lactic acid bacteria are capable of causing stuck fermentations, while other strains appear to be harmless. However, it was not previously known whether or how these strains interact one with another. In this study, more than 500 harmless strains of lactic acid bacteria were tested in a model system in combination with strains that cause stuck fermentations. Among these harmless strains, a group of beneficial strains was identified that restored ethanol production to near normal levels. Such beneficial strains may serve as an alternative approach to the use of antibiotics in fuel ethanol production.

Improvement of iturin A production in Bacillus subtilis ZK0 by overexpression of the comA and sigA genes.

This study provides new perspectives on improving iturin A production by Bacillus subtilis. Our newly engineered strains could be applied to commercial fermentation by enhancing yields of iturin A and reducing the rate of 'stuck fermentation'. Increased production would facilitate more widespread application of this powerful antibiotic.

Properties of a Wine Yeast Antagonist, Saccharomyces cerevisiae T206. A Review

Regardless of the type of killer yeast, winemaking may be adversely affected by a single type of killer yeast. In this review we present the properties of a single K2 strain, Saccharomyces cerevisiae T206, which was isolated from a stuck fermentation in a South African winery. This zymotidal strain has demonstrated its potential as a wine yeast antagonist and may be differentiated from other NCYC killer strains of S. cerevisiae on the basis of CHEF karyotyping and mycoviral RNA separations. Resolution of its genomic DNA into 13 chromosome bands, ranging from 0.2 to 2.2 Mb, has been reported. The resident viral-like particle in strain T206 yields pancreatic RNase-sensitive L and M double-stranded RNA species of 5.1 and 2.0 kb, respectively. The latter can be cured using cycloheximide to produce a K-R- derivative. In microscale vinifications the K2 toxin of strain T206 demonstrates a lethal effect on sensitive mesophilic wine yeasts over a narrow pH range of 3.2 to 3.5, disrupting the cell wall structure and perturbing cytoplasmic membranes. Contrary to known fermentation trends, the challenged fermentations are neither stuck nor protracted as strain T206 competes for substrates and induces over 65 % cell death in enriched Hanepoot grape juice media. Mucoid secretions of mesophilic wine yeasts, induced by nutrient limitation, appear to restrict the K2 killer effect. However, the supplementation of 1 to 100 ppm bovine submaxillary gland mucin in nutrientenriched grape juice media also depresses the K2 killer effect of strain T206 in mesophilic wine yeast starter culture strain, S. cerevisiae VIN7. Preliminary results suggest that mucoid secretions either affect the level of toxin production by strain T206 or block the cognate K2 receptor on the cell wall of challanged yeasts.

Selection of non-Saccharomyces yeasts to be used in grape musts with high alcoholic potential: a strategy to obtain wines with reduced ethanol content

Ethanol content of wine has increased over the last decades as consequence of searching phenolic maturity, requiring increased grape maturity. This may result in the production of wines with excessive alcohol levels (sometimes more than 15% (v/v)), sluggish and stuck fermentations and excessive volatile acidity. Many strategies to reduce ethanol in wines are being studied, and microbial methods have some additional advantages. However, because of the broad intra- and interspecies variability, new selection criteria should be included. Therefore, the goal of the present work was to design and evaluate a simple and integral procedure for non-Saccharomyces yeast selection. This strategy allowed selection of yeasts that presented successful implantation in grape must with high alcohol potential and their use in co-cultures could reduce the ethanol in wines. A total of 114 native non-Saccharomyces yeasts were assayed to determine their respiratory, fermentative and physiological characteristics of enological interest. Hanseniaspora uvarum BHu9 and BHu11, H. osmophila BHo51, Starmerella bacillaris BSb55 and Candida membranaefaciens BCm71 were selected as candidates to design co-culture starters.

Microbial Succession in Spontaneously Fermented Grape Must Before, During and After Stuck Fermentation

The microbial succession in spontaneously fermenting Riesling must was investigated from the beginning (pressing) until the end (sulphuring) of the fermentation in two harvest years (2008 and 2009) at a Moselle winery (Germany). In both years, the fermentation was interrupted by a stuck period. The length of the stuck period varied considerably (20 weeks in 2008 and one week in 2009). Different yeasts (Candida, Debaryomyces, Pichia, Hanseniaspora, Saccharomyces, Metschnikowia, Cryptococcus, Filobasidium and Rhodotorula) and bacteria (Gluconobacter, Asaia, Acetobacter, Oenococcus, Lactobacillus, Bacillus and Paenibacillus) were isolated successively by plating. The main fermenting organism was Saccharomyces uvarum. Specific primers were developed for S. uvarum, H. uvarum and C. boidinii, followed by the determination of the total cell counts with qPCR. The initial glucose concentration differed between the two years and was 116 g/L in 2008 and 85.4 g/L in 2009. Also, the fructose concentrations were different in both years (114 g/L in 2008 and 77.8 g/L in 2009). The stuck period appeared when the glucose/fructose ratio was 0.34 and 0.12 respectively. The microbiota changed during the stuck period.

Front cover: Stuck at work? Quantitative proteomics of environmental wine yeast strains reveals the natural mechanism of overcoming stuck fermentation

Front cover: Stuck at work? Quantitative proteomics of environmental wine yeast strains reveals the natural mechanism of overcoming stuck fermentation

The use of dextrose in winemaking

In this work the benefit of using dextrose instead of sucrose was investigated. Trials with stuck fermentation could show the potential of compensating glucose / fructose imbalances by adding dextrose. Trials on sparkling wine production showed that under stressful conditions the second fermentation started earlier and finished more complete, when dextrose was used instead of sucrose in the tirage liqueur. In general the sensory property of the later product was not changed when dextrose was used as alternative to sucrose. Several trials on must chaptalization showed in general no clear differences in terms of fermentation kinetics. The aroma analysis showed as well, that the fermentation aroma was not clearly different when dextrose used as sugar source.

Biofilm formation and ethanol inhibition by bacterial contaminants of biofuel fermentation

Bacterial contaminants can inhibit ethanol production in biofuel fermentations, and even result in stuck fermentations. Contaminants may persist in production facilities by forming recalcitrant biofilms. A two-year longitudinal study was conducted of bacterial contaminants from a Midwestern dry grind corn fuel ethanol facility. Among eight sites sampled in the facility, the combined liquefaction stream and yeast propagation tank were consistently contaminated, leading to contamination of early fermentation tanks. Among 768 contaminants isolated, 92% were identified as Lactobacillus sp., with the most abundant species being Lactobacillus plantarum, Lactobacillus casei, Lactobacillus mucosae, and Lactobacillus fermentum. Seven percent of total isolates showed the ability to form biofilms in pure cultures, and 22% showed the capacity to significantly inhibit ethanol production. However, these traits were not correlated. Ethanol inhibition appeared to be related to acetic acid production by contaminants, particularly by obligately heterofermentative species such as L. fermentum and L. mucosae.

Evolutionary engineering of a wine yeast strain revealed a key role of inositol and mannoprotein metabolism during low-temperature fermentation.

BackgroundWine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes.ResultsWe used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 °C. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1Thr108) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins.ConclusionsIn this study we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1755-2) contains supplementary material, which is available to authorized users.

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation.

At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu2+ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu2+ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu2+ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.

The ICY1 gene from Saccharomyces cerevisiae affects nitrogen consumption during alcoholic fermentation

article i nfo Background: Saccharomyces cerevisiae is the main microorganism responsible for alcoholic fermentation. In this process, the consumption of nitrogen is of great importance since it is found in limiting quantities and its deficiency produces sluggish and/or stuck fermentations generating large economic losses in the wine-making industry. In a previous work we compared the transcriptional profiles between genetically related strains with differences in nitrogen consumption, detecting genes with differential expression that could be associated to the differences in the levels of nitrogen consumed. One of the genes identified was ICY1. With the aim of confirming this observation, in the present work we evaluated the consumption of ammonium during the fermentation of strains that have deleted or overexpressed this gene. Results: Our results confirm the effect of ICY1 on nitrogen uptake by evaluating its expression in wine yeasts during the first stages of fermentation under low (MS60) and normal (MS300) assimilable nitrogen. Our results show that the mRNA levels of ICY1 diminish when the amount of assimilable nitrogen is low. Furthermore, we constructed strains derived from the industrial strain EC1118 as a null mutant in this gene as well as one that overexpressed it.

Physicochemical property changes during the fermentation of longan (Dimocarpus longan) mead and its aroma composition using multiple yeast inoculations

Mead is a traditional drink obtained from diluted honey with the appropriate yeast inoculations. However, the production of mead is time-consuming, and the process often takes several months to accomplish. During the elaboration of mead, sluggish and stuck fermentations often occur and lead to the production of undesirable flavours. To improve the condition of longan mead production in Taiwan, multiple yeast inoculations with different strains of yeast were examined in this study. The physicochemical properties and mead aroma compounds were measured. Longan mead without multiple inoculations, but with added yeast nutrients, displayed a sharp increase in ethanol content compared with the inoculated groups without yeast nutrients. However, the physicochemical properties of every mead reached almost the same endpoint at the end of the fermentation time (day 22). Moreover, differences among the groups were observed in their aroma compounds. Some aroma compounds, such as 2-methyltetrahydrothiophen-3-one, 1-propanol and 2-methylbutyl acetate, were only found in the double-inoculation groups, whereas other aroma compounds, such as phenethyl acetate and linalool, were only found in the single-yeast inoculation groups. These results demonstrate that multiple inoculations have a positive effect on longan mead fermentation. This technique could assist local producers in elaborating a longan mead of acceptable quality. Copyright © 2013 The Institute of Brewing & Distilling

Identification of a yeast strain as a potential stuck wine fermentation restarter: a kinetic characterization

Restarter yeast strains are used to consume residual sugar (mainly fructose) in stuck wine fermentations. Forty-three yeast strains were evaluated initially. Strain ITD-00068 showed the highest values for the maximum growth rate, maximum fructose consumption rate, and maximum ethanol production rate (0.143, 0.268, and 0.231 h−1, respectively). Therefore, it was selected for further molecular and kinetic analyses. RFLP analysis identified this isolate as Saccharomyces cerevisiae. Strain ITD-00068 consumed 100% and 36% of the fructose present (at 20°C and 30°C, respectively), when cultured in medium with 12% (v/v) ethanol. The fructose consumption rate was reduced by 97% at 30°C, when ethanol was initially present in the media. S. cerevisiae ITD-00068 was tested in stuck fermentation conditions and was able to complete fermentation in 144 h in a commercial red wine, demonstrating that S. cerevisiae ITD-00068 is a potential candidate for use as a restarter in wine stuck fermentation at 20°C.

Metabolomic Comparison of Saccharomyces cerevisiae and the Cryotolerant Species S. bayanus var. uvarum and S. kudriavzevii during Wine Fermentation at Low Temperature

Temperature is one of the most important parameters affecting the length and rate of alcoholic fermentation and final wine quality. Wine produced at low temperature is often considered to have improved sensory qualities. However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase, and sluggish or stuck fermentations. To investigate the effects of temperature on commercial wine yeast, we compared its metabolome growing at 12°C and 28°C in a synthetic must. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae. This is the case of the cryotolerant yeasts Saccharomyces bayanus var. uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the metabolome of these species growing at 12°C, which we compared with the metabolome of S. cerevisiae (not well adapted at low temperature) at the same temperature. Our results show that the main differences between the metabolic profiling of S. cerevisiae growing at 12°C and 28°C were observed in lipid metabolism and redox homeostasis. Moreover, the global metabolic comparison among the three species revealed that the main differences between the two cryotolerant species and S. cerevisiae were in carbohydrate metabolism, mainly fructose metabolism. However, these two species have developed different strategies for cold resistance. S. bayanus var. uvarum presented elevated shikimate pathway activity, while S. kudriavzevii displayed increased NAD+ synthesis.

Phenotypic analysis of mutant and overexpressing strains of lipid metabolism genes in Saccharomyces cerevisiae: Implication in growth at low temperatures

The growing demand for wines with a more pronounced aromatic profile calls for low temperature alcoholic fermentations (10–15°C). However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase and sluggish or stuck fermentations. The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. The aim of this study was to detect lipid metabolism genes involved in cold adaptation. To do so, we analyzed the growth of knockouts in phospholipids, sterols and sphingolipids, from the EUROSCARF collection S. cerevisiae BY4742 strain at low and optimal temperatures. Growth rate of these knockouts, compared with the control, enabled us to identify the genes involved, which were also deleted or overexpressed in a derivative haploid of a commercial wine strain. We identified genes involved in the phospholipid (PSD1 and OPI3), sterol (ERG3 and IDI1) and sphingolipid (LCB3) pathways, whose deletion strongly impaired growth at low temperature and whose overexpression reduced generation or division time by almost half. Our study also reveals many phenotypic differences between the laboratory strain and the commercial wine yeast strain, showing the importance of constructing mutant and overexpressing strains in both genetic backgrounds. The phenotypic differences in the mutant and overexpressing strains were correlated with changes in their lipid composition.

Very high gravity ethanol fermentation by flocculating yeast under redox potential-controlled conditions

BackgroundVery high gravity (VHG) fermentation using medium in excess of 250 g/L sugars for more than 15% (v) ethanol can save energy consumption, not only for ethanol distillation, but also for distillage treatment; however, stuck fermentation with prolonged fermentation time and more sugars unfermented is the biggest challenge. Controlling redox potential (ORP) during VHG fermentation benefits biomass accumulation and improvement of yeast cell viability that is affected by osmotic pressure and ethanol inhibition, enhancing ethanol productivity and yield, the most important techno-economic aspect of fuel ethanol production.ResultsBatch fermentation was performed under different ORP conditions using the flocculating yeast and media containing glucose of 201 ± 3.1, 252 ± 2.9 and 298 ± 3.8 g/L. Compared with ethanol fermentation by non-flocculating yeast, different ORP profiles were observed with the flocculating yeast due to the morphological change associated with the flocculation of yeast cells. When ORP was controlled at −100 mV, ethanol fermentation with the high gravity (HG) media containing glucose of 201 ± 3.1 and 252 ± 2.9 g/L was completed at 32 and 56 h, respectively, producing 93.0 ± 1.3 and 120.0 ± 1.8 g/L ethanol, correspondingly. In contrast, there were 24.0 ± 0.4 and 17.0 ± 0.3 g/L glucose remained unfermented without ORP control. As high as 131.0 ± 1.8 g/L ethanol was produced at 72 h when ORP was controlled at −150 mV for the VHG fermentation with medium containing 298 ± 3.8 g/L glucose, since yeast cell viability was improved more significantly.ConclusionsNo lag phase was observed during ethanol fermentation with the flocculating yeast, and the implementation of ORP control improved ethanol productivity and yield. When ORP was controlled at −150 mV, more reducing power was available for yeast cells to survive, which in turn improved their viability and VHG ethanol fermentation performance. On the other hand, controlling ORP at −100 mV stimulated yeast growth and enhanced ethanol production under the HG conditions. Moreover, the ORP profile detected during ethanol fermentation with the flocculating yeast was less fluctuated, indicating that yeast flocculation could attenuate the ORP fluctuation observed during ethanol fermentation with non-flocculating yeast.

Identification of genes related to nitrogen uptake in wine strains of Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is the main microorganism responsible for wine fermentation and its development influences the quality of wine. A problem affecting these types of fermentations, generating important losses in this industry, are the slow or stuck fermentations which may result from low nitrogen availability in the must. Therefore, several studies have been directed towards identifying genes involved in nitrogen metabolism using high throughput strategies which include subjecting the yeast to changes in the type or concentration of the available nitrogen source. However, this type of approach can also generate responses in the yeast that do not necessarily alter the expression of genes related to nitrogen metabolism. In this work, by using intraspecific hybridisation of wild wine yeast strains we obtained genetically and oenologically similar strains with differences in the consumption of nitrogen sources. Using the same must, the global expression patterns of these yeasts were compared by microarrays, the analysis of which identified 276 genes that varied in their expression between the strains analysed. The functional analysis of the genes with a known function indicates that some participate in nitrogen metabolism, alcoholic fermentation, ion transport and transcriptional regulation. Furthermore, differences were observed in the expression of genes which have been partially associated to nitrogen, as in the case of ZRT1 and ATO2. Interestingly, many of the genes identified have no known function or have not been previously associated to this phenotype.

English

Yeasts used in fermentation processes are subject to different stress conditions, including low pH, high initial substrate concentration, accumulation of toxic compounds (e.g. ethanol), and temperature fluctuations. The fermentation of mescal is conducted under variable environmental conditions throughout the year. One of the most important environmental factors is temperature, as autumn and winter fermentations occur at low temperatures, but fermentation temperatures are much higher in summertime. The aim of this work was to compare the response of Saccharomyces cerevisiae strains isolated from agave fermentations to different stress conditions and two different medium culture one mimicking mezcal production, using agave must and other synthetic must development for analysis in wine industry. The strains isolated from agave were compared with a commercial strain used in wine elaboration, which exhibits good tolerance to the different stresses found in industrial fermentations. All strains grew in the presence of glucose and fructose irrespective of the sugar concentration, and low pH did not affect the formation of colonies. Differences in growth were observed among the strains at low temperature and high concentrations of ethanol. Only 28% of the tested strains exhibited good tolerance to high ethanol concentrations, a desirable trait for avoiding stuck fermentations. The strain ITD00185 was able to grow in alcoholic stress condition, consuming sugar and producing ethanol in agave must. This strain shows tolerance to the different stress conditions and may be a useful starter culture for agave fermentation, the potential of the native strains to be used to improve other industrial fermentation processes that involve low temperatures and high ethanol yields. Key words: Ethanol, tolerance, Mescal, native strains.

Reassessment of phenotypic traits for Saccharomyces bayanus var . uvarum wine yeast strains

Among Saccharomyces yeast, S. cerevisiae and S. bayanus var. uvarum are related species, sharing the same ecosystem in sympatry. The physiological and technological properties of a large collection of genetically-identified S. bayanus var. uvarum wine strains were investigated in a biometric study and their fermentation behavior was compared at 24 °C and 13 °C. The variability of the phenotypic traits was considered at both intraspecific and interspecific levels. Low ethanol tolerance at 24 °C and production of high levels of 2-phenylethanol and its acetate were clearly revealed as discriminative technological traits, distinguishing the S. bayanus var. uvarum strains from S. cerevisiae. Although some S. bayanus var. uvarum strains produced very small amounts of acetic acid, this was not a species-specific trait , as the distribution of values was similar in both species. Fermentation kinetics at 24 °C showed that S. bayanus var. uvarum maintained a high fermentation rate after Vmax, with low nitrogen requirements, but stuck fermentations were observed at later stages. In contrast, a shorter lag phase compared with S. cerevisiae, higher cell viability, and the ability to complete alcoholic fermentation at 13 °C confirmed the low-temperature adaptation trait of S. bayanus var. uvarum. This study produced a phenotypic characterization data set for a collection of S. bayanus var . uvarum strains, thus paving the way for industrial developments using this species as a new genetic resource.